UA68345C2 - A method for pest control of the plants - Google Patents

Abstract

The invention concerns a receptor to vegetative insecticide protein of a VP3-class, sequence of DNA which codes the receptor, an antibody to a receptor, a method of identification of homologues of the receptor, a method of identification of compound as chemical ligand of the receptor, a method of pest control of the plants, including use of chemical ligand or antibody to the receptor.

Description

Description of the invention

The present invention relates to a new class of proteins for controlling plant pests. 2 Plant pests are a major cause of loss of commercially important crops worldwide, leading to both economic hardship for farmers and malnutrition for local populations in many parts of the world. A wide variety of chemical pesticides are widely used in agriculture to fight pests and destroy them. However, there is also considerable interest in the development of effective alternative pesticides. 70 The fight against various pests through the use of biological molecules is possible only in a limited number of cases. The best known examples of biomolecules for pesticidal use are 3-endotoxins from Bacillus sp. Ipygypdiapsis (VO), which is a gram-positive, spore-forming microorganism.

Vi varieties are known to produce more than 25 different but related 5-endotoxins. Vi strains produce 5-endotoxins during sporulation, the use of which is limited because they are active only against a very limited number of many insect pests.

The limited specificity of B-endotoxins depends, at least in part, both on the activation of the toxin in the insect's body (Naideg, M. 7. ei ai.,, 1986, Eig. U. Viospet. 156:531-540), and on its ability to bind to specific receptors present in the epithelial cells of the midgut of insects (Noitapp, S.R., 1988, RMAB 85:7844-7848). Thus, the ability to control specific insect pests through the use of 5-endotoxins currently depends on the ability to find a suitable 5-endotoxin with the desired spectrum of activity. In many cases, such 5-endotoxin is unknown, and it is not even possible to say with certainty whether it exists at all.

Plants are also commonly infected by fungi and bacteria, and many species of microorganisms have evolved the ability to exploit the various niches provided by growing plants. In addition to infection by fungi and bacteria, many plant diseases are caused by nematodes that live in the soil and infect the roots, usually causing serious damage when one type of crop is cultivated for several years in a row on the same plot of land.

The severity of the destructive process caused by the disease depends on the aggressiveness of the phytopathogen and the host, and one of the goals of most plant breeding programs is to increase the resistance of the host plant to the disease. New sources and combinations of genes designed to provide disease resistance usually have only a limited period of successful application in many crop-pathogenic systems due to the rapid development of phytopathogens that overcome resistance genes.

Thus, it becomes clear that scientists must constantly be in search of new ways to protect the environment

Зз5 Cultures from plant pests. The present invention has identified a new class of proteins that can be used to control plant pests.

Programmed cell death is a process in which evolutionary stimuli or the environment activate a genetic program that culminates in cell death (dasorzop, M.O. et al., 1997, Sei! 88: 347-354). This genetic potential exists in most, if not all, multicellular organisms. In the case of "invertebrates, programmed cell death plays a dual role, being an integral part of both the process of plant development and the mechanism of response to infections, in particular, those of viral origin (Siet, K.). et al.,, 1991, Science 254: 1388-1390). Programmed cell death occurs in several "" different ways, leading to apoptosis, atrophy, or differentiation. Apoptosis is one of the best characterized types of programmed cell death, which encompasses cytological changes including

Membrane-bound 3 apoptotic bodies and cytoplasmic vesicles, as well as molecular changes such as

Ge") endonucleolysis, which is characterized by the formation of fragments of oligosomal length (Mah, O.I apa 5igazzeg,

A. 1996, RMAB5 93:2239-2244). | although the general apoptotic phenomenology is almost invariant in different

Among organisms, it is interesting to note that for many insect cells cytoplasmic vacuolization and d) swelling, rather than condensation, are cytological features associated with apoptotic processes (Vouep, iz 50 I.O., ei aiYi., 1996, Misgo5. Kevs. Thesppids. 34:202-217). It was found that the new class of proteins described in this invention causes programmed cell death and has a pesticidal effect. sl The present invention relates to MIRZA(c) proteins, including their homologues. Also provided by the invention are domains of MIRZ-class proteins, including a toxic domain and a stabilizing domain. The best option according to the invention is the toxic domain of the MIRZA(a) protein and their homologues. Another optimal option is antibodies to MIRZ-class proteins, better - to MIRZA(c)-protein.

The invention also provides hybrid toxins comprising the toxic domain of a MIRZ-class protein. In the iF) optimal variant, the hybrid toxin is a chimeric protein having a toxic core domain that is functionally connected to a heterologous stabilizing domain. In another optimal variant, the hybrid toxin includes an antibody or an immunologically active fragment thereof, which is immunologically recognized by the MIRZ receptor because it is functionally connected to the toxic domain of other proteins, and the toxin domain is derived from many cytotoxic proteins, including, but not limited to, Vasiy5 toxins, including endotoxins and vegetative insecticidal proteins.

The invention also encompasses plants comprising a DNA sequence encoding a protein of the MIRZ class, preferably -

MIRZA(c)-protein. Preferably, these are plants selected from the group consisting of corn, sorghum, wheat, sunflower, tomatoes, various types of cabbage, cotton, rice, soybeans, sugar beet, sugar cane, tobacco, barley, and canola. In the best version, the plant is corn.

The invention also provides microorganisms that include a heterologous DNA sequence that encodes a protein

MIRZ-class, better - MIRZA(c)-protein. In the optimal version, the microorganism is selected from the group consisting of bacteria, baculoviruses, algae and fungi. In another optimal variant, the microorganism is selected from the group consisting of Vasia5, Rzepdotopaz, Siamirasieg and Kpig2gobisht. The invention also covers entomocidal compositions that include microorganisms that include a heterologous DNA sequence that encodes a MIRZ-class protein, preferably a MIRZA(c)-protein.

The invention also relates to plants and microorganisms that also include a second DNA sequence that encodes a second insecticidal protein. Particular preference is given to second DNA sequences that encode 70 b-endotoxin, that encode another protein of the MIRZ class, or that encode a protein of the MIR1 or MIR2 classes. In an even better embodiment, the 5-endotoxin is active against an insect selected from the group consisting of Eridorieg and Soieorieg.

In a preferred embodiment, the 5-endotoxin is active against Ozyhipia or Oiabgoiis. In another preferred embodiment, the second DNA sequence encodes a 5-endotoxin protein selected from the group consisting of Stu!, Stu3, Stub and Stud. In an even better version, the 5-endotoxin is selected from the group consisting of StuAa, StuAB, StuAs, 75 Stu1vB, Stu1C, Stu10O, StuEa, StuRa, StuA. Stgu9A, Stub9S and Stu9V. 5-endotoxins selected from the group consisting of SgtutAb, Stu1Va and Stu9s proteins are considered the best.

The invention also provides a method of controlling insects by contacting insects with an insecticidal amount of a MIRZ-class protein, preferably a MIRZA(c)-protein, or an insecticidal amount of a chemical ligand with a MIRZ-class protein receptor. In one optimal variant, the insects are contacted with a transgenic plant that includes a DNA sequence that expresses a MIRZ-class protein, preferably a MIRZA(c) protein. (c)-protein, or including a DNA sequence that expresses a MIRZ-class protein, preferably -

MIRZA(c)-protein. In yet another optimal variant, the transgenic plant includes a DNA sequence that expresses the MIRZA(a) protein. In another optimal variant, the insect is selected from the group consisting of So|Ieoriega, Oiriega, Nuteporiega, I eridoriega, MaiPorpada, Notoriega, Neptiriega, Ohipgoriega, Tpuzaporiega, o

Oeptariega, Isoriega, Aporishga, Zirpopariega, Tgisporiega and Asagi. In the best version, the insect belongs to

SoiIeoriega or I eridoriega. In another best option, the insect is selected from the group consisting of the epsilon bollworm (Aadgoiis irvip), the autumn migration worm (Zrodoriega ytydiregda), the ground bollworm (5. ehidtsa), the yellow-striped migration worm (5. oti(podaiii ), the southwestern corn butterfly (Oiaigaea is) dgapaiiosePa), sugar cane fireflies (0. sasspagaiiiv), scoops (Neiissomegra 7gea), the Mediterranean corn butterfly (Bzezatia popadgoiidevs), nii scoops (Tysporiivzia pi), scoops (Apiiiisagvia dettaiiiii5) , cabbage moth (Rishega huiosiea) and tobacco moth (Neioipiz migezsepv). co

The invention also provides a method of insect control, according to which the transgenic plant or co-organism also includes a second DNA sequence that encodes a second insecticidal protein like those mentioned above. (se)

The invention also provides recombinant DNA sequences that encode the MIRZA(c) protein, including their homologues. In another preferred embodiment, the DNA sequence is a synthetic sequence that has been modified for optimal expression in a plant, particularly in cases where the DNA sequence has been optimized for expression in maize. Preference is also given to DNA sequences that include both a synthetic part and a natural part. Preferably, the DNA sequence encoding the MIRZA(c) protein has been optimized for tc expression in maize. Another optimal option is DNA sequences that are homologous sequences of the DNA encoding the MIRZA(c) protein. In particular, preference is given to DNA sequences that, under moderately stringent » conditions, hybridize with the mrzZA(c) coding sequence. Another embodiment of the invention is a recombinant DNA sequence that expresses a MIRZ-class protein, preferably a MIRZA(c) protein, under the control of a heterologous promoter, or when the coding region is included in the genome of an organism in which it is not naturally expressed, or ( 2) is expressed at a higher level than natural. o The invention also relates to the method of identification and isolation of homologues of the MIRZA(c) protein, or the DNA sequence that encodes the above-mentioned protein. o The invention also provides for expression cassettes that include a promoter functionally connected to the 20th DNA sequence that encodes a protein of the MIRZA class, preferably a MIRZA (c) protein. In one of the optimal options, the promoter is selected from the group consisting of the main optimal for certain tissues and tissue-specific promoters for expression in plants. In the best version, the promoter is selected from the group consisting of ubiquitin, PEP-carboxylase (phosphoenolpyruvate carboxylase), I RT and MT. promoters In another optimal variant, the promoter functions in a microorganism. 22 The invention also provides a receptor for a MIRZ-class protein and a DNA sequence. In one version

Gee! the receptor of the invention includes a death domain and a repetitive ESE motif. In an even better version of the invention, it is a receptor for MIRZA(a). The best option is the sequence of the receptor protein shown in ZEO IU MO:9, ko and its homologues. The invention also covers DNA sequences that encode these receptor proteins, for example, the DNA sequence shown in 5EBEO IJOO MO:8 and its homologues. The cDNA for the MIRZ receptor is contained in plasmid 60 pSIV7113, which was deposited under the Budapest Agreement in the Collection of Patent Crops of the Agricultural Research Service (IKK)

Mogpyegp Kediope! Kezeagsp Sepieg, 1815 Mogii Opimegeiu Zigeei, Reogia, Shipoise 61604, O5BAI) March 29, 1997. under inventory number B-21676. The invention also covers antibodies to the MIRZ-class protein receptor.

The invention also provides for a method of identifying a compound as a MIRZ receptor chemical ligand that has pesticidal activity, which includes subjecting a cell, preferably an insect cell, to the action of a test compound and analyzing the aforementioned cell for apoptotic activity. In another variant of the invention, the method includes measuring the specific binding between the MiIRZ receptor and the test compound. The best option is

MIRZ receptor ligands identified by this method.

Definition "Plant pest" means any organism associated with plants which, as a result of this association, causes harmful effects on the health and vitality of plants. Plant pests include, but are not limited to, fungi, bacteria, insects, and nematodes. As used by us, the term "plant" includes whole plants and parts of plants, such as roots, stems, leaves and seeds, as well as cells and tissues in plants or parts of plants. The "MIRZ class of proteins" includes MIRZA(a), MIRZA(B), MIRZA(c) and their homologues. The term "tomolog" throughout the text means that the specified protein or polypeptide has a certain defined relationship to other representatives

MIRZ-class proteins. This defined relationship includes, but is not limited to, 1) proteins that are at least 70,905, better - 8,095, best - 9,095 identical at the sequence level to another member of the MIRZ class of proteins, while also retaining pesticidal activity, 2) proteins that enter in a cross-reaction with antibodies that immunologically recognize another representative of the MIRZ-class of proteins, C) proteins that enter into a 7/5 cross-reaction with the receptor of another representative of the MIRZ-class of proteins and retain the ability to cause programmed cell death and 4) proteins that at least at 7095, better - at 8095, best - at 9095 are identical at the sequence level to the toxic central region of another representative of the MIRZ class of proteins, while also retaining pesticidal activity.

The term "hybrid toxin" is used to denote a genetic fusion that has domains that are functionally linked in such a way that, upon translation, a functional chimeric protein is formed that generally has the properties of the individual domains. The term "domain" is used to denote an area or part of a protein that provides a function that can be recognized, or a structure that participates in the overall functionality of the protein. It is recognized that this definition also covers a DNA sequence that encodes a protein domain.

The term "heterologous" is used to indicate that a protein, polypeptide or nucleotide sequence has a different natural origin in relation to a given host. For example, if the gene mrzAda) with

Vasiyiv (pygipdiepsis is genetically transformed into a plant cell, the gene is described as heterologous in relation to its current host, which is a plant cell). In addition, if the mrzZA(a) gene from Vasiyya5

Ipyhypdiepsis is genetically transformed into the bacterium Rzeidotopaz, so the gene is also described as heterologous in relation to Rzeidotopaz. The term "heterologous" is also used to indicate that one or more domains present in a chimeric protein, polypeptide or nucleotide sequence differ in nature from other domains present. For example, if the toxic domain from the MIRZ "

MIRZAT(a) protein is fused to the binding domain of MIR1TA(a) protein to create a functional c insecticidal protein, the chimeric fusion will have domains that are heterologous to each other. In addition, a heterologous chimeric protein or polypeptide comprising a fusion of the toxic domain from the MIRZA(a) protein with the co-binding domain from the MIR1TA(a) protein, which, when expressed in a plant, should also be considered "heterologous in relation to the host plant.

The term "chimeric" is used to indicate that a protein, polypeptide or nucleotide sequence consists of domains, at least one of which has an origin that is heterologous to the other domains present. These chimeric proteins or polypeptides are encoded by chimeric nucleotide sequences that have been fused or ligated, resulting in a coding sequence that does not occur in nature. Such fancy designs can also be called "recombinant". . The term "expression cassette" used by the authors means a DNA sequence capable of directing gene expression in plant cells, including a promoter operably linked to an amino acid coding region operably linked to a terminal region. A gene can be chimeric, meaning that at least one component of the gene is heterologous with respect to at least one other component of the gene. Gen too

He" can occur in nature, but it must be obtained in a recombinant form, which is used for genetic transformation of a plant or microorganism. Figure 1: The amino acid sequence of the receptor for MIRZA(a) is translated from cDNA. Some 2) features of the protein are shown: dotted line - signal peptide (amino acids from 13 to 35); italics - the domain that 5p fills the hypothetical death domain (amino acids 81-205); underlining with a double line - sequences with significant homology to sequences that contain generalizing domains of death; bold font - SKO-motive, sp which is repeated six times, filling ESE-motives; underlining with one line - sequences that are repeated in ESE motifs.

Arthropod Pests 5B In terms of the present invention, pests include insects and arachnids selected from

SoiIeoriega, Oiriega, Nuteporiega, I eridoriega, MaiIorpada, Notoriega, Neptiriega, Opipgoriega, Tpuzaporiega,

F) Regtauriega, Isoriega, Aporishga, Zirpopariega, Tgisporiega and Asai, in particular, Soieoriega and I eridoriega. The list of pests of the main crops is given, for example, in Tables 1-10 on pages 13 to 20 of MO 96/10083, to which we refer. Other pests are listed in Tables 1-8 below. Such boron pests are included in the scope of this invention. b5

Table 1: and eriyoriega (butterflies and moths)

Maize Cruciferous (broccoli, cabbage, Bezatia popadgoides, butterfly cabbage, fodder cabbage) corn Mediterranean Alpiodeia garae, mermitida

O5ipipia Pythasaiyi5, butterfly Viegih bgazzisae, white cabbage corn Asian Tpsporishzia pi, scoop no 0 Richeia huyuzieya, cabbage moth

Cotton Zroayoriega ehidtsa, ground scoop

Neiissochuegra aptidega, sovka Adtgoiiyiv irzyop, sovka epsilon cotton Adgoiiz zedegt, sovka winter

Mateziga sopidiga, sovka berta

Fig

So zirrgevzvaiiv, oligurikarisova // Asian grape, Epdoriga uyeapa, leafy zeirornpada vr. grape

Tomatoes Fruits and nuts of deciduous trees

Neiissomegra hea, Sudia rotopeia fruit-eater

Zrodoriega ipidiregda, autumn Riaiupoia kizeizaiyiv, pistovyka o derived apple worm

Zroyoriega otiipodaiiii, derived yellow-striped worm Pepper yu zZroborivga rgaveisa, derived Ozyhipia pibiyaiv, butterfly « yellow-striped western European corn worm

Zrodorivga epdapia, derivative Zrodorivga echidna, ground scoop about June as southern Zroyoriega epdapia, derivative so (Se) - p. a (o) (ee) (95) d» sl ime) bo b5

Adgoiiz irbyop, scoops epsilon worm southern

Repatta zashsia, scurvy scoop

Raraireta pen, scoop Kk artoplia

Tpenoriivia pi, sovka ni i

Keyepa usoregvisena, Ovilipia pyrya moth/lv5, European corn butterfly

Mapdysa zechia, the brazier of Sapoia

Mapaisa diipdietasiiaia, brawny ; Dila and RIshepya huiovivia, five-spotted cabbage moth

RIitonptava oregoshietsa, potato moth

Cane

Riaigava zassnagaiiyiv, a fire of sugar cane

Table 2: So/eoriega (Beetles)

Potato rice

Oyeta ogugaye, rice beetle and eriipogagza desetiipeaa, Colorado potato beetle sch

Tomatoes Erich sisytep5, flea beetle o and eriipoiagza deseti/peagia, potato Colorado potato beetle Netisteriiye tetpopiiy5, larvae

Erynkh pipireppiv, beetle-flea of blacksmiths o sherekhata Meiaproish" 5yr., larvae of blacksmiths "

Cruciferous (broccoli, cabbage, Sapoia with cauliflower, fodder cabbage)

RNuioyigeiga stisieegae, flea hidden seed cabbage so cruciferous REuyoigeia sgispegae, flea

Rpupoigeiga rizya, black cruciferous western flea «

Pepper and -

Aptpopoti»h eijdepii, weevil with pepper. a (o) (ee) (95) d» sl ime) bo b5

Adgoiiz irbyop, scoops epsilon worm southern

Repatta zashsia, scurvy scoop

Raraireta pen, scoop Kk artoplia

Tpenoriivia pi, sovka ni i

Keyepa usoregvisena, Ovilipia pyrya moth/lv5, European corn butterfly

Mapdysa zechia, the brazier of Sapoia

Mapaisa diipdietasiiaia, brawny ; Dila and RIshepya huiovivia, five-spotted cabbage moth

RIitonptava oregoshietsa, potato moth

Cane

Riaigava zassnagaiiyiv, a fire of sugar cane

Table 2: So/eoriega (Beetles)

Potato rice

Oyeta ogugaye, rice beetle and eriipogagza desetiipeaa, Colorado potato beetle sch

Tomatoes Erich sisytep5, flea beetle o and eriipoiagza deseti/peagia, potato Colorado potato beetle Netisteriiye tetpopiiy5, larvae

Erynkh pipireppiv, beetle-flea of blacksmiths o sherekhata Meiaproish" 5yr., larvae of blacksmiths "

Cruciferous (broccoli, cabbage, Sapoia with cauliflower, fodder cabbage)

RNuioyigeiga stisieegae, flea hidden seed cabbage so cruciferous REuyoigeia sgispegae, flea

Rpupoigeiga rizya, black cruciferous western flea «

Pepper and -

Aptpopoti»h eijdepii, weevil with pepper. a (o) (ee) (95) d» sl ime) bo b5

Table 3: Notorivga (Aleurodids, aphids, etc..)

Rice Melon

Miparaguaia Indepe Vetivia agdepiyoii, white-winged bodaiea Tshtsyega Vetisia habasi, white-winged batatova and aode!rpnaah zipageniv

Carrots 70 Tomatoes SamayenNa aedorosiiii, aphid

Mutiz regsisae, peach-carrot aphid

Musgovirpyt eirpogiiae, potato aphid Sapoia

Tpivygodez uarogapogyt, white-winged Viemisogupe bgazvaye, greenhouse cabbage aphid

Vetivia yarasi, sweet potato whitefly

Vetivia apuepinoii, bicarpica Vegetables

Cruciferous (broccoli, cabbage, cauliflower, aphid bean cabbage, fodder cabbage)

Vhemisogupe rgazvzisae, aphid Sugar beet cabbage Retrnidiv rorshiimepae, root sc

Mugiz regzisae, aphid sugar beet peach aphid and)

Pepper Fruits and nuts of deciduous trees

Mugykh regvisae, aphid Buzarpi" rialiadipeg, pink peach aphid o

Potato "

Etroavsa igarae, Sugar cane leafhopper with potato zasspagozuype zasspapooga, fly with

Mugy5 regvisav, peach cane aphid West Indian s

Mastozirpyt eyrpogb/ae, aphid oirpa Pama, yellow potato aphid of sugar cane

Ragaynoga soskege!iiyi, pistoblishka « potato - p. a (o) (ee) (95) d» sl ko bo b5

Table 4: Netirivga (Bugs)

Tomato bugs

Asgtosietite Riiage, green bug

Eyzvepiziv zeguiv, brown bug

Table 5: Opiyoriega (Grasshoppers, crickets and cockroaches)

Wheat

Meiiaporinz zapodiipirev, a filly

Table 6: ririega (flies and mosquitoes)

Tomatoes Carrots

Mpotuza Ipyu!ii, moth-miner Ryna gozaye, carrot fly

Mpotuta zayuae, vegetable miner

Zsgobirairia arzoitsia, miner Sugar beet tomato Teiapore tuoraeyu!tiv

Cruciferous vegetables (broccoli, cabbage, cauliflower, cabbage)

Beiia gaiyisit, cabbage fly IF) «

Table 7: Tnuzaporiega (Trils co

Tomatoes Peppers (oo)

Egapkiipivya ossidepiakiv, thrips TIpirz rai!ti, melon and wheat thrips

EgapkiipivNa Tizsa, tobacco thrips Potato

Tnprz zabasi, tobacco thrips Tipre raiti, melon thrips "

Cruciferous vegetables (broccoli, cabbage, cauliflower, fodder cabbage) with Tpprev yabasi, tobacco thrips (22) (ee) (95) 50 ml

F) name) 60 b5

Table 4: Netiriega (Bedbugs)

Tomato bugs

Asgovivtit Niage, green bug

Eizspiei5 zeguiv, a brown bug

Table 5: Oporiega (Grasshoppers, crickets and cockroaches)

Wheat

Me!aporiiz zapodtsipirev, filly

Table 6: Ririega (flies and mosquitoes)

Tomatoes Carrots

Opotuga ipioiiii, moth-miner Rvima flies, carrot fly occupies, miner vegetable

Zsgobira!rsha abzoisha, miner Sugar beet tomato Teiaporz tuoraeiyu?ttiv

Cruciferous vegetables (broccoli, cabbage, cauliflower, cabbage) Vegetables

The ray hangs around, the larva of the Omiotuga fly occupies, the vegetable miner of the cabbage spring

Oeia gayisit, cabbage fly "

Table 7: Truvaporiega (Thrips) co

Tomatoes Pepper co

Egapkiipivia ossidepiakiv, thrips TRpr» rai!ti, melon and wheat thrips

Etapkiipiva Tizsa, tobacco thrips Potato

Tplprz habasi, tobacco thrips Tipre ra!ti, melon thrips

Cruciferous (broccoli, cabbage, cauliflower Z s cabbage, fodder cabbage) "» Gabassi dash, tobacco thrips p (o) (ee) (95) iz 50 sl

F) ko bo b5

Table 4: Netirieta (Bugs)

Tomato bugs

Asgozyvtite Niyage, green bug

Eizspiziih vegliv5, brown bug

Table 5: Oproriega "(Grasshoppers, grasshoppers and cockroaches)

Wheat

Mei! Iaporiiv zapotsipirez, a filly

Table 6: Oiriega (flies and mosquitoes)

Tomatoes Carrots

Opotuga ip, moth-miner Rviia flies, carrot fly

Opotuzga is busy, vegetable miner

Zegovira!riia abzoishcha, miner Sugar beet tomato Teiapore tuoravyuttiv

Cruciferous (broccoli, cabbage, cauliflower with 79 cabbage, fodder cabbage) Vegetables Ge)

Reiia Bgazvisaye, fly swatter (Llotuga zayuavye, cabbage spring vegetable miner

Repya gayisit, cabbage fly IS o) «

Table 7: Truzaporiega (Thrips) p

Tomatoes Peppers)

Egapkiipisya ossidepiakiv, thrips Tipirze raiti, thrips melon «so wheat

Agapkiipivna Tizsa, tobacco thrips Potato

Tiprz iabasi, tobacco thrips Tra ra!ti, melon thrips "

Cruciferous (broccoli, cabbage, cauliflower, kohlrabi) Tinirv Yabashi, tobacco thrips (o) (ee) (95) iz 50 sl

F) ko bo b5

Table 4: Netirivga (Bugs)

Tomato bugs

Asgtosietite Riiage, green bug

Eyzvepiziv zeguiv, brown bug

Table 5: Opiyoriega (Grasshoppers, crickets and cockroaches)

Wheat

Meiiaporinz zapodiipirev, a filly

Table 6: ririega (flies and mosquitoes)

Tomatoes Carrots

Mpotuza Ipyu!ii, moth-miner Ryna gozaye, carrot fly

Mpotuta zayuae, vegetable miner

Zsgobirairia arzoitsia, miner Sugar beet tomato Teiapore tuoraeyu!tiv

Cruciferous vegetables (broccoli, cabbage, cauliflower, cabbage)

Beiia gaiyisit, cabbage fly IF) «

Table 7: Tnuzaporiega (Trils co

Tomatoes Peppers (oo)

Egapkiipivya ossidepiakiv, thrips TIpirz rai!ti, melon and wheat thrips

EgapkiipivNa Tizsa, tobacco thrips Potato

Tnprz zabasi, tobacco thrips Tipre raiti, melon thrips "

Cruciferous vegetables (broccoli, cabbage, cauliflower, fodder cabbage) with Tpprev yabasi, tobacco thrips (22) (ee) (95) 50 ml

F) name) 60 b5

Table 8: Asap (ticks)

Tomatoes

Teigapuspiz peeing, two-spotted spider mite

Asiyuorz iIusoregzvisi, destructive mite 5iepeogiagzoletiv raiidiv, mite 70 strawberry

Citrus fruits

Rapopusnpyh si, red citrus mite

Vgemirairiv iem/izi, flat citrus mite

Rnuyosorigaiga oiviuoga, rusty citrus mite

Fruits and nuts of deciduous trees

Rapopausniz yiti, European spider mite 6)

Teigapsnyv 5p, spider mite

From the point of view of this invention, pests also include fungal plant pathogens. A list of fungal pests of major crops is given in Table 9. Such pests are included in the scope of this invention.

