UA82485C2 - Insecticide protein toxins from photorhabdus - Google Patents

Abstract

Proteins from the genus Photorhabdus are toxic to insects upon exposure. Photorhabdus Iwmnescens (formerly Xenorhabdus lununescens) have been found in mammalian clinical samples and as a bacterial symbiont of cntomopathogenic nematodes of genus Heterorhabditis. These protein toxins can be applied to, or genetically engineered into, insect larvae food and plants for insect control.

Description

Description of the invention

This invention relates to toxins isolated from bacteria and to the use of these toxins as insecticides.

Many insects are widely regarded as pests by homeowners, picnickers, gardeners and farmers, and others whose investment in agricultural products is often lost or diminished by insect damage to field crops. In particular, in areas where the growing season is short, significant insect damage can mean the loss of all profits for farmers and a severe reduction in yields. Short supply of specific agricultural products inevitably leads to higher prices for food processors and then for end consumers of food plants and products derived from these plants.

Reversal of insect damage to agricultural crops and flowers Eliminating the problems associated with insect pests is usually based on the use of strong pesticides and insecticides with broad spectrums of toxicity. These synthetic products face a sharp rejection of the entire population as too cruel in relation to the environment and the objects that are exposed to the action of such agents. Thus, outside of agriculture, householders would be content to keep insects away from their homes and picnic areas, without exterminating these insects.

The intensive use of chemical insecticides has raised environmental and health concerns among farmers, insecticide companies, government agencies, public interest groups, and the general public. The development of less stringent pest control strategies has been stimulated by public concern about the environment and the development of biological agents that use biological mechanisms to control insects. Biological protection means are a promising alternative to chemical insecticides.

Organisms at every level of evolutionary development have developed means to enhance their own progress and survival. The use of biological molecules as tools of defense and aggression is known in the animal kingdom and in the plant world. In addition, the relatively new tools of genetic engineering allow the modification of biological insecticides to implement specific solutions to specific problems.

One such agent, Bacillus (Pygypsis (BO), is an effective insecticidal agent and, as such, is widely used commercially. In fact, the Bacillus insecticidal agent is a protein that has such limited toxicity that it can be used on human food crops in harvest day. (Hu

For non-target organisms, Vi toxin is a non-toxic, easily digestible protein.

Another known class of biological insect control agents is some genera of nematodes, which are known to be vectors for the transfer of insect-killing bacterial symbionts. Nematodes containing insecticidal bacteria inhabit insect larvae. These bacteria then kill the larvae. Nematodes multiply in the killed larvae.

Nematode offspring eat the killed larvae from the inside. The progeny of nematodes obtained in this way, containing co-bacteria, can then inhabit other larvae.

In the past, insecticidal nematodes in the genera Zieipegpeta and Neyegogparai have been used as insect control agents. Probably, each genus of nematodes is a feeder for a specific type of bacteria. In nematodes of the genus "

A negative symbiotic bacterium is Rpoiyugpavraz Ishtipezsepv. 70 Although these nematodes are effective insect control agents, obtaining, maintaining, and disseminating nematodes for insect control is currently difficult, expensive, and ineffective. It was known in this field that an insecticidal toxin can be isolated from RPIioiogpardis Iptipizzepe, which is active only when injected into the larvae of lepidoptera and antoptera insects. This made it impossible to effectively use the insecticidal properties of these nematodes or their bacterial symbiont. A more practical, less time-consuming, large-scale method of delivering an insecticidal toxin that would retain its biological properties after delivery would be useful. The discovery of toxins with co-oral activity produced by the genus Ripoiogpardivz would be highly desirable. Isolation and use of these toxins is desirable due to their effectiveness. Prior to applicants' discoveries, such toxins had not been isolated or characterized. because 20 Native toxins are protein complexes that are produced and secreted by bacterial cells of the genus

Squirrels that grow and attract attention are the proteins that are produced by the species Riogiogpardyz Ishtipezsepv. Protein complexes with a molecular size of approximately 1000 kDa can be divided into numerous component proteins by analysis using electrophoresis in PAAG-DSN. These toxins do not contain hemolysin, lipase, phospholipase C, or nuclease activities. They show significant toxicity when introduced to a number of insects. 25 This invention provides an insecticidal protein that can be easily administered, as well as expression of the toxin in a heterologous system.

This invention also provides a method of delivering insecticidal toxins that are functionally active and effective against a wide range of insects.

The aims, advantages and features of the present invention will become apparent from the following description. 60 Brief description of drawings

Fig. 1 is an illustration of the coincidence of cloned DNA isolates used as part of the gene sequence for the toxin of the present invention.

Fig. 2 - a map of three plasmids used in the sequencing process.

Fig. 3 is a map illustrating the relationship of several partial fragments of DNA. because Fig. 4 is an illustration of homology analysis between protein sequences of TerA proteins; and TsavV;.

Fig. 5 - phenogram of strains of RPIoiogpardy. The relatedness of the strains of RIioiogpardis was determined using her-"SK. The upper part of Fig. 5 measures the percent similarity of the strains based on the scoring of the her-RSK products (ie, 0.0 (no similarity) - 1.0 (10095 similarity)). On the right the axis numbers and letters represent the different strains that were tested: 14-UU-14, Nt-Nt, NO-NO, 7-UuUHx-7, 1-MUH-1, 2-Uuh-2, 88-НРВ8В, МО- 1-MS-1, 4-UUH-4, 9-MUH-9, 8-MUH-8, 10-UUH-10, MMIK-MUMIC, 3-MUH-3, 11-MUH-II, 5-MUH- 5, 6-МУХ-6, 12-МУХ-12, хи4-УуУХх-14, 15-МУХ-15, НЭ-НЭ, 82-82, 48-52-АТСС43948-АТСС43952. Vertical lines dividing horizontal lines, indicate the degree of relatedness (as read from the intersection of the extrapolated vertical line with the upper axis) between strains or groups of strains at the base of the horizontal lines (eg strain MU-14 at about 60905 is 70 similar to strain HO and Ht).

Fig. 6 - illustration of genomic maps of strain MU-14.

This invention is directed to the discovery of a unique class of insecticidal protein toxins from the species

Pesticides with oral toxicity to insects. A unique feature of Rpoiogpardis is its bioluminescence. Rypoyugpardiz can be isolated from a variety of sources. One of these sources is nematodes, more specifically - nematodes of the Negyegogpabaiyiz species. Another similar source is isolated from clinical samples from human wounds, see Rapteg ei ai., 1989, 9. Siip. MisgobioI., 27 years, 1594-1600). These saprophytic strains were deposited in Ategisap

Toure Siyige SoPesiop (CoskmShe, MO) ATSS MoMo43948, 43949, 43950, 43951 and 43952)|) and incorporated herein by reference. It is possible that other sources could capture the bacteria Ripoiugparadives, which produce insecticidal toxins. Such sources in the environment could be either living in or on the ground, or aquatic.

The genus Rpoyugpavrdiv is taxonomically defined as a member of the family Epiegobasieegiaceae, although it has some characters atypical for this family. For example, strains of this species do not reduce nitrates, produce yellow and red pigment and have a feature such as bioluminescence. This last trait is not known to other members of the family

Epiegorasiegiaceae. RIPOiogpardiz was only recently described as a species distinct from Hepogparatz

Ivoetage ei ai., 1993, Ipi U. Buzi. Vasiegioi., 43, 249-255). This differentiation is based on DNA-DNA hybridization studies, phenotypic differences (e.g., presence (Rpoiogpardis) or absence (Hepogpardis) of catalase and bioluminescence) and nematode host family (Hepogparatsv; 5ieipegpetaisae, (8)

Rpoiyugpavradiv; Negyegogparaiidae). Comparative analyzes of fatty acids in cells | (Chapze ei ai., 1990, I ei.

Arri. Misgobioii., 10, 131-135; BylyKki ei aiYi., 1990, 9. Sep. Arri. MisgorioI., Collection, 393-401| confirm the differentiation of Rpoyugpavadiz from Hepogpavrashv. In order to establish that the collection of strains described here consists of strains of Ripoiogpardys, these strains were characterized on the basis of recognizable characters that define Ripioigpardys and distinguish it from other Epiehoraceae and from species of Hepogpardys (Rapgteg, 1984, Vegdeuz Mapia! oi buvieetis Vasiegioiodu, "

Mo|.1, year 510-511; AKpigwi apa Voetage, 1988, 9). Sept. Misgobio!Y., 134, 1835-1845; Voetage ei ai., 1993,

Yippee 9. Buvi. VasiegioI., 43, pp. 249-255), which are included here as a reference). The following characters were investigated: gram-negative rods, body size, colony pigmentation, inclusion bodies, presence of catalase, ability to reduce nitrates, bioluminescence, growth on selective media, growth temperature, survival under anaerobic conditions, and motility. Fatty acid analysis was used to confirm that all these strains belong to the same genus Rpoiogpavrashva.