Table 9: Fungal diseases of plants IS o)

Cob mold "

Ear mold Sirbetgena sirbegeia geae so b. zappipetsi s vil kolossya Ahregoy5 Azregoyishv Pamiv z TSI reg reg. | with

A. ragaziysiv

Ear mold Oiriodia Biriosdia taua/v5 - s. and? (22) (ee) (95)

Cheka" sl

F) name) 60 b5

0. taste enemy

Ear mold Ryzapchshgt Ryzapyt topiyope

E. topi. magician. Zyboshypape

Rotten stalks

Stem rot Ruitisht Ruiyisht arpapidetva!ya

Stem rot Apipgaslovze SoLeyufspyt dgatipisoia

S. Tysytapepviv siotegeia ogatipisoia

Stem rot Oiriodia Oiriodia tausiiv 0. geae-tauaiiv t5 5ieposagre!a tauaiiv

Musgodiriodia geae

Zriaepa taudiv 5. 2eav. 0. tasgosrogha

Stem rot Ryvagicht Eizapyt topcyope

Stem rot of Ssibbehein b. praises about overbaked

Wilting and Decaying of Stewart Em/pia Viemapnia

Leaf diseases

Leaf disease of northern corn Ezhegoyyit shtisit h

Leaf disease of southern Wiro/apv tawais corn

Gray spots on the leaves of Segsozrog geae-taudih o

S. Goguli mag. Taudiv so

Zo Leaf disease Apiygaspose Sopegyutspyt dgatilisoia ise)

Common rust Rissipia sogavi

R. Taudaiv "

Southern rust Rissipia roiiuzoga 70 Risvaeota roiiuzogyt n-s Sedge head brpacefipesa heyapa ; » Common sedge uviyado tauyi» 16 Carbon spots on leaves Neitipipozropyt satopyt (22) (ee) (95) iz 50 sl (F, ime) 60 b5

The eye-shaped spots of Karayen geae

False powdery mildew Regopozsiegozrog zogupi

False sorghum powdery mildew

Brown-striped powdery mildew 5siehornivpoga hauswiav

Soft floury dew of sugar / Regopovsivgozroga zasspa of canes

Soft Philippine powdery mildew. Regopovsivg rinirripepviv

Javanese soft powdery mildew

Bropiapect

Soft powdery dew Kadaziyap../ RegopozsIegozroga peiegorodopi

Mild powdery mildew Ogatipisoia Zsiegogroga dgatilisoga

Rust Rissipia dgatiliv T.5yr. Ipiysi

Rissipia hesopaia i.5yr. (yNsi

Rissipia zipioptii

Sazhki Tena ipysi o

Tena sopygomegza

Teva ipais oyu 5Schado niysi "

SHotgosuziiv Igisi

Rotten roots, root necks of those Sagitappotusev dgatips and) diseases Ruiyit srr. ee)

Rizapyt sitogyt "I

E-examination dgatipavgyt

Rizapyt auepaseit

Ogesiviste Iliisi-hereliiv « du Kppigosiopia 5yr. in s. ipysi

Zsivegorpiyoga tastochroga o Mixed fungal diseases of Beriopa ipisi

ChK» 50 Zeriopa podogyt sp Proteins of the MIRZ class are secreted into the environment by Vasiyz5 species during the vegetative stages of development. MIRZA is a representative of a new discovered class of proteins that exhibits insecticidal activity against many insects

And eridoriega, including the epsilon scoop (Aadgoiiz irsiop), the autumn migration worm (Zrodoriega Ihydiregaa), the ground scoop (5. ehidtsa), the yellow-striped migration worm (Z. otypodaiyi), the southwestern corn butterfly (Oiaigaea dgapaiozeiMa ), sugar cane fire (0. vzasspagaiiiv), cotton scoop

F) (Neiisomegra gea), the Mediterranean corn moth (Zezatia popadgoiidev), the moth (Tgisporivia ka pi), the moth (Apiisagzia dettaiia|iv), the cabbage moth (RishieMya huiovieia) and the tobacco moth (Neioipiv migezsepv). Some of these insects and Eridoriega proved to be very resistant to other insecticidal proteins, such as b-endotoxin. For example, the RDA for StuTA(c), one of the most effective 5b-endotoxins against the epsilon bollworm, requires more than 6000 ng/cm? (Masipiozy ei ai, U. Ipmeperg. Raiyoi. 56:258-266 (1990)). In contrast, MIRZA(a)-protein is required 260 times less to kill 5095 larvae of the epsilon bollworm. So

MIRZATS(a) protein has a unique insecticidal effect.

The present invention offers a new representative of the MIRZ class of proteins, the MIRZA(c) protein isolated from strain 65 АВ5Б1 (рСИВ7112, deposited on March 28, 1997 under ICC inventory number! В-21675; all cultures are deposited in accordance with the Budapest Agreement in the Collection of Patent Cultures Agricultural Research Service (ASR), Mogpiegp Kediopa!Kezeagspj Sepieg, 1815 Mopi Opimegeiu 5igeei, Reogia, Shipoi 61604,

ZA), as described in BEO and MO: 5-6.

The MIRZA(c)-protein encoding the DNA sequence was identified and isolated using polymerase chain reaction (PCR) technology. In particular, primer sequences can be created that recognize invariable or variable regions of the coding sequence and then used to screen DNA samples obtained from known or unknown strains.

It has been recognized that many approaches exist for the identification and isolation of homologues in the MIRZ class of proteins and the DNA sequences that encode them, and these approaches are known to those skilled in the art. 70 DNA and protein sequences for MIRZA(a)- and MIRZA(c)-proteins are given in Table 12.

Table 12: Comparison of MIRZAZ) (upper row) with MIRZA(s) (lower row)

PTU TE LETTER EE TT TT TI II

1.

HY TI TI ET TA TENT TT and

З1 ТРЕМООБОМОИБОКОСУМОВИМОМИАООМИМТЕС5КЕТИКИАМЕОМОМИ, 100

AND

191.

PI TENT TET TT NO TT ATIT ITIT

201

GO TEN TT TIN TI o 151 OKYOITIMKLILIMUTOTEITRAHOVIKUUMEKREEITKATETIAZKUKKIU 200 201 5RARIBOESTEBTETSAKOUTKMOUOSEEEEUIKTENOUMUUEMMOvSVBATCK 250

PIT CT. TT TETU TZ TET ET A TET TI ET 201 5RAVIKOETS5BEGTETSPAKZUTOMOUOZEEKU S MTENOUMUSMMNMOKSVVATK 250 yuyu « 251 TABEBITKEMUKTZOBEMOMUUME SIM TASOAOAEBTITTOEKIISSAYU 300 Geo)

PTY A ETTA AND TT A PBX

251 ТАБЕЦДІТКЕНУКТЯСЗЕУСМУУМЕСІУСТАГОАОАЕСТОТРСЕКІІ СТАЮ 300 со 301 ІРУТ5ІММЕНІМКЕКЕЕЕВУМІЮРТЬБКТЕ5МНРМУАКУКОЗОЕРАКМІМЕ 350 325 ПЕТ ЕТ ПИ ТЕТ ЕЕ АТ ПТ ЕТ ЕТ ТТ ТТ (Се) 301 ІРУТ5ІММЕНІМКЕКЕЕЕКУМІШРТІЗМТРОМРМТАКУКСВРЕОАКМІУЕ 350 351 АКРОНАЦІСЕБІЗМОБТТУЄКУТЕАКІКОМУОУТКОЗЬСЕМІХСОМОКИ: 400

PTU TT TET TP ET ET TT TT TI

400

ПЕТТІ с 401 СРООБСОІУУТММІУЕРМЕУУІТКІРЕТККМКТІКУБУТАМЕХУОБУТОКІ 450 "» 451 О1ЛАКККУВ5ЗЕАВУВТЬБАМООСУУМРІХЛЛІБЕТЕСТРІМОЕССОАОЕМЕ 500 " ПИТИ ГТ ОТ АТ ТИТА 451 РОМККЕКУЄЗ5ЕАВЕУЕТОЬЗАМООСУУМРІСУІЗЕТЕМТРІМСЕСТЬОАОЕМ5 500 501 ВІІТІЬТСК5УТВЕСЬСАТООЗМКЕТКЦІУРРОСЕІ5МІУЕМСВІБЕОМОЕ 550

F PTY ET TITIT TT TI T TAT TAT TITA U

501.

PYTY TEO JSC TAT ET TET TE d) 551 RICAKAMKMAUUYUNTOSUYUSTKATSUNKOSSIVORISOKOKRKTEUUYOUT 600 of them 50 601 UKSKROINKOENTOUTNUERTYMVNVOUTINKARTTOTKOUUTK 650

PEPEPPTEO TT TT NT ET TT TET TT sl 601 UKOKRBINICOEMTOUINUKOTMMEOUOTIMNKAETTSTROKSUUTIIK 650 651 BOMOOEAMSRMEIITBETURBZEKIIERECINTYAMYTOTOZTMIVOMTTX 700

WASH TT ETO TAT TT ETO TT S

700

IEE TT in TT TETU

GF) 701 OSOVSIBKOMYOGOBEZTUVUTEZUZSOAKUVIVMOKEUOREKKOI 746 ime) and

Polypeptide domains of MIRZ-m/as proteins

It was shown that the MIRZA(a) protein undergoes proteolytic processing when mixed with the intestinal fluids of 60 insect larvae. When intestinal fluids isolated from epsilon scoops were mixed with purified MIRZA(a), four major MIRZA(a)-derived proteolytic products with molecular weights of approximately 66, 45, 33, and 22kDa could be identified. The 22kDa disk includes the M-terminal part of the MIRZA(a) protein from amino acid 1 to amino acid 198 of the ZE IYUO MO:2. The bbqDa disk includes the rest of the MIRZA(a) protein from amino acid 200 to amino acid 789 of ZEO IU MO:2. Disks 45 and ZZkDa are obtained by proteolysis from the bbkDa disk and include amino acids from 412 to amino acid 789 and from amino acid 200 to amino acid 455 of 5EO IU MO:2, respectively.

The ZZkDa disk is the main component of the MIRZA(a) protein, which remains after an incubation period of more than two hours. This ZZkDa domain of "toxic bark" (amino acids from 200 to 455 5EO IU MO:2) of the MIRZA(a)-protein completely preserves its insecticidal properties against many insects and Eridorieega. Similar results are obtained by incubating MIRZA(a) with intestinal fluid isolated from the fall worm, another insect sensitive to

MIRZATS).

In addition to the measles toxic domain, the MIRZA(a) protein also has a C-terminal stabilizing domain. The role of the stabilizing domain was studied using mutants of the MIRZA(a) protein and MIRZA(c) protein, none of which exhibit insecticidal properties when consumed by insects sensitive to MIRZA(a). When carrying out 70 similar studies in the intestinal fluid of epsilon scoops with MIRZA(a)-mutants, in particular, with a mutant

MIRZA(a) protein containing three point mutations located in the carboxy-terminal domain (amino acid 742 (E-,0); amino acid 770 (5.»P) and amino acid 784 (U-»H)) was detected , that the protein was completely hydrolyzed. Similar results were obtained for the MIRZA(c) (ZEO IU MO:6) protein isolated from AB5B1, which is 9695 identical to the MIRZA(a) protein, but lacks the carboxy-terminal domain of MIRZA(a). However, both the 75 Mutant and the MIRZA(c) protein are active against the ZI-9 insect cell line. These results indicate that the function of the carboxy-terminal domain of MIRZ-class proteins is to provide stability for the protein in the gut of a susceptible insect.

Hybrid toxins that include the MIRZ site and a heterologous site

Toxins, enzymes, transcription factors, antibodies, cell binding components or other protein domains

Can be functionally connected to the new proteins of this invention by the formation of genetic fusion frameworks, which, being translated by ribosomes, produce a fusion protein with combined properties of MIR and another component used for fusion. Additionally, if the protein domain fused to the MIR has affinity for another protein, nucleic acid, carbohydrate, lipid, or other chemical reagent or factor, a ternary complex may be formed. This complex will have the same properties of all its components. A similar explanation can be used for the formation of complexes of four or more components. These complexes are used as insecticidal toxins, pharmaceutical, laboratory reagents, diagnostic reagents, etc. Examples of the use of such complexes are fusion toxins for the treatment of potential cancers, reagents for enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. oh

The hybrid toxins of the invention include chimeric proteins having a domain of the toxic core, which is heterologous to the stabilizing domain. Hybrid toxins are also formed by combining an antibody or its immunologically active fragment, which immunologically distinguishes the MIRZ receptor with a toxic domain from other proteins. The toxin domain is derived from many cytotoxic proteins. These include, but are not limited to, Vasiy5 toxins, including endotoxins and vegetative insecticidal proteins. See, for example, US Patent Application No. 08/037,057, filed March 25, 1993. and US Patent Application No. 07/951,715, filed September 25, 1992, to which the authors refer. Other toxins include catalytic ribosome inactivators such as gelonin, Rzeidotopase exotoxin A, or phytolaccin (the structure of Rzeidotopase exotoxin was well characterized in the work of Spatsanagu et al., 9U. Vioi. Speth. 265:16303-16310 (1990)); disruptors of cell metabolism, such as ribonucleases (see, for example, Magiapi ey a). Maishge 347:737-741 (1990)); Vagpase toxin (or RE-Vaig), a chimeric toxin derived from Rzepdotopase exotoxin A and ribonuclease (see Rheumatology, Sci. 64:1017-1023 (1991)); hydrophilic peptides that form pores in membranes (see Egopiys apa UMeiIv, p. and U. Reriide Rgoievip Kev. 37:2-6 (1991)). "" MIRZA's principle of action

MIRZA(a)-protein has shown its activity against many plant pests. For example, histopathological observations show that the use of MIRZA by susceptible insects such as the epsilon bollworm

Gee! (Adhoyz irsiop) and the autumn derivative worm (Zrodoriega Iygidiregda), causes intestinal paralysis at ration concentrations of 4 ng/cm 2, with complete lysis of intestinal epithelial cells, leading to larval death at concentrations over 4 ng/cm?. In less susceptible insects, such as the European corn moth (95) (Ozyhipia pibiiaiv), no pathology develops after the use of MIRZA(a). While proteolytic processing of their 50 MIRZA(a) protein by midgut fluid obtained from susceptible and non-susceptible insects is comparable, immunolocalization studies show that MIRZA(a) binding is restricted to intestinal cells of susceptible insects. Thus, the list of insect hosts for MIRZA is determined by its ability to bind to intestinal cells. Histopathological observations show that the epithelial cells of the midgut of susceptible insects are the main target for the insecticidal protein MIRZAZ), and their subsequent lysis is the main mechanism of mortality. o Programmed cell death is an active process of self-destruction that is important for the development and maintenance of multicellular organisms (Siet, K.). her a). Zsiepse 254: 1388-1390 (1991)). Cells undergoing apoptosis, which is a form of programmed cell death, form membrane-bound apoptotic organisms and activate endogenous nucleases, which cleave chromatin into separate fragments. because ZE-9 cells of the insect 5. Igidiregaa exposed to the action of the MIRZA(a) protein undergo cytological and molecular changes, including membrane protrusion, significant vacuolization, and endonucleolysis, which indicate programmed cell death of the apoptotic type. Histological studies showed that the MIRZA(a) protein is targeted to the midgut epithelial cells of susceptible insects, causing cytological changes that include significant vacuolization and swelling before cell lysis and larval death. These cells of the midgut 65 also undergo endonucleolysis when exposed to the MIRZA(a) protein, which is evidenced by the detection of DNA fragmentation. These results indicate that MIRZA(a) exerts its insecticidal properties on susceptible insect cells by inducing programmed cell death of the apoptotic type.

The receptor for MIRZA is highlighted

The immunohistochemical results presented above show that MIRZA(a) has the ability to bind to the apical membranes of midgut epithelial cells and that this binding initiates a process that ultimately ends in cell lysis. This means that there are one or more proteins located in the apical membrane that recognize MIRZAd) and bind to it, acting as a receptor. This receptor signals interaction with MIRZATSsa) and initiates the process of apoptosis. Thus, through the receptor, the insect cell responds to

MIRZATS). 70 To isolate this receptor by screening, a cDNA library is selected from mRNA isolated from the midgut tissue of the epsilon bollworm. The purpose of this screening is to identify and isolate DNA sequences that encode proteins that interact with MIRZA(a) in a two-hybrid system (see RieviIdv. 5. apa opo, O.-K. Maishge 340:245-246 ( 1989)). As a result, this approach made it possible to identify and isolate cDNA, the encoded protein of which actively interacted with the MIRZA(a) protein. This cDNA is 1.75 kb in length. (ZEO IU MO:8) 7/5 encodes a protein of approximately 48 kDa (396 amino acids; see ZEO IU MO:9). The cloned cDNA is nearly the same size as the mRNA encoding the cDNA, as evidenced by Northern blotting. Part of the DNA sequence that codes for the first 5 to 20 amino acids may be missing. The following features can be recognized in the encoded cDNA protein (see Figure 1): 1) it contains a signal peptide; 2) it contains a domain homologous to the so-called death domain (Reiipvzieii, E. Ei ai. Tgtepaz ip Biospet. 20:342-344 (1995)); and 3) it contains EsE-like

Motives or repetitions (Rapii, MU. U. ei aiI.,, Appy. Keu. Viospet. 62:453-481 (1993)). A search of protein databases using the MIRZA(a) receptor revealed homology with a family of extracellular glycoproteins known under the names Tepazsipz (Reagzop, S.A. ey a. EMVO 3. 7:2677-2681 (1988)) or Nehargaspiop (Miez. Yu 9.

Vioi Spent 266:2818-2823 (1991)). This family of proteins contains ESE-like repeats, interacts with complex ligands, and plays a role in cell adhesion and/or signal transduction. The combination of the death domain and the chain of repetitive ESE motifs observed in the MIRZ receptor is unique among the receptors of programmed cell death. and)

In addition, a part of the MIRZA(a) receptor has homology with the so-called "death domain. The death domain is a motif 60-70 amino acids long, which is involved in the interaction between proteins and is common to proteins with various cell functions (Reipvivipy, E. ei aI. Tgepavz ip Biospet. 20:342-344 (1995)). Some proteins containing death domain motifs include receptors associated with apoptotic processes. These include, for example, the GRaz receptor (Vgakerysis , S. eyai. EMVO 3. 11:943-950 (1992)) and tumor necrosis factor (TME) - (Tapadia, GI A. evya!. Sei! 74:845-853 (1993)).

Homologs of the MIRZA(a) receptor can be identified and isolated in various ways, for example, by hybridization of nucleic acids. Southern blotting can be performed on DNA samples taken from insect or fungal cells subjected to enzyme restriction, agarose chromatography, and blotting on nitrocellulose and/or nylon filters. Southern blotting can be probed with full or partial nucleic acid encoding the MIRZA(a) protein receptor under conditions of moderate hybridization and washing. Genes can be easily cloned and sequenced from a cDNA or genomic library. A size-selected genomic library can also be obtained to enable cloning of the desired gene(s). The above-mentioned technical protocols for conducting experiments are readily available (see, for example, Moiesshciag Siopipo, A I arogaiog Mapiai, Zesopa z s Edyiop, Moiv. 1-3, ZatbgookK eii aiY. (edvz.) Soid Zrgipd Nagrog I arogaiog Rgezv, Soii Zrgipd Nagrog, MU (1989) and references). ;" Antibodies to MIRZACda) and its receptor

Polyclonal and monoclonal antibodies to the MIRZ protein or its receptor, including their fragments, which immunologically recognize parts of each of the proteins are offered. Antibody and monoclonal antibodies

He" of this invention can be prepared using the MIRZ protein or its receptor as an antigen.

Antibodies of the invention include polyclonal and monoclonal antibodies, as well as their fragments, which retain the ability to bind the MIRZ protein or its receptor. It is noted that an antibody, monoclonal 2) antibody or its fragment is able to bind a molecule, if they are able to specifically react with a molecule to bind the molecule to an antibody, monoclonal antibody or its fragment in this way. The term "antibody" (AB) or "monoclonal antibody" (Mab) includes the entire molecule, as well as its fragments or sp-binding regions or domains (such as, for example, Bar and (ar) 5-fragments) capable of to hapten. Such fragments are usually produced by proteolytic cleavage, such as papain or pepsin. Or hapten-binding fragments can be formed using recombinant DNA technology or synthetic bv Chemistry.

Methods of preparation of antibodies of this invention are generally known to specialists. For example, see

F) Apiirodiez, A I arogaiogu Mapia!i, Ei Naiozhm apa Oamii I ape (eds.) Soid rgipd Nagbrog I arogaiogu, MM (1988), and also references of the authors. Typical works that outline the general principles of immunology include: Kieip, .).

IptypoYodu: Te Zsiepse oi SeiI-Mopsei! Oivsgitipayeyop, add. UMyeu 5 Bopv. MM (1982); Oeppei, K., ei ai. because Moposiopa! Apiirodiez, Murgidota: A Mem Oitepviop ip Vioiodisa! Apaiuzez, Riepyt Rgevv, Moscow State University (1980); apa

Satreii, A. "Moposiopa!

Vioiodu, Moi. 13, Vygdop eyi ai. (Eaz.), Eivemieg, Atwiegdat (1984). See also MoMo US Patents: 4,609,893; 4,713,325; 4,714,681; 4,716,111; 4,716,117 and 4,720,459.

It was recognized that by the methods described by the authors, it is possible to obtain antibodies specific to a specific 65 MIRZ protein or its receptor. A subset of Mab-lines that has the described binding specificity can be used as a source of messenger RNA for cDNA cloning for a specific monoclonal antibody.

The cloned DNA can then be sequenced by methods known to those skilled in the art. See, for example,

ZatbrgooK ei aiYi., MoYiIesshag Siopipd: A Igarogayugu Mapiai, 2pa. Eaiiop, So Zrgipd Nagrog I aroghaiogu Rgezv,

MU (1989) mine. 1-3, and authors' references. The protein sequence of the binding site from the selected MAbB can be excluded from the nucleic acid sequence.

One method of using the antibodies and monoclonal antibodies of the invention includes, among others, the formation of hybrid toxin molecules. That is, being connected, the monoclonal antibody or antibody fragment retains its binding properties, and the toxin part retains its cytotoxic properties.

Various methods of obtaining antibody genes are known. One way is to clone a randomly selected 7/0 antibody gene library in phase and screen the library for the ability to bind the MIRZ protein or its receptor. Another possible approach is to produce monoclonal antibodies that bind to

MIRZ protein or its receptor, and then antibody genes from such lines are cloned. In this case, the second method is used. Antibody genes can be cloned from hybridoma cells using primers to conserved DNA sequences in constant regions and framework regions of variable regions and amplified for cloning /5 Using polymerase chain reaction (PCR). See Miiiiiv ey ai., Me. Epgutoi., 155:335-350 (1987); Episp. A database for heavy chain and light chain sequences of mice, compiled in the work of KaBbai ei ai., 05 Oeri Neay apa Nitap Zegmisev. 05

Somuegptepi Rhipipd ObPisez (1991), was successfully used to produce isotype-specific and degenerate primers for gene antibody cloning. (supplement to her AIY. VioLesppoiodu 9:88-89 (1991)). In addition, there are known methods of cloning smaller fragments of antibodies (Bar), which have the binding properties of the original antibody. Full antibodies are large molecules (150kDa), but much smaller Bar and Em antigen-binding fragments (12kDa-5OkDa) have been found to retain full binding affinity.

Single-chain Em-fragments (ssEm), in which the MP and MI domains are connected by a hydrophilic and flexible peptide, have been successfully used to direct enzymes and toxins to specific cells (Viga, Zsiepse 423:423-426 sch ov (1988); Nysiop, RMABZ 85:5879-5883 (1988)). Single Mi domains (Oarv) and single complementary determining regions of 20 amino acids in length, called minimal recognition units (MRIs), have also (8) been used for antigen binding (Uuaga, Maishge 341:544-546 (1989); Tashb, 9. Vioi. Speth 264:259-265 (1989); MUShiatv, RMABZ 86:5537-5541 (1989)). Thus, it is possible to reduce as much as possible the binding domain specific for MIRZ or its receptor. Polymerase chain reaction technology and specific oligonucleotide primers are used to clone immunoglobulin genes or areas of immunoglobulin genes. PCR primers specific for both heavy and light I9M chains and three isotypes were selected from the Kabay database described above. Primers for the region encoding the MNO-terminal end of the generated variable region were designed to stimulate in the first framework region and were designed with a certain degeneracy that allowed them to be used as "universal co primers". 3' and primers used for specific PCR amplification of variable regions were constructed from conserved sequences of the first constant domain (CDN) of both light and heavy chains. Another 3-primer is used for immunoglobulin isotypes IdsC1, DSZ and I9M. Isotypes IdC2A and IdC2B can be amplified with the same primers used for IdDO1. The variable regions of the antibodies are cloned into a light and heavy chain expression vector containing the signal peptide of the endoplasmic reticulum and the constant regions of IdDO1 light and heavy chains, respectively. The sequences of the primers used for PCR cloning of light and heavy variable regions of mouse immunoglobulin can be found in the published literature (Soyota et al. Vio/Hesppidnez 11:152-156;" (1991); dopez ei. Vioespoiodu 9 :88-89 (1991)). Oligonucleotides were created on a DNA synthesizer 3808 from

Arriei Viosuvietvs (Arriei Viosuvietv, Rovieg Spiu, SA) using the standard conditions described below.

PCR primers include restriction sites and after amplification and cleavage can be cloned into a vector

Ge" expression of plants under the control of a promoter capable of expression in plants. Restriction sites that are not present in the sequenced antibody genes were selected. Another method of using the polyclonal and/or monoclonal antibodies of the invention involves stimulating 2) apoptosis by targeting the MirzA receptor with antibodies. The interaction of antibodies produced against proteins located on the surface of cells, which are involved in the control of cell growth, eventually leads to the initiation of apoptosis by preventing the binding of the above-mentioned receptor with its natural ligand(s). For example, an anti-APO-1 antibody completely blocks the proliferation of leukocytes carrying the APO-1 protein and initiates apoptosis in these cells (Tgtashiy, V.S. ei aYi. Zsiepse 245:301-305 (1989)). In addition, the activity resulting from the interaction between a given receptor and a ligand is mimicked by substituting the ligand for antibodies raised against the receptor. For example, adding a certain amount of anti-base antibodies to the carrier cells

The Eaz receptor on their cell surfaces mediates apoptosis in the same way as it would when adding a ligand

F) ERaz-receptor (POP, M. eiai. Sei! 66:233-243 (1991)). ka The receptor for MirzaZA(a) isolated from the souk epsilon has homology to a family of extracellular glycoproteins known as Tepazsipz, in particular to Tepazsip-X (Vgiviomu, 9. ei aiYi. U. SeiiIi Visi. 122:265-278 (1993 )). Tepavsip-X is known to be involved in cell-cell adhesion and signal transmission. The lack of functionality of Tepazsip-X, either through mutation or deletion of the gene, leads to death. Thus, antibodies raised against different domains of the receptor for Mirza(a) either effectively block binding of the receptor to its ligand(s) or mimic the interaction of Mirza(a)-protein that initiates apoptosis. This approach extends to different receptors with similar biological functions. In this aspect, antibodies produced against insect cell receptors involved in basic growth processes and cell interactions induce apoptosis and are used as a strategic means of insect control.

Screening of new insecticidal substances whose effect is apoptosis

The materials described in this invention are used for screening chemical ligands having pesticidal properties that cause an apoptotic reaction. Chemical ligands include small organic molecules, peptides, and proteins. In one embodiment of the invention, insect cell lines are used as model insect organisms for screening compounds that are insecticidal due to their ability to initiate apoptosis. These cell lines are subjected to high-throughput screening by growing the cells in multiwell plates and exposing them to various compounds. Yeast is also used as a model organism. Using the procedures described by the authors or known to specialists, it is determined whether the compound is pesticidal due to the induction of apoptosis.

One of the ways to identify compounds that initiate an apoptotic reaction through interaction with a known receptor is the use of identified receptors that participate in signal transduction initiated in apoptotic insect cell lines. These receptors are transformed into heterologous cell lines, creating isogenic lines, one of which contains a gene for the expression of a specific receptor, and the other does not have and does not express such a 7/5 Gene. These cell lines are subjected to high-throughput screening, and transformed cell lines expressing the receptor show different responses to compounds that initiate apoptosis through their specific interaction with the aforementioned receptor.

The present invention also encompasses the determination of biochemical and/or molecular markers that specifically identify insect cell lines undergoing apoptosis. For example, it is possible to isolate specific cDNAs that arose during the apoptotic process in specific insect cell lines. Although the death bark pathway appears to be phylogenetically invariant (Madaya, 5. Sei! 88:355-365 (1997)), the signal transduction pathway from the receptor to the death bark may be different in different organisms. RNA mediators that are differentially expressed in apoptotic insect cells are identified by many available methods, for example, by differential display (Vaceg, Yu. ei ai). Mysievis Asii Kevs. 21:4272-4280 (1993)) or subtractive SC libraries (Botteg, N. ei a. EMVO 4. 9:605-613 (1990)). Proteins encoded by differentially expressed cDNA are used as markers for apoptosis in specific insect cell lines. and)

Transgenic plants comprising a DNA sequence encoding a MIRZ-class protein

A host plant expressing at least one of the sequences of the invention is more resistant to attacks by plant pests and thus has a greater ability to resist losses associated with such attacks. A plant is any type of plant that can be genetically transformed by methods known to specialists. These methods of plant transformation are discussed below. Host plants include, but are not limited to, the crops listed above that are affected by pests. with

PLANT EXPRESSION CASSETTES

The methodology of building expression cassettes in plants, as well as the introduction of foreign DNA into plants, are described in the specialized literature. Such expression cassettes may include promoters, terminators, enhancers, leader sequences, introns, and other regulatory sequences operably linked to the coding sequence of the pesticide protein. It is also recognized that promoters or terminators of MIRZ genes can be used in expression cassettes.

The genes of toxins derived from microorganisms can also differ from those of plants. Genes of plants «

They differ from the genes of microorganisms, in which their transcribed RNA does not have a defined sequence with ribosome binding adjacent to the initiating methionine. Thus, the genes of microorganisms can be amplified by incorporating a eukaryotic consensus translation initiator into ATO (Kozak, Sci. 44:283-292 (1986)). In ;" the Siopiesp catalog (1993/1994, p. 210) suggests the sequence ЗСТООАСС АТОСТС (5ЕБО ИЙУО МО:---) as a common initiator of translation for the expression of the cydA gene of E. soybean in plants. In addition, the work of Dozpi (Myseiis siy Kev. 15: 6643-6653 (1987)) contains a comparison of many sequences of plants adjacent to the ATO and

Ge" is offered by consensus TAAASA ATOOST (5EO AND MO:--). In case of difficulties with the expression of microbial open reading frames (ORFs) in plants, the inclusion of one of these sequences in the initiating ATO can improve co-translation. In such cases, the last three consensus nucleotides may be unsuitable for inclusion in 2) the modified sequence due to their modification of the second AA residue. The optimal sequences adjacent to the initiating methionine boron may be different in different plant species. By examining the sequence of corn genes entered in the SepVapk/EMVI database, it is possible to determine which nucleotides adjacent to ATO should be modified to enhance the translation of the toxin gene introduced into corn.

In addition, it has been shown that removal of inappropriate splice sites can enhance the expression and stability of the introduced genes. Genes cloned from non-plant sources and not optimized for expression in plants may contain motifs that can be recognized in plants as 5 or 3 splice sites. Therefore, the process of transcription can be prematurely terminated with the formation of truncated or delegated mRNA. Toxin (F) genes can be engineered to remove these inappropriate splice sites using many different technologies. For example, several available methods are used to identify potential splice sites in a DNA sequence. First, potential splice sites can be identified by computer analysis of the DNA sequence. Consensus sequences that identify splice sites are known to those skilled in the art. See, for example, boodaiII, (3.9. apa Riiiromzhis". MU, EMVO 3. 10, 2635-2644 (1991) apa

Vgomup, 9U.MU.5., MysiIviis Asii Kezeagspy 14, 9549-9559 (1986). Alternatively, the splice sites actually processed by the plant can be identified by comparing the PCR analysis of the cDNA derived from the gene with the actual gene products. Products showing splicing ability are shorter than expected. These 65 smaller products are then cloned, sequenced and the exact splice site determined. It is also recognized that a combination of computer research and RSC analysis can be used.

The novel toxin genes of the present invention, either as native sequence or as optimized synthetic sequences as described above, can be functionally fused to a variety of promoters for expression in plants, including constitutive, inducible, temporally regulated, evolutionarily regulated, chemically regulated, tissue selective and tissue-specific promoters for the preparation of recombinant DNA molecules, that is, chimeric genes. Optimal constitutive promoters include Samm 355 and 195 promoters (Pat

US Patent No. 5,352,605, issued to Fraley (Rhieu et al.) on October 4, 1994). An additional optimal promoter is derived from any one or more actin genes that are expressed in most cell types. Promoter expression cassettes, described in the work of MeseiYigoui ai. (My. Sep. Sep. 231: 150-160 (1991)), can be easily modified to express a new toxin gene and are particularly suitable for use in monocot host plants.

Another optimal constitutive promoter is derived from ubiquitin, which is another gene product that accumulates in many cell types. The ubiquitin promoter has been cloned from several species for use in transgenic plants (e.g., sunflower Vipei et al. 5siepse 79: 87-94 (1991), maize /5 7 Spgiviepsepi a. Riapi Moyes. Vii. 12: 619-632 ( 1989)). The maize ubiquitin promoter has been developed in transgenic monocot systems, and its sequence and vectors constructed for monocot transformation are described in patent publication EP 0 342 926. The ubiquitin promoter is suitable for the expression of novel toxin genes in transgenic plants, particularly in monocots.

Other promoters that are used for the expression of a new toxin gene in plants, in particular, in corn, are, for example, tissue-specific or tissue-selective promoters, such as those described in UUCHO 93/07278; chemically inducible promoters described in EP-A 0 332 104, to which the authors refer in its entirety.

In addition to promoters, transcriptional terminators are also available for use in chimeric gene sequences using the new toxin genes of this invention. Transcriptional terminators are responsible for termination of transcription outside the transgene and its correct polyadenylation. Suitable transcriptional terminators and terminators that function in plants include Samu c55 terminator, (ti-terminator, and) nopaline synthase terminator, pea grs5 EO9 terminator, and other terminators known to those skilled in the art. They can be used both in monocots and in dicots.

Many untranslated leader sequences derived from viruses, such as those described in, for example, MO 96/10083, are also known to enhance expression, and they are particularly effective in dicotyledonous cells.

In particular, the leader sequences from Tobacco mosaic virus (TMM, (ie "O-sequence"), Maize chlorotic mosaic virus (MSMU) and Alfalfa mosaic virus (AMSU) have been shown to be effective in enhancing the expression of c (see, for example, Saieya ). Mysi. Asia Kev. 15: 8693-8711 (1987); Z5Kygezki ei a). Riapi Moyes. Vioi 15; 65-79(1990)).

Various intron sequences showed increased expression when adding up to 5 regulatory regions, especially in monocot cells. For example, it was found that the introns of the Adn1 gene of corn significantly enhance the expression of the natural gene with its female-related promoter when introduced into corn cells (Sayyiz ei aiI., Sepez JuOmeior. 1:1183-1200(1987)).

OPTIMIZATION OF mir3-GENES FOR EXPRESSION IN PLANTS

The pesticidal genes of the invention can be optimized for enhanced expression in plants. See, for example, "

ERA 0359472; ERA 0385962; MO 91/16432 and Regpiak ei aiY., Rhos. May Asai. si. 88:3324-3328 (1991). In this way, coding sequences optimized for expression in plants can be synthesized. and In one variant of the invention, miRZzA(a) is obtained according to the method described in US Patent Application Ser. "" Me07/951,715, to which the authors refer. This procedure uses the optimal maize codons, i.e. the single codon that most often codes for this amino acid in maize. Optimal codon

Corn for a specific amino acid can be obtained, for example, from a known gene sequence

Gee! corn The use of the maize codon for 28 maize genes is mentioned in the work of Migtau ei ai., Mysiewis

Asiid Kezeagsy 17: 477-498 (1989), to which the authors refer. Examples of synthetic sequences created with the help of optimized codons of corn are presented in ZEO IU MO:7 (MIRZA(a)), ZEO IU MO:19 g) (MIRZA(B)) and BEO IU MO:20 (MIRZA(c) ).

Thus, nucleotide sequences can be optimized for expression in any plant. It has been recognized that all or any portion of a gene sequence may be optimized or synthesized. 4 That is, synthetic or partially optimized sequences can also be used.

PLANT TRANSFORMATION

Recombinant DNA molecules can be introduced into a plant cell in a number of ways recognized by those skilled in the art. Specialists know that the choice of method may depend on the type of plant intended for transformation, that is, on whether it is monocotyledonous or dicotyledonous. Appropriate methods of cell transformation (F, plants) are described, for example, in MUUO 96/10083 and MUUO 97/46105, respectively, including microinjection, co-electroporation, Adhorasiegisht-mediated transformation, direct gene transfer and ballistic particle acceleration using devices from Adgaseya5, Ips., Madison, Wis., and Europe, Ips., for Wilmington, Delaware.

The optimal option is the method of transformation of protoplasts for corn, described in Application EP 0 292 435, as well as in US Patent Mo5,350,689, to which the authors refer in their entirety. One of the best options for introducing the expression cassettes of this invention into wheat by bombardment with microparticles can be found in US Patent No. 5,610,042, which is hereby incorporated by reference in its entirety. 65 Plant transformation is carried out using a single DNA molecule or multiple DNA molecules (ie, co-transformation), and both of these technologies are suitable for use with the expression cassettes of the present invention. There are many vectors for plant transformation, and the expression cassettes of the present invention can be used with any such vectors. The choice of vector depends on the optimal method of transformation and the species intended for transformation.

There are many vectors for transformation using Adgurasiegisht (Shtegiasiepz. They usually carry at least one extreme T-DNA sequence and include vectors such as rVIM19 (Wemap, Mysi. Asidz Kezv. (1984)). In one of the optimal variants the novel toxin gene of this invention can be introduced into any of the binary vectors pSIV200 and pSIV2001 for use with Adgorasiepyt, the construction of which is described, for example, in

MO 95/33818 (example 35) (see also EP 0 332 104, example 19). 70 An additional vector used for Adgobasiheit-mediated transformation is the Resiv10 binary vector, which contains the gene encoding kanamycin resistance for selection in plants, right and left extreme T-DNA sequences and includes sequences from the multihost plasmid pKK252, allowing it reproduce in E. soii and Adgorasiepyt. its structure is described in the work of Koivivip ei a). (Sepe 53:153-161 (1987)). Different derivatives of pSIV10 were constructed, which include the gene for hygromycin B phosphotransferase, described 75 in the work of Sg; her and her (Sepe 25: 179-188 (1983)). These derivatives allow the selection of cells of transgenic plants only on hygromycin (pSIV743) or hygromycin and kanamycin (pSIV715, pSIV717).

Methods using direct gene transfer or Adgorasiegit-mediated transfer are usually, but not necessarily, carried out with a selectable marker that can provide resistance to an antibiotic (eg, kanamycin, hygromycin, or methotrexate) or a herbicide (eg, phosphinonitricin). The choice of a selectable marker for plant transformation, however, is not key to the invention if the expression of this resistance and its biochemical activity does not affect the choice of the method of conversion of protoxin to toxin for use in conditional fertility.

Different antibiotics or herbicide selection markers may be selected for some plant species. Selection markers commonly used in transformation include priI-gea, which confers resistance to kanamycin and similar antibiotics (Mezzipu 5 Miegpta, Sepe 19: 259-268 (1982); Vemap et al., Maishge 304:184-187 (1983)), a rag gene that provides resistance to the herbicide phosphinonitricin (MUpye ei ai., and)

Mysi Asidz Kez 18:1062 (1990), Zrepseg ei aiI., TpeogArry Sepei 79:625-631 (1990)), pri-gei which provides resistance to the antibiotic hygromycin (Viospipdeg 5: Oiddetapy, Mo! Se Vio! 4: 2929-2931), anti-hey, which provides resistance to methotrexate (Votsgotsiz ey a, EMVO 3). 2: 1099-1104 (1983)), the mannose phosphatisomerase gene, which allows the selection of mannose as a carbon source (EP 530 129, MO 94/20627).

One such vector used for direct gene transfer in combination with selection using the herbicide Vasia (or phosphinonitricin) is pSIV3064. The basis of this vector is the plasmid pSIV246, which includes the Samm 355 promoter in a functional fusion with the 55 gene of E. soybean, and the transcriptional terminator SaAMM 355 is described in MO 93/07278 published according to the PCT application, to which the authors refer. Other co c5 The selected marker used is obtained by functionally combining the ubiquitin co promoter, synthetic PAT gene and pos terminator. One example of a vector including this marker is the pSIVU804 plasmid.

An additional transformation vector is reOs35, which uses the dihydrofolate reductase (ONEK) gene of E. soybean as a selectable marker that provides resistance to methotrexate and the construction of which is described, for example, in " 40. UMO 95/33818 (example 35). with с Another transformation vector is the vector ro! 2 (Question a). Maishge 338, 274-276 (1989)), which contains the gene for hygromycin phosphotransferase (pri) Zigeriotuses, functionally connected to the sequences 355;" promoter and 355 terminator.

Transgenic plants can also be identified using a marker that is amenable to quantitative analysis. Examples of such markers that are used in the invention are p-glucuronidase, green

Gee! fluorescent protein and C1 and B-reg regulatory genes of the maize anthocyanin pathway. In addition, transgenic plants expressing the MIRZ protein can be identified by screening them for insecticidal activity without the need for quantifiable or selectable markers.

Ge) Transformation of maize with a DNA sequence encoding a MIRZ-class protein, preferably a MIRZA(c) protein according to 5or With any of the above-mentioned methods, is easily achieved by microparticle bombardment of either underdeveloped zygotic embryos or embryogenic callus of type I, which can be serially bred. 4 For transformation using underdeveloped zygotic embryos, the ear is self-pollinated, and after about 10 days, underdeveloped zygotic embryos are obtained. Approximately eight hundred underdeveloped zygotic embryos are distributed among different target plates containing a medium capable of inducing and

Support the formation of embryogenic callus. Underdeveloped zygotic embryos are immediately transferred to the same medium, which additionally contains 1295 sucrose. After 5 hours, immature zygotic embryos (F) are bombarded with a plasmid or plasmids using the VioKcad ROZ-1000/Ne device. The plasmid or coplasmids include a selectable marker, such as a gene conferring resistance to phosphinonitricin, or a marker amenable to quantitative analysis, such as green fluorescent protein, and the gene encoding the MIRZ-class protein prepared for introduction and expression in corn as described above.

Plasmid or plasmids are deposited on micron gold particles practically according to the published procedure proposed by VioCaid. The particles are injected using a burst pressure of 1550 psi of helium. The composition of plasmid and gold particles is added twice to each target plate. Since, in one variant of the invention, the plasmid or plasmids include a chimeric gene encoding resistance to 65 phosphinonitricin, this substance can be used to select transformed cells and migo. When used, the selection agent is used at a concentration of 1Omg/l on the day of gene injection and increased to 4Omg/l approximately one month later. Embryogenic callus obtained in this way can be regenerated in the presence of phosphinonitricin when using a selected marker. Plants are obtained from selected lines of embryogenic callus. The regenerated plants are analyzed for resistance to susceptible insects. All Voslins that are resistant to insects also express an introduced chimeric gene encoding a protein or proteins of the MIRZ class, as evidenced by the detection of the MIRZ protein in plants using the EHIZBA enzyme-linked immunosorbent assay. Transform plants that are resistant to insects and express

MIRZ protein.

For the transformation of corn using embryogenic callus type of callus is obtained from 7/0 underdeveloped zygotic embryos using standard methods of cultivation. For the introduction of genes, approximately 300 mg of callus is prepared type by chopping with a scalpel or reseeding for 3-5 days before gene introduction. Before introducing the gene, the prepared callus is placed on a semi-solid culture medium, which also contains 1295 sucrose. After approximately 4 hours, the tissue is bombarded using the ROZ-1000/Ne biolistic device from VioCad. The plasmid or plasmids include a selectable marker, such as a gene conferring resistance to phosphinonitricine or a quantifiable marker, such as green fluorescent protein, and a gene encoding a MIRZ-class protein prepared for introduction and expression in maize according to the description presented above. Plasmids are deposited on micron gold particles using a virtually standard protocol proposed by VioCad. Approximately 16 hours after the introduction of the gene, the callus is transferred to a standard culture medium containing 290 sucrose and, in the case

The use of a selected marker, up to mg/l of phosphinonitricin. The callus is transplanted for selection for 8 weeks, after which the callus, which continues to develop, is transferred to a standard regenerative medium for plant formation. The regenerated plants are analyzed for resistance to susceptible insects. All plants that are resistant to insects also express the introduced chimeric gene encoding a MIRZ-class protein, as evidenced by the detection of the MIRZ protein in the plant by the EI IZA test. Plants that are resistant to insect pests and express the MIRZ-class protein are transformed.

ADDITIONAL ELEMENTS OF INSECT CONTROL i)

The pesticidal proteins of the invention can be used in combination with Bi 5-endotoxins or other insecticidal proteins to increase the susceptibility of insects. In addition, the use of MIR-proteins of this invention, preferably MIRZA(c)-protein, in combination with Bi 5-endotoxins or other specifically defined insecticidal elements has a specific application for preventing insect resistance and/or combating it. -

Different insecticidal crystal proteins Vasiy5 (ypiypdiepviz) have been classified based on their activity spectrum and sequence similarity.

Chem. 53: 242-255 (1989), divided the insecticidal crystal proteins known at that time into four main classes. co

In general, the main classes are determined by the spectrum of activity when Stu 1-proteins are active against I eridorieg, "o

Stu2-proteins are active against Eridorieg I and Oirieg, Stu3-proteins are active against Soieorieg, and Sgu4d-proteins are active against Oirieg.

In the coding of the main classes, b-endotoxins are grouped according to sequence similarity. Stu1 proteins are usually produced as 130-140kDa protoxin proteins, which are proteolytically cleaved to produce active toxin proteins of approximately 60-70kDa. The active part of 5-endotoxin is contained in the MH-terminal part of the full-length molecule. The above-mentioned Nopye apa Mupieieu divided the known Sgu1 proteins into six groups, TAa, TAB, 1Ac, 18, 1C, and 10. Since then, proteins classified as StuEa, StuRa, Stu9A, Stu9cS, and Stu9B have also been characterized.

The spectrum of insecticidal activity of a single 5-endotoxin from Vasiyshv Ipygipdiepsis is significantly narrowed, and this 5-endotoxin is active against only a few insects. Specificity is the result of the effectiveness of different

Ge) stages of production of active toxin protein and, as a result, its ability to interact with epithelial cells of the digestive tract of insects. In one optimal variant, the expression of MIR proteins in the transgenic plant is accompanied by the expression of one or more Bi 3-endotoxins. Particular preference is given to (95) VE b-endotoxins described in US Patent Application Serial No. 07/951,715, to which the authors of iz 50 refer.

It is well known that many b-endotoxin proteins from Vasiyv5 (pygipdiaepsis are really expressed as sl protoxins. These protoxins are dissolved in the alkaline medium of the insect intestine and proteolytically converted by proteases into a toxic fragment of bark (Noye apa MUpieieu, Misgorio! Chem. 53: 242- 255 (1989)).

For β5-endotoxin proteins of the Stgu!-class, the toxic fragment of cor is found in the M-terminal half of the 29 protoxin. The scope of this invention provides that genes that encode any form of full-length protoxin

GF) or a truncated toxic fragment of the cortex of new toxin proteins can be used in the transformation of plant vectors to provide insecticidal properties to host plants. o Other insecticidal elements include protease inhibitors (serine and cysteine types), lectins, o-amylase, peroxidase and cholesterol oxidase. Other miR-genes, such as miR'A(a) and miR2A(a), as described in US Patent Application Serial No. 08/463,483, to which the authors refer, are also used in the present invention.

This co-expression of more than one insecticidal element in a single transgenic plant is achieved by genetically engineering a plant to contain and express all the necessary genes. Alternatively, Parent Plant 1 can be genetically engineered to express MIR proteins. A second plant, Parent Plant 2, can be genetically engineered to express an additional insect control element. Through the crossing of the parent plant

1 with Parent Plant 2 produce offspring that express all the genes introduced into Parent Plants 1 and 2.

Recombinant microorganisms that include genes and proteins of the MIRZ class

It was recognized that the isolated genes of this invention, which encode a MIRZ-class protein, better - MIRZA(c)-protein,

They can be transferred to any host microorganism and give this host insecticidal properties. Various hosts for the novel genes of the present invention are selected as being suitable for cloning, for characterizing the form and function of the gene or encoded protein, for use as fermentation hosts to increase production of the toxin protein, for more efficient delivery of at least one toxin protein to into the organism of an insect pest or the introduction of a new toxin gene into the organism of pathogenic insects such as baculovirus (nuclear polyhedrosis virus, e.g. Amtsiodgarniez saiotis) to increase their effectiveness.

By design, the aforementioned hosts are to be applied in the environment or to a plant or animal to control insects. Host microorganisms are selected that occupy their place in the "phytosphere" (phylloplane, phyllosphere, rhizosphere and/or rhizoplane) of one or more crops. These microorganisms are selected for their ability to successfully compete in a specific environment with wild microorganisms, they provide maintenance and expression of the gene expressing the polypeptide pesticide, and preferably they provide improved protection of the pesticide from degradation and inactivation in the environment.

Such microorganisms include bacteria, algae and fungi. Particularly interesting are microorganisms such as bacteria, for example, Bacillus, Saccharomyces cerevisiae, Adtepeiischt, Revepdotopaz, Egpmipia, Zegtaya, Kierzieia,

Hapitopaz, 5igeriotusev, KPihorisht, Kpodorzeydotopaz, Meipuyiv, Adtrbasiegisht, Asefobrasieg, I asioradiIsv,

Agiphorasieg, Agoiorasieg, I eisopovios and AIIsaiiidepev; fungi, in particular, yeast, for example, Zasspagotusev,

Stguriosossis, Kipumegotusevs, Zrogoroiotusevs, Kpodoiotia and Aigeoravzidiit. Of particular interest are such species of phytospheric bacteria as Vasishz5, Rzeidotopaz zuhipdae, Rzepdotopaz Pogezsepz, Zeitaia tagsezsepv,

Aceforasieg huipyt, Aadhorasiegia, Kpodorzeidotopaz zrpegoiidez, Hapitopaz satrezigiv, KPihorisht peioii, sch ov Lsaiydepez epihorpiz5, Siamirasieg hui and Agoyurasieg mipiapai; and phytospheric yeast species such as

KPodoyotia gibga, KK. adishipiv, K. tagipa, K. ashgapiasa, Sguriosossiz airidiz5, S. ayitsepe, S. Iashtsgepiy, (8)

Zasspagotusev gozei, 5. rgeigiepviv, 5. segemiviae, Zrogoroiiotusev goztsev, 5. odogiv, Kisumegotusevz megopae and Aigeoravzidiit roiIshapv. Pigmented microorganisms are of particular interest.

Suitable host cells for processing pesticide-containing cells to prolong the activity of the toxin in the

Cells when applying the treated cell to the environment of specific pests can include either prokaryotes or eukaryotes, which are usually limited to cells that do not produce substances toxic to higher organisms, such as mammals. However, organisms that produce substances that are toxic to higher organisms may also be used if the toxin is unstable or the level of application is low enough to avoid any possibility of toxicity to the mammalian host. As hosts, co-prokaryotes and lower eukaryotes, such as fungi, are of particular interest. Examples of prokaryotes, both Gram-negative and Gram-positive, are Epiegorasipaseae such as Eussppepspia, Egpmipia, ZppideMPia, ZaItopeia, and Rgoiecv,

VasiPaseae; KPi2obriseae, such as Kpigoriit;

Zripiaseae, such as rpoiiobasiegisht, 2utotopav, Zeitaia, Aegotopaz, Mirgio, YuOeviyomirgio, ZrigPit;

It is asiorasiaceae; Rzecdotopadaseae, such as Rzepdotopaz and Aseyorasieg, Agoiorasiegaseae and Mihorasiegaseae. "Eukaryotes include fungi such as Rnusotuseiez and Azotuseiez, which includes yeasts such as Zasspagotusev sv)s and Zspigozasspagtotusev; as well as Vasidiotuseifev yeast, such as Kpodoiogtsia, A!ygeoravzidiyt, Zrogoroiotusev, etc. ;" Particularly interesting characteristics in the selection of the host cell for production are the ease of introducing the protein gene into the host cell, the availability of expression systems, the efficiency of expression, the stability of the protein in the host, and the presence of additional genetic possibilities. Characteristics associated with the use of microcapsules as a pesticide include protective qualities for the pesticide, such as thick cell walls, pigmentation, and intracellular packaging or inclusion formation; leaf affinity; non-toxicity for mammals; because attractiveness for use by pests; ease of destruction and assimilation without harm to toxins, etc. Among 2) other characteristics - ease of formulation and application, economy, stability during storage, etc.

Especially interesting host organisms are such species of yeast as Kpodoiogtsia, Achtsgeoravzidiit, Zasspagotusev, pi and Zrogoroiiotusev; phylloplane organisms such as Rzepdotopavus, Egpmgipia, and Riachoracepyta; or other similar sp organisms, such as Euspepspia, I asiorasiyiz, Vasiyshv and others. Among the specific organisms - Rzepdotopaz aecygdipozva, Rvecydotopaz Ppogezsepz, Zasspagotusev sehemizide, Vasiiv (pippdiepviv, EvsPegispia "soy,

Vasiyiz with, etc. 5B There are many methods available for introducing a gene expressing a pesticide protein into a host microorganism under conditions that allow stable maintenance and expression of the gene. For example, they can

F) to construct expression cassettes that include the desired DNA sequences, functionally linked to the transcriptional and translational regulatory signals for the expression of the DNA sequences, and a DNA sequence homologous to the sequence of the host organism in which the integration occurs and/or the replication system , which functions in the host when integration or stable maintenance occurs.

Transcriptional and translational regulatory signals include, but are not limited to, promoter, transcription initiation site, operators, activators, enhancers, other regulatory elements, ribosome binding sites, initiation codon, termination signals, etc. See, for example, US Patent No. 5,039,523; US patent

Mo4,853,331; ERO 0480762A2; ZatrgbooK ei aiY. vyrga; MoiIesshag Siopipd, A I aroghaiogu Mapia!. Mapiaiiov ei ai. 65 (eds) Soiy 5rgipd Nagbrog I aroghagu, Soiy rgipd Nagrog, MU (1982); Admapseid Vasieegiai Sepeiyisv, Yuamiv ei ai. (eds.) Soia brgipd Nagbrog I arogayugu, Soyd 5rgipd Nagrog, MU (1980) and references to them.

New genes or their recombinant forms can be transformed into such hosts using a number of generally accepted methods. One of these optimal methods is electroporation of a microorganism cell, described for example as the method of Dauer (Oozhmeg) (US Patent No. 5,186,800). Another optimal way is the method

Shurter (Zspipeg ei aI.) (Moi. Sep. Sepei. 218: 177-181 (1989)), also described in US Patent Application Serial No. 07/353,565, which is hereby incorporated by reference in its entirety.

Genes encoding MIRZ-class proteins can be introduced into microorganisms that reproduce on plants (epiphytes) or in plants (endophytes) to deliver MIRZ-class proteins to pests intended for destruction. Many types of bacteria are able to live in the vascular tissues of plants. Most of these endophytes and 7/o epiphytes have little physiological effect on plant growth and productivity.

Bacteria that form colonies in the root, for example, can be isolated from the desired plants by methods known to specialists. For example, the Vasiyya5 segei5 strain, which forms colonies in the root, can be isolated from the roots of the plant (for example, see .). Napadeivtap, 5. KaiPe!, E. Mezieg, Y. UUypaegispy apa S. Ogats, Arri.

Epmigop. Misgobryoi 56:713-718, (1990)). MIRZ-genes can also be introduced into Vasiliyz segeiz forming /5 Colony in the root, using standard methods known to those skilled in the art. Yes, a gene that codes for a protein

MIRZ-class, which is derived from strain AB88, can be introduced into root colony-forming Vasiy5 segue by conjugation using standard methods (). (zopgaie7, V. Vgom/p apa V. Sagiop, Rgos.

May Asad All together. 79:6951-6955, (1982)).

The new genes of the invention can also be introduced into root colony-forming Vasiy5 by 2o electrotransformation. For example, mirzA(a) can be cloned into a shuttle vector, such as pHNTZ3101 (0.

It's egesiiz ei ai, REM MisgobioY. Ienv., 60:211-218 (1989)). The shuttle vector RNTZ3101, which contains the coding sequence, can be transformed into the root colony-forming Vasiyi5 by electroporation (0.

She exesiiv her aI. 1989, REM5 Misgobioi. And av. 60:211-218). It is also possible to use Vasiyiz tedayenita, which forms colonies in cotton. high school

Another example is the endophyte Siamirbasieg hui from the genus/species, which contains phytopathogenic bacteria that slow plant growth. This bacterium can grow to very high levels in the vascular system of i) plants. 5-endotoxin was injected into this endophyte, which was then inoculated into the plant, which provided effective control of the corn moth. Other endophytes are also known.

Expression systems can be constructed in such a way that MIRZ proteins are secreted outside the cytoplasm of gram-negative bacteria, for example, E. soii. The advantages of secreting MIRZ proteins are that (1) it can increase the level of expressed MIRZ protein and (2) it can facilitate the efficient clearance of C

MIRZ protein. co

Secretion of MIRZ proteins of E. soybean can be provided, for example, by fusing the appropriate signal peptide of E. soybean with the amino-terminal end of the MIRZ signal peptide or replacing the MIRZ signal peptide with the BE signal peptide. soybeans Signal peptides recognized by E. soya are found in proteins that are secreted in He)

E. soyii, for example, OtrA-proteins (U. OPhauer. N. Kitiga, M. Takapaga, M. Mavzii apa M. Iposue, EMVO 3., 3:2437-2442 (1984)). OtrA is the main protein of the outer membrane of E. soybean and, thus, its signal peptide should be effective in the translocation process. Also, there is no need to modify the OtrA signal peptide before processing, as is the case with other signal peptides, for example, the lipoprotein signal peptide (O. Oillatsa, R. Magsi, and M. Ipotsue, Medicines and Epigenetics 153:492 (1987)). - c Yes, unique Vat restriction sites can be introduced at the amino-terminal and C-carboxy-terminal ends of MIR coding sequences using standard methods known to those skilled in the art. or AZ (uU. sNgaueb,

N. Kitiga, M. TaKapaga, N. Nvishpd, U. Mavyi apa M. Ipotsshue, EMVO )3., 3:2437-2442 (1984)), thereby forming an otrA: MIR fusion gene that is secreted into the periplasmic space. Other restriction sites in the polylinker

Ge) riM-P-otrA can be deleted by standard methods known to those skilled in the art, so that the MIRZ amino-terminal amino acid coding sequence is located directly after the cleavage site of the otrA-signal co-peptide. Thus, the secreted MIRZ sequence in E. soya should be identical to the natural one

OO MIRZ sequences.