Currently, the bacterial genus Ryoigpardyz consists of a single identified species, Ryoigpardyz Istipezsepz "

ATSS Ture zigaip Mo29999, Roipag eyi ai., 1977, Metaioiodisa, 23, 97-102). Numerous related strains have been described in the literature (for example, AKpig8i ei ai.,, 1988, 9. dep. Misgobioii., 134, 1835-1845; Voetage ei ai., 1993, Ipi 9. Buvi. Vasiegioi., 43 , pp. 249-255; Ryi; ei aiYi.,, 1990, Arri. Eepmigop. Misgobryo!., 56, 181-186). with Numerous strains of Ripoyugpardys have been described here. These strains are listed in Table 18 in the Examples. Since there is currently only one species (Ishtipezsep5v) identified in the genus RIoiogpardyz, the characters of the Ishtipezsepz species were used to characterize the strains described here. It cannot but be foreseen that in the future other species of Rpoiogparadis may be discovered, which will have some characteristic features of the Ishtipezsepv species, as well as some distinctive properties that are not currently identified as a feature of Rpoyugparadis Ishtipezsepv. However, the scope of the present invention includes any species or strains of Ppoiogpardis that produce proteins that have their functional activity as insect control agents, regardless of other features and distinctive properties.

In addition, as shown herein, bacteria of the genus RIogpardis produce proteins that have the functional co-activity defined herein. Of particular interest are the proteins produced by the species RIPoiogpardis sl Iyutipezsepv. This invention should not be limited to the strains described herein. These strains illustrate for the first time that the proteins produced by various isolates of Riojopardys are toxic when treated with insects. Thus, the strains described herein and any mutants thereof, as well as any two strains or species of the genus Rpoiogpardis having the functional activity described herein, are included in the invention described herein.

Several terms are used here that have a specific meaning:

GF) By "functional activity" is meant here that the protein toxins act as insect control agents, in the sense that they are active orally or exhibit toxic effects or are capable of disrupting or impairing nutrition, which may or may not cause the death of the insect . When an insect comes into contact with an effective amount of a toxin delivered through the expression of a transgenic plant, via prepared protein compositions, sprayable protein compositions, a bait matrix, or other delivery system, the results are usually death of the insect, or the insects do not eat the source , which makes these toxins available to insects.

The term "Ripiogpardyv toxin" means any protein produced by a strain of the organism Rpiogipardivs 65 that has functional activity against insects, and the Ripiogpardiv toxin can be prepared as a spray composition, expressed in a transgenic plant, prepared in the form of a matrix baits, be delivered by baculovirus or by any other acceptable host or any other delivery system.

The protein toxins discussed here are generally referred to as "Insecticides". By insecticides, it is understood that these protein toxins have the "functional activity" previously defined herein, and are used as insect control agents.

The term "oligonucleotides" refers to a macromolecule consisting of a short chain of nucleotides or RNA or DNA. This length can be at least one nucleotide, but typically the length is in the range of about 10 to about 12 nucleotides. Determining the length of an oligonucleotide is 70 well known to those skilled in the art and is not a limitation of the present invention. Thus, oligonucleotides can have less than 10 or more than 12 nucleotides.

The term "toxic" or "toxicity" as used herein means that toxins produced

Ripoyugpavadyzv, have the "functional activity" defined here.

The term "genetic material" as used herein refers to genes, nucleic acid, DNA or RNA.

Fermentation broths from the selected strains, which are described in Table 18, were used to determine: the extent of production of insecticidal toxins by the species P. iologpardivs, the insecticidal spectrum of these toxins, and to provide starting material for the purification of toxin complexes. The strains characterized here have been shown to have oral toxicity to numerous insect orders. Such insect orders include (but are not limited to) SoiIeoriega, Notoriega, Ieridoriega, Oiriega, Asagipa, Nuteporiega, and Oisiuoriega.

As with other bacterial toxins, the rate at which bacteria mutate in a population results in the production of many related toxins that differ slightly in sequence. The toxins described herein are those toxins that form protein complexes that are toxic to various insects exposed to them as described herein. More preferably, these toxins are active against Ieridoriega, SoiIeoriega, Notoriega, BOiriega, s Nuteporiega, Risiuoriega and Asagip. This invention relates to the production of protein toxins homologous to the protein toxins produced by the strains described herein and any strains derived from them, as well as any protein toxins produced by P. These homologous proteins may differ in their sequence, but they do not differ in function from the toxins described here. Homologous toxins are understood to include protein complexes between 300kDa and 2000kDa and consisting of at least two (2) subunits, wherein the subunit is a peptide that may be the same as or different from the second subunit. A variety of protein subunits have been identified and listed in the Examples of the present invention. Typically, α subunits of the protein are between about 18 kDa and about 23 kDa in size; between about 160kDa and about "U 23OkDa; 100 kDa - 160 kDa; approximately 8OkDa - approximately 100kDa; and approximately 5OkDa - approximately 8OkDa.

As discussed above, some strains of Riogiopardis can be isolated from nematodes. Some nematodes, elongated cylindrical parasitic worms of the Metaioada type, have evolved the ability to use insect larvae as a more acceptable medium for growth. The insect larvae provide a food source for the growing nematodes and an environment where they reproduce. A dramatic effect after larval infestation by some nematodes is the death of the larvae. The death of larvae is due to the presence (in some nematodes) of bacteria that produce an insecticidal toxin, which stops the growth of larvae and inhibits feeding activity. "

Interestingly, each genus of insect-parasitic nematode appears to host a specific type of bacterium that is uniquely adapted for symbiotic growth with that nematode. Since the beginning of this study, the name of the bacterial genus Hepogpardys has been changed to Hepogpardys and Rioiogparaivs. Bacteria of the genus Rioiogpardys have been characterized as symbionts of the nematode Negegopabys, while species of Hepogpardys are symbionts of Bieipegpeta species. This modification in nomenclature is reflected in this application, but the modification in nomenclature in no way changes the scope of the invention described herein. The peptides and genes described here are named according to the guidelines recently published in Moigpa! oi Vasiegioiodu "Ipvigisiope (about Ashoga", r.i-hi (Chap. 1996)), which are incorporated herein by reference. The following peptides and genes were isolated from strain MU-14 Rpoyugparaiv. their Nomenclature of peptides/genes of the toxin complex (Tc) from

ZES PO Moshcha Genomny district 111 o yu

Genomic rat 1 in

Geyuomnyi rai, 1 bo TssA YissA 8

Genomic Rat 1; (the sequence in parentheses indicates the internal amino acid sequence, which was obtained on the basis of trypsin cleavage products 70)

The above sequences are grouped based on genomic region. The IsBA gene was expressed in E. soybean in the form of two protein fragments TerA and TeorA, as shown in the Examples. It may be advantageous to have proteolytic cleavage of some sequences to produce higher activity toxins for commercial transgenic applications.

The toxins described here are quite unique in that these toxins have a functional activity that is key to the development of insect control strategies. In the development of an insect control strategy, it is possible to slow down or bypass the process of protein degradation by injecting the protein directly into the body, bypassing the digestive tract. In this case, the protein that is introduced into the body will retain its function until it is denatured, nonspecifically destroyed, or eliminated by the immune system in higher organisms. Injecting insects with an insecticidal toxin has potential application only in the laboratory and, moreover, only in the case of large insects that are easy to inject. The observation that the insecticidal protein toxins described here exhibit their toxic activity after oral ingestion or contact with the toxins allows the development of an insect control strategy based solely on the possibility of incorporating these protein toxins into the insect diet. Such a strategy leads to the creation of insect lures. everything

Toxins of RIPOiogpardis can be administered to insects in a purified form. Toxins can also be supplied in amounts from about 1 to about 10 ug/L of broth. The amount may vary depending on the conditions of preparation, conditions of the inoculum source, methods of toxin release, etc. Toxins can be administered in the form of an exudate secretion protein or a cellular protein that was originally expressed in a heterologous prokaryotic or eukaryotic host. The hosts in which proteins are expressed are, as a rule, bacteria. Eukaryotic Io) hosts could include (but are not limited to) plants, insects, and yeast. Alternatively, these toxins can be produced in bacteria or transgenic plants in the field or in insects using a baculovirus vector. Typically, these toxins are administered to insects by incorporating one or more toxins into the insect's food. ch.i

Complete lethality to feeding insects is applicable but not required to achieve useful toxicity. If insects avoid the toxin or stop feeding, this avoidance of food will be beneficial in some applications, even if these effects are sublethal. For example, if insect-resistant transgenic crops are desired, reluctance of insects to feed on those plants is as valuable as lethal toxicity to those insects, since the ultimate goal is to protect the plants, not to kill the insect.