If the MIRZ natural signal peptide is not required for the proper assembly of the formed protein, such signal sequences can be deleted and replaced by the otrA signal sequence. Unique Watni sl restriction sites can be introduced at the amino ends of the proprotein coding sequence directly after the coding sequence of the MIRZ signal peptide and at the carboxy terminus of the MIRZ coding sequence. These

Cotton fragments can then be cloned into rIM-IP-otrA vectors as described above.

General methods of using the strains of the invention for pesticidal control or engineering other organisms as pesticidal agents are known to those skilled in the art. See, for example, US Patent No. 5,039,523 and EP 0480762A2. o MIRZ can be fermented in host bacteria, and the resulting bacteria are processed and used to spray microbes in the same way as strains of Vasiyz (Pipdiepsis have been used in insecticidal sprays. In the case of MIRZ, which is secreted from VasiШ5, the secretion signal is removed or because mutated using procedures known to those skilled in the art. Such mutations or deletions prevent the secretion of the MIRZ protein(s) into the growth medium during fermentation. The MIRZ proteins are retained in the cell, and the cells are then processed to obtain the encapsulated MIRZ protein. To this end any suitable microorganism is used.Rscedotopase was used to express Bacillus endotoxins5

Ipihypdiepsis as encapsulated proteins, and the resulting cells were processed and sprayed as 65 insecticide (N. Saegpeg ei a!. 1993, |Ip Admapsei Epdipeegei Reziisidesz, I. Kit ea.).

In this way, different strains of Vasiyiv (pygipdiaepsis) are used. Such strains of Vi produce endotoxin|(i)

protein(s) as well as MIRZ. Or, such strains can only produce MIRZ. VasshShivz zybBiiv strain with insufficient sporulation produces a high level of StuzA endotoxin from Vasiyiz (Pyhipdiepviz (Adaizze, N. apa

Iiegesiiv, O., "Ehrgeziop ip Vasiiv 8zyBiiv oi She Vasiiv (pygipdiepviv StuyshA iohip depe iv poi derepdepi op a zrogtsiaiop-zresiiys vta vzidta Tasiog apa iz ipsteasei ip a zroOA tpshapi, u. Vasiegioi., 176:4734-4741 (1994) ).

Crops to be protected within the scope of this invention include, for example, the following types of plants: 70 cereals (wheat, barley, rye, oats, rice, sorghum and other similar crops), beets (sugar beet and fodder beet), fodder grasses (grasses, fescue, etc.), stone fruits, seeds and berries (apple, pear, plum, peach, almond, cherry, strawberry, raspberry and blackberry), legumes (beans, lentils, peas, soybeans), oilseeds plants (rapeseed, mustard, poppy, olives, sunflower, coconut, castor, cocoa beans, peanuts), cucumber plants (cucumber, squash, melon), fibrous plants (cotton, flax, hemp, jute), citrus fruits (orange, lemon, grapefruit, tangerine), vegetables (spinach, lettuce, asparagus, cabbage and other Vgazvzisae, onions, tomatoes, potatoes, paprika), and ashgasede (avocado, carrot, cinnamon tree, camphor), deciduous and coniferous trees (for example, linden, yew , oak, alder, poplar, birch, spruce, larch, pine) or such plants as corn, tobacco, nuts, coffee tree, sugar cane, tea, grapes, hops, banana and rubber plants, as well as ornamental plants (including composites).

Microorganisms that have been genetically modified to contain a pesticide gene and protein can be used to protect crops and products from pests. In one aspect of the invention, whole, ie unlysed, cells of a toxin (pesticide) producing organism are treated with reagents that prolong the activity of the toxin produced in the cell when the cell is used in the environment of the pest(s).

Or pesticides are produced by a heterologous gene in host cells. Expression of a heterologous gene p

As a result, directly or indirectly, it leads to the production and preservation of the pesticide inside the cells. These cells are then treated under conditions that prolong the activity of the toxin produced in the cell when i) the cell is used in the environment of the pest(s). The resulting product retains the toxicity of the toxin.

These naturally encapsulated pesticides can then be formulated according to traditional technologies for application in the environment - the host of the pest, for example in soil, water or plant leaves. See, for example, ERA 0192319 and its references.

The active ingredients of this invention are, as a rule, used in the form of compositions on areas occupied by crops, or applied to plants to be treated, simultaneously or alternately with other compounds. These c compounds can be fertilizers or a source of micronutrients or other compositions that promote plant development. They can also be herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides or, if necessary, mixtures of several of these compositions together with other agriculturally acceptable carriers, surface-active substances or adjuvants that are traditionally used in formulation Suitable carriers and adjuvants may be solid or liquid and correspond to substances traditionally used in formulation, for example, natural or regenerated minerals, solvents, dispersants, wetting agents, tackifiers, binders or "fertilizers" The optimal methods of application of the active ingredient of this invention or the agrochemical composition of this invention, which contains at least one of the pesticidal proteins produced by the bacterial strains of this invention, are application to leaves, covering seeds and application to the soil. The number of methods and application rates depend from the intensity of the attacks x or other pests.

Entomocidal compositions, which include a recombinant strain of Vasiliiz (pygipdiepv

The present invention also provides an entomocidal composition comprising a recombinant strain VasiPsh5

Ipippdieppziz, which contains at least one of the new toxin genes in recombinant form or its derivatives or co-mutants together with such an agricultural adjuvant as a carrier, diluent, surface-active substance 2) or an adjuvant that facilitates application. The composition may also contain another biologically active compound selected from fertilizers, sources of micronutrients, compositions for plant growth, herbicides, insecticides, fungicides, bactericides, nematicides and molluscicides, as well as mixtures thereof. The compositions may include from 0.1 to 9995 by weight of a recombinant strain of Vasiyz (a pygypdiepzyz containing at least one of the novel genes in recombinant form, or derivatives or mutants thereof, from 1 to 99.995 by weight of a solid or liquid adjuvant and from 0 up to 2595 by weight of surfactant Recombinant strain VasiShiz (pygypdiepsis containing at least one ov of the new genes in recombinant form, or a composition containing it, can be introduced into a plant or crop in need of protection, together with some other insecticides or chemicals (1993 Stor Rgogesiop

F) SpetisaFrom Keiegepse, Spetisa! apa RNpagtaseciisa! Rgezz, Sapada) without loss of efficiency. It is equivalent to other commonly used materials used in agriculture for spraying, but it cannot be used in spraying solutions with a strong alkaline reaction. It can be used as a powder, suspension, wetting powder or in any other form suitable for use in agriculture.

Recombinant strain Vasiiiiv (pygypdiepsis) containing at least one of the new genes in recombinant form is usually used in the form of entomocidal compositions and can be applied to areas or plants to be treated simultaneously or alternately with other biologically active compounds. These compounds 65 Can be fertilizers or micronutrient source or other compositions that promote plant development.They can also be herbicides, insecticides, fungicides, bactericides, nematicides,

molluscicides or mixtures of several of these compositions, if necessary - together with other traditionally used carriers, surface-active substances or adjuvants that facilitate application. Suitable carriers and adjuvants may be solid or liquid and correspond to substances commonly used in the formulation, i.e. natural or regenerated minerals, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers. Entomicidal compositions, compositions or mixtures containing the recombinant strain VasiShv5

Ipihypdiepsis, which contains the novel gene in recombinant form as an active ingredient, or a combination thereof with other active ingredients and, if necessary, with a solid or liquid adjuvant, is prepared by a conventional 7/0 method, for example, by homogenization and/or trituration of active ingredients with diluents such as solvents, solid carriers and, in some cases, surface-active compounds (surfactants).

Solvents, carriers, surface-active substances, surface-active compounds, etc., which are traditionally used in the development of compositions and which can be used within the scope of this invention, are described, for example, in MO 96/10083.

Another optimal characteristic of the entomocidal composition of this invention is the stability of the active ingredient when applied to plants and soil. Possible causes of loss of activity include inactivation by UV light, high temperature, leaf exudates, and pH. For example, at a high pH level, in particular, in the presence of a reducing agent, 5-endotoxin crystals dissolve and become more suitable for proteolytic inactivation. A high pH level in the leaf can also be important, especially if it is pH 8-10 on the surface of the leaf. The development of the entomocidal composition of the present invention can be applied to solve these problems either by including additives that help prevent the loss of the active ingredient, or by encapsulating the material in such a way that the active ingredient is protected from inactivation. Encapsulation is carried out chemically (MeScige apa Zpnazpa, 1992) or biologically (Vagpez apa Sittipoz, 1986). Chemical encapsulation involves the process of encapsulating the active ingredient with a polymer, while biological encapsulation involves the expression of endogenous toxins in a microorganism. For biological encapsulation, a whole microorganism containing 5-endotoxin protein is used as an active ingredient in the composition.

Adding UV protection can effectively reduce the damage from exposure. Inactivation due to high temperature can also be combated by incorporating a suitable additive. IS)

Entomicidal compositions, which usually contain from 0.1 to 9995, preferably from 0.1 to 9595, of the recombinant strain

Vasiyiv (pygypdiepsis containing at least one of the new genes in recombinant form, or its combination with other active ingredients, from 1 to 99.995 solid or liquid adjuvant and from 0 to 2595, preferably - from.j8 "o 0.1 to 2095, surfactant. Since commercial products are optimally prepared as concentrates, the consumer generally uses diluted formulations of much lower CO 3335 concentration. Entomicidal formulations may also contain other ingredients such as stabilizers, (Ce) antifoams, regulators viscosities, binders, tackifiers, as well as fertilizers or other active ingredients to obtain the desired effect.

Ways to fight insects «

In light of the foregoing description of the invention, it will be appreciated that there are several ways in which insects 470 can be controlled using MIRZ-class proteins as an insecticidal element, either alone or in combination with auxiliary insect control elements such as 5 - endotoxins. Any method of introducing the MIRZ protein for consumption by a susceptible insect leads to the death of this insect. is" In one variant of the invention, plants are transformed with a gene encoding a MIRZ-class protein. Expression of the protein can occur at any time during the growth and development of the plant, depending on the characteristics of the insect to be controlled. For example, the MIRZ-class protein according to the invention can be expressed in roots, (22) stems, leaves, seeds, pollen, etc. It provides the advantage of expressing the protein only in the cells or tissues where the target insect feeds. Consumption by a susceptible insect of cells or tissues of a plant expressing the MIRZ protein leads to the death of this insect. In one embodiment of the invention (95), the MIRZ protein is expressed in the stems or petioles of a plant to control the epsilon bollworm. Their 50 plants are grown either in the field or in greenhouse conditions. Seeds containing the MIRZ protein can also be protected from insects during storage. sl EXAMPLES

Examples 16 to 18 on pages 73 to 82 of MO 96/10083 describe the isolation and biological specification of Vasiliyz strains (pygipdiapsis AB88 and AB424, purification and characterization of the MIRZA(a) protein, cloning of the mirza(a) and mirza(Bv) genes and identification of novel if genes by hybridization. Reference is made to the above-mentioned examples in their entirety. The examples below also describe the materials and methods used in the embodiment of the invention and the results of that embodiment. They are offered by way of illustration and by way of enumeration should not be construed as limiting the claimed invention.

Example 1: The presence of miR3-like genes and miRZ-like proteins in isolates of Vasilia5 60 Other isolates of Vasilia5, except AB88, showed insecticidal activity against Ieridoriega larvae when testing the supernatants of the cultures used. Some isolates that were active against the epsilon bollworm were analyzed for the presence of MIRZ-like genes and the production of MIRZ-like proteins.

A standard polymerase chain reaction (PCR) assay was used to determine whether Bacillus isolates active against upsilon scoops contained a miR3-like gene. Using a pair of primers RSK 65 / ZUM110 (5-SOSA TTA ATO To SS TO-3; BO IU MO:17) and SMU111 (5-SAT TAS SAT STO So ASA SAO-3;

ZEO IU MO:18), found that all isolates active against the epsilon scoop produce 728 bp. mirZ gene product,

which was equal in size to that produced by the type strain, AB88. One Vasishv5 isolate, AB5B1, which was not active against the epsilon bollworm, produced a miRZ product of the same size. None of the other Bacillia5 isolates inactive against the scoop epsilon produced the ROK miR3 product.

Analysis of the production of MIRZ protein was carried out using a standard Western blotting procedure.

Antibodies produced against MIRZA(a)-protein, described in the previous example, were used to detect immunoreactive proteins. Aliquot samples of supernatants of cell-free sporulated cultures were electrophoresed with sodium dodecyl sulfate in a polyacrylamide gel (5O5-RACE) using standard methods. After that, standard western blotting was performed to determine the presence of 70 MIRZ-like proteins. All Vasiyiv5 isolates, which had 728 bp. of the RSK product and were active against the epsilon scoop, produced an 8O0kDa protein that was immunoreactive to the MIRZA(a)-antibody. Isolate AB5O1, which had the correct size of the RSC miR3 product but was not active against the epsilon scoop, produced an immunoreactive protein that was truncated, suggesting that this may account for the lack of biological activity against the epsilon scoop.

Example 2: Characterization of the Vasiyz strain (pygypdiepsis AB51 containing a miR3-like gene

The Vasiyiz strain (pygypdiepsis, designated as AB51) revealed the content of MIRZ-class proteins in Western blotting using rabbit polyclonal antibodies against MirZA(a). The mirZ-like gene was cloned into pK5, which produces pSIV7112. This gene was designated as mirZA(c) . The DNA sequence for mirZa(c) is described in ZEO I MO:5, and the sequence of the encoded protein is described in ZEO IU MO: 6. The MIRZA(c) protein is 746 amino acids long, 43 2o amino acids shorter than its homologues (MIRZa) and MIRZA (B).

Example 3: Production of antibodies to MirzA(a)-protein

Antiserum against the purified MirzAd(a) insecticidal protein was produced in rabbits and goats. For rabbits, nitrocellulose-bound protein (50 μg) was dissolved in OM5O, emulsified with complete Freund's adjuvant (Oiso), and administered subcutaneously twice a month for three months. For goats, active, soluble, pure MirzA protein (30 μg) was administered intramuscularly twice a month for three months. Blood was taken from them 10 days after the second and third injections, and serum was recovered from the blood samples (For example, E. apa Gape, 0. i)

Apiirodiev: And Mapia! Iarogayugu, Soy Zrgipd Nagrog ar. Rgezv, Moscow State University, 1988). The antisera were then fractionated by affinity chromatography using staphylococcal protein A, and the resulting

The IDS fraction was further purified by filtration through a column containing immobilized lysate of E. soybean (Mi, S... yu zo eiaI. Arri. Epmigop. Misgobrioi. 63:532-536 (1997)).

The specification of rabbit and goat antisera was made by analyzing the MIRZA(a) protein by Western blotting. -

Proteins were separated using 505/RACE and transferred to nitrocellulose. Nitrocellulose stains were blocked in a mixture of 20 mM Ttiv-HCI, pH 7.5 / 0.15 M mass / 0.02956 MaMaz / 595 non-fat dry milk. Then they were revealed using either rabbit or goat antibodies against MirZa(a) at a concentration of 20 ng/ml or 10 ng/ml, respectively. Alkaline phosphatase-conjugated anti-rabbit goat anti-rabbit antibody or anti-rabbit anti-rabbit antiserum was used as secondary antibodies at a concentration of μg/ml (Kigedaaga 85 Regitu I Arogaifgiev,

Ips.). Bromochloroindoyl phosphate and nitrotetrazole were used as a substrate for the alkaline phosphatase reaction.

Both rabbit and goat anti-MirzA(a) antibodies are polyclonal. Anti-MirzA(a) antibodies obtained from a goat have a higher titer than those obtained from a rabbit. In the experimental approach, anti-Mirza(a) antibodies "

Rabbits should be used as diluted 1/500 of the original serum (200ng/ml). By comparison, with anti-MirzA(a) antibodies obtained from a goat, can be diluted up to 1/2000 (1O0Ong/ml) from the original serum. . If the antibodies obtained from the rabbit only recognize the M-terminal part of the MIRZA(a) protein, then the antibodies, and? obtained from goats, react with epitopes present along the entire length of the MIRZA(a) protein.

Example 4: Construction of expression cassettes in plants

Expression cassettes in plants consist of promoters that can stimulate the expression of the coding

Ge" sequences either constitutively or tissue-specifically, coding sequences that must be expressed, and terminating sequences that allow polyadenylation of mRNA and its proper co-translation. 2) Promoters selected from the DNA sequences of the present invention include constitutive promoters, such as the 5p promoter from the maize ubiquitin gene (SpHgiziepzep ei a). Riapi Moi. Vioi 12:619-632, 1989) (rsSIV8029, Figure 4; rSIV8055, Figure 5; rSIV80b, Figure 6), and tissue-specific promoters, such as promoters from sp metallothionein-like gene of maize (de Rgatopa, A. REV5 290:103-106 , 1991) (pSIV8030. Figure 7; pSIV8O56, pSIVO805), which provides optimal expression for roots, from the RERS gene of corn (Niazreiy. K.I. apa Sgsha,

MIND. Riapi Moi. Vio 12:579-589, 1989) (pSIV5535, Figure 8; pSIVO807), which provides specific expression for two Green tissue, and from the non-specific lipid transfer protein of barley STRA (pSIVO819, Figure 9) (Moiippa, A. apa Sagsia-Oitedo, R. Riapi.). 4:983-991, 1993), which provides optimal expression for stems. (F) All sequences used in the present invention contain a terminator sequence derived from 355 ka Samu and intron 9 derived from the maize PERS gene to enhance gene expression. Plasmids pSIV8029, pRSIV8O55, and pSIV9806 contain the Mo1 intron of the maize ubiquitin gene located between the maize 6o ubiquitin promoter and the mirzA(a) gene. The sequence including the coding sequence of the mirzA(a) gene, the Me9 intron, and the 355 termination sequence was engineered into a recipient plasmid carrying different promoters as double WatnNi-Esoki hydrolysates.

Expression cassettes in plants were used as such in plant transformation experiments, or they were linearized through the use of restriction enzymes that ligate to the Atr" gene of a framework plasmid. 65 In some experiments, fragments including the promoter, the desired gene, an intron, and a terminator were isolated from the rest of the plasmid framework by restriction digestion and purification of the fragment. In these cases, purification of the fragment was carried out as follows: X00μg of DNA is cleaved with the appropriate enzyme and separated on a 0.895 agarose gel. The desired fragment is identified, excised from the gel and purified using a Yuigaroge MiProge filter (0.45μm). The filtrate containing the fragment is precipitated with sodium acetate and ethanol.

The fragment is resuspended in TE and used in transformation experiments.

Example 5: Insecticidal activity of maize expressing MIRZA(a)

Maize expressing the MIRZA(a) protein was tested for insecticidal activity against the insect species listed below in the Table using the following procedure. One to four fragments of 4 cm each were cut from the leaves of transgenic and control corn. Each leaf fragment was placed on a moistened filter disc in a 7/0 Z0Homm Petri dish. Five newborn specimens of the tested species were placed on each leaf fragment, with a total number of 5-20 larvae tested for each plant. Petri dishes were incubated at 30"C in the dark. Mortality was assessed after 48-72 hours. The results are shown in Table 16.

Shyudniki where 0001

Sovkaipsilon (Aotivivyut 00000000

Autumn derivative worm vrodoretya tdrenya) 00001000

Sugar cane fire (emwa zasta) 0000001000

Western corn-sucking butterfly (Bianava sgapoyueeiv) 1000 nshishyudnyuterayureya 0000 sch

Zvzemnasova (in porridge 11111111111100000001000

Posadny chereyakhovtosmuayetyi (5 otiluzai 00001000 o

Example 6: Expression of mirZA(a) in corn. Transformation of inbred lines of elite corn Sira СО00526 and 2154 with the help of the MirZ gene was achieved using bombardment with particles of callus tissue | type For transformation using - embryogenic callus type callus was obtained from zygotic embryos using standard methods and co-inoculated 1-2 days before bombardment. Callus tissue was prepared for bombardment by placing 20 fragments with a diameter of 3-5 mm in the form of a ring on a culture medium containing 1295 sucrose. Callus c5 tissue was placed on the medium for four hours before bombardment. The DNA used for co-transformation of corn callus was circular plasmid DNA, linear plasmid DNA, or purified DNA fragments containing the Mir3 gene under the control of various plant promoters. In experiments using a selectable agent, the gene provided resistance to phosphinonitricin or growth in the presence of mannose. Fragments of plasmids or DNA isolated by filtration were precipitated on 0.µm particles "

Gold according to the published procedure from VioKaai I arogaigiev, Negsciez, SA. The gold particles were fed by applying a burst pressure of 50 psi of helium. Each target plate was bombarded twice with DNA-coated particles. After six to twenty hours after the bombardment of СО00526, the callus was transferred to ;" standard culture stabilizing medium. Seven days after bombardment, the tissue was transferred to a medium containing the selection agent Vazia at a concentration of 100 mg/l. Vavzia is a commercial composition of glufosinate ammonium produced by Noespivi. Callus 2154 was stored on 1295 sucrose throughout

He" 1-7 days after bombardment and transferred to a standard culture medium containing 20-ZOmg/l

Vavzia on the 7th day. 2154 and CO00526 callus were inoculated in the presence of ZO or 10 ug/l Vauzia, respectively, for 10 to 8 weeks. The selected tissue was inoculated with lower levels of Vauzia (5-40mg/l) for a period of approximately five to ten weeks to allow the tissue to be harvested and then transferred to a standard regeneration medium without selection for plant production. In total, 1,295 bombarded callus fragments produced a transformed callus that underwent Vazvzia selection. Individual transformed sp calli usually regenerate, giving 20-30 plants.

Events were obtained in experiments in which no selection was applied. In these experiments, callus were grown for a period of 9-10 weeks on stabilizing medium before being transferred to regeneration medium. Event 1337 is an example of a transformed MIRZ event resulting from a marker-free transformation experiment amenable to selection or quantitative analysis by screening plants for

F) insecticidal activity. ka Transformed calli were also obtained in experiments in which mannose selection was applied. In these transformations, the mannose-6-phosphate isomerase gene under the control of the ubiquitin promoter of corn pSIV9818 60 was bombarded with the Mir3 gene. Mannose at 0.5-1.590 was included in the stabilization medium for a period of twelve weeks and was not included in the regeneration medium.

Transgenic plants were evaluated for the expression of MIRZA(a) protein by insect bioassay and solid-phase enzyme-linked immunosorbent assay (ELISA). Leaf fragments were taken from plants at the 2-4 leaf stage for evaluation using epsilon bollworm and autumn cutworm bioassays. Bioassays were carried out with 65 Using ten newly hatched larvae placed in vessels with fragments of leaves. The percentage of mortality in 72 hours was calculated. Tissues of transgenic plants were also analyzed by EG I5ZA analysis using standard protocols for quantitative determination of MirZ protein levels in various plant tissues.

Plant tissues were taken, and Table 17 shows characteristic events and their corresponding insect bioassay results.

Transgenic maize was transformed with different plasmids containing the Mir3 gene under the control of different promoters, such as the maize PEP carboxylase (PEP) promoter, the maize ubiquitin promoter (Bi) and the maize metallothionein-like promoter (MT). The gene of the selected marker was the PAT gene under the control of the maize ubiquitin promoter in rOVIAS. Among the characteristic events listed in Table 17, events produced with different plasmids or plasmid-derived DNA fragments are shown. DNA fragments were obtained using restriction enzyme cleavage and size fractionation using electrophoresis in 0.876 agarose gels. DNA fragments were excised from the gels, frozen, minced, and purified by filtration through 045 μm Juroghe Miroghe filters followed by ethanol precipitation. Transformed events of maize were obtained with the help of circular DNA plasmid pSIV5535, containing the Mirz3 gene, under the control of the PERS promoter of maize. Events were also transformed with linear DNA plasmid pSIV5535 and pSIV8029 containing the Mir3 gene under the control of the maize ubiquitin promoter. 7/5 Additional events were obtained by bombarding purified DNA restriction enzyme fragments containing only the Mir3 gene with a promoter. Fragments that correspond to the Mirz gene include: 4906Br EsokI/NipaiYi fragment from RSIV5535 with the maize PERS promoter; 5142rr Krpi/Nipai fragment from pSIV8030 with MT. promoter; 4597

Krpi/Nipai fragment of pSIV8029 with a maize ubiquitin promoter; 4818Br Nipai fragment of rsSIV8O55 with a corn ubiquitin promoter; 5364 Nipai fragment of pSIV8056 with MTI promoter; 5964 Ausi fragment VSIVU8O5 with MT. promoter; 5418rr Azsi fragment of pSIV9806 with ubiquitin promoter of corn and 5727rr

The Ausi fragment of rSIV9807 with the RERS promoter of maize.

Event Mo Plasmid used Chimeric gene with higher pSIV8O29 SHORImirzA(a) pSIVVoZo MT mirzA(a) o 30 "

Example 7: Insecticidal activity of corn containing Mirz and B 53-endotoxins

MIRZA(a) has weak activity against European corn moth (ECB). In order to provide plants capable of fighting against many insects of the Eridoriega series I, corn containing the mirzA(a) gene was crossed with corn containing sgu1B, which is active against ESV. Progeny from the cross were subjected to bioassay 35 for resistance to ESV and fall-derived roundworm (HAMU) as described in Example 1. Results are shown in ise)

Tables 18. Approximately 3495 progeny were not active against any of these species, 15.4956 were active only against ESV, 23.195 were active only against EAMU, and 27.995 were active against both species. Plants active against both species contained both MIRZA(a) and Stu!B proteins. Similar results were obtained with the use of other VI 5-endotoxins, in particular StutAbB or Stu9s. 40 - p. that 45 Example 8: Lysis with the help of MIRZAdSa) of epithelial cells of the middle intestine of susceptible insects (o) Experiments with consumption and cleansing of the intestine. The temporal sequence of symptoms after the consumption of a diet containing MIRZA(a) by susceptible insects - larvae of the upsilon bollworm (VSMU) in the second stage of development - was recorded from the time of the first introduction until the death of the larva. Larvae fed this diet exhibited (95) active feeding followed by continuous intestinal parastalsis. But adding iz 50 MIRZA(a) protein to the diet had a significant effect on eating behavior. When added in concentrations not higher than 4ng per cm? the larvae continued to feed from time to time for periods of 10-20 minutes. The presence of blue sl in their intestines indicated nutrition, but the content of the cleansed intestines varied greatly, judging by the decrease in the amount of feces. When 4ng of MIRZATS(a) per cm 2 was added to the diet, the development of larvae was markedly impaired after 48 hours of the incubation period, but mortality was not observed. At concentrations of 4 Ong MirzA(a) per cm? larvae developed intestinal paralysis after consuming a small amount

HF) of the diet, and no excrement was observed, which indicated an almost complete absence of bowel cleansing. Under these cue conditions, approximately 5,095 deaths were observed after 48 hours. At concentrations higher than 4Ong MIRZA(a) per cm? the larvae died after only a few bites, without excrement, and the mortality reached 10095. When conducting similar experiments with the autumn worm, also a susceptible insect, a similar pattern of behavior was observed. But the European corn moth did not change its feeding behavior even when added

MIRZATSa)-protein to the diet in concentrations of not less than 400Ong MIRZATSa) per cm.

Histological observations of the effect of MIRZA(a)-protein. Histopathological observations of exposure

MIRZA(a)-protein at VSMU was carried out with larvae in the second and third stages of development, which were fed with a diet containing MIRZA(a). Analysis of the VSMUU intestinal section showed significant damage to the midgut epithelium, which indicated that the midgut tissue is the main site of action of the MIRZA(a) protein.

No damage was seen in the foregut and hindgut. Epithelial cells of the midgut of control larvae were tightly connected to each other, showing no signs of damage. Sections of larvae fed for 24 hours on a diet containing MirzA(a) showed that the distal ends of the epithelial cylindrical prismatic cells swelled and became bulging. Although goblet cells showed some morphological changes, they did not show signs of damage at this stage. Degeneration of epithelial cylindrical prismatic cells continued, and after 48 hours. after eating a diet containing Mirza(a), the lumen was filled with the remains of destroyed cells. Goblet cells also showed signs of damage after 48 hours, but both cell types were still attached to the basement membrane. Upsilon larvae died after 72 hours, and sloughing of the epithelial layer was completed. If a similar histopathology was observed in the fall armyworm, the European corn moth did not show any tissue damage under the same experimental conditions.

IP mimo immunolocalization of MIRZA(a) protein. The larvae of the epsilon bollworm and the European corn moth in the third stage of development, which were fed with an artificial diet with the addition of 100-200ng of MIRZA(a) per cm 2, 75 were used for immunocytochemical characterization of MIRZA(a) bound to fragments of the midgut. Bound MIRZA(a) was visualized using a rabbit anti-MIRZA(a) antibody pre-purified through protein A sepharose and immobilized E. soybean columns (Mi, S.b. ei ai. Arri. Epuigop.

Misgobryo!i. 63:532-536, 1997). Binding of MIRZA(a) was detected in the epithelium of the midgut of the epsilon bollworm, with no binding to the midgut of the European corn moth. Fragments of 20 midguts of epsilon larvae fed a control diet did not show binding

MIRZA (a). Apparently, MIRZA(a) binding is specifically associated with apical microvilli and most associated with cylindrical prismatic cells, with no evidence of a signal in goblet cells.

Example 9: MIRZA(a) and MIRZA(B) cause apoptosis in insect cells

MIRZA(a) and MIRZA(B) were shown to be apoptosis-inducing proteins resulting from Ha 25 characterization of insecticidal activity targeting insect cell lines (51-93) derived from Brodariega ygidiregda, an insect susceptible to MIRZA(a). MIRZA (a) showed insecticidal activity against the cell line and9) insects, if its presence was ensured throughout the experiment. When ZE-9 insect cells were temporarily exposed to MIRZA(a) and MIRZA(B), the viability of their cells was significantly reduced, even with such a short period as 5 min. When the incubation time exceeded 10 minutes, the effect of MIRZA(a) and MIRZA(B) on insect cell viability was maximal over a period of b hours, showing a 9095 decrease in cell viability. Cytological changes occurring in 5E-9 cells temporarily exposed to MIRZA(a) were monitored by microscopy. Small protrusions appeared on the surface of the treated cells in the time interval from s to 15 minutes after they were exposed to the action of the MIRZA(a) protein. At this stage, the mitochondria of the cells remained functionally intact, as shown by staining with rhodamine 123, a dye that accumulates in mitochondria with an active membrane potential (doppzop, I.M. ei aYi. Rhos. Mai. Asad. Zsi. OBA 77:990-994 , Ge) 1980). These protrusions eventually disappeared, and the cells entered a phase of active vacuolization, which lasted for another 30 to 100 minutes. In the last stages of insect cells, they drum before disintegration. For a single cell, the whole process takes 1 to 2 hours. All these cellular events correspond to the data of previous studies of cells undergoing apoptosis, "especially if we take into account that the programmed death of cells during the transformation of certain insects 70 is accompanied by cellular vacuolization and swelling (Zspmagig, H.M. ei aI. Rgos. Mai. Asai. si. OBA - p 90:980-984 (1993)). Recent studies have shown that the distribution of phospholipids in the plasma membrane is affected in the first stages of animal cells that undergo apoptosis (Magip, 5. ., ei aiYi. U. Vkhr. Mea.:i82, 1545-1556, 1995) , in particular, the incorporation of phosphatidylserine (P5). This process can be visualized through the use of Appehip M, an anticoagulant protein with a high affinity for phosphatidylserine (P3). When processed

Ge») MIRZAd(a) 5E-9 cells were incubated with Appehip M, the incorporation of PO manifested itself in the membranes of insect cells already 5-10 minutes after exposure to MIRZAd(a), which may have marked the beginning of apoptosis. One of the main molecular events that is a criterion for apoptosis is endonucleolysis, which ultimately leads to double-stranded DNA breakage, releasing oligonucleosome-sized fragments of 200 nucleotide pairs and several times more. We investigated the occurrence of endonucleolysis in 5E-9 cells treated with MIRZAd) using the above method of detection and analyzing DNA by electrophoresis in an agarose gel. Based on the ability of the Klenov enzyme to incorporate modified nucleotides using DNA ends obtained by DNA fragmentation, 5E-9 insect cells showed signs of endonucleolysis as early as 30 minutes after their exposure

MIRZATS(a)-protein. This stage coincides with the appearance of membrane-bound subcellular apoptotic organisms visualized under microscopic observation. These early signs of endonucleotic activity were confirmed by the detection of DNA fragments in agarose gels, which are characterized by chromatin

F, stairs at slightly later stages of the process. These results confirmed the signs revealed by cytological observations, according to which 5E-9 cells cause programmed cell death of apoptotic type after exposure to the MIRZA(a)-protein. in MIRZA) and MIRZA(B) proteins were identified on the basis of their insecticidal properties against some insects of the genus I Eridoriega. Thus, we needed to know whether the MIRZZA(a) protein induces an apoptotic pathway in the intestinal cells of susceptible insects after ingestion and thus exerts its insecticidal properties by initiating an active cell death process. Histological and histochemical studies showed that the MIRZA(a) protein is specifically targeted to the cylindrical prismatic cells of the epithelium 65 of the midgut of susceptible insects, causing changes in the cells characterized by protrusions on the membrane and active vacuolization, which leads to cell death. These cytological changes are caused

MIRZATSa) in insect intestinal cells, similar to those described above for 5E-9 cells. Therefore, we tried to check whether midgut epithelial cells of susceptible insects undergo endonucleolysis after consumption of a diet containing MIRZA(a) by detecting (SimiMPeg, O., ei aYi. Mayishge 381:800-803 (1996)) fragmentation. DNA. When midgut tissue fragments of epsilon larvae were fed a diet containing MIRZAc(a) or a control diet, nuclear staining, which indicated fragmentation

DNA was detected only in the cylindrical prismatic cells of the epithelium of the middle intestine exposed to the action

MIRZA(a)-protein. This result suggests that the MIRZA(a) protein induces the process of endonucleolysis in the epithelial cells of the midgut simultaneously with the cytological changes reported earlier. 7/0 We concluded that the MIRZA(a) protein also exhibits its insecticidal properties by activating the apoptotic type of programmed cell death of the midgut epithelium of susceptible insects.

Example 10: Isolation of the receptor for MIRZA(a) scoop epsilon

The Epsilon bollworm is sensitive to MIRZA(a) and thus this insect was used to isolate the MIRZA(a) receptor. The midgut of epsilon larvae in the third stage of development was selected by dissection and 7/5 immediately frozen in liquid nitrogen. One gram of midgut tissue was used for isolation

mRNA according to the protocol described for the cDNA two-hybrid library construction kit from Siopiesp (1997). Ten micrograms of poly A" RNA were taken as the starting material. When synthesizing the first strand, both random and binding oligo(at)»va(A/C/5) primers are used in a separate synthesis with MMI reverse transcriptase. The second strand of cDNA was obtained by optimal ratio of activity

DNA polymerase and RNase H activity in the enzyme cocktail of the second chain. The newly synthesized double-stranded cDNA is then ligated with EsoKI-MoiI-ZaiI adapters. The cDNA was ligated into RAJ (Miiauspapaeg, 5. ei a. SG OMTESNpodtsez IX-3:8-10, 1994), which provides an activation domain. The MirzA(a) gene was introduced by engineering into the polylinker site of the rOOVTO9 plasmid in the frame with the ZAI-4 DNA binding domain (Vagiei, R.Yi. and a).

Seiiishiag Ipiegasiope ip Oemeyuortepi Rgasiisai Arrgoasi, pp. 153-179, Ohoga Opimegeyu Rgez5, 1993). with

Recombinant roVvtTO-mirZza(a) was transformed into a yeast strain (c(0M1:171 (S, sb. apa Riazppe, M.

SeiIp51:121-126, 1987) by electroporation (Evigisp, 9.9U. ei aiYi. VioTesppidshez 16:610-612, 1994). i9)

Transformed yeast was selected in a minimal environment without tryptophan (Vagie!i, R.Yi.

Ipiegasiope ip OemeIuortepi: Rgasiisai Arrgoasi, pp. 153-179, Ohgoga Opimegeiu Rgevzv, 1993). Expression

MIRZA(a) protein in recombinant yeast was confirmed by Western blotting. Yeast strain 5OU1:171-MIRZA(a) was transformed using the epsilon scoop cDNA library presented in rOAbIO. OM1::171 has a NIZZ marker under the control of SAIG4 recognition sites. The НИЗ5 gene allows selection of positive M growth for clones transformed by two interacting hybrid sequences. After seeding more than 200,000 recombinant clones, only one was able to grow in minimal medium without histidine. Plasmid DNA of a positive yeast colony was isolated by the yeast lysis buffer method (Kaizeg, R. so apa Aetseg, V. VioTesppidnez 14:552 (1993)) and subjected to electroporation in E. soya. The insert containing the cDNA was subcloned in EsoKiI site rVishezsgiri (Zigaiadepe) and sequenced by the method of dideoxytermination (Zapdeg ei ai.,

Russia May Asad All together. OBA, 74: 5463-5467 (1977)), using Keadu RKIZM sequencing kits

Keasiop YuOyuue YuOeohu Teptipayog Susie Zedcepsipud Kiiz and RKIZM bZedshiepase? Tegttipayug Juocibrie-5igapdea OMA "

Zedntepsip Kiya and analyzed on an AVI 373 automatic sequencer.

An alternative method of identifying clones encoding protein(s) that interact with MirzZA consists of - with the cultivation of yeast transformed with the "Epsilon scoop DNA library present in pOAVIO. After the transfer of the above-mentioned population of transformed yeast on nitrocellulose filters, they were subjected to screening with " » using biotin-labeled MirZzA protein, after which incubation was carried out with a solution containing streptavidin together with alkaline phosphatase (AL). After active washing, the clone or clones expressing a protein with the ability to bind to MirzZA were visualized using the substrate -

Ge") of 5-bromo-4-chloro-3-indoyl phosphate (VSIR) in combination with nitrotetrazole blue (MVT). AR catalyzes the formation of insoluble precipitates, which develop as dark spots on nitrocellulose filters. Experimental procedures are described in detail in the work of ZatrgbooK. in, hey a). ip Moiescshiag Siopipu; I arogagu Mapiai, yr. oz 12-21-12.24, Soia rgipd Nagrog I arogaiogu, 1989. After re-cultivation of plates with yeast for a day, the developed spots were compared with the yeast colonies in the original plates. These colonies were grown, their plasmids isolated, and their inserts characterized as described above. sl Example 11: Insect cells, transformed with the gene for the receptor, show apoptosis under the action

MIRZA(a)-protein

The receptor of midgut cells of the scoop Epsilon for the MIRZA(a)-protein was cloned in KhpoI-Watni -saup vector cosmid tay 2 (ZreeK, M. eyi a). Sepe 64: 173-177 (1988)) and the recombinant sequence was used to transform the cell line Zsppeiideg 2 (52) of the Ogozorp species using the method

F, coprecipitation of calcium phosphate (Siet, K.U. apa MiMeg. I.K. Moi. Sei. VioY. 14: 5212-5222 (1994)). tai 2 ko carries the selectable marker iiei (tetracycline) for bacterial transformation and peo (neomycin) for transformation of Ogozorpia cells. The selected peo marker is expressed under the control of the Hg5p70bo promoter of the Ogozorpia species. The transformed 52 cells were selected in the 52 medium of the Ogozorpya species from 1095 fetal seroalbumin with (3418 (Img/ml) at 330"C (see the 1997 SIVSO catalog). After 45 days of selection in the above-described medium, several stably transformed 52 cell lines were formed.

The sensitivity of transformed 52 cells to MIRZA(a) was tested by adding MIRZA(a)-protein (at a final concentration of 1.7 μg per ml) to the medium containing transformed 52 cells and was previously shaken at 42"C for 30 minutes. The induction of apoptosis in the transformed 52 cells was confirmed by microscopic observations and by using the TAO5 kit, as well as the kit for detecting apoptosis of the Aroriosis Oe(esiop) (for a detailed description, see the 1996 catalog of Themidep saiodce).

Example 12: Isolation of receptor homologues from other insects

Epithelial cells of the midgut of epsilon larvae have a receptor that is recognized

MIRZA(a)-protein. Receptors of other insects susceptible to MIRZA(a) are isolated by identifying DNA sequences using Southern blotting. DNA is prepared, enzyme is restricted, passed through agarose gels and subjected to blotting on nitrocellulose and/or nylon filters. These filters are probed with the cDNA encoding the scoop epsilon receptor under mild hybridization and washing conditions.

Genes with less than 5095 similarity to the scoop epsilon receptor to MIRZA(a) were identified. Southern blotting /0 could also be probed for partial cDNA sequences that encode specific domains such as the death domain or ESE-like motifs to isolate genes containing similar domains, even if they are functionally distinct from the scoop epsilon receptor to MIRZATS(a).

Isolation of homologues to the epsilon scoop receptor to MIRZAd(a) is carried out with the help of two hybrid systems described in the work of Rieid5, 5. apa Zopao. OK. Mayge 340:245-246 (1989). Isolated mRNA is obtained from the required /5 organisms, cDNA is synthesized and cloned into ROAVIO or equivalent plasmids. The cDNA library is co-transformed with rVoO carrying the mirZA(a) gene (or homologues of this gene) and release the putative receptors in yeast by activation of a marker based on the protein-protein interaction between the mirZA(a) protein (or homologues) and the putative receptor .

Homologs of the epsilon scoop receptor to MIRZA(a) are isolated by expression of cDNA libraries isolated from the desired organisms, cloned into appropriate expression vectors, and transformed into host cells, such as yeast or insect cells, that are unable to bind and/or be sensitive to MIRZA (a). Transformed cells are screened based on their acquired ability to bind mirza(a) or undergo an apoptotic response when incubated with mirza(a). In this case, the MIRZA(a) protein is used as a probe, and its binding is monitored either with anti-MIRZA(a) antibodies or with labels such as biotin attached to the

MIRZAa).

Example 13: Screening of new compounds that induce apoptosis in insect cells i)

Model cell lines for various insect orders (some examples include 51-9 cells for Eridoriega order I, Colorado potato beetle, SoiIoriega order, 52 species of Jugozorpia, Oiriega order) are used to screen new compounds that act by inducing apoptosis. Cells are grown in multiwell plates, which are used for high-throughput screening of thousands of compounds (both high and low molecular weight). The compound(s) are added as a single component or as mixtures. The compound(s) that cause apoptosis are identified as follows: 1) membrane protrusions are observed in the cell membrane, 2) when using specific proteins with a high affinity for phosphatidylserine, such as Appehip-U, bound to a visible marker, reorganization is detected phosphatidylserine containing membrane lipids, 3) clumps of cytoplasm are observed in the cytoplasm of cells, 4) active mitochondria can be visualized by using vital dyes such as rhodamine 123, which accumulate in mitochondria, 5) DNA fragmentation is detected either by DNA analysis in agarose gels, by EI IZA-analysis of nucleosome release or by detection of IP past single-strand breaks in DNA. All these cytological and molecular features are signs of apoptosis. "

The scoop epsilon receptor to MIRZA(a) is transformed into the 52 cell line. Thus, isogenic 52 lines can be obtained with and without the above-mentioned receptor. These cell lines are used to screen compounds that provide a differential response due to the presence of the above-mentioned receptor. 52 cells transformed, ;" that undergo apoptosis under the action of certain compounds are identified as above. The difference in the response of transformed and non-transformed cells indicates that the action of the compound is mediated by the cloned receptor. Similar approaches are applied to insect cells transformed with homologous receptors

Ge» receptor scoops epsilon to MIRZATS).

All publications and patent applications mentioned in this description indicate the level of training of specialists to whom this invention relates. All publications and patent applications are hereby incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated as being incorporated by reference. Although the present invention has been described with certain details used by way of illustration and example for better understanding, certain changes and modifications may be contemplated within the scope of the appended formulae.

LIST OF SEQUENCES

(1) GENERAL INFORMATION: 5B (1) GENERAL INFORMATION: () PROVIDER: (F, (A) NAME: MOMAKTIZ DO ka (B) STREET: Zspmuaggmaidavee 215 (C) CITY: Basel vo (D) COUNTRY: Switzerland (E ) POSTAL CODE (21IR): 4058 (E) PHONE: z4161 324 11 11 (Z) FAX: 4161 322 75 32 (Ii) TITLE OF THE INVENTION: A new class of proteins for the control of plant pests 65 (its) NUMBER OF SEQUENCES: 20 (m ) FORM OF COMPUTER READING:

(A) MEDIA TYPE: Floppy disk (B) COMPUTER: IVM RS (C) OPERATING SYSTEM: RO-B0O5/M85-605 (D) PROGRAM: Raiepiip Keyeaze 241.0, Megwiop 21.30 (2) INFORMATION FOR 5EO IO MO 1: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 2378 nucleotide pairs (B) TYPE: nucleic acid 70 (C) STRANDING: single (D) TOPOLOGY: linear (its) TYPE OF MOLECULE: DNA (genomic) (its) HYPOTHETICAL OPTIONS: NONE (xx) CHARACTERISTICS: (A) NAME/CODE: CO (B) LOCATION: 9..2375 (D) OTHER INFORMATION: /mark- "Natural DNA sequence encoding the MIRZA(a) protein from AB88 contained in pSIV7104" (xi) SEQUENCE DESCRIPTION: ZEO IU MO 1:

ASATSAAS ATO AAS AAO AAT ACT AST AAA TTA ASSOASA ASA bSS TTA CsA 50

Mes ABi PCuz ABp o ABp TI Was Pec bek Tpt Ago A1a es Ro s (8)

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1 5 10

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ACC STA SAS ODA ATT OTTA Avb JSC SAS SAS TTA STA STA JSC OAT ATT TST 194

TI rez Abr obich t1e ey was Ap bip b1ip Petz Bay Azp AvroT1e 5eh 0 55 bo

OST AAA TT AT o sbo STO AAT OSA AOS OTTA AAT SAT STT ATS OSA SAS 242 bsiu Former Avr biu Uua1i Aep Siu beg Hey ABp ABr Pec I1e dia vip 65 70 75 75 soA AAS TTA AAT ASA SALA TTA TST AAS SAA ATA TTA AAA ATTObSA AT 290 biu ABp rep AvBpoTpg o bio res beg o Puv biz I1e her Gu 1716 Aza ABp vo 85 90

SAA SAA AAT SAA STT TTA AATOSAT STT AAT AAS AAA STO SAT OS ATA 338

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AAT So ATO STR SOb STA TAT STA SST AD JSC OASS TST ATO TTo AST 386

Ap oTph Mes tsets AtYa Uai Tukhk ep Rrgo GuB I11ee Tweet Beh Mee cem Beh 115 120 125

SAT STA ATO ALA SAA AAT TAT OSO STA AST STO SAA ATA BAA TAS TTA 4z4 Ha

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AT AAA SAA TTO SAA SAS ATT TST SAT AAS TTO SAT ATT ATT ATT AAT STA 482

Beh Ppuv beep rap beep beach TI1ie beg A5r uv Pem Avr 116 I1e Ap Uai 145 159 155

IF) from AVT OTA STI ATT AAS OTST ASA STT ASTOSAA ATT ASA SST SO TAT Sad 530

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Ach I1e pu Tuk Ma1i Abp siy Pu5 Re beach biz beach TBg o Re Aia TBg so 175 180 185 190

SALA AST AST TSA AAAA OTA AAAA AS SAT SOS TST SST OSA OAT TT OSTI 626 so bio Tig bek bek Tsuv Ma1! Mu pub Avr o biu beg Rgo Aza ABr I1ie Gets 1985 200 205

OJSC BAS TTA AST SAS TTA AST OSAD STA CO ADA AST STA ASA ADA JSC 5674 «

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ABr Uai ABr biu Rpe Siy RBe Tukh rey AB5p Tag o Re Ni Ar o ma1i Mes v! 225 230 235 a

STA Oba AAT AAT TTA TT bob SST TSA OST TTA AAAA AST OSA TSO ODA 770 uai s1u Avp Avpoles Rpe b1u AgYa beg dia Pecs uz Tpg A1a bek bits 240 245 250 (o)

TTA ATI AST AAAA SAA AAT STO AAAA ASA AST SOS AST SAS sto OSA DAT 818 (ee) Pe I11e TBgoPCuv bij Ap Ua! Puz TBgobet biu bBeg bi Ua1i biu ABp 255 260 265 27 (95)

Sg" sl ko bo 65

STT TAT AAS OTTO TTA ATT OTA TTA ASA OST STO SAA SSS SA obst TT Voe

Uai Tut Aep Re em Iie Ma! cem TE Aia cep Sip Aia bip Aza Re 275 280 285 2 STT AST OTTA ASA ASA TOS SbA DAA TTA TTA SOS TTA OSA SAT ATT SAT 914

Bec Tk Pec Tik To Suz Acad puv rap this biu Pec Aia Avr I1e Ar 290 | 295,300

TAT AST TST ATT ATO AAT SAA SAT TTA AATOAAb bAA AAA САС SA, TTtT 962

Tug Tig o Bek I1ie Mes ABp o bich Ni ces ABpoPuv S1its Buv 013 610 Re 305 310 315

ASBA STA AS ATS ST SST ASA STT TST AAT AST TT TST AAT SST AT 1910

Aku aii ABpoi11e Pez Rgo Tk Ge beg ABp otTpPt Rpe Beg ABp Rho ABP 320 325 330

TAT SA AVA STT ALLA ZA AST SAT baAA SAT SA VAS ATO ATT STO BAHA 1058

Tut A1a Ppuz mai uv siu Beh Avr b1its A5r A1a Gu Mes tie uai siy 135 340 345 350

OST AAA SSA sOA SAT OSA TTO ATT 005 TTTOZAA AT AST AAT SAT TSA 1106

Aia tsuv Rgo b1u Ni Aza Pec I1e S1u Rpe bi I1e Beg ABpP Avr 5eg 355 zvo 365

ATT ASA STA TTA AAA STA TAT SAS OST AAS STA DAA SAA AAT TAT SAA 1154

I1e TpI Uaii is a pub Ua! Tu biz Aza There was a thing Guv b1p AbpoTuh s1ip 370 375 zvo

STO AT AAS SAT TSS TTA tob o bAA OTT ATT TAT OT SAT ATO SAT AAA 1202 uai Avr GUB Avr beg Pem betg sii Mai I1e Tut siu A5r Meii Ar Gu 385 390 3935 s

TTA TTO TOS SSA SAT SAA TST SAA SAA ATS TAT TAT ASA VAT AAS AtTA 1250 (o) iep bets Suv Rko Azr b1p bek sii bip I11e Tut Tut Tpg Up Avzp I12 400 405 410

STA TTT SSA AAT SAA TAT STA ATT AST DAH ATT SAT TT AST AVA АХА 1298

Uuai Bpe Rgo Avp si) Tuk Ua) T1e TiBh Tsu5 I1e A5r Re TNg Tsuv Buv yuyu 415 420 425 az «

ATO AAA AST OTTA doA TAT SAS STA ASA SO AAT OTTT TAT SAT OTST OTST 1346

Mes tsuv Tk o Bep Agu tTuk biy Uai Togo Aza ABp o Rpe Tut Ar bek Yeeg so 435 440 445 (ee)

ASA SA SAA ATTOSAS TTA AAT AAS AAA AAA STA SAA TSA AST SAA oso 1394 tTBh siu biz i11e Ar Gremsh Ap Buv bue Buv Uai! biz beg beg 010 Aia (se) 450 455 460

SAS TAT ASA ASOo TTA AST OST AAT SAT SAT sob OTO TAT ATO sSos tTTA 1442 biz Tug Ahd Tph ren Beh A1Ta A5p ABr Ar osSiu Ua! Tukh Mee Rgo Gets 465 470 475 «

OST OT ATS AST SA ASA TTT OTTO AST SSO ATI AAtTosos TT ososS sto 1490 - siu Mai I1e bek bis Tph Re Hey TPg Rko I1ee Avzgp o biu Rpe siu this s 480 485 490 . it SAA OST SAT SAA AAT OTSA ASA TTA ATTOAST OTTA ASA TOT AAA TSA TAT 1538 sip A1a ABr Si Avp o beh Ago Pech T1e TBgoBechp Trg Sub GuB Bek Tug 495 500 505 510 b TTA ABA SAA STA STO STA OSA ASA SAS TTA AOS AAT AAA SAA AST AAAdA 1586 res Ag bish Ber Teo Pem Aia TBbu Avr rets Bek ABp o Tsuv bi TOK o Gub so 515 5720 525 (95)

Sg» sl ko 60 65

TTO ATS SRS SSO SSA ST o ssST TTT ATT ASS AAT ATT STA bAb o AAS obo 1634

Bec I1e Ma1 Rko Rgo bek s1iu Re I1e beg Adep I1e Ua1i biz AbBp biu 30 535 5iIO

TOS ATA SALA SAS SAS AAT TTA sao SSO tob AAA SA AAT AAT AAb AAT 1582

Bek t1e beach bits Avr dep Gays b1its Rgo Tgo Puv Aza Ap o OR Buv Ap 545 550 555

SSS TAT STA SAT SAT ASA SOS Both STO AAT SOA AST AAAA OST TTA TAT 1730

A1a Tukh Mai ABr Niv TiK Siu Siu Ua1i Ap Ss1u TK oBPuv d1ia pes Tuk 5BO 565 570

STT SAT AAb SAS OSA SA ATT OTSA SAA TTT ATT OSA SAT AS TTA AAA 1778 tuai niv Puv Avr sS1iu Siu I1e bet bip Rre T1ie biu Avr uv Gech tsu 575 ZVO 585 50

SSO AAA AST SAb TAT STA ATS SAA TAT AST OSTT AAA OSA AAA OSST OTST 1826

Rto Puv Tit s1iz Tuk Ua1i Iie beep Tu Tih Ua1i! Was in the village of Guv Rko Beg 595 600 605

ATT SAT TTA AAA SAT SAA AAT AST BOTH TAT ATT SAT TAT ODA SAT ASA 1874

Iiee NiB ray PuB AvBr o b1i AB TVg o b1u Tuk I1ie Niv Tug bij Azr TVg 610 615 620

AAT AAT AAT TTA SALA SAT TAT SALO AST ATT AAT AAA SOT TTT AST ASA 1922

Ap Ap odep rap bi ABr o Tut bip Tato i1e ABp o Guz Ako Rpe TVg o Toh 625 630 635

SOA AST SAT TTA AAb o ObBA STO TATO TTA ATT OTTA DAA AST OSAA DAT OSA 1970 b51u Tpg ABr oGeh Puv Siu Mai Tut Pcech I1e cem Puz bBei bip Ap o biu 640 645 659 s 29 SAT SAA OST TOS OSA SAT AAS TTT ATT ATT OTTO BACH ATT AST OST TST 2018 Ge)

ABr b1ch Aza Tgr siu AbBr ABp oRre 116 Iie Hey) bi) I1e betg Rgo beg 655 660 665 ' 670

SAA AASOTTA TTA AST SSA SAA TTA ATT AAT ASA AAT DAT TOS ASO AST 29066 biz Puv em Ped bei Rgo bich em I1e Avp tri Avp o AZp Tur TvVk Beh IF) 675 evo 685 "

Sat saA TSA AST AAT ATT ASS OOT AAT ASA STO AST OSTT TAT SAB SSA 21141

TpPt biu beg TEt ABP I1ie beih Siu Ayep Tu Gay Tit Pec Tukh bip p1u so 680 695 700 (ee)

SA SSA boss ATT STA AAA SAA AAS OSTI SAA TTA SAT AST TTT TSA AST 2162 s1u Aga b1iu K1e this Bub5 bip Avp rez bip iets A5r bek vpe bek Teh («e) 705 710 715

TAT ASA STO TAT TTTOTST STO TOS BOA SAT ObST AAT STA AS ATT DoA 2210

Tut Atu Uaii Tut Rpe beh Ua! Beh b1iu ABr Aza Avp Ma1 Ag I1Ze Ag 720 725 730 « 70 WAT TST ASO SAA STO TTA TTTOSAA AAA ASA TAT ATS AOS OST OST AAA 2258 -

Av Beh Ag ba Mai Pem Rrpe bij Guz Ago Tut Mes beg biu d1ia puh s 735 740 745 150 :z» SAT STT TST SAA ATO TTS AST ASA ADA TTT SAS ADA SAT AAS TTT TAT 2306

Avr Mai Bet Jan Meb Rre TP Tit Was Rre bi Was Ar o ABo Re Tug 755 1760 765

FATA SAb STI TST SAA SO AAT DAT OTTA TAT osot SOT SST ATT STA SAT 2154

I1e biz Be) Beh bip b1u ABp o ABp otsey Tut b1iu Siu Rgo i1eee Uai No 710 715 780 (ee) syu TTT TAS SAT TS TST ATT AAS TAD 2378 - Rne Tug ABr Uai Zeg Iie uv 785 sl (2) INFORMATION FOR 5EO IO MO:2: () SEQUENCE CHARACTERISTICS: 59 (A) LENGTH: 789 amino acids

HF) (B) TYPE: amino acid (H) TOPOLOGY: linear de (her) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: ZEO IYUO MO:2: bo b5

Mek dvp Guz ABpoAepoTBg o Puv rey beg TptI Agu A1ia Hey Rgo Bei Re 1 5 10 15

I1ie dvr Tut Re Ap siu Ize Tut biu Rie A1a Tit biu I1e Puv ABr 20 25 30

I1e Meb Avp Mes I1e RBe pub TPh Abr Tvyh b1iu Siu Avr Gay Tig Gay 35 40 45

Ayer sich I1e rem Tsuv Avp bip bip red Petz Avp Ar o I11e Beh S1u tsTsuvye 50 55 60

Hey ABr s1iu Uai Ab5p biu bei Ted ABpoABr Pe i11e Aza bip zu Avp 65 70 75 vo

Tes AvpoTu bij Gez beg Muye Si) i1e ep tsub5 I1e Aza Avp biz bij 85 90 95

Ap osip Mai Bez ABp o Ayer UMa1i Aepbp o Avp oHCsuv Pecs Avzr Aza YIT1e ABp Tig 100 105 110.