There are many other ways in which toxins can be introduced into the food of insects. For example, it is possible to tamper with the larvae's food source with a toxic protein by spraying the food with a solution of the protein, as described here. Alternatively, the purified protein could be genetically engineered into an otherwise harmless bacterium, which could then be grown in culture and either applied to a food source or introduced into the soil in an area where insect control is desired. It would also be possible to introduce the protein obtained by genetic engineering directly into the insect's food source. For example, the main source of food for many insect larvae is plant material.

As a result of the inclusion of genetic material, which encodes the insecticidal properties of Rpoiogpavratsv toxins, to

Go | genome of a plant that is eaten by a particular insect pest, the adult insect or larvae die after feeding on this plant. Numerous members of monocotyledonous and dicotyledonous genera have been transformed. Transgenic agronomic crops, as well as fruits and vegetables, are of commercial interest. Such crops include (but are not limited to) corn, rice, soybeans, sapoia, sunflower, alfalfa, sorghum, wheat, cotton, peanuts, tomatoes, potatoes, and the like. There are several ways to introduce foreign genetic material into plant cells and to obtain plants that stably retain and express the introduced gene. Such methods include the acceleration of genetic material, which is applied to microparticles, for introduction directly into cells (0.5. Rayepiz 4945050 IU SogpeiYi and 5141131 IU Oom/ EIapso). Plants can be transformed according to the technology using Adgobasiegisht, see O.5. Raiepi 5177010 yu Mpimegeyu oi Toiedo, 5104310 (0 Texas A apa M, Eigoreap Raiepi Arriisayop 013162481, Epgoreap Raiepi

Arriisaiope 120516, 15941881 and 176112 in Zspyregooi, 0.5. Districts 5149645, 5469976, 5464763 and 4940838 and

Ф) 4693976 yuyu Zspiriegooi, Eygoreap Raiepi Arriisayopev 116718, 290799, 320500 yu MakhRiapsk, Eygoreap Raiepi ko Arriisayope 604662 apa 627752 o darap Torasso, Eygoreap apa Raiepi Arriisayope 02671524 apa 5, 029.

Raiepi 5231019 (about Sira Seyidu, O.5. Raiepiv 5463174 and 4762785 Yuyu SaYidepe and ).5. Rayepiv 5004863 and 5159135 Yuyu 60o Adgaseishv|. Other transformation technologies include "mpizKegv" technology, see O.5. Rayepiv 5302523 and 5464765 (0 7epesa). Electroporation technology has also been used for plant transformation, (see UMO 87/06614 by Vouse Tpotrzop Ipviyshe, 5472869 and 5384253 by OekKkaev, UMO 9209696 and MO 9321335 by ROZI. All of these transformation patents and publications are incorporated by reference. In addition to numerous transformation technologies plants, it is possible to vary the type of tissue brought into contact with foreign genes. 65 Such tissue may include (but is not limited to) embryogenic tissue, callus tissue of types I! and II, hypocotyl, meristem, etc. Almost all plant tissue can be transformed during dedifferentiation using appropriate methods known to those skilled in the art.

Another variable is the choice of the marker that selects it. A more suitable specific marker is determined by the discretion of the skilled artisan, but any of the following selectable markers can be used, along with any other gene not named here that could function as a selectable marker. Such selectable markers include (but are not limited to) the Tp5 transposon aminoglycosylphosphotransferase (Ari II) gene, which encodes resistance to the antibiotics kanamycin, neomycin, and 5418, as well as genes encoding resistance or tolerance to glyphosate, hygromycin, methotrexate, phosphinothricin (bialofos), imidazolinones, sulfonylureas and triazopyrimidine herbicides, such as chlorsulfone, bromoxynil, /o dalapon, etc.

In addition to the marker that is being selected, it may be desirable to use a reporter gene. In some cases, a reporter gene can be used without a selectable marker. Reporter genes are genes that are not normally present or expressed in the recipient organism or tissue. A reporter gene often encodes a protein that confers some phenotypic change or enzymatic property. Examples of such genes /5 are given in |K. UMeyvipu ei ai., App. Kew. Sepoys, 22, 421 (1988)), which is incorporated herein by reference. A more acceptable reporter gene is the glucuronidase gene (5І5).

Regardless of the method of transformation, the gene is more preferably included in the gene transfer vector, which is suitable for the expression of Ppoiogpardiv toxins in a plant cell by including a plant promoter in this vector. In addition to plant promoters, promoters from various sources can be effectively used in plant cells to express foreign genes. For example, you can use promoters of bacterial origin, such as octopine synthase promoter, nopaline synthase promoter, mannopine synthase promoter; promoters of viral origin, such as the tobacco mosaic virus promoter (355 and 195), etc. Plant promoters include (but are not limited to) ribulose-1,6-bisphosphate (RBF) carboxylase small subunit (MS) promoter, beta-conglycinin promoter, phaseolin promoter, AN promoter, heat shock promoters, and tissue-specific promoters. Promoters may also contain some enhancer sequence elements that can improve the efficiency of transcription. Typical enhancers include (but (8) are not limited to) Aap intron | and Aap intron 6. Constitutive promoters can also be used.

Constitutive promoters regulate continuous gene expression in all cell types and at all times (eg, actin, ubiquitin, CaMmM 355). Tissue-specific promoters are responsible for gene expression in plant-specific cell types or tissues, such as leaves or seeds (eg, zein, oleosin, napin, ACP), and these promoters can also be used. It is also possible to use promoters that are active during a certain stage of plant development, as well as active in plant tissues and their organs. Examples of such promoters include (but are not limited to) pollen-specific, embryo-specific, corn silk-specific, cotton fiber-specific, root-specific, endosperm-specific, seed-specific promoters, etc. co

In certain circumstances, it may be desirable to use an inducible promoter. The induced promoter is responsible for the expression of genes in response to a specific signal, such as: a physical factor (heat shock genes); light (RBF-carboxylase); hormone (Et); metabolites and stress. Other preferred transcription and broadcast elements may be used. Numerous plant-specific gene transfer vectors are known in the art. "

In addition, it is known that in order to obtain high expression of bacterial genes in plants, it is better to rearrange bacterial genes from c in such a way that they are more efficiently expressed in the cytoplasm of plants. Maize is one such plant for which it is more appropriate to reengineer the bacterial gene(s) prior to transformation to increase the expression level of the toxin in that plant. One of the reasons for this rearrangement of genetic engineering is the low content (33-35 of native bacterial gene(s)) (and subsequent deviation towards high

In the content of JSC). This causes the formation of sequences that mimic or duplicate the regulatory sequences of plant co genes, which are known to be very rich in A-T. The presence of some A&T-rich sequences in the DNA of the gene(s) introduced into plants (for example, TATA-box regions normally present in gene promoters) that may cause aberrant transcription of the gene(s). On the other hand, the presence of other regulatory sequences present in the transcribed mRNA (for example, polyadenylation signal sequences "AAyAAAH" or sequences that are complementary to small nuclear RNAs involved in co-pre-mRNA splicing) can cause RNA instability. Thus, one of the goals in the construction of the bacterial sl gene (genes) that it (them) rearranges, and which, more appropriately, is called a plant-optimized gene (genes), is the formation of a DNA sequence that has a high content of Zh-C, and, more preferably, a sequence close to the sequence of plant genes that encode metabolic enzymes. Another goal of constructing plant-optimized gene(s) is to produce a DNA sequence that not only has a higher

SS, but it must be produced by modifying the sequence changes in such a way as not to interfere

GF) broadcasts.

Ф An example of a plant with a high C3-C content is corn. The table below illustrates how high the C content is in corn. It is believed that, like corn, the --C content in other plants is also high. 60 protein-coding regions of maize genes 65 Spare proteins a The number of genes in the class is given in parentheses.

For the data in Table 1, the coding regions of the genes were selected from SepWapk (Keyase 71), and the base composition was calculated using the MasMesiogtm program (IVI, MemNamep, ST). Nitron sequences were not taken into account in these 75 calculations. Gene sequences of reserve proteins of groups I and II were distinguished by a noticeable difference in the composition of the bases.

Due to the flexibility provided by the redundancy of the genetic code (that is, the fact that some amino acids are encoded by more than one codon), the evolution of the genomes of different organisms or classes of organisms has resulted in differential use of redundant codons. This taxonomic "codon shift" is reflected in the average base composition of protein-coding regions. For example, organisms with a relatively low c-C content use codons with A or T in the third position of redundant codons, while codons with a high content of C-C use codons with a C or C in the third position.