Mee rei Ato Ua! Tuk Pep Rgo IB I1e TPg beg Mee Gay bet Avr Mai 115 120 125

Mes uz Sip Abo o Tukh Aza Pec beg Pec bip Z1e bi Tut Grec beg Guz 130 135 140 bip without b1ip 514 i11e bek Ar Guye Pe Ar 11e I1e Avp Ua1i A8p uai 145 150 155 160 cem I1e Ap Beh Tyh GPem Tit bi i11e Tx Rgo Aza Tukh bip Agu 116 165 170 175 p

There was Tuh Ma! Ap bi Buv Rpe siy Si pe TiPh RBe Aia Tk si: TBg d) 180 185 190 zek Beh Puv Mai Gub5 Puv Avr s1u bek Rgo Aia ABr I16 Pei ABr p1h 195 200 205

Pem TBI bi rechsh TBI bij this A1ia pub bei Mai Tu gub A5p Ar Uai yuyu 210 215 220 -

Ar b1)1U Rpe b1Ch Rpe Tuk Grec ABp Tpg Re Ni Ayer Mai Mes uai cS1uU 225 230 235 240 i)

ABp o Avp ore Rbe siu Agu beg Aza rey Puz TNu A1a bet sii cem i11ie (ge) 245 250 255 (Se)

TrtI pub biz Ayep Ua! pub TBg o bei biu beg Sich Ua) Siu A5p Uai tuk 260 265 270 20 - s . a (o) (ee) (95)

Сг» sl ime) 60 65 -4О-

Azp o Rpe Pec I1e Uaiii pem Tph A1a Pech bip Aza bip A1ia Re cem TBg 275 280 285 cem Tit oTrg Sub Ato Buv em Hey biu em Aza Azr o T1e ABr Tut TECH 230 295 300 sec I1ie Mes dep bij Nibv res Abp Buv sii puv bii bi RBpe Ago Uai 325 310 315 320

Avp o i11e rep Rgo TiK rep beg Ab5p Tit Rpe Beh Azp Rto Ap Tukh Aza 325 330 335

Mus Ma1 tsue Siu Beh Avr Si ABr Aza pub Meg T1e Ua1i bij A1a Buv 340 345 350

VBho s1u Nib5 A1ia rep i1e biu Rpe sich T1e beh ABp ABr bek I1e Tg 355 360 365

Uuvai rech tsu Mai tuhk sim Aza Buv cem uv sip Ayep Tuh bip Uai ABr 370 375 Zo

Tu ABr Beh cem bet bij Ma1i I1e Tut S1u ABr Mei ABr uv Tem this 385 390 395 400

Suv Rgo ABr beep beg bij beep IT1e Tug Tut TPzh AbBp Ap o I1e Uai Rre 405 410 415

Rgo ABp obi Tut Ua1 I1e Tapg rub t11e A5r Re TPg o bue Buv Mee Buv 420 425 430

TPg thing Ago Tut bi az! TBg Aza Abp Re Tut Avr beg beg tig 01u s 435 440 445 o bis 716 Abr ce Azp o HCubB Buv Buz Ma! Si beg bek siyu A1a bitz Tut 459 455 460

Ah TBg opech bek A1ia Ap oABr o Avr siu Ua! Tuhk Mes Rgo tsez siu Uvi 465 470 475 480 yu

I1e bet sich Tit Re Ptsep Tu Rto I1e Azp b13u Re biu Pesh sSip dia « 485 450 435

Avr bij Avp bek Agu Becp I1e TBh Peo Tvg Suz Buv bek Tuk cem AgF i.

TO) 5О5 510 (ee) bish Bey rec rey Zia Trg Avr ope bek Ap o Guz Sid TBbgopuye Bey 1126 515 520 525 (Se)

Uai Rgo Rgo Beh Siu Rpe Iie 5eg Avp I1e Uuai bits Ab5p biu bek Ii 530 535 5ab b1p bij Avr Avporei Si) Rgo Ter otsuv Aza Ap Ap Gubie Avp dia Tut « 545 550 555 550 -v ,

Uai ABr Niv Tk Siu biu Uai Ap siu Trh muye Aza rem Tukht Ma1i Niv s 555 570 575 :z» Was OR sb1u b1u I1ie Bet bip Rpe I1e S1u d5r Shuv Pec uv Rkgo Buv

BO 585 590

TVVt bish Tug Uai I1ie sat Tut TVi Uai TsuB biu tsuv Rgo beg I1e No 585 500 605 (o) I so Ted uv ABr bij Abp o TVg b1iu Tut I1e No Tug Sich Avr TVgoABP ABo 516 615 620 (95)

Avp ope bij Avr o Tut bip Tik I1e AvpoGuv Agud RBe Tk otak siu Tg chi» 625 530 635 640 sl ko 60 65 -A1-

Avr pes pub siu ma1i Tut Pez T1e Mvy Guz begt bip Avp b1u Ar bi 6545 650 655

Aza Tgr b1u Ar Abp Re 11e I1ie Pe bij T1e bek Rgo bek siy Buv bbo 665 670

Tez pechu Bek Rho bimi Pec Ii11e ABp Tah ABp o ABp TgroTVg bek Tit b1iu 675 6vo 685

Zeg Tph Avp I1e beg siu Abp TygoBez Tygotsem Tut bip S1u s1u Ago 630 595 700 s1u i1e em Puv bip Avp o rets bip Bech Azr bek Re bek Tit Tut AgF 705 to 715 720

Wai Tut Rre beg Ma! bZeh b1iu Ayer Aia Ayep Ma1i Ako I1e Aku Abp bek 125 130 735

AkYa biz Uai Besh Re bitz rub Agu Tut Mee beh Siu A1a Tsuv Avr Uai 7140 745 75o

Bek bi Me Rpe tTNh TrI Tsuv Re bij Buv ABr Ap o ROE Tug I1ee ish 755 7650 765

Graham beg sip Siu Avp ABpobem Tug Siu s1u Rgo i116e Mai Nibv Rpe Tug 770 775 780

Aur Ma! back I1e Buv 785 (2). INFORMATION FOR 5EO IO MO:Z: c (2) INFORMATION FOR 5EO IO MO:3: сч () SEQUENCE CHARACTERISTICS: o (A) LENGTH: 2612 nucleotide pairs (B) TYPE: nucleic acid (B) STRENGTH: single oyu ( D) TOPOLOGY: linear (her) TYPE OF MOLECULE: DNA (genomic) AND (her) HYPOTHETICAL OPTIONS: NONE s (2) FEATURES: (A) NAME/CODE: СО5 (ee) (B) LOCATION: 118..2484 s (GL) OTHER INFORMATION: /label- "Natural DNA sequence encoding MIRZA(B) from AB424" (xi) SEQUENCE DESCRIPTION: ZEO IYUO MO:3:

ATTOALTTO ATAAAAASTT ATOASTOTTT AATAATSAST AATTASSAAT DALOAATTAA bo

SAATASAAST TTASAASAAA TAASTOTTAS DAAAAAATS TOAAAAOSAH BATOSAAS 117 «

ATO AAS ABC AAT AAT AST AKA TTA AOS ASA ASA OSS TTA SSA dat TT 165 shi s Mes ABP pu Ap Azp Taho pub Bez BbBeg TNK o Ago Aza Ge Rgo bet Rpe 790 795 oo vo z (22) (ee) (95) i" 50 sl

F) ime) 60 65 -dD2-

ATT SAT TAT OTTO AAtT SOS ATT TATO SbA TTT o bSS dST SOT ATS AVA SAS 211

Ize Avr Tukg Rrpe Ap b1iu Iie Tut biu Rpe dia Trg b)u 112 Guz Ar 810 815 829

ATT ATO AAS ATO ATT o TTT AAA SYA SAT DSA ST SOT SAT STA ASS OSTA 251

I1e mes Ap Me I1e Rpe Ppuz Tig Avr o TpgoSiu b1u ABr Ge Tag Gay 825 830 835

SAS SALA ATT TTA AD OAAT SAS SAS STA STA AAT SAT ATT OTST OST AAA 308

Ar osia Yej1e besh o tsuz Ayep bip bij o Ppez Gray Ab5p o AvBroYiz1e Bet biu tsu 840 845 850 to TTO SAT SOS STO AAT o ObA DOS TTA AVT OSAT STT ATS OSA SA OSA AAS 157 yeech Ar obiu Mai Ap s1u Beg Pech Avp Ar Brad T Aza sbip 01u ABp 855 vo 865

TTA AAT ASA SAA TTA TST AAb SAA ATA TTA AAAA ATT SA AAT OAA SAA 405

Mrets Ap oTbg bi Pec beg Puv bij I1e Pep pub I1e Aia Ap ob1u b1p 870 875 vo v85

JSC SAA STT TTA JSC PAT STT JSC AAS DAA STO bAT CO ATA JSC So 53

Avpobip UMai Dey Avp o Avr oMa1! AvpoAvp o Guv ces Avr o A1a I1ie ABp o Toh 890 895 900

ATO STT sob STA TAT STA SST AAA ATT AS TST ATO Ta JSC OBAT OTA 501

Met Petz AkYaa Whoa1! Tug Brep Rgo Tsuv I11e Tiig Beh Mebs rec Beh ABr Uai1i 305 910 915

ATS AAA SAA AAT TAT So STA AST STO SA ATA SAA TAS TTA AST DAH 549

Mee pu5 pip Up Tukhk A1ta Pes bek Pec bip i11e biz Tud rez Bek Gu 920 925 930 s

SA OTTO SAM OSAS ATT TST SAT AS OTTO SAT ATT ATT AAT STA AAT STA 537 sip rem b1ip si) T1e Bek Der uv rem Avr T1e T1ie ABp a! Aa Ua1yi (o) 935 940 945

STT ATT AAS TST ASA STT AST SAD ATT ASA SST SSO TAT SAA AsS ATT 645

Tetsi IT1e Avp bBet Tit Pec TNg Si I1e TBgobgo Aza Tut bip Ak 11e 950 955 960 965 IF)

AvA TAT STO AAS OSAA AAA TTT SAS SAA TTA ASTOTTT OST ASA SAA AST 693 "I

Was here Mai Avp obi Guz Rre biz b01iu Ge) Tk Rpe Aia Tpg o beach Tbg 970 975 z80 Geo)

AST TSA AAA STA AAA AS SAT SOS TST OST OSA SAT ATT SOT SAT SAS 7141 (ee)

Bek Behg ShPuB Ma! tsuv Guv Avros1iu Beh Rgo A1a Ar t11e Akd Ar si 985 990 9395 (Te)

TTA AST SAS TTA AST OOAA STA SSS o AAA dST STA ASA DAd DAT SAT hundred 789 yivy TP ob1ich pei TpI biz red Aza Buz beg Uai1i TNrg tsuv A5p Ar Uai 1000 1005 1010 «

SAT OST TT bAA TTT TAS STT AAT ASA TTS SAS SAT STA ATO STA So 837 du Ar b1u Rpe Si Rre Tuk Gets Ap Tr o rde Niv Ar uai Mes uai Siu - s 1015 1020 1025

AAT AAT TTA TTS obo SOT TSA OST TTA AAAA AST OSA TO SAA TTA ATT 885 "z ABpoABp oGech Rpe biu Agu bek A1a Pec Id/e Trg dia deh si Grez I1e p 1030 1035 1040 1045

AST AVA SAA AAT STO ALA ASA ASTOSOS AST SAS STO SA AATOSstTtT TAT 933

TNg was Sich Abo Ua! uz Togo obeg o biu beg 414 Uai biu Ap Uai Tug (is) .1050 1055 1060 (ee) (95)

Sg» sl ko 60 65 -AZ-

AASOTTS STA ATTOSTA TTA ASA OST STO SALA OSA AAAA OST TTT ST AST 981

Ap Rpe Pec I1e ua1y yep Tbg A1ia Pec o bip dia Guye Aza Re pe» Tag 1065 1070 1075

TTA DSA SSA TOS SSA AVA TTA TTA SbS TTA SSA SAT ATT SAT TAT AST 1029 rech Tvg oRgo Suv Aga Puz rep Pec biu rech A1a Avr I1e Azr Tut TiK 1080 1085 1090

TStT ATT ATO AAT SAA SAT OTTA DAT AAS SAVA SAS SA TTT ASA STA 1077 bek Iie Meb AbBp obi Nibv Pecs Ap buh b1i Buv bij si Rpe Ago Mai 1095 1100 1105

AVS ATO STO SST ASA SstTtT o TST AAT AST TTT TST AAtT SST AAT TAT OSA 1125 vV5p oi11e Pem Rgo Trg o Gets beg ABp o TBg o RBe bek Avp Rgo A5p Tut A1a 1110 1115 1120 1125

AVA STT AAA OSA AST SAT SALO SAT OSA AAb ATO ATT STO ZAA OST AAA 1173 uv Mai pub Siu behg Avr sii Ayer A1a Tuv Mes I1e Ua1 Sish Aza Buv 75 1130 1135 1140

SSA OSA SAT OSA TTO ATT o obo TTT bAA ATT AST AAT OAT TOA ATT АСА 1221

Rko biu Niv A1a Gez I1e b1u Re siy I)e Zeg Up Avzr Bek I1e Tig 1145 1150 1155

STA TTA AAA STA TAT SAS OST AAS STA AV SAA AAT TAT SAA OTO OsAT 1269 wai cem Buv Uai Tuk Si Aia uz Pecs Tsuv bip Azp Tuk b1ip Ua1i Apr 1160 1165 1170

AASOSAT TOS TTA TO SAA sSTT ATT TAT OSS SAT ATO SAT AAA TTA TT 1317 uv Avr begt Pec bBeg biz Uai T11e Tut Siu AvBr Meb Ar Guz rem em 1175 1180 1185 s

TOS SSA SAT SAA TST OSA SAD ATS TAT TAT DSA AAT AAS ATA STA TT 1365

Suv Rko ABr Sip Bek siu bip I1e Tut Tug Ti ABp Azp I1e Uai Rie o 1190 1195 1200 1205

SSA AAT SAA TAT STA ATT AST AAAA ATT SAT TTS AST ALLA AAA ATO AAAd 1413

Rgo Aep osi) Tut Ua1i I1e Tphi pub I1e Avr Rpe Tikh GuB Puv Mee Tsuv 1210 1215 1220 that)

AST OTTA ASA TAT SAS STA SA Sat AAT TTT TAT SAT TST TST DSA Sad 1461 "I

Tvt Getch Agu Tut bish UMai Tit Aia ABp o Rpe Tug Avr beg Beh Tig b01u 1225 1230 1235 so

SAA ATT SAS TTA AAT AAS AAA AAA STA SAA TSA AST SALA So SAS TAT 1509 (ee)

Sish I1ie Avr Pec Ap ogGub Buv Tsuv Uai biy beg bBeg bi Aia s1ts Tug z 1240 1245 1250 Ge)

ASA ASO TTA AST OST AAT SAT SAT o sos sto TAT ATO SSO TtTA sot st 1557

Ako Tpg o esh Zeg A1a ABp o Avr Avr Siu Ma) Tut Mes Rgo ey s1u Ua1 1255 1260 1265 «

ATS AST SAA ASA TTT OTTO AST SSS ATT AAT OSos TTT bos hundred garden sst 1605

I1e back battle TPk Rre Pec Trg o Rgo IZie Avp siu Rpe Siu ep Sip A1a - 1270 1275 1280 1285 s and SAT SAA AAT TSA AbA TTA ATTOAST OTTA ASA TST AAAA TOA TATO TTA SA 1653 ;z Ar Si ABp bek Ago Gem t11e TNg this tTrg Sub PUB back Tuc cem Aka 12990 1295 1300

SAA STA STO STA OSA ASA SAS TTA AOS OAAT AAAA SAA AST AAAA TTO ATS 1701 bij ep rec pes Aia Trt ABr o Pay bek Ap Tsuv bi) Taho pub Gvts I1e b» 1305 1310 1315 (Fe) Sto ss sSsA ASsT Ost TTT ATT ABS JSC ATTOSTA SAS AAS co-op TOS ATA 1749 (95)

Sg" sl ko bo 65 -YES-

wai Rgo Rgo back biu vre I1e bZet Avp T1e Mai bi» ABp sb1u bek I1e 1320 1325 1330

SAA SAS SAS AAT OTTA SAS SSo TOS AAA bSA AAT AAT AAS AAT Oso TAT 1797 siy Si ABr dp rem sim Rgo Ter o PCuye Aza Ap o Ayep HCuv ABp Aza Tut 1335 1340 1345

STA SAT SAT ASA SOS ss Sto AAT SbA AST AAAA OST TTA TAT STtT SAT 1845 7uai ABr Nib TpgosSiu siu uai Avp Siu Trg o Guz A1a Hep Tuk Uai Niv 1350 1355 EhzBo 1365 710 AAS SAS OSA SSA ATT OTSA SAA TTTAS ATT ObSA SAT TTA AKA SOS DAH 1893

Was Avr biu s1u I1e beh sip Rpe I1e siu ABr PCu5 Pe tsuv Rgo Gu 1370 1375 13980

AST SAS TAT STA ATS SAA TAT AST STI AAA OSA AAA OSST tot ATT SAT 1941

TBg bits tuh uai Iiiiye sip Tu Trg Maiii TsPuv SsS1u tsuv Rko bek I1e No 1385 1390 1395

TTA AAA SAT SAA AAT dST OSA TAT ATT SAT TAT TAT SAA SAT ASA AAT AAT 1989

You were Avrob1ich Azop TrgoSiu Tut I1e Nieu Tut Si Avr TI Avp o dp 1400 - 1405 1410

DAT TTA SAA SAT TAT SAA AST ААТ ААА СсТ ТТТ о AST АСА ОСА AST 2037

AzpoPets bi Abr Tut b1p Ti I1e Avp o Puv Ago Rpe TrgotTvbg 51u TOg 1415 1420 1425

SAT TTA AAb SOA STO TATO TTA ATT OTTA AAA AST SAA DAT SZA SAT SAA 2085

Ar Ptsech Tsuv siu Mai Tut Pech I1e em Puz Beh biy A5p biu Ar sich 1430 1435 1440 1445

OST tob OA OAT AAS o TTT ATT ATT OTTO SAA ATT AST SST TST SAA AAs 2133 s

A1ia Ttr b1u Ab5r ABp Rpe I1ie Iie Pesh Siy I1ie Beg Rgko Bek b) Was 1450 1455 1460 (o)

TTA TTA AST SSA SAA TTA ATT AAT ASA AAT AAT TO ASO AST ASO sOA 2181 yitsez yemsh Zeg Rgo bi rem I1e Avp Tk o Avp Avp Tgr TBgobeg Tr b1u 1465 1470 1475

IF)

TSA AST AAT ATT AOS OST AAT ASA STO AST STT TAT SAS OA Spa SoA 2229 bZeg TI Azp I1e Bek biu Avp TI Pep Tpgotsep Tug bip b1u 01Uu AgU «I 1480 1485 1490 (ze) bo ATT STA AAA SAA AASOSTT SAA TTA SAT AST TTT TSA AST TAT AbA 2277 biu ch11e ep Buv Sip ABpoiez bip this Avrobek Re bek Tit Tut Aga o 1495 1500 1505

STO TAT TTS TST Sto TOS OSA SAT OST AAT STA ASS ATT ASA AAT TS 2325 i-o

Ua1i Tug Rpe bBeit UMa1i bek Siu A5r A1i1a Ap o wai Aku I1ie AkCh Ayep behg 1510 1515 1520 1525 vOos o sAv STO TTA TTT ObdDA AAV AbA TAT ATO DOS OST OST AAA SAT OST 2373 « vho sich Mai Pec Rpe bish Ptsuv Agu Tut Meb beg biu A1ia uz Ar Mai 1530 1535 1540 shsh s TST OAA ATO TTS AST ASA ALLA TTT SAS AAA SAT o AAS OTTO TAT ATA SA 2421 beg b5ich Mes RiBe TPk Tpt o Puvie Rpe si Gu ABr AB Re Tuk I1e pich "from 1545 1550 1555 p

STT TStT SAA bbb AAT o AAT TTA TAT OST OST SST ATT STA SAT TITT TAS 2469

Tech bBeg sSip b1iu Avp Ap Gech Tu biu sSiu Rko IЙ1e Mai Niv Re Tukh 1560 1565 1570 (o)

SAT hundred Tst ATT AAS TAASBATSOSO ATSTAATATT AASASTTTTT АСААССТАТТ 2524

Go) Avr Chai Bek I1e tsu s 1575 iz 50 TSTTOTATAA TOTOSTTSAT TATOSAAAAAA SASAATTTTo TTTOSTAASA TOTATATATA 2594 sl SSTSASTSAT TAAAASOSAA TSAASSTT 25612 (2) INFORMATION FOR 5EO IO MO:4: () SEQUENCE CHARACTERISTICS: o (A) LENGTH (79) amino acids TYPE: amino acids ko (G) TOPOLOGY: linear (y) TYPE OF MOLECULE: protein bo (hi) SEQUENCE DESCRIPTION: ZEO IYUO MOa: 65 -DB-

Mes dep o tsuv Avp Or Tvh Guv Tey beg TlI Agu Aza Te Rgo 5eh Rpe 1 5 10 15

I1e Advr Tukh Rbe Abp o sSiu I1e Tut biu Rpe A1a Tk s1u I1e Gu ABr 2 25 30 20

Iiiie Mebs Avp Mes ITe Re Ppuz Tit Avr TBg z1iu biu Azr Ge Trh Gay 15 40 45

Avr sich I1e rech Puv Avpobip bip o Pem Tez Ayep o Abr Ii11e Beh b1u Guz 5O 55 BO tem Ar biu Uai Ayep Si1u beg Pecs AbBp A5r Tem 116 A1a bip S1u Abp 65 70 75 8o cem ABpP tTpg Si Hey bek Buv 011 I1ie Gets Bbu5 IchI1e A1a ABp bi bip 85 930 95

Ap obSip Ua1 Pec db5p Ar Uuai Aep Ap o guv Bay Der A1a 116 dBp TBg 100 105 110

Mee Pec At Uaiii Tut Bez Rgo Pu5 I1e TI beh Mee Pec Beh Ar Ua1i 115 120 125

Mee puB beep o ABp Tut Aia Graham bet pep sip I1e pizza Tukh Ppem beg Gu5 1320 135 140 bip cem bSip biz 11e Beg ABr gub Be Avr I1e 11e Ap Ua1i ABp Uai 145 150 155 160

Bec I1e Ap obet TVh Pec Tbti biz I11e Te Rgo dia Tut Sip Aga Iie s 165 170 175 schi c) tsuv Tut Ua1 A5p Sich PuB Rpe bi b): rem TI Rpe A1a Tu bich TBg 180 185 190 begt bek pu Ua! tsuvz tsuv Avr Siu bBeg Rgo A1a Avr I1e Agd Avr bij 195 205 205 yu rem TE bij rech Tpt bish Bez A1ia Gub becht Mai TI Guz Ap o Avr chai 210 215 220 M

OR both Rpe bip Rpe Tuk Pec Ap TB Re Ni Ar Ua) Mes Uai sSiu o 225 230 235 240 (ee)

ABp Avp o rec Re s1i1u AkYa bek Aia tse Guv Tpg dia bek bi tsech Iie 245 250 255 (Se) - p . a (o) (ee) (95)

Сг» сл ко 60 65 -4В-

Tit Il/uv bij Ap Uai uv Tak obeg o biu Zeg bij Ma1 bS1iu Ap Ma1 Tut 250 265 270

Azp Re Pec I1ie Mai Pec tt dia Ge» bip Aia pub Aza Re Gei TEG 275 280 285

Hey TNg o Rgo Subv Ago It was Geo Siu Bey A1a Abr 116 Ar Tug tih 290 295 300 bek I1e Meb Ap si Ni Ptsets A5p o puz siy guv biz si) Rpe Ago Ma! 305 310 315 320

Ag oi1e rep Rgo Taho Pvy beg Abp TPg Rre Beh Up Rgo Avp Tut Aza 325 330 335

Buv Ma) tsubz S1u bZeg ABr beach Avr A1a Pubv Meii ІI1e Ua! January A1ia Was 340 345 350

Rgo Siu Nib Aia Pecs I1e Siu Rpe 611 I1ie Beig Ap Avr Beh I1e TBg 155 360 365

Uai Bech tsuB Ua! Tukhk bitz Ata tsuv tse Idu beep Ayep Tut beep Uai A5r 370 375 380

Was ABr bet Pes Bek 010 Ua1 T1e Tuk S)iu Avr Mei Avr uv Pecs Gay 385 390 395 400

Suv Rgo Ar bip bZeg siu bip IZe Tug Tug TVti ABp A5p o 11e MUai Riye 405 410 415

Rgo Avpobich Tut Uaii I1e TiI tsuv i11e ABvr Re TpPh gub was Mes Buv Ge 420 425 430 2 o

Tit rep Ak Tug o b1m Ua1 Tk ATa ABporRPpe Tuh Ar o beg behg Tit s1U 435 440 445 bich i1ie Avr cem Ap obuz Was in my mind! fight running back Sicp dia si Tug 450 455 460 iv)

Agu Tvg te Veg Aza ABP ABr Avr Siu Uai Tut Meb Rgo Gets biu Uai "I 465 470 475 480 (ze)

I1e Beg bi Tph RBe Pec Tg o Rgo I1e Ap b1iu Rape s1iu Pay soi Aia 485 490 495 (oo)

Avr bi ABp o beg Ato ep i1e Tbt Ped Taho Suv Muz bet Tut tsem Aga I«o) 500 505 510 sich rech tsech Gesh A1ia TBt Avr Gei beh Ayep Buv Si Tk Buv Bez I1e 515 520 525 «

Mai Rgo Rkgo bek biu Rre I1e begt Ap I1e Ua1 Sits Ayp b1iu Bek Iie 20 530 535 540 - s bij 030 AvBr ABp rep 01 Rgo Tgr pub d1a Azp Ap o Guye Azp dia Tug z» 54v 550 55 50 i y

Maiii Ar niv Tit Siu b1iu ua1i Ayep biu Tit Buv A1a Tep Tukh Uai Niv 555 570 575 b Buv ABr s1u s1u Ize bek pip Re Iiye sSiu ABr tsuz ei Buv Rko Tuv

BO 585 590 so Tph bij Tug Muai I)ye bip Tukh Tng uai uv S1u Buv Rgo Beh I1e Niv (95) g» sl ko 60 65 -D7-

595 600 605 cem Iluv Avrobits Ayp Tbt Siu Tut I11e Ni5 Tukh b) Avr Tph ABp ABY 610 615 620

ABpobez bi dvr o Tukh bip TVg o i1e Ap o was Agu RBe Trh Tpgosiu tTag 525 530 635 640

ABr rech Guz Siu Ma1i Tuk Pec I1ie rem tsuv bek bip Azp b1u A5r vich 645 650 655

Aza Tur siu Ayer dp Re I1e Iiee ep sii I1e Beh Rgo bek b1its Was 66o 665 570

Pec rech bek Rgo biz Gay T1e Aep TBgoAzp Aep Ttr TBk obeg Tk o sS1iu 675 bo 685

Beg TiBI Avp o ITe beg Siu Ap Tit Ge Tbg Pec Tug biu biu b1u AgYa 690 595 tvo s1u I1e this Buv bip Abp o BPep bip Bez ABr beg Rie betg Tig Tut Aga 705 710 t15 7120 chai Tut Rre beg Ua)i Beg biu Avr A1a Ap o Uai Agu I1e Agu Avp beg 725 tzo 735

Ako biz Uai Prep Rre C13 Guv Agd Tuk Mes bek biu A1ia Guv5 Avr Uuai 740 745 750 bek bis Mee Rre Tik Tik o MPue Rpe b) Tsu5 Avr Aup o RBe Tuk 116 61 755 760 765 s

Te Beg bip o biu Avp Abp Ped Tug biu S1u Rgo I1e Uai Ni Re Tuh ge) 770 775 780

Azr Maii bet I1ie Hug! 785

IC c) zo (2) INFORMATION FOR 5EO IO MO:5: () SEQUENCE CHARACTERISTICS: - (A) LENGTH: 2364 nucleotide pairs s (B) TYPE: nucleic acid (C) STRENGTH: single co (G) TOPOLOGY: linear Ge (her) TYPE OF MOLECULE: DNA (genomic) (her) HYPOTHETICAL OPTIONS: NONE (2) FEATURES: (A) NAME/CODE: СО5 « (B) LOCATION: 56..2295 shsh (D) OTHER INFORMATION: /product - "UIRZA(c)-protein" s (xi) SEQUENCE DESCRIPTION: ZEO AND MO:5: z (22) (ee) (95) i» 50 sl

F) name) 60 65 -AB-

AATASAATTT ASSASOSATA ASTOTTATASAA ASAATASTO ASSASOSASA TOAAS ATO z8 mech

AAS AAS AAT AAT OST AAA TTA ASS OASA DSA boss TTA SSA STO TTT ATT 106

ABp Puv Ap oAzp o Asia Guye Ped bet Togo Atu Aia em Rgo Beh RBe I1e 5 10 15

SAT TAT TT AAT SOS ATT TAT SSA TT bSS АStT SstT ATS AAA САС ATT 154

Aver Tuk Rpe Ap o biu I)e Tukh sSiu Rpe Aza Tit s1u I1e Pu5 Avr I1e zo 20 25

ATS AAS ATO ATTOTTT AAA DSS SAT ASA Sst SST SAT STA OSS STA SAS 207

Meb Avp oMei Iie pe BuB TBt Ar Tig s1u S1iu Avr this Aza Hem Avr 35 40 45

SAA ATT TTA SAS AAT SAS SAS STA STA AAT SAT ATT TST OST AAA TTo 250 siv i11e bep si) ABo sSip Sip pesh Ge) Ap Avr I1e beg biu pub Gay 50 55 bo 65

SAT co STO AAT SSA DOS TTA AAT SAT STT ATO OSA SAS OSA AAS OTTA 238

ABr s1u Mai Avp o biu Bek Gay Up Avr Bay I1e A1a bip b1iu Avp Pec 70 75 vo

AVT ASA OAA TTA TST AAS SALA ATA TTA ADAM OATT OSA AAT SAA SAA AAtT 46

Ap oTpgob1iy Pec back u bish I11e ts u IZ1e Aza Avp o Sits Sil ABp 85 90 95

SAA STT TTA AAT SAT STT AAT o AAS ALA STO SAT OSS ATA AAT AS ATO 354 se sip Ma1! Pem ABp o AvroMai ABpoABp o Puv cem ABr Aza I1ie ABp Trg Mee 100 105 110 o

STT So STA TAT STA SST Ada ATTOASS TST ATO TTO AST SAT OTA ATO 442

Tem Akd Ua! Tut Pem Rgo Guv I1e Tru betg Mes tets bek Avr Ua1 Mee 115 120 125

IF)

AAA SYA AAT TAT SSO STA AST STO SAA ATA bAA TAS TTA AST DAH SAA 490 «

Buv s1ip Avp o Tut A1ia Per bBet Pecs sil Ize bis Tukh rem behg TubB bip 130 135 140 145 (ze)

TTO SAA SAS ATT TST SAT AAb OTTO SAT ATT ATT AAT STA AATOSTA SsTt 538 so iep bip Si IZ1e Zek ABr tsu Pecs ABr I1e I1e Avp Ua1i ABpoua1 Bey 150 155 160 (Se)

ATT AAS TST ASA STR OAST SAV ATT ASA SST SOS TAT SAA ASO ATT ADA Vb

I1e Abp obet TrI rey Tk biz I1e Trh rgo A1ia Tut bip Ago I1e Guv 165 170 175 «

TAT STO ALAS OSAA AAA TTT SAS SALA TTA AST o TTT OST ASA SAA AST ASst 634

Tuh Mai Abvp Si Puv RNe s1h battle Pec Tu Rde ALia TI battle Tpg beg - 180 185 190 s and TSA AAA STA AAA AAbOSAT SOS TST SST SSA SAT ATT OSST SAT SAb TTA 682 a Beh Mu Ua! Puye tsu Avr biu bBet Rgo Aiia AvBr I1e Ag ABr Sich rech 195 200 205

AST SAS TTA ASTOSAA STA SSS AAA ASTOSTA ASA SAA AAT SAT STO SAT 730

F Bek bis ep Tph Sich Pech D1ia tsuv beh uai Tih siy ABp Avr Mai Ar 210 215 220 225 (ee)

OST TTT SAA TT TAS STT AUTO ASA TTS SAS SAT STA ATO STA SSA AAtT 778 s b1u Rre s1its Rrpe Tug Pech Avp TvygorRre Niv Avr UMai Meb wai Siu dB -230 235 240

60 65

AAT TTA TTS bob sot OTsA OST OTTA AAAA AST OSA tob SAA TTA ATT st 826

Ap Gay Rpe Siu Ag 5eg dia rep tsubv Tx A1ia behg bij Pech I116 TBg 245 250 255 9 AAA SALO AAT STO ALLA ASA AST SOS AST SAS STO OBA DAT STT TAT AAS 874

Was from Avp oUai Was Tvt bek o siu beg Sim Mai b1u Ayep Mai Tuk ABp 260 265 270

TS STA ATTOSTA TTA ASA SST STO SAA bSA SAA OST TTT STT AST TTA 922

Re Pec Iie Uuai rech Tpg A1ia rep beep dia beep A1ia Rppe Gei TBg o Bec 275 280 285

DSA SSA TOS SSA AAA TTA TTA SOS TTA SSA SAT ATT SAT TAT AST OTST 970

Twig Rgo Suv AkYa o pub this thing b1iu Pecs Aia ABr I1Ee Ar Tut Tig Beg 230 235 30 305

ATT ATO AAT OSAA SAT OTTA AAT AAO SAA AAA SAS SAA TTT Aa STA LAS 1918

T1e mee dyap Si Ni Gei AbpoIluz 010 Puv biz beach Rpe Ago Uai ABp 310 315 320

ATO STO SST ASA STI TST AAT AST TTT TST AAT SST AAT TAT OSA AAA 1066

I1e ey Rho Tph Pec beg Avp o Tbu Re bBeg ABp Rgo Azp Tut Aia Gu 325 330 335

STT AAA SSA AST SAT SAA SAT OSA AS ATO ATT STO SDA OST AAA SSA 1114

Mai tsuv sziu Bek Avr Si Avr A1ia Tuv Mes 116 Uai 011 Aia Bu5 Rgo 340 345 350

SPA SAT OSSA TT ATT sob TTT OSAA ATT AST AAT SAT TSA ATT ASA OTA 1162 s siun niv A1i1a Pes I1e biu Rpe bip IT1e beg ABp ABr beg I1e Trg Uai1 29 355 zvo 365 o

TTA AAA STA TAT SAS OST AAS STA ALA SAMA DAT TAT SAA OTO JSC Av 1210

Getch pu Uai Tut 010 A1a tsubye Pei Guv bib Aby Tukh b10 Ua) ABr Bu 370 375 380 385

IF)

SAT TOS TTA TS SAA STT ATT TAT SOS SAT ATO SAT AAA TTA TT TOS 1258

Avr Beh Pechp Beh bij uai I1e Tuk Siu A5r Mee Avr Tsuz ep Gay Suye « 390 395 400 (ze)

ССА SAT САА TST o ОБА САВА ATS TAT TAT DSA AAT AAS ATA STA TTT OSSA 1306

Rho Ar beep back s1u s1p I1e Tut Tu Tbg ABp Abp I1e Uai Re Rkgo so 105 410 415 (Se)

AAT SAA TAT STA ATT AST AAAA ATT SAT OTTO AST AAAA AAA ATS DAH AST 1354

ABp biz Tukh Mai Ii Tpg puv I1e Ar Rpe Tpt Buv Buv Mee Buv Tig 420 1425 430

TTA AbA TAT SAS STA ASA SSO AAT TTT TAT SAT TST OTST ASA SSA SAD 102 h cem Ak Tut bij Ma1i TBgoAsia Avp Re Tug Avr bet beg Tit Siu Jan shsh 70 435 440 445 s a ATT SAS TTA AAT AAS AVA AAAA STA SAA TSA AST SAA SSO SAS TAT AHA 1450 a I1e A5r Pes AvBp o Buv tsu tsuv Uaii b1ich bek bek sich dia si Tukh Aga 450 455 460 455

ASO TTA AST OST AAT SAT SAT sSo STO TAT ATO SOB TTA ST hundred AtTe 1498

FO Tph rep Beg Aia Avp A5r Azr o Siu Uai Tuk Mes Rgo cez biu Muaii Iie .470 475 4vo (ee) (95)

Sg» sl ko 60 65

AST SAD ASA TT TTS AST SSS ATT AAT sob tTTtT SOS Sto SAA OST SAT 1545 beg biz Trgorre ep TpI Rko I1e A5pobiu Re p1u Pay bip Aia Ar 485 490 435

SAV AAtT o TSA ASA TTA ATT AST TTA ASA TST DAH TSA TAT TTA ASA bAd 1554 bitz dep beg Ago tse ii Tygopesh Tr o Suv Buv bek Tut Mei Ah 61 500 5O5 510

STA STO STA OSA ASA SAS TTA ASS VAT AAA SAA AST AAAA TO ATS ste 1642 pech Pec rey A1Ta Tit Avr Pec bBeg Azp PCuv bi TP Buv Hem I1e Ma1i 515 520 525 710

SS SSV ASTtT SsT TT ATT AbS AAT ATT STA SAS AAS person ToS ATA OAA 1690

Rgo Rgo beg sbiu Rie I1e bBeg AB5p I1e Ua1! b1c Aep b1iu beg I1e bi 530 5I5 540 5AB

SAS SAS AAT TTA SAS SSO tob LADA SA AAT AAT AAS AAT SSO TAT STA 1738 Jan Ar AbBp oBGechcs bim Rgo Tkr rub Aia Ap o dBpogub Ap oAia Tut Ua

BO 555 5BO

SAT SAT ASA POS BOA OTO AAT SOZA AST AAA OST TTA TAT OSTI SAT ААо 1786

ABr Niv TI biu s1iu Ma1! Ap b1iu Tig Guye Aia Pec Tug Uai Nib tsu 555 570 575

SAS OSA ZA ATT TSA SAA TTT ATT o bOA SAT AAS TTA AAAA SOS AAAA AST 1834

Avr s1u s1u I1ie bet bip Rbe I1e b1u A5r tsu This TGuv Rho Tsuv Tih 580 585 I 590

SAS TAT STA ATS SAA TAT AST OTTO AAAA OSA AAAA SST TST ATT SAT TTA 1882

Sich tuk Ua1! T1e 5ip Tut Tlpk o wai Buv Z1u Buv Rgo bek Iie NiB Hey Ge 595 6o0 605 ' o

AAA SAT SAA AAT AST o OZA TAT ATI SAT OTAT SAA SAT ASA AAT AAT DAT 1930 uv Avr biz Ap TI biu Tut I1ie Niz Tuk b1h Avr TBg o AB Avp o ABp 610 615 620 625

TTA bAA SAT TAT SAA AST ATT AAT AVA SST TTT AST ASA ObA AST SAT 1978 t)

Tez sich ABr Tut bip TVi I1e Ap oBuv Ato Re Tbg o Trt b1u Toh Ar 63 535 640 in

TTA AASb o ObA STO TAT OTTA ATTOTTA AAA AST SAA AAT OSA SAT OSAA OST 2026 i) cem Buv siu Ma1i Tut Mep I1e Te tsu Bek bip ABp o b1iu AbBr s) A1a 545 650 655 so

TO OA bAT AAS TTT ATT ATT o TTS OAA ATT AsST SST TST SALA AAb TTA 2074 ise)

Tgr S1u A5r ABp RBe I1e I1e cem sb1h I1e Beg Rgo back 01i Was Gay 660 665 670

TTA AST OSC SAA TTA ATT AAT ASA AAT AAT TOS ASO AST AS SSA TSA 2122 «

Ted bBeg Rgo sich Pet I1e Avp TNK Abp Ap o Ttr TBgobet Tpg o siu 5eg 20 675 VO 685 - s AST AAT ATT ASS SOT AAT ASA STO AST OSTI TAT SAS SA SbA SSA oso 270 a Tit AvpoT1e Beh biu ABp TBh Bred Tpg o Hep tTuk beep siu Siu dko siu a 690 695 700 705

ATT STA AAA SAYA AVS OSTI SAA OTTA SAT AST TT OTSA AST TAT DSA sto 2218

I1e cem Tstsuv bip ABpotiei sip Pe AvBrobeh Rpe eh Tyh TUug At Mai1i b 710 715 120 so TAT TS t

TST STO TSS boA SAT OST AAT STA ASS ATT ASA AAT TST dob 2266 o Tut Rpe Beh Uai Zeg biu Azr A)a Ap UMai Ag I1e Ago Abp o beg Atgu 725 730 735 iz 50

SALO STO TTA TTT SAA LA AAS SAT ATA TA oS sosost s su Mai rem RBe siy Gue Tsuv Ar I1e val VTOTTTSToA 2318 740 745

AATSTTSAST ASAAAATTSA AASATAASTT STATATASAE STTTST 2364 (2) INFORMATION FOR 5EO IO MO:6:

Ф) () CHARACTERISTICS OF THE SEQUENCE: ka (A) LENGTH: 747 amino acids (B) TYPE: amino acid vo (D) TOPOLOGY: linear (its) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: ZEO AND MO:6: 65

Mes Up oGuye Avp o AvBp Aza Guye Bey beg Trg Ago Aza rez Rgo beh RNe 1 5 10 15

Iiiie Ar Tut RiBe ABp bsiu I1e Tug Siu RbBe A1a TE biu I1ee Tssuv Avr 20 25 30

I1ie Meb Avp o Mes I1e Rre Puz Tiph Avr Tit s1u Siu Avr Gen Aza Gech 35 40 45

Ar bi I112e Hey b)iy Azp sip bip Hemsh Pem Ap Ahr o I1a Beh s1u Guz 50 55 Bo I 710 yeech Avr b1iu Ua) Abp biu beg Pec Azp Ayer Pec I1e Aza sip b1iu Ap 65 7o 75 vo

Graham Avp oTpt 01 cem bet Gu b1Ch i1i6 Pecs Gu T1e A1a A5p bich siy 85 90 95

ABp Sbip wai em Avp o Azroua) Azp Ap Guz rep Azr Aza 71e AvBp TTG 100 195 110

Meb Gem Ago Uai Tut Pem Rgo buye I1e Tit beg Mes Pec bek Ar Ua1 115 120 125

Me tsu sip AvpoTukh Aia pey beg o Ptsesh bip I1e Si Tukh ey bBet Buz 130 135 1410 bip o rem sip biz I1e beh Avr Pub Pes Ar o i11e t11e6 dep Ua1i Aep Uai 145 150 155 160 s

Pech I1e Azp beg tTigoPech Tk obich t)iee Tk Rgo Aza Tu si Ago I1e 165 170 175 Ge)

Was Tut Uai ABpobim Guye Rre b3i sich Ge Taho Rbe dia tTrg obi TE 180 185 1980

Zek bei Guv Mai Tuz Tuz Avr b1iu bet Rgo Aia ABr I1ie Agu Avr S1M Is) « (ze) (ee) (Se) - s . a (o) (ee) (95) g» sl ime) 60 65

195 200 205

Pech bZeg bimM rec Tph bib Pech A1a Gub Bet Ma) tTrt obip Ap AvroUai 210 215 220

ABr b1u Re bi Rne Tut iem ABp TBg Re Ni Ayer ua1i Mes uai b1U 225 230 235 240

ABp AzpoPez Rpe Siu Ago Beg A1a yeezch pub Trg Aza bZeg 013 Geo I1e 245 250 255

Trg tsu 01st Avp Mai Guz Tato beg b1u beg sich Uai b1u Ap Ma! Tukh 260 2655 270

Ap oRBe Bay T1ie wai besh Tk oAia Pep bip Asia Sip A1ia RBe ybem tok 215 280 285

Tem TBg Rgo Suv Agu gub cem rep b1u Gay A1a Ar Y1e Avr Tug TB 290 295 300

Beh I1e Me A5p o bich Ni Gech Ap tsu 51y Was b1z b1ts WBe dgo uai 305 310 315 320

ABpot1e tech Rgo TBh Petz beg Avp o TBu RBe Bek Avp Rgo Abp o Tug Aia 325 330 335

Guz Mai Ide siu bek Avr s1its Avr A1a Was Mee Iie uai Sitz Aza Was 140 345 350

Rgo s1iu NiB A1a Ptsep I1v Ssiu RBe b1its I1e beg ABp Der bek I1e TPg sem 355 360 355 o

Uai this Puv Ma1 Tut bij Aza yuyB rez was bip Azp oTuh sip Uai Ar 370 375 380

Tuz Ar bek Ptsep bBeh Si Uai t1ie Tut b1iu Aev Mei Abr o Tsuv ep Pec yu 385 390 395 400 «g

Suv Rgo ABr Sip beg biu Sip I1e Tut Tut TPg Ab5p ABpP oI11e Ua1i Rpe 405 410 415 (ze)

Rgo ABp s1iZ Tut Ma1i I1e t tsuv I1e Azr Re Tig Buz 1/5 Me Buv so 420 425 430

Tng tsech At Tut Sim Uai Tnu dia Ap Rle Tukh Avr beth Bek Tk b1U i-y 435 440 145

Si I1e A5r iey AvpoMtsu5 Guv tsuB Ma! 01p bBeg bBei 514 A1ia 514 Tut 450 455 460 «

Aha TI yech beg Aia ABp o ABr Ar biu Uai Tut Mee Rkgo ey b1u Mai shsh 465 470 475 480 s p T1e bek sits Tit RBe tsets TI Rgo i11e Azp biu Rre biu Pec sip A1a "» 485 4190 495

ABr ob1ich ABp bBeg Ag Pei I1e Tph Pech Tpg Suz Buv Beg Tut Pez AkF 500 505 510 (o)

Siv re pcetsp prep. A1ia Tit ABr Tets beg Abp o tsuz bi Tpg Buye Pets iT1e (ee) (95)

Sg» sl ko 60 65

515 520 525 uai Rtgo Rkgo bek biu RNe her Beh Ab5p Iie Uaii Si Abzp s1u beg I1Ee 530 535 540 bim sich ABr Azpopech 010 Rgro Tgrotsue Aia Ap doro uv Ap A1ia Tut 54A5 5B5O 555 5voO

Uai Ar Niv Tph biu b1iu uai A5p siu Tig Tsuv A1a Gech Tukh Ua1i No 5655 570 575

Guz Ar Siu biu I1e beg bip Rie 116 C1u Ar GuvB Pec Buv Rgo Buv 580 585 590

Tg biz Tut Ua1i I1e beep Tuh Trg Uai pu s1u was Rko beht I1e No 595 600 605

Tiech Puye Avr bij Avp tru b1iu Tuk Y16 Niv Tuk 014 der TI dvo ABp, 610 615 620

Ap o pechp bi Ar o Tug bip Taho I1e Azp o Guz Ago Rpe Ts Tig osiu Tag 625 630 635 640

AvBr Gay Puv siu Ua1! Tut Brets I1e Pecs Guz behg bip Ayep b1u Abvr b1y 6545 650 5655

A1ia Tur b31u Avr Avzp Rpe I1e 116 Bei bi I1e bBeg Rgo beg bii Buv bo 665 570

Graham Pez beg Rgo bij Bez T1e Azo o TBt Ap Aza Tgrotrt beg Tik s1u s 675 vo I: o

Beh tru Avp i1e beg biu Ab5p Tpi Ppem TNh Pec Tukhk sip S1u biu Aga 650 695 7o0 siu I1e cep Guv sbip Abp o rec bip ley Ar o beg Re beg Tit Tuk Aga 705 710 715 720 IS c) uai Tukhk Rie bek Ma1i begt Siu Azr Aza A5p Ua! Agu Iziye Ag Ap beg "I 125 739 735 so

Ako sich Mai tsep Rbe b1its Bu5 Buv ABr oK1e 740 145 g)

Zo (2) INFORMATION FOR 5EO IO MO:7: ise) () SEQUENCE CHARACTERISTICS: (A) LENGTH: 2403 nucleotide pairs (B) TYPE: nucleic acid « (C) STRANDING: single (D) TOPOLOGY: linear Z c ( i) TYPE OF MOLECULE: other nucleic acid "" (A) DESCRIPTION: /dess - "Synthetic DNA" " (ii) HYPOTHETICAL OPTIONS: NONE (2) FEATURES: (A) NAME/CODE: tiss Teaishige

Me. (BU) LOCATION: 11..2389

Go! (D) OTHER INFORMATION: /sign: "optimized maize DNA sequence encoding MIRZA(a)" (xi) SEQUENCE DESCRIPTION: ZEO IYUO MO:7: (95) и» 50 sl

F) name) 60 65 -5D-

SSATOSASSA ATSALSATOA ASAASAASAA SASSALOSTO ASSASOSOSO SOSTOSSSAYE Because

STTSATSSAS TASTTSAASO SSATSTASOS STTOSSSASS SOSATSAASS ASATSATSAA 120

SATSATSTTS ABSASSSASA SSSSOSSOSOA SSTOASSSTO SASSASATOS TOAASAASSA 180

SSASSSTOSTS АASSАSATSA ССОССААССТ ОСАСООССТО ААССОСАССС TOVASSСССТ 240

САСССССАС СОСААССТСА АСАСССАССТ САССАОСАС АТСССТТААСА ТОСССААССА 300

OSASAASSAS STOSTOAASO ASOTODASAA SAAOSTOSAS SSSATSLASA SSATOSTOSO 3BO

SSTOTASSTO SSSAAASATSA SSASSATOST SASSSASSTO ATOSAASSASA ASTASOSOST 420

SASSSTOSAS ATSSASTASS TSASSAASSA OSTOSASSAS ATSASSASSASSA ASSTOSASAT 480

SATSAASSTO AASSTOSTOA TSAASSASSAS SSTOASSSSAE ATSASSSSOSS SSASSASSSO 540 75 SATSAASTAS STOAASSASA ASTTSSAASA SSTOASSTTTO SSSASOSASA SSASSASSAA voy

СОТОААСААС САСОССАСССССОСССАСАТ ССТОСАССССССТОАССОАОС ТОАССсАОоСст 66o

SSSSAAASS STOASSAAASA ASSASSTOZA SOSSTTOSAS TTSTASSTOA ASASSTTTSSA 720

SOVSoUSVTS STOSOSAASA ASSTOTTOSS SSSSASSOSS STOAASASSO SSASSOAAOoST 780

SATSASSAAO SASAASSTOA ASAASSASSSSS ASSSOASOTO SOSAASOTOT ASAASTTOST 840

SATSSSTOSTO ASSOSOSTOS ASSOSSSSASSSS STTOSTOASS STOASSASSST STSOSAAOST 500

ОСТОбОССТОо ОСССАSATSS ASTASSASSAS SATSATOAAS САССАСТТСА АСАОСАСАА 96o

ССАСОАСТСССССТОААСА ТОСТОСССАС ССТСАССААС АССТСАССА АСССБААСТА 1020 o

ССССАЛОСТО ААСОССАОСО АССАССАСОССАСАСТАССТСОССОСТА ВССССОСССА 1080

SOSTTSSATS SOSTTSBASA TSASSAASSA SASSATSASS STOSTSAVOS TOTASSASoS 1140

СААССТСААС САААСТАСС АССТОСААА ССАСАССТТС АСССАССТОА TСАССССА 1200 ю

SATSSASAAS STOSTOTTSTS SOOASSASAS SOAOSAAATS TASTASASSA ASAASATOST 12650 «

STTSSSSOAAS OASTASTTOA TSASSAASAT SSASTTSASS AASAASATSA ASASSSToSo 1320 so

STASSASSTO ASSSSSAAST TSTASSASAS SASASSASSOS SASATOSASS TOLASYASAA 1380 so

СААСОТОСАС АОСАОСОАСо СССАСТАССО САСССТОАСС ОССААСОАСо АСОБСОТСТСА 1440 со счи САССССАСТ СОССТСАСТСССАСАССТ СССОАССССО ATSAАССОСТ ТТОбСССТОСА 1509

СОССОАССАС ВАСАСССССССТ TOATSASSST САССТСТАЛО АССТАССТОС сосАССТТОосТ 1560

ССТАССАСССАССТОАОСА АСАССАСАССАДОСТОСАТССТОССАСССА СССОСТТСАТ 1620 «

САССААСАТС СТОСАСААСС ОСАССАТСОА ССАССАСААС STOOАСССОТ ОСААОСССАА 1680 2 с САСААСАС ОССАССАССО ССОССТОААС СССАССАdos СОССОСАСТ 1740 with ОСАААОСАС ООСООSATSA СССАСТSAT СООСОАСААС СТОААССССА АСАСОСАСТ 1800

ССТОАТССАС ТАСАСССТОА АСОССААОСС АТССАТТСАС СТОААССАСС АСААСАССсс 1860 б 7 СТАСАТОСАС ТАССАССАСА ССААСААСАА ССТОСАССАС ТАССАСАССА ТСААСААОСС 1920 со СТТСАССАСС ОССАСССАСС ТОААСОСССТ СТАССТОАТС СТОСААСАССС АСААСОСССА 1980 о ССАССССТОО ССССАСААСТ ТСАТСАТОСТ ССАСАТСАОС ССОАСССАСА АССТОСТОАС 2040 їз 20 сссосАОСТо АТСААСАССА АСААСТОСАС САССАССООС АССАССААСА ТСАССООСАА 2100 сл САСССТСАСС СТОТАССАСО ССбОССОССо САТОСТОААЄ САСААССТОС АССТОСАСАС 2160

СТТСАССАСС ТАССОСОТОТ АСТТСАСССТ ОАССОСССАС СССААССТОС ОСАТССОСАК 2220 ря САСССОССАС СТОСТОТТСЯ АСАВОДОСТА САТСАССОСС СССААОСАСО ТОСАОСОАСАТ 2280 о СТТСАССАСС ААСТТОСАСА АССАСААСТТ СТАСАТССАС СТОАСССАВО ССААСАВОСТ 2340 кю СТАССОССОС СССАТСОТОС АСТТСТАСОА СОТОДОСАТС ААСТТАВССТ АСАССТСАСА 2490 тет 2403 6о (2) ІНФОРМАЦІЯ ДЛЯ 5ЕО ІО МО:8: () ХАРАКТЕРИСТИКИ ПОСЛІДОВНОСТІ: (A) LENGTH: 1638 nucleotide pairs (B) TYPE: nucleic acid (B) STRENGTH: single bo (D) TOPOLOGY: linear

(her) TYPE OF MOLECULE: cDNA (ii) HYPOTHETICAL OPTIONS: NONE (2) CHARACTERISTICS: (A) NAME/CODE: СО5 (B) LOCATION: 44..1191 (ГЛ) OTHER INFORMATION: /product "Translation of cDNA encoding MIRZA (a) receptor" (xi) SEQUENCE DESCRIPTION: ZEO IYUO MO:8: t AST OSA ToS sosS bos Sto SAS SAA TTS ssSo oSs oso tob AS ATO 46

Mass 1 5 19 15

TAS TST ASA AtTA TTT OTTO STO sStTtT STO ATA sto tot OST sTT AAb Ost 54

Tut beg Agu Iie Re Rpe Pen Pes Mai I1ie Ua1! Su5 AZia Uai Bub5 A1a 20 25 zo

TST Sto TTT AST STA DAT sto TAT SAT SAT AAS SSS SAA AST SAA ATT 142 bet pe Re tik oUai Ap uai Tut Azr oder Ap o Rgo bim Tpg obitiv 35 40 45

ССО AST AST STA AAA OSOoS TsST AAS СОС САА bAb TOT САС САб Soo tot 130

A1ia bBegt bet this pu s1u Sub Ap Rko beep 0101 Suv Abr beep Agu Suye 50 5 at 20 . with (8)

IS c) "so s (Se) - s ;" (22) (ee) (95) i» 50 sl

F) ime) 60 65 -5c-

SST AbA STS AAb TTT sSssS ost bos osS tot sto AAtT bot SOS TosS A 238

Ah Aga em tsuv Rae Rgo biu biu A1a Sub Ma) Ayep S1iu Ag Suv TuB 65 70 75

TST SAS AASOTTS ST AST STA AAAA SAT o bAS sto tot o sttT o alA bab sst 286

Suz AhroAvp o Rde em back tea! tsu ABr o ABr o Ma1 beg Uai 013 Jan Rgo in 85 Zo 95

SSS o dtTt Sto AAV SAT OTTO STO TSA TTA SAA OST baAA SAS OSA SSO AAA 334 dia Iie em MCsuv Avr Pec wai beg Pem bitz Aza bish s1ip A1i1a Aza Buv 100 105 110

AosT ASA TOS AbA AS OAbA STO TOT OAS oso sto ROS sot sosS STA SAS 382 bek Aga Suv Agd Ap AtYa Ma1i Suz ABr o A1a Ua) Suv AgYa A1a rep No 115 120 125