It is assumed that the presence of "minor" codons in the mRNA of a gene can reduce the absolute rate of translation of this mRNA, especially when the relative abundance of charged tRNAs corresponding to the minor codon is low. A corollary of this is that the reduction in translation rate by individual minor codons would be at least additive for multiple minor codons. Thus, mRNAs with a high relative content of minor codons will have correspondingly lower translation rates. This rate is reflected in i) the synthesis of low levels of the encoded protein.

To rearrange the bacterial gene(s), the codon shift of the plant is determined. Codon bias is the statistical distribution of codons that a plant uses to code for its proteins. Having determined this yuyu

Displacement, find the frequency of these codons in the gene(s) of interest. It is necessary to determine the primary, more acceptable codons for the plant, as well as the second and third choices of more acceptable codons. The amino acid sequence of the protein of interest is reverse translated such that the resulting nucleic acid sequence encodes the same protein as the native bacterial gene, but this resulting nucleic acid sequence corresponds to the first more acceptable codons of the target plant. A new sequence se

335 is analyzed for restriction enzyme sites that may be created by this modification. Identified co sites are further modified by replacing codons with more acceptable second and third choice codons. Other sites in this sequence that may affect transcription or translation of the target gene are the 5' splice sites or

From exon: intron, poly A joining signals or RNA polymerase termination signals. This sequence is further analyzed and modified to reduce the banding frequency of TA or CC doublets. In addition to these doublets, blocks of sequences with C or C having more than about 4 residues can affect the transcription of this w-in c sequence. Therefore, these blocks also modify by replacing codons of the first or second choice, etc., with the next and more acceptable codon of choice. More preferably, the plant-optimized gene(s) contain "" about 6395 first-choice codons, about 2295-about 3795 second-choice codons, and 15905-090 third-choice codons, with a total percentage of 10095. The most preferred plant-optimized gene (genes) contains about 6395 first choice codons, at least about 2295 second choice codons,

Go! about 7,595 third-choice codons and about 7,595 fourth-choice codons, with a total percentage of 10095. The method described above enables one skilled in the art to modify gene(s) foreign to a particular plant such that these genes are optimally expressed in plants The method is further illustrated in a pending prior application (0.5. 60/005405 filed October 13, 1995) that

Incorporated here by reference. (22) Thus, to construct a plant-optimized gene(s), the amino acid sequence of the sp toxins is back-translated (translated) into a DNA sequence that uses a non-redundant genetic code that is determined from the codon offset table that is compiled for the DNA sequence of the gene for a particular transformed plant. The resulting DNA sequence, which is completely homogeneous with respect to codon usage, is further modified to produce a DNA sequence that, in addition to having a high degree of codon diversity, also contains strategically located restriction enzyme recognition sites, a desired base composition, and non-(F) containing sequences. which can interfere with the transcription of this gene or the translation of the mRNA product. It is theoretically suggested that bacterial genes can be more easily expressed in plants if these bacterial genes are expressed in plastids. Thus, it is possible to express bacterial genes in plants, because without optimizing these genes for plant expression, and obtain a high level of protein expression, see T.5.

Raiepov Momo4762785, 5451513 and 5545817, which are incorporated herein by reference).

One outcome in terms of commercial use of transgenic plants is resistance management. This is especially true of Vasiyiv toxins (pPygypdiaepsis. There are numerous companies commercially

Vasiyiv (pygypdiepsis, and there has been considerable concern about Vi toxins becoming resistant. One strategy 65 to manage insect resistance may be to combine toxins produced by Riojugparadiv with toxins such as Vi, insect vegetative proteins (Sira Seidu) or other Toxins Such combinations could be prepared for use by spraying or they could be molecular combinations.

Plants can be transformed with insect toxin-producing Ryoigpardy genes and other insect toxin genes, such as Vi.

IV Eshgoreap Raiepi Arriisayop 0400246A1) described the transformation of 2 VE genes into a plant, and these 2 genes could be any other two genes. Another method of obtaining a transgenic plant containing more than one insect resistance gene could consist of keeping two plants, each of which has an insect resistance gene. These plants could be backcrossed using traditional plant breeding techniques to produce a plant containing more than one insect resistance gene. 70 In addition to obtaining a transformed plant that has a plant-optimized gene(s), there are other delivery systems for cases where rearrangement of the bacterial gene(s) is desired. According to the same type, a genetically engineered easily secreted protein toxin fused together with an insect-attracting molecule as a source of nutrition and insecticidal activity of the toxin can be genetically engineered and expressed in bacteria or eukaryotic cells using standard, well-known methods. 7/5 After purification in the laboratory, such a toxic agent with a built-in "bait" can be packaged in standard insect traps.

Another delivery scheme is the inclusion of the genetic material of the toxins in the baculovirus vector.

Baculoviruses infect specific insect hosts, including those affected by the toxins of Riogiogparatsv.

Infectious baculovirus carrying an expression construct for the toxins of P. spp. could be introduced into the zones

Insect infestation for intoxication or poisoning of infected insects.

The transfer of insecticidal properties requires that the nucleic acid sequences encoding the amino acid sequences of the Riogiopardys toxins be integrated into a protein expression vector suitable for use in the host in which the vector will reside. One of the methods of obtaining a nucleic acid sequence encoding a protein with insecticidal properties is the isolation of native genetic material that produces the toxin of Pioiogpardis, using the information deciphered from the amino acid sequence of the toxin, the larger parts of which are given below. As described below, (o) methods for purifying proteins responsible for toxin activity are also provided.

Using the data on the M-terminal amino acid sequence, which are given below, it is possible to construct oligonucleotides complementary to all or part of the DNA bases that encode the first amino acids of the yuzo toxin. These oligonucleotides can be radioactively labeled and can be used as molecular probes to isolate genetic material from a genomic genetic library constructed from genetic material that has been isolated from strains of RPI. The genetic library can be cloned in plasmid, cosmid, phage or phagemid vectors. The library can be transformed into EzsPegispia soya and screened for toxin production by transformed cells using antibodies produced against c

With TOXIN, or direct tests for toxicity in relation to insects. co

This approach requires obtaining a set of oligonucleotides because the degenerate genetic code allows one amino acid to be encoded in the DNA of any of several three-nucleotide combinations. For example, the amino acid arginine can be encoded by triplets COA, COS, CO, SOT, ASA, and ASO. Since it is impossible to predict which triplet is used at these positions in the toxin gene, oligonucleotides with each potentially characteristic triplet should be prepared. More than one DNA molecule corresponding to the c subunit of the beam may be required to construct a sufficient number of oligonucleotide probes to detect all protein subunits required for oral toxicity. Based on the amino acid sequence of the purified protein, the genetic material responsible for the production of toxins can be easily isolated and cloned, in whole or in part, into an expression vector with

Using any of several methods well known to those skilled in the art of molecular biology. A typical co-expression vector is a DNA plasmid, although other vectors are also contemplated, including (but not limited to) cosmids, phagemids, and phages. In addition to traits necessary or desirable for plasmid replication, such as replication efficiency and resistance to antibiotics or another form of selectable marker, such as the bug gene Zygeriothus Ppuygossorisys or mygidospgotodepevs, protein expression vectors often additionally require a 5p expression cassette that includes a cis- active sequences necessary for transcription and translation of the target gene. (og) The cis-acting sequences required for expression in prokaryotes differ from similar elements required in eukaryotes and plants.

A eukaryotic expression cassette requires the presence of an upstream transcriptional promoter (5) relative to the target gene, a transcription termination region such as a poly-A attachment site, and an upstream ribosome binding site from the first codon of the target gene. In bacterial cells, a commonly used transcriptional promoter that could be incorporated into a vector is a promoter that binds

GF) RNA polymerase 17. As described above, promoters effectively enhance mRNA transcription. Also against the flow

F transcription (on the left) from the target gene, the vector may have a nucleotide sequence encoding a signal sequence, which is known to direct a covalently bound protein to a specific compartment of the host cell, such as the cell surface.

Insect viruses, or baculoviruses, are known to infect and adversely affect some insects. The effect of these viruses on insects is slow, but the viruses do not stop the insects from feeding. Therefore, viruses are not considered to be useful as insect control agents. The introduction of the genes of the toxins of Rpoyugpardis into a baculovirus vector could become an effective means of transmitting toxins with a simultaneous increase in the lethality of such a virus. Additionally, since different baculoviruses are specific to different insects, a specific toxin can be used to selectively target specific insect pests. A particularly valuable vector for toxin genes is the nuclear polyhedrosis virus. Vector vectors using this virus have been described in the literature and can now be selected as vectors for the transfer of foreign genes in insects. Recombinant virus-toxin gene can be constructed in an orally transmissible form. Baculoviruses often infect

Insects through the mucous membrane of the midgut. The toxin gene, embedded behind the strong promoter of the virus's envelope protein, will be expressed and quickly kill the infected insect.

In addition to the insect virus or baculovirus or transgenic plant delivery system for the protein toxins of the present invention, these proteins can be encapsulated using Vasiyv5 Tpihipdiepov technology, such as (but not limited to) (Sh.5. Raiepov MoMo4695455, 4695462, 4861595), which are included herein as a reference Another 7/0 delivery system for the protein toxins of the present invention is to introduce this protein into a bait matrix, which could then be used in aboveground and belowground test sites. Examples of such technology are provided in (but not limited to) (RZT Raiyepi Arriisayop UUO 93/239981, which is incorporated herein by reference.

As described above, it may be necessary to modify the sequence encoding the target protein when expressing it in a non-native host, since the acceptability of the codons of other hosts may differ from the acceptability of the 7/5 Codons of Rpoijugpardis. In this case, translation may be completely ineffective in the new host unless compensatory modifications are introduced into the coding sequence. In addition, modifications of the amino acid sequence may be desirable to avoid inhibitory cross-reactivity with proteins of the new host, or to improve the insecticidal properties of the protein in the new host. A genetically modified toxin gene may encode a toxin that exhibits, for example, enhanced or attenuated toxicity, altered development of insect resistance, altered stability, or modified specificity for a target species.

In addition to the Ripoiyuogparaiv genes that encode its toxins, the scope of the present invention includes related nucleic acid sequences that encode amino acid biopolymers homologous to the toxin proteins, and which preserve the toxic effect of the Riiogpardis proteins in insect species after oral ingestion.

For example, the toxins used in the present invention are likely to initially inhibit the feeding of larvae of sch ov before death occurs. By manipulating the nucleic acid sequence of the Rpoiogparaiz toxins or its regulatory sequences, genetic engineers, inserting the toxin gene into plants, could modulate its potency or mode of action, for example, to retain feeding-inhibiting activity while eliminating absolute larval toxicity. This change could allow the transformed plant to survive until harvest without causing an unnecessary dramatic impact on the ecosystem due to the destruction of all yu zo Kom ah targets. It is assumed that all such modifications of the gene encoding the toxin or the protein encoded by this gene are within the scope of the present invention. co

Other contemplated nucleic acid modifications include the addition of guide sequences to “direct the toxin to specific parts of the insect larvae to enhance efficacy.

ATSS strains 55397, 43948, 43950, 43951, 43952 were deposited in (Ategisap Toure Siyige SoPesiop, 12301 s

RagkIamup Ogime, Koskmshe, MO 20852 BA). Amino acid sequence and nucleotide sequence data for the native M/U-14 toxin (ATCC 55397) are provided below. Isolation of genomic DNA for toxins from bacterial media is also exemplified here.

Standard techniques and methods of molecular biology are used and described in this application. Additional information can be found in IZatrgookK 3., Egiivssp, ER, apa Mapiaivs, T. (1989), Moiesshiag Siopipd, A « Gabogagu Mapiai, Soia Zrgipu Nagrog Rgezz), which is incorporated herein by reference. The examples use the following abbreviations:

Tris-tris(hydroxymethyl)aminomethane; with DSN sodium dodecyl sulfate;

EDTKU - ethylenediaminetetraacetic acid;

IPTGe- isopropylthio-beta-galactoside; co X-hal-5-bromo-4-chloro-3-indolyl-beta-O-galactoside;

TSAB - cetyltrimethylammonium bromide; ko t.p.O. - thousands of base pairs; their aATP, STR, STR, attTR, I-2'-deoxynucleoside-5-triphosphates of adenine, cytosine, guanine, thymine and inosine, respectively; with ATP: adenosine-5-triphosphate. sl Example 1

Purification of toxin from R. istipezsepz and demonstration of toxicity after oral delivery of purified toxin

The insecticidal toxin of the present invention was purified from the MU-14 strain of R. Ishtipezsepz, ATSS Assezviop Mo55397.

Stock cultures of R. istipezsepz were kept in Petri dishes containing 295 Proteose Peptone MoZ3 (i.e. RRZ, Oso (F) I arogayugiez, Oeigoii MI) in 1.595 agar, incubated at 252C and recultured weekly. Colonies of the primary form of these t bacteria were inoculated in 200 ml of RRZ broth supplemented with 0.595 polyoxyethylene sorbitan monostearate (Twin 60, Zidt Spetis! Sotrapu, 51. I otsiv, MO), in a one-liter flask. Broth cultures were grown for 72 hours at 302C on a rotary shaker. Toxin proteins were extracted from cultures grown in the presence or absence of Tween; however, the absence of Tween can affect the shape of the bacteria that grow it and the profile of the proteins produced by the bacteria. In the absence of Tween, a variant shift may occur, as the molecular mass of at least one identified toxin is shifted from about 200kDa to about 185kDa. 65 72-hour cultures were centrifuged at 10,000 x 9 for 30 minutes to remove cells and cell remnants (debris). The fraction of the supernatant containing insecticidal activity was drained and adjusted to 50 MM

KoNRO, by adding a suitable volume of 1.0M K.NRO). The pH was adjusted to 8.6 by adding potassium hydroxide. Then this fraction of the supernatant was mixed with OEAE-Zerpaseya! (Rpagtasia I KV Vioiespoiodu), which was balanced by 50MM KoNRO,. Toxic activity was adsorbed on OEAE. Then this mixture was poured into a 2.6 x 400 cm column and washed with 50 mm of CoHRO at room temperature at a flow rate of 34 Oml/h. until the effluent reached a constant baseline UV absorbance at 280 nm. The column was then washed with 150 mM KS until the effluent again reached a constant baseline at 280 nm. Finally, the column was washed with 30 mM KCl and fractions were collected.

Fractions containing the toxin were pooled and sterilized by passing through a sterilizing filter, which was a 7/0 membrane filter with 0.2 micron pores. Then the toxin was concentrated and equilibrated to 100 mm KRO,, рНб,ЗО, using an ultrafiltration membrane with a molecular weight cut-off of 1О0kDa at 4 es (Sepigurger 100, Artichoke Oimiviop-MU.K., Sgase apa Sotrapu). A sample (Zml) of the toxin concentrate was applied to the upper part of a gel filtration column (Rpagtasia KV Vioyesppoiisdu). The elution buffer was 100MM

KRO, pHb, 9, flow rate 17 ml/hour, at 42C. The effluent was monitored at 28O0nm.

Fractions were collected and tested for toxic activity. The toxicity of the chromatographic fractions was tested in a biological test using larvae of Mapdis zechia. Fractions were either applied to insect food (wheat germ food for the unpaired silkworm, ISM Viospetis! Oimiviop - ISM Viotedisai!5, Ips.), or injected by intrahemocellular injection of 5 μl of the sample through the first false leg using a 30-gauge needle.

The weight of each larva in the toxin-treated group was recorded at 24-hour intervals. Toxicity was assumed if the insect stopped feeding and died within a few days of eating the treated insect food, or if death occurred within 24 hours of fraction injection.

Toxic fractions were pooled and concentrated using Sepigurger-100 and then analyzed by HPLC using a gel filtration column of 7.5 mM hboOsm T5K-ZEI (-4000 5UU with 100 mM potassium phosphate, pHb.9, elution buffer flow rate 0, 4ml/min This analysis revealed that biloctoxin C was contained in a single sharp peak that eluted from the column with a retention time of approximately 33.6 minutes.

This retention time corresponds to a molecular weight of 100OkDa that was estimated. Peak fractions were collected for further purification, and fractions that did not contain this protein were discarded. The peak eluted from the HPLC column absorbs UV light at 218 and 280 nm, but does not absorb light at 405 nm. It was shown that absorption at 405 nm is a sign of xenorhabdin antibiotic compounds. IS)

Electrophoresis of combined peak fractions in non-denaturing agarose gel (Mebarpog Adagose, EMO

Viorgodisiv) showed that there are two protein complexes in the peak. Peak material that was buffered in 50MM oo

Tris-HCl, pH 7.0, was separated on a 1.595 agarose concentrating gel buffered with 100 mM Tris-HCl at pH 7.0 and a 1.995 agarose separating gel buffered with 200 mM Tris-borate at pH 3.0 under standard conditions buffer conditions (anodic buffer 1M Tris-HCIII, pH 8.3; cathodic buffer 0.025M Tris, 0.192M glycine). with

Electrophoresis was performed at 13mMA direct current at 15923 until the control dye, phenol red, reached the end of the gel. Two protein bands were visualized in agarose gels with Coomassie staining (Sootavvi VgiShapi reshe).