AAS OASS ASsST sot boss ToT ST SAT OSA SAA TOS AS OTOT AST LAT DA 430

Avp o TBg beg biu A1і1a Suv uMa1i ABr siu Sip Su pub Sud Tit ABpoGue 130 135 140

ATS AST OSA OSA SAT ATT STO TST SAT SST OST baAA TS STA SOS AST 478

I1a Bek A1a Siu Ar I1e Ua1i beh Avzr Rgo Aza bii bei Per Ago TBh 145 150 155

TST AAS SST ATA ASO TST SAS OAA SALA TOT ASA ASA AVT SOS SAT ААА 526

Sub ABp Rgo I1e Ag Sub Avr o sSish bip Sub Agu Aku Ap biu Niv si 160 165 170 175

TTT OST ST TOS To AL OSA SAA TOS A TOT SAT TAS TI STO A 574

Rpe piu Ua!) Sub RNe uv C1u bip Suv uv Sub Ar Tut Rpe Pec Gu se 180 185 1950 o

SAA OAA ST AT OAA SST SAA OT ASA AOS STT SSA AAA dSOTOS As 6522 bi bi Uai Azr bi; Rko bii Mai TBgobeg Pes Rgo Bu5 Ap oSuv Ap 195 200 205

SSS SAA OAS TOT SAS SAS SOT TOT SOT AP hundred VAS TtTe sss o sstT bos 670 o

Rko bip bij Sub Abvrob1p Aku Sub Agu Aga Gech Buv Re Rgo Siu biu 210 215 220 Z

SSS tot OT AS ooo SOS TOS AAS TOT SAS AAS TI TT AST OSA ss 718 o

Aza Suz Mai Avp siu Aku Suv Gu Sub ABr Ap Re Rre beg A1a Ssiu s 225 230 235 (Se)

SAT ATT STO TOTO SAT SST boss SAA TOS STA SOS TST TOT AS SST ATA 766

ABr I1e Ud1i beg Avr Rgo Aza b1iu bek Bei Agu beg Su Ayep Rugo I1e 249 245 250 255

AS TST SAS SA SAA TOT ASA ASA AAT SOS SAT sSAA tTtTtT osot stt tes 814 «

Atu Suv Avr o Siy Sip Sub Agu Agu ABpobiu Niv bi Rpe biu Mai Suv 260 265 270 - s TTS AAA SOA SAA TOS AAS TOT SAT TAS TTE STO AASOTSA SALA STA SAS 862 "» Rpe tuye siu bip Suv Guz Suv Azr Tuk Riv Pec Azp bZeg bits uai A5r and 275 280 285

OST STI AAT SAS TTT SST SAA SSO SOS TsA ADA SOS TAS TOS LAS TtTA 310

Aia Mai Avpobiy RBe Rto bip A1a biu beg tsu Agu Tut Suv Ayep Pec

Those! 290 295 z0o (ee) (95)

Sg» sl ko 60 65

AS SAA TOS VAS SAS So TOS SSS JSC SST TTIS TAT OAT AST OST ASA 858

Tigobip Suye AbzpoSip TpgoSuv A1a Ap Ago Rpe Tut Azr b5eg Aza Ag 305 310 315

STO ATS SAS OBOS TOb TOS dAA TOS TAS AST AAb ATO SAA AbA SAS SAT 1006

Ua1 I1e Ni siu Ter Suye Guv Sub Tut bet tsuv Meb bitz Ako bip ABr 329 125 330 335

OSA TST SSA TTA AAS SAT STO AST SAS SAT SALO AAT SAA sTT TStT AAS 1054

A1ia bek Rgo Mei Ap o Avr o Ua1i! Tpg obi Abo bi Ap bib Uuai beg Ap 340 345 150

SAT ATS STO ABS AST STT OSA SAC STO STO TST SAT STO TSA SST aso 1102

Avr I1e cem Ak Tvg Uai A1a bi bits Pes bek Abr Ua! Beh Rgo AhkdF 355 360 365

SSS TOS AAA TSA So AOS TOS AAT SAA bSA tot SosS sosS TS TAS OT 1150

A1a Suv Puv Beg A1a Bet Suv Azp beep Ahcha Sub5 Ago Aza Re Tukh Riye 370 375 380

AVA SOA ss TO TT SOS TTT Oba SBA TS SAA TOS TISTA 1191 uv biu bi1u Ttr Suv Agu Re s1u Ag Sat s1p Sat Be 385 390 395

AAAATTASTAT SATATATOAA TITTOTATTA TTOSSTTTAAT TOTOTTATOT TTAAAAAASA 1251

TAATOTSTIS ATTTTASAAA AAASTASSTT SASTAAASSO SAASAATTAA STASTASTTA 13511

ATTATTAAST ASTASTTAAA TTATTOATOSA TTATOSATTAT STTASTASTA STTAATTATA 1371 sem

ATSATSAAST ATTAASTAST ASTTAATTAT TAASTASTAES TTAAATTATT SATOATTATO 1431 Ge)

ATTATSTTAS TASTASTTTAA TTATTOTTIS TTATAATAAT STASTATOTT SSTASTAST 1491

TAATAATAAS SSTISTSASA AAAVATTTAA AATTAAATAA TTSTATSALA SATAAATAAT 1551

AASTOAAATA AAVATTTATA ASASAAAAAAA DAAVASTSOA SOSOBSSOSO AATTSSATAT 1611 «g

SAASSTTATS SATASSSTOV ASSTOSA 1638 co (2) INFORMATION FOR 5EO IO MR:9: (ee) () SEQUENCE CHARACTERISTICS: co (A) LENGTH: 396 amino acids (B) TYPE: amino acids (G) TOPOLOGY: linear (her) TYPE OF MOLECULE: protein « 20 (xi) SEQUENCE DESCRIPTION: 5EO IYUO MO: 9: z beg s1u bBeg Rho ziu Pem bip 013 Rpe A1ia Aza AZcha bek Tr Meb Tug s 1 5 19 5 . "" Bek Ak I1ee Re Rbe Pes Pec Uai I1ie Mai Sub A1a Uai uv A1i1a bek 20 25 30

Iets Rpe Ti Ma)! Ap Mai Tuk Avr Avr ABp oRgo Si TptI Si i11e A1a

Ge») 35 , 40 4145 (ee) (95) g» slime) 60 65

Bek bek pcech Puv biu Sub Ayep Rgo Sip bi Suz A5r b1ip Ago Suv Agu 50 55 60

AgCh ate Was Rbe Rgo siu siu A1ia Sub Ma! Abp S1u dk Suv5 Buv Suv s 65 70 75 vo

Avr ABp o RNe Pes bek Uai Puv AbBr Ab5r Maiii Beh Ua! 91 Beach Rgo A1a 85 90 95

I1e Gea GuvB Agro yeech Ma) beg Pes bip Aza siy bij Aza Aza Bu5 beg 160 105 110

Aha Suv AgYa ABpoAgo Ma! Suv Azr dia Ma1i Suv Ata Aza Petz niv Ap 115 120 125

Tig bBeg siu A)a Sub Ua) Aver C1u Sip Suv Ptsuv Suv Tik Avp rub I1e 130 135 140

Beg Aza biu ABr Iie Ua1i bek ABr Rgo AD1a b1i beg iem Ak Tig o Suv 145 150 : 155 160

ABpoRgo I1e Aku Suv ABr bi sip Suv Aga At ABp os1i1u niv s1ii RBe 165 170 175 s1u Ma1! Suv Rpe tsuv siu bip Sub tsu Su Ar Tut Riye yeech Ppub5 1 180 185 190 bij Mai Ar bib Rgo biz Ua1i TBg beg Pec Rgo uv Avp Sub AB rRgo 195 200 205 s bip br Suv Avr bip Aga Sub Aga Aku Tez Puye Rbe Rgo b1u p1u A1a Ge) 210 215 220

Suv Uai ABp S1u Agu Sue Buv Sue Avr Ap Re Rpe beg A1a Siu Avr 225 230 235 240

IC c)

I1e Mai beg ABr Rgo A1a b)ch bei rech Ato bek Suk Ap Rho i11e6 Ahg5 245 250 255 «

Suv Avr sitz bip Suz Agu Aku Ap biu Ni5 bis RBe s1u Mai Suv Re so 260 255 27 (ee)

Buv biu bip Su Guv SuB Avr Tut Rre Gay ABl o beg Si Mai Ar Aza 275 280 285 (Se) uai Avp obi Re Rgo bip Aza biu beg tsuv Agu Tut Suv Ap oGec TEG 290 235 390 bip Sub ABpobip Tr Sub dia A5p Ago Re Tugt Avr beh Aza Akd Uai « 305 310 315 320 -v s t1ie Ni s1u Tgr SuB PCuv Suv Tukh Bet Buv Mesii Agd bip ABr Aza 325 330 335 ;" " bek Rgo rech Ayap ABr Ua)! TI Sich Ar si) Ap biz Uai beg A5p Avr 340 345 350

T1e rez Ag Tvt Uai Aza 010 bim pey beg Avr ouaii bek Rho Ag Aza (2) 355, 360 365 (ee) syu Su tsuv Beg A1ia Beh Suv dp Sip A1a Sub Agu Aza Rpe Tug Rpe tsu 370 375 380 i» 50 s1u b1u Ter Suv Aga Re o1u Aku Sub sip Su Vre 385 390 395 sl (2) INFORMATION FOR 5EO IO MO:10: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids o (B) TYPE: amino acid (C) CHAIN STRENGTH: single co (H) TOPOLOGY: linear (s) TYPE OF MOLECULE: peptide 60 (s) HYPOTHETICAL OPTIONS: NONE (m) TYPE OF FRAGMENT: M-terminal (s) SOURCE OF ORIGIN: (A) ORGANISM: Vasiiiz (pygipdiepviv (B) STRAIN : АВВ5 65 (2) FEATURES: (A) NAME/CODE: Peptide

(B) LOCATION..14 (D) OTHER INFORMATION: /mark- "M-terminal amino acid sequence of the protein known as anion exchange fraction 23 (minor)" (xi) SEQUENCE DESCRIPTION: ZEO IU MO: 10: hva bi Rgo Re Te Bek dia Haa Haza Haa s1ip Haa Haa Haa (2) INFORMATION FOR 5EO IO MO: 11: to () SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRENGTH: single (D) TOPOLOGY: linear t (m) SOURCE OF ORIGIN: (A) ORGANISM: Vasiiiz (pygipdiepviv (xi) SEQUENCE DESCRIPTION: ZEO IU MO: 11:

Haa sSits Tug 01 Avp oUai bij Rgo RBe Ua! Beg A1ia Haa 1 5 10 (2) INFORMATION FOR 5EO IO MO: 12: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRENGTH: single ch 29. (huTOPOLOGY: linear o ( m) SOURCE OF ORIGIN: (A) ORGANISM: Vasiiiv (pygidiepviv (xi) SEQUENCE DESCRIPTION: ZEO IU MO: 12: oyu

Mes Avp osuv ABpoAvp TT Tsuv em Rho Tr Ag A1ta Gey Rgo 1 5 10 - (2) INFORMATION FOR 5EO IO MO:13: co () SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids co (B) TYPE: amino acid (Se ) (B) CHAIN STRENGTH: single (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: peptide « (ii) POTENTIAL OPTIONS: NONE (m) TYPE OF FRAGMENT: M-terminal - c (m) SOURCE OF ORIGIN: a (A) ORGANISM : Vasiiiz (pygipdiepviv ,» (B) STRAIN: AVZ88 (their) CHARACTERISTICS: (A) NAME/CODE: Peptide (B) LOCATION: 1..15 (o) (D) OTHER INFORMATION: /mark- "M-terminal amino acid sequence of 35 kDa MIR, active against co-Adgoiiz irviiop" (hi) SEQUENCE DESCRIPTION: 5EO IYUO MO: 13:

Via cem beht bij Or Trgob1u Buv ABrosiu Siu tuh I1e wai RKO and 1 5 10 15

Her. 70 (2) INFORMATION FOR 5EO IO MO: 14: sp () SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRENGTH: single 55. (d) TOPOLOGY: linear

GF) (m) SOURCE OF ORIGIN: 7 (A) ORGANISM: Vasiiiz (pygipdiepviv (xi) SEQUENCE DESCRIPTION: ZEO IU MO: 14:

Me Avr dep Abp Rgo Avp i1e Ap b1c4 ba 5 (2) INFORMATION FOR 5EO IO MO: 15: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acids bo (C) STRENGTH: single

(TOPOLOGY: linear (s) MOLECULE TYPE: peptide (s) POTENTIAL OPTIONS: NONE (m) FRAGMENT TYPE: M-terminal (m) FEATURES: (A) NAME/CODE: Peptide (B) LOCATION: 1..9 ( SG) OTHER INFORMATION: /mark- "M-terminal sequence of ZOkDa delta-endotoxin" 70 (xi) SEQUENCE DESCRIPTION: ZEO IO MO: 15: es Avr Aut ABP go ABpP t11e Avp 61 (2) INFORMATION FOR 5EO IO MO: 16 : () SEQUENCE CHARACTERISTICS: t (A) LENGTH: 11 amino acids (B) TYPE: amino acid (B) CHAIN STRENGTH: single (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: peptide 20 (i) POTENTIAL OPTIONS: NONE (m) TYPE OF FRAGMENT: M-terminal (m) SOURCE OF ORIGIN: (A) ORGANISM: Vasiyiz (pygypdiepviv (m) CHARACTERISTICS: ch 29. (DU NAME/LCODE: Peptide o (B) LOCATION: 1..11 (GL) OTHER INFORMATION : /mark: "M-terminal sequence from bOkDa delta-endotoxin" (xi) SEQUENCE DESCRIPTION: ZEO IU MO: 16: oyu 30 mos VBp Ma1yi Hey dev Beh siu Ag4d Tit TPg o I1e Y so (2) INFORMATION FOR 5EO IO MO: 17: so () CHARACTERISTICS OF THE SEQUENCE:

Zo (A) LENGTH: 17 nucleotide pairs ise) (B) TYPE: nucleic acid (B) STRANDING: single (D) TOPOLOGY: linear « (her) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: /devs - "sequence primer" Z c (ii) POTENTIAL OPTIONS: NONE "» (xi) SEQUENCE DESCRIPTION: ZEO IU MO: 17: и SOATTAATSt TososTe 17 (2) INFORMATION FOR 5EO IO MO: 18: () SEQUENCE CHARACTERISTICS: (o) (A ) LENGTH: 21 pairs of nucleotides (B) TYPE: nucleic acid (B) STRENGTH: single (95) (D) TOPOLOGY: linear 1" 50 (its) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: /devs - " primer sequence" sl (ii) POTENTIAL OPTIONS: NONE (xi) SEQUENCE DESCRIPTION: ES IU MO:18:

SATTAOSATS ToSObvSСА at 21 (2) INFORMATION FOR 5EO AND MO: 19: 59 () CHARACTERISTICS OF THE SEQUENCE:

HF) (A) LENGTH: 2370 pairs of nucleotides 7 (B) TYPE: nucleic acid (B) STRENGTH: single (G) TOPOLOGY: linear 60 (its) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: /dess - "synthetic DNA encoding MIRZA(B)" (ii) POTENTIAL OPTIONS: NONE (xi) SEQUENCE DESCRIPTION: ZEO IO MO: 19: b5

(xi) DESCRIPTION OF THE SEQUENCE: 5EO IU MO:19:

АТСААСААСА АСААСАСАА ССТОАССАСС СОСОСОСТОС ССАОСТТСАТ СОАСТСТСТС 60 и АВССОСАСТ ASSOSTTSOS САССОСАСТАССАСАСТСА TSAАСАСТСАТ СТСААСАСС 120

SASASSSOSO OSSASSTOAS SSTOSASOAO ATOSTOLASA ASSABSASSST SSTOAASSAS 180

АТСАБСОССА АССТОСАСОС ССТСААССОС АСССТОААСО АССТОАССОС ССАСОССААС 240 70 СТСААСАССО АССТОАОСАА ОСАСАТОСТ ААСАТСОССА АССАССАСАА ССАССТОСТО 300

ААССАССТСА АСААСААССТ ССАСОССАТС ААСАССАТОС ТОСОССТОТА ССТОСССАДо 360

ATSASSASSA TOSTOSASSSA SSTSATOLAYE SASAASTASS SOSTSASSST SSSATOSAS 420

TASSTOASSA ASSASSTOSA SOASATSASS SASAAOSTOS ASATSATSAA SSTOAASSTo 480

STOATSAASA OSASSSTOAS SSASATSASS SSOOSSTASS ASSOSATSAAA STASOTOAAS 540

САСЛАСТТСО ААСАССТОАС СТТСОССАСССА САСАССАССА bСААССТОАА СААССАСССС бо МОССССБССССАСАСТССТОСА ССАССТСАССССАСССАССО AOSTOSOSАА САСССТОАСС 66o

AASVASSASS TOSASSSSTT SOAOTTSTAS STOAASASST TOSASSASSST SAToSTOSOS 720

ААСААССТST ТОССССОСАС СОСССТСААС АСССССАССО AOSTSATSAS СААССАСААС 780

СТОААСАССА СССОСАССОА ОСТОСОСААС СТСАСААСТ TOSTOATOST ССТОдоСОСС 840 s schi СТОАСАСССА АСОССТТССТ САСОСТСАС СССТСТОССА AbSTOSTObS ССТОССССАС z00 (o)

АТССАСТАСА ССАССАТСАТ СААСОСАССАС ТТСАВСААСО АСАССАСЗА СТТОСОСОТе 9560

ВАСАТОСТОС СОАСОСТоАС СВАСАССТТС АСАВССССА АСТАСОССАА ССТСААССОоС 1020 ю

АОСсАССАСО АСОССААСАТ SATССТОСАС ССТААСССОС СССАССОСТТ SATOBOSTTS 1080 «

SASATSASSA ASSASAOSAT SASSOSTOSTO AASSSTSTAS ASSSSADOST SAASSASAAS 1140 (ze)

TASSASSTOO ASAASSASAS STTOAOSOAS STOATSTASO SSOASATOSA SAASSSTOoSTOo 1200 (he)

TSTSSSSSASS ASASSSSOOSA AATSTASTAS ASSAAASAASA TOSTOTTOSS SAASSASTAS 1260 (Se)

STOATSASSA ASATSOASTT SASSAAASAVS ATSAASASOS TOSOSTASOA bOTSASSosSS 1320

ААСТТСТАСС АСАССАОСАЄ СССОСАСАТС САССТСАВСА АСАДСААССТ ССАСАОСАОС 1380 « 20 САСССССАСТ АССССАСССТ САСССССААС САССАССССС ТСТАСАТОСС АСТОбОСОТО 1440 -в с АТСАСССАСА ССТТССТОАС ССССАТСААС СОСТТТОССС ТОСАСССССА ССАСААСЬОС 1500 :з» СОССТОАТСА СССТСАССТО ТААСАССТАС СТОССССАСС ТОСТОСТАбС САССОАССТОо 1560

DOSLAVSAVOS ASASSALOST SATSOTOSSA SSOASSSOST TSATSASSAA SAToOTOsSAE 1620

Ге») 75 ААСООСЬОСА ТОСАССАССА СААССТОСАС ССОТОЗААСО ССААСАДСАХ СААСОССтТАС 1680 о СТОСАССАСА СССССОССсТ СААССССАСС ААСОСОСТОТ АССТОСАСАА ССАСобссоС 1740 со АТСАСССАСТ ТСАТСОСССА СААОСТОААС СССААСАСОС АСТАССТОАТ ССАСТАСАСС 1800 їх 20 СТОСВАССОСА АСССАТОСАТ ТСАССТОСААС БАССАСААСА ССООСТАСАТ ССАСТАССАЄ 1860 с САСАССААСА АСААССТОСА ОСАСТАССАС АССАТСААСА АССОСТТСАС САСССОСАСС 1920

САССТОЛАСО ОСОТОТАСТ SATOSTОААС АСССАСААСО СССАСбАСОоС STOSobСssАС 1980

ААСТТСАТСА ТОСТОСАСАТ САСССССАЄС САСАЛССТОС ТОАССССОСА ОСТОАТСААС 2040 (ФІ АССААСААСТ ССАССАССАС ССОСАССАСС ААСАТСАСОС ССААСАСОСТ САСССТОТАС 2100 ко САСОобССОСС СССССАТОСТ САВОСАБААС СТОСАОСТОС АСАОСТТСАС САССТАССОС 2160 бо СТСТАСТТСА ОССТОАССОС ССАСОССААС СТОСОСАТОС бСААСТоССо СсАсоТОоСсТо 2220

TTSAASAASA SSTASATOAS SOSSOSSSAAS SASSTSASOS ASLTSTTSAS SASSAASTTS 2280

SASAASSASA ASTTSTASAT SSASSTSSASS SASOSSSAASA ASSTOTASOS SSOSSSSATS 2340 65 STOSASTTTST ASOASOTOAS SATSAASTAS 2370

(2) INFORMATION FOR 5EO IO MO: 20: () SEQUENCE CHARACTERISTICS: (A) LENGTH: 2,241 nucleotide pairs (B) TYPE: nucleic acid (C) STRENGTH: single (D) TOPOLOGY: linear (her) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /dess - "synthetic DNA encoding MIRZA(B)" 70 (ii) POTENTIAL OPTIONS: NONE (xi) SEQUENCE DESCRIPTION: ZEO IU MO: 20:

АТСААСААСА АСААСОССАА ОСТСАССАСССОССОСССТСОССОСССРСТССКИЙ

ААСОСОСАСТ АССОСТТООС САССОСАСТАААОСАСАТСА TOААСАСТСАТСТСААСАСС 120 7/5 САСАССОСОССССАССТООСССТОСАССАСАТОССАСАСАСТАССАССТССТОААССАС 180

ATSASSOSSOSA ASSTOSASSS SOTOAASSOS ASSSTOAASSO ASSTOATSOS SSASOSSSAAS 240

STOAASASOS AOSTSASSAA SSSATOSTTT AASATSSSSSA ASSASSASAAA SSASOTOSTO 300

ААССАССТОА АСАСААССТ ОСАСОССТС ВАСАССАТОС ТОСОССТОТА ССТОСССАdo 360

ATSASSASSA TOSTOAOSSA SSTOATSAAS SASAASTASO SOSTOASOST SSSATOSAS 420

TASSTOASSA ASSASSTOSA SSASATSASS SASAAOSTOS ASATSATSAA SSTOAASOSTS 480

STSATSAASA SSASSSSTSAS SOASATSASS SSOSSSSTASS ASSSSATSAA STASSTOAAS 540 s 729 САОААСТТСО AASLOSTSAS STTSOSSSASS САСАССАОСА OSAASOTOAA САЛССАСООС 6oo o

AbСССбОСОС АСАСССОССА СОАССТОАОС САССТОАССО АССТОССОАА САСССТОАСС 660

САААСОАССТОСАССОСТ ССАСТСТСТАС STOAАСАССТ TOСАССАССТ SATOSTObSOS 720 points

AASAVSSToT TObOSSOSAS SSSSSTOAAS ASSOSSASSSO AOSTOATSAS SAASSASAAS 780 «

STOAASASSA SSOSSASSOA SSSTOSSSAAS STSTASAAST TOSTOATSST SSTOdssoss 840 so

STOSABOSSS ABOSSTTOST SASSSTOASS SSSTOTSOSA AOSTOSTOSo SSToSSSbAS 900 so ze ATSSASTASA SSASSATSAT SAASSASSASSAS TTOAASAASS ASAASSASsSA STTSSOSSTo 960 s - s. and? (o) (ee) (95) sh» sl ime) 60 b5

ААСАТССТОС СОАСССТОАС СААСАССТИС АССААССССА АСТАСОССАА СОТОААвооС 1020

MOSSASOASO ASSSSALSAT SATOSTSSAS SSTALOSOSO OSSASOSOTT SATOSOSTTS 1080

SASATSASSA ASSASSASSAT SASOSTOSTO AAOSTSTASO ASOSSAAOST SAABSASAAS 1140

TASSASSTOS ASAASOASAAb STTSASSODO STOATSTASO OSSASATOSA SAASSSTOSTO 1200

TSTSSSSSASS ASASSOSSSSSA AATSTASTAS ASSAAASAASA TOSTSOTTOSS SAASSASTAS 1250 /0 STOMTSMOSA ASATOSASTT SASSMASAVS MTSAASASOS TOSOSTASSA SOTSASSSVSS 1320

AASTTISTASO ASASSASSAS SOSSSASSATS SASSTOAASA ASAASALOST SASASSAOS 1380

SMOSSSSAST ASSOSASOST SAOSOSSSAAS SASSASOSSO TSTASATOSS ASTOSosSsto 1440

АТСАССОАСА ССТТССТОАС ССОАТСААС ООСТТГОЗССС ТОСЛОБОССА СОАСААСССС 1500 19 svostedtsya SOSTOASSTO TAASAOSTAS STOSOSOMOS TOSTOSTAYUS SASOSASSTO 1560

АССААСАСО АСАССААОСТ SATСОТОССА ССОАБОСОСТ TSATSСАССДА SATOSTOSAS 1620

AASOOSAVSA TOSASOASSA SAASSTOSASAS SOSTOSAVOU SSAASAASAAA SAASOSSSTAS 1680 stosASSASA SSOBSSOSOT SAASOOSASS AASOSOSTOT ASOTOSASAAA OZASSoSoss 01740

MTSMOSSVOT TSATSOOSSA SALOSTOAAS SSSAVSASOS, VSTASOTSAT SSASTASSASS 1800, ZTOAASOOSA AOSSATSSAT TOLOSTSAMS SASOMSAASA SSFOSTLSAT SSASTACEAS 1860, p

ОБ , ЗАСАМССАМССМ ВСААССТСОССОАСТССССССАССАСТСМСА ДОСОСТСССССССССССАССС 1920 o

САССТСВАСО ОССТОТАСТ САССТСТСАМС АСССАСАЛСО ОССАСОЛОСС СТобОСССАЭ 1980

AASTISATSA TSSTOSASAT SASSSSSSASS SASLASSTOS TOAOSSSOSA SSTSATSAAS 2040

АССААСАСТ ОЗАССАССАС СОССАССАСС ААСАСАСССС ОСААСАСОСТ САСOSTОТАС 2100 that)

I sovvsooss SSOSSATSTSST SAAOSASAAS STOSASSSTOSS ASASSTTTSAS SASSTASSOS 2160 in

СТОТАСТТСА осотОоАОосСоо СОАСОСсСААС сТОоСОСАТОС осАвстоссо ссАстостосте 2220 o

TTSSASAASA ASSASALSTA S 2241 so (Se) « - s ;» (e)) (ee) (95) iz sl (F) ko vo 65

Fig. 1 1 20 .

ZazrROHOERAAAZTMUZNIRR MUSAUKAVEK UM at 40 6o

MUVOMRETEIABBI KaSMROESPRONSNAI KEROESA 80 100 7 SUMOVSKSOMEI EUKORUZMEERAPKOI.M5IEAEO 120 140

AAKONSAMNUSrRAMSNAINMTosASURISOSKSTM - 160 z» KIBABOLM5ORAEBI.ATSMRINSOEOSNAMaNEESU o 180 200

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Claims (10)

7 Formula of the invention 1. A receptor for a vegetative insecticidal protein of the MIRZ class, which is encoded by the DNA sequence presented in ZEO IO MO: 8 or by the DNA sequence that hybridizes with it under strict conditions. bo 2. The receptor according to claim 1, which includes a death domain and an epidermal growth factor repeat motif (ESB). 3. The receptor according to point 1, which has the sequence presented in ZEO IU MO: 9. 4. The DNA sequence presented in 5EO IJOO MO: 8, or the DNA sequence that hybridizes with it under strict conditions, which encodes a receptor for the MIRZ-class protein. 5. Antibody to the receptor according to point 1. 6. The method of identification of homologs of the receptor for the MIRZ-class protein through the DNA sequence that encodes the receptor for the MIRZ-class protein, which includes: (a) hybridization of the DNA sequence according to item 4 with DNA obtained from the organism under study, detection of 70 hybridization with DNA from the specified organism, and the selection of the gene that hybridizes from the specified organism; or (b) obtaining a DNA sample from an organism using primers to a DNA sequence according to item 4, obtaining a reaction product and then isolating a DNA sequence encoding a receptor for a MIRZ-class protein from the specified organism. 7. The method of identifying a compound as a chemical ligand of the MIRZ receptor, which has pesticidal activity, which 7/5 Includes subjecting the MIRZ receptor according to point 1 to the action of the test compound and analyzing the interaction between the receptor and the test compound. 8. The method according to item 7, in which the MIRZ receptor is expressed in cells and the analyzed interaction is programmed cell death. 9. The method according to item 7, in which the analyzed interaction is specific binding between the MIRZ receptor and the test compound. 10. A method of combating insects by contacting insects with an insecticidal amount of a chemical ligand obtained by the method according to item 7, or an antibody according to item 5. Official Bulletin "Intellectual Property". Book 1 "Inventions, useful models, topographies of integrated circuits about Microcircuits", 2004, M 8, 15.08.2004. State Department of Intellectual Property of the Ministry of Education and Science of Ukraine. i) IS) « (ze) (ee) (Se) - with . and? (o) (ee) (95) sh» sl ime) 60 b5