The band that moved more slowly was called "protein band 1", and the band that moved faster was called "protein band 2". Two protein bands were present in approximately equal amounts. Coomassie-stained agarose gels were used as a template for precise cutting of two protein bands from unstained portions of gels. The excised areas containing these protein bands were macerated and a small amount of sterile water was added. As a control, a portion of the gel that did not contain protein was also excised and treated in the same manner as the portions of the gel that contained protein. Protein was extracted from gel pieces by electrophoresis at 100 MM

Tris-borate buffer, pHe8.3, at 100 volts (DC) for two hours. Alternatively, the protein was passively eluted from the gel pieces by adding an equal volume of 50 mM Tris-HCl, pH7.0 to the gel pieces followed by incubation at 302 for 16 hours. This allowed the protein to diffuse out of the gel into the buffer, which was then collected.

The results of insect toxicity tests using HPLC-purified toxin (peak g» 33, min.) and agarose gel-purified toxin demonstrated the toxicity of these extracts. so 20 | injection of 1.5 μg of HPLC-purified protein kills within 24 hours. Both protein bands 1 and 2 obtained from agarose gels by passive elution or electroelution were lethal upon injection. The protein concentration, sp, determined for these samples was less than 5 ng per larva. A comparison of weight gain and mortality between groups of larvae injected with protein bands 1 and 2 shows that protein band 1 was more toxic when delivered by injection. 29 When the HPLC-purified toxin was applied to larval food at a concentration of 7.5 μg per larva, the increase in larval mass was stopped (24 larvae were studied). The larvae began to feed on the food, but after eating only a very small portion of the food that had been treated with the toxin, they began to show pathological symptoms caused by the toxin and stopped eating the food. Insect excrement became colorless, and most larvae showed signs of diarrhea. Significant insect mortality was observed when several doses of 5 μg 60 were applied to the feed during a period of 7 - 10 days.

Band 1 obtained by separation on agarose significantly inhibited the increase in the weight of larvae at a dose of 20 ng per larva. Larvae receiving similar concentrations of protein band 2 with feed were not inhibited and gained weight at the same rate as control larvae. Twelve larvae were fed the eluted protein and 45 larvae were fed agarose pieces containing the protein. These two data sets show that protein band 1 was orally toxic to Mapdis zechia. In this experiment, probably protein band 2 was not toxic to Mapadis zechia.

Further analysis of protein bands 1 and 2 by electrophoresis in PPAG-DSN under denaturing conditions showed that each band consisted of several smaller protein subunits. Proteins were visualized by staining

Coomassie followed by silver staining to achieve maximum sensitivity.

The protein subunits in these two bands were very similar. Protein band 1 contains 8 protein subunits of 25.1, 56.2, 60.8, 65.6, 166, 171, 184 and 208 kDa. Protein band 2 had an identical profile except that the 25.1, 60.8, and 65.bkDa proteins were not present. Proteins of 56.2, 60.8, 65.6, and 184 kDa were present in the protein band 1 complex in approximately equal concentrations and accounted for 8095 or more of the total protein content of this complex. 70 The native HPLC-purified toxin was further characterized as follows. The toxin was thermolabile, as it lost its ability to kill or inhibit weight gain when injected or fed to larvae of M. zechia after heating to 602 for 15 minutes. Tests for lipase activity, phospholipase C activity, nuclease activity, or erythrocyte hemolytic activity have been developed and performed with purified toxin. None of these activities were detected. Antibiotic 7/5 zone inhibition tests were also performed, and the purified toxin did not inhibit the growth of gram-negative and gram-positive bacteria, yeasts, or filamentous fungi, indicating that it is not a xenorhabdin antibiotic.

The native HPLC-purified toxin was tested for its ability to kill insects other than Mapadisa zechia. Table 2 lists the insects killed by HPLC-purified R. istipezceps toxin in this study. yu

From vyvynya yazha | Row | Ridtavid | I'll deliver it to you

Example 2 Applicability as an insecticide

Next, the applicability and toxicity of Rpoijugpavrdiz Ishtipezsepz were characterized. Culture broth 89 strain MU-14 Rpoiyuogpardyz Iptipezsepe was obtained in the following way. The production environment was 295 Vasio "

Rgoieose Reriope" Mitbreg Z (RRZ, Oyiso I arogayogiev, YuOeigoii, Mispidap) in deionized MiSh-S" water. Culture flasks for inoculation, containing 175 ml of medium, were placed in a partitioned into three parts

B5OOml flask with a Delong neck, close the Carias and autoclave for 20 minutes at a temperature of 2502 C (121.12). The production flasks contained 50Oml in a 2.8L flask, which was partitioned into three parts of 500ml each, with a Delong neck, closed with a cork made of Zpip-eiva organosilicon foam. they were autoclaved for 45 minutes at 2502 (121.12). Culture for seeding was incubated at 28 2C at 150 rpm. in a rotating and shaking thermostat with an eccentricity of 2 in. After 16 hours of growth, 195 seed cultures were placed in a production flask and allowed to grow for 24 hours before harvesting. The production of 2s toxin probably occurs during the logarithmic phase of growth. The broth containing the microbes was transferred to a centrifuge bottle on ll, and the cellular biomass was precipitated (30 minutes at 2500 rpm at 4 s "» IK.S.R.--1600| NO-4i Koiog KSZBogua! sepiptide, The primary broth was cooled at 42C for 8 - 16 hours and re-centrifuged for at least 2 hours (under the conditions described above) to further clarify the broth by removing possible mucopolysaccharide that (se) precipitates on standing. ( An alternative processing method combined both of these steps and involved the use of 16-hour centrifugation for clarification under the conditions described above.) The broth was then stored at 4 C until dc bioassay or filtration.

FT» RIoiogpardis culture broth and the protein toxin(s) purified from this broth have shown activity (mortality and/or growth inhibition, reduced hatching of adults) against a number of insects. More specifically, activity was found against the long-bearded flea (larvae and adults),

GA of the Colorado potato beetle and the lawn worm-like larvae of the Japanese beetle, which are members of the order of insects

SoiIeoriega. Other members of the order SoiIeoriega include wireworms, canola borers, earthworms, seed-eating beetles, and long-nosed beetles. Activity was also observed against the aster leafhopper, which is a member of the Notoriega order. Other members of the Notoriega include cicadas, pear leafhoppers, apple leafhoppers, cutworms, whiteflies, and shrisse beetles, as well as numerous host-specific thrips species. The broth and the purified fractions are also active against the small scoop, the cabbage scoop, the epsilon scoop, the tobacco scoop,

Kkz grain grinder, cotton bollworm and apple borer, which are members of the 1st series of eridoriega. Other typical members of this order are the true moth, southern barnyard firefly, leafhoppers, 60-cabbage moth, American cottontail moth, American cottontail, American ringed cocoon, meadow butterfly, and grass moth. Activity was also observed against the common fruit fly and mosquito larvae belonging to the order Oirieega. Other members of the Oiriega order are the gall pea, carrot fly, spring cabbage fly, turnip fly, onion fly, weevil, house fly, and various species of mosquitoes. Activity was observed against the woodpecker ant, the Argentine ant, which are members of a series that also includes b5 Richter's ant, house ant and small ants.

The described broth and fraction can be used to reduce the insect population and have been used in the method of inhibiting the insect population. The method may include introducing an effective insect-inhibiting amount of the described active ingredient into the insect cell. The results are shown in Table 3.

The activity against the larvae of the long-bearded flea was tested as follows. Rpoogpardy culture broth (which was sterilized using a sterilizing filter, cell-free) or fractions purified by HPLC were applied directly to the surface (-1.5 cm7) of 0.25 ml of artificial feed in the form of 3 µm aliquots after dilution in control medium or 10 mM phosphate buffer, pH7.0, respectively. Tablets with feed were allowed to air dry in a sterile laminar box and the wells were infested with one neonate of Biabgoiis ipadesitripsiaga pozhmagai (maize long-bearded flea (Southern), SK), hatched from a surface-sterilized egg), and second-instar larvae of ZSK grew on artificial feed, or larvae of the second instar Oiabgoiis migoiiega migdiega (UUeviegp sogp gooiuogt (western long-bearded flea), MUSK) were grown on corn seedlings growing in Meigotih". held at 272C for a suitable period of time (4 days for neonates and adults of ZSK, 2 - 5 75 days for larvae of M/SK). Mortality and mass were scored as indicated. Generally, all studies used 16 insects per mortality in the controls was as follows: newborn larvae "5905, adult beetles - 5905.

Activity against the Colorado potato beetle was tested as follows. Riojopardivz culture broth or control medium was applied to the surface (-2.0 cm?) of 1.5 ml of standard artificial feed, located in the wells of a 24-well plate for tissue culture.

Each well received 5 µl of the broth being tested (treatment) and the wells were allowed to air dry. After that, individual larvae of the second instar of the Colorado potato beetle (I eriipoiagza desetiipeaia, SRV) were planted on food, and mortality was evaluated in points after the 4th day. In all studies, 10 larvae were used per treatment. Mortality in control reached 3,395. with

The activity against larvae and beetles (adults) of the Japanese beetle was tested as follows. Lawn (5) worm-like larvae of the Japanese weevil (Rorisha |aropis, 2nd, 3rd instar stage) were collected from the infected lawn and kept in the laboratory in a mixture of soil with peat and pieces of carrots, which were added as additional feed. Lawn beetles were caught in pheromone traps placed in some locations and kept in the laboratory in plastic containers with maple leaves as food. After applying the undissolved IV) culture broth of Rpoiogpardis or the control medium to the artificial food for the long-bearded so flea (ZOmcl/1.54 cm 7, for beetles) or to pieces of carrots (for larvae), insects of both stages were planted one at a time in wells with food and observed mortality and nutrition. In both cases, a clear decrease in feed intake (and excrement production) was observed. with

Activity against mosquito larvae was tested as follows. The test was performed in a 9b-well microtiter

From a tablet. Each well contained 200 µL of aqueous solution (Rpoiogparaiv culture broth, control medium or water) and approximately 20 day-old larvae (Aedes aeduria) A well would be used for each variant. The results were recorded 2 hours after infection, and they did not change during the three-day observation period. No mortality was detected in the control. "

Activity against common fruit fly was tested as follows. The medium for Ogozorpiya teiapodavzieg was prepared from 5095 purchased dry powder and 5095 liquid (water, control medium or culture broth - with Ripoijugpavdyz). This was done by placing 8.00 ml of dry medium in each of three rearing bottles "" for one variant and adding 8.00 ml of the corresponding liquid. After that, ten larvae of " Ogozorpya teiapodazieg" were planted in each bottle. The vials were kept on a laboratory table at room temperature under the light of fluorescent lamps located on the ceiling. Pupae or adults were counted after 3, 7 and 10 days of exposure. Inclusion of culture broth in the food medium for fruit fly larvae resulted in a small (1795) but significant reduction in day 10 hatching of adult t individuals compared to water and control media (395 reduction).

The activity against the aster leafhopper was tested as follows. The test with internal acceptance for the aster leafhopper (Musgoviewiez g» zvemehip) is constructed in such a way as to allow only the ingestion of the active substance, without any other external contact. The reservoir for the active starter solution/"feed" was prepared by making 2 holes in the center of the bottom part of the Petri dishes, size 35x1Omm. A square of Ragaiyit M'"2" was placed on the top of the cup and secured with an "0" ring. A 1-ounce plastic cup was then infested with approximately 7 leafhoppers and the indicated reservoir was placed on the cup with the parafilm facing down. The test solution was then added to tank through the holes. In tests using undissolved culture broth" Ripoiogpardivz 22 broth and control medium were dialyzed against water to reduce control mortality. Mortality was recorded on day 2, when 26,595 deaths were observed in the control. In tests using purified fractions (200 mg of protein per ml), sucrose was used at a final concentration of 595 in all variants to improve the survival of leafhoppers. The test was carried out in a thermostat at 282C, 7090 relative humidity with a photoperiod of 16 hours of light/8 hours of darkness. Mortality was determined at 72 hours. Mortality in controls was 5,590. 60 The activity against the Argentine ant was tested as follows. An aliquot of 1.5 ml of 10095 culture broth

Repoiugpardy, control medium, or water were pipetted into 2.0 mL clear glass vials. The vials were sealed with a piece of cotton swab moistened with a solution of the appropriate variant. Each vial was placed in a separate Petri dish of the size of 1 cm with 8 - 12 adults of Argentine ants (Iiperiyeta Ppitiye). There were three repetitions in each variant. The bioassay cups were kept on a laboratory table at room temperature under the light of fluorescent lamps located on the ceiling.

Mortality was recorded after 5 days of exposure. Mortality in control reached 24,905.

The activity against the woodpecker ant was tested as follows. Workers of the black woodpecker ant (Satropoiis reppzuimapisiz) were collected from trees on the Yuom/Eiapso estate in Indianapolis, IM. Tests with Ryoigpardis culture broth were performed as follows. Each plastic bioassay container (7 1/8 in. x 3 in.) contained 15 workers, paper liner and 1O0ml of broth or control medium in a plastic dosing cup. A cotton swab delivered the appropriate solution to the ants through a hole in the dosing cup lid. All solutions contained 595 sucrose. Bioassays were kept in the dark at room temperature and evaluated at 19 days. Control mortality was equal to 995. In the tests in which purified fractions were delivered, 7/0 artificial ant feed was used, which was mixed with the appropriate solution (purified fraction or control solution) at a ratio of 0.2 ml of solution per 2.0 g of feed in a plastic test tube The final protein concentration of the purified fraction was less than 1 Ωkg per gram of feed. 10 ants per variant, water source, substrate and treated feed was placed in packed (sealed) plastic containers and kept in the dark at 272C in a humidified thermostat. Mortality was estimated at 75 points on the 10th day. No mortality was observed in the control.

The activity against the larvae of various lepidopterous insects (I eridoriega) was tested as follows. The culture broth of RIPOiogpardis or purified fractions was applied directly to the surface (-1.5 cm?7) of 0.25 ml of standard artificial feed in the form of aliquots of ZOmcl after dilution in the control medium or 10 mM sodium phosphate buffer, pH 7.0, respectively. The food tablets were allowed to air dry in a sterile laminar box and the wells were infested with individual newborn larvae. Eggs of the corn borer (Ozyhipia pibiiaii) and corn borer (Neiisomegra gea) were obtained from commercial sources and hatched in the laboratory, while larvae of the small borer (Zrodoriega echidtsa), cabbage borer (Tgisporivia pi), tobacco borer (Neiioipiz migezsepz), and apple borer larvae (I azreugezia rotopegia) and epsilon scoops (Adgoii5 irsiop) were collected by themselves. After infection with larvae, the food tablets were sealed, placed in He humidified rearing chambers and kept in the dark at 27 °C for the incubation period. Determination of mortality and mass in points was carried out on the 5-7th day for the Riojugparadiz culture broth and on the 4th-7th day for the purified fraction. As a rule, in all studies, 16 insects were used for each variant.

Control mortality ranged from 4 to 12,595 in the case of the control medium and was less than 1,095 for the phosphate buffer. Іо) со з and fractions of the purified toxin on mortality and growth inhibition of various rows/species of insects z со cotton 000001

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Applicability as insecticides when applied to the soil

It was shown that the culture broth of strain Mu/-14 Rioiogpardis Ishtipezsepz is active against the long-bearded flea when applied directly to the soil or to a soil mixture (Meigothich"). Activity against newborn ZSK and M/SK in Meigotich" was tested as follows (Table 4). The test was carried out using corn seedlings (Opiedy Adgizeedz bgapa Si 614), which were germinated in the light on wet filter paper for 6 days. After the roots reached a length of 3 - bcm, a single seed/seedling was planted in a transparent plastic cup containing 591 ml of 50 g of dry Meigotih." After that, twenty newborn ZSK or

M/SC was placed directly on the roots of the seedling and Meigotih was closed." When infected, the seedlings were then 7/0 moistened with a diluted broth solution, the total volume of which reached 5 Oml. After wetting, the cups were sealed and left at room temperature in the light for 7 days. After that seedlings were washed to remove all Meigotih" and the roots were cut and weighed. Activity was expressed as the percentage of corn roots remaining relative to control plants and as leaf damage caused by feeding larvae. Damage to the leaves was assessed in points visually and /5 Expressed as -, zh, as or kn, and - means no damage, and t means significant damage.

The activity against newborn 5SK in the soil was tested as follows (Table 5). The test was performed using corn seedlings (OpieFeid Adgyseeaz Vgapa SI 614), which were germinated in the light on wet filter paper for 6 days. After the roots reached a length of 3 - bcm, a single grain/seedling was planted in a transparent plastic cup with 59 ml of 150 g of soil, which was taken from a field in I erapop, IM, where corn was grown last year. This soil was not previously treated with insecticides. After that, twenty newborns

ZSK was placed directly on the roots of the seedling and covered with soil. After infection, the seedlings were moistened with a diluted broth solution, the total volume of which reached 500 ml. After soaking, the uncovered dishes were incubated in a high relative humidity chamber (80905) at 782. The seedlings were then washed to remove all soil and the roots were cut and weighed. The activity was expressed in the form of percentages of the roots of

Residual corn relative to control plants and leaf damage caused by larvae feeding on it. Leaf damage was scored visually and expressed as -, k, -- or i, with - indicating no damage and і-- indicating severe damage. yu: on the larvae of the long-bearded flea after soaking with broth after infection (Meigothich") (ee)

Stupid pedant 0111 z ve 00000000 ojiv tyu, sch zv Seredovishche (fozhovyuyu 00000000 oyayateoyugyu 100 so

Buzhyunvaezhosu 20000000 oyavisosvy 1

Broth (vegetables 000ya00000000-000000 ovoya h 2 Blshna dovtyuutya Zadnya 11111111 no s va 00000000 yamoy tyu, y" Broth (fovovyuyu 00020000

Bulyunozzhovyu 10510000 zvo vv so yu

The influence of the culture broth of strain M/-14 Rpoiogpardyv Ishtipevsepv so vd 00000010 oomeyustm o e Buleyun ovo 00201000 ojyutoy 105

Bull fodder 10201110 hunting | m5 o The activity of the culture broth of the strain MU-14 Rpoiyugpardyvz Ishtipezsepz against lawn worm-like larvae of the second age stage in Meigotih" was observed in the tests carried out as follows (Table 6). Approximately 50 g of dry Meigotih" was added to a transparent plastic cup for 59 Iml. Then Meigotih" was permeated with a total volume of 5Oml 5095 (vol./o6) of a diluted solution of Rpoyugpavrdyz broth, the initial broth was diluted with water, 60 and the 5095 broth was prepared by adding 25ml of the initial broth to 25ml of water to obtain a total volume of 5Oml. 195 (mass/ volume) of a solution of Rgoieose Reriop Mo3 (RRZ), which is a 5095 dilution of the concentration of the normal medium, was used as a control broth. After impregnation, 5 lawn worms of the second age stage were placed on the upper surface of the moistened Meigotich". Healthy larvae quickly hid in Meigotich". Larvae that did not hide within 1 hour were removed and replaced with fresh larvae. The cups were sealed and placed in a thermostat at 282C in the dark. After 7 days, the larvae were removed from Meigotich" and their mortality was assessed in balach Activity was expressed as a percentage of mortality relative to the control. before infection (Meigothich")

Example 4

Applicability as an insecticide when applied to a leaf

The activity of the broth of R. iojopardis against the grain borer was observed when the broth was applied 12 directly to the surface of the leaves of corn (Table 7). In these tests, Rpoiogpardivz broth was diluted 100-fold with culture medium and applied by hand to the surface of cut corn leaves at 6.OmcL/cm of leaf surface. The leaves were air-dried and cut into equal strips of approximately 2 x 2 inches. Leaves were twisted, secured with paper clips, and placed in 1-ounce plastic measuring cups with 0.25 inches of 295 agar at the bottom to ensure humidity. Twelve newborn grinders were placed on a folded sheet, and the cups were hermetically closed. After incubation for 5 days at 27 °C in the dark, the samples were scored for leaf-eating damage and the larvae were extracted. o grain grinder after applying the broth to the cut leaves of corn before infection with ge) that

The activity of the culture broth against newborn larvae of the tobacco weevil (Neioipiz migezsepv) was shown using the leaf immersion methodology. Fresh leaves of cotton were cut off from the plant, and leaf disks (cuts) were cut out with a 18.5 mm hole drill. Discs were individually immersed in control medium (RRZ) or culture broth of strain MU-14 RVIyoyogpardyvz Ishptipezsepe, which was concentrated approximately 10-fold using Artisop (Wemepu, MA) with a tangential filtration system Rgoykh M12 with a 1OkDa filter. Excess liquid was removed and a straightened paper clip was secured through the center of the disk. The clip was then secured (plugged) into a 1 ounce plastic dosing cup containing approximately 2.0 mL of 195 agar. This was done in order to suspend the disc above the level of the agar. After drying of the leaf disk, one newborn larva of the scoop was placed on the disk and the cup was closed.

The cups were then sealed in a plastic bag and placed in a darkened thermostat at 272°C for 5 days. After this period of time, the remaining larvae and leaf material were weighed to determine the extent of leaf damage (Table 8). Example 5. Part A

Characteristics of the peptide components of the toxin

In further analysis, the protein subunits of the band toxin, which was isolated as described in Example 1, 22 were separated on a 795 pol and acrylamide gel for electrophoresis with a ratio of 30:0.8 (acrylamide:bis-acrylamide) containing DSN. This gel matrix gives the best separation of large proteins. o The gel system used to determine the subunit molecular weights of Lane 1 and Lane 2 in Example 1 was an 1895 gel with a ratio of 38:0.18 (acrylamide:bis-acrylamide), which allowed for a wider size range to be resolved but gave poorer separation of high molecular weight components. because in this analysis 10 rather than 8 bands were obtained. Table 9 shows the calculated molecular weights of 10 bands obtained and directly compared the molecular weights that were determined under these conditions with the molecular weights of the previous example. Not surprisingly, additional bands were detected under different separation conditions in this example. Variations between previous and new molecular weight determinations were to be expected due to differences in assay conditions. In the analysis of this example, it was believed that the larger values of the molecular weights are more accurate than the values in Example 1, due to better resolution. However, these estimates are based on analysis by electrophoresis in PAAG-DSN, which is generally not analytically accurate and provides estimates of peptides that may be further altered by post- and co-translational modifications.

The amino acid sequences for the M-terminal parts of five of the 10 obtained peptides were determined. Table 9 provides a comparison of the molecular weight of the proteins and sequences that were identified. In the case of ZEO IU

Mo2, some analyzes suggest that the proline in position 5 may be asparagine (azp). In the case of ZEO IU MoZ3, some analyzes suggest that the amino acid residues at positions 13 and 14 are both arginine (aha). In the case of ZEO IU

Mo4, some analyzes suggest that the amino acid residue in position b may be alanine (aa) or serine (zeg/. In the case of ZEO IU Mo5, some analyzes suggest that the amino acid residue in position C may be aspartic acid (azr). This is analytically accurate.

Example 5. Part B

Characteristics of the peptide components of the toxin

The new M-terminal sequence, ZEO IU Mo15, AIa SiIp Avzr Su Avp Sp Avr Tig Re Zeg Su Avp Tig, was obtained by further M-terminal sequencing of the peptides that were isolated from the native purified by sc 29 HPLC toxin as described above in Part A of Example 5. This peptide is encoded by the isaA gene. The peptide called Tsad, starts at position 254 and extends to position 491, where the peptide TsaA, ZE IU Mo4 begins.

The size of this peptide, which is determined on the basis of the gene sequence, is 25240Da.

Example 6

Characterization of the peptide components of the toxin o

In yet another analysis, the bleaching complex of the toxin was re-isolated from the medium for growing c

Istipezsepsis was isolated (after cultivation without Tween) by ammonium sulfate precipitation (1095 - 8090) followed by ion exchange chromatography (Mogo C) and two gel filtration steps. These conditions are similar to those used in Example 1. During the first step of determining the size of Ha molecules (gel filtration), a second biologically active peak of approximately 100 41OkDa was detected. Based on protein measurements, this fraction was 20-50 times less active than the major (or primary) active peak of approximately 860-100kDa (native). During this isolation experiment, a smaller active peak of approximately 325-50kDa was also obtained that retained much of the original biological activity. It was assumed that the 325kDa peak relates to or is formed from the 8b0kDa peak. "

In this analysis, a 5 bkDa protein was obtained. The M-terminal sequence of this protein is given in ZES IU Mob. It is noteworthy that this protein shares significant identity and conservation with ZEO IU Mo5 at the M-terminus, suggesting that these two proteins may be encoded by different members of gene families and that the proteins produced by each gene are similar enough to be active in the insecticidal complex of the toxin.

A second prominent protein of 185 kDa was consistently present in an amount comparable to that of protein C in Table 9, and may be the same protein or protein fragment. The M-terminal sequence of the 185 kDa protein is shown in Fig. 5EO of IO Mo7.

Additional data on M-terminal amino acid sequences were also obtained from selected proteins. None of the identified M-terminal sequences is apparently identical to the protein identified in their Table 9. Other proteins were also present in the isolated preparation. One of these proteins has a defined molecular structure

The mass of 108 kDa and its M-terminal sequence are shown in the ZEO IYUO Mo8. The second such protein has a determined molecular weight of 800 kDa, and its M-terminal sequence is shown in ZEO IU Med. sl When analyzing the protein material in the active peak of about 325 kDa, bands of about 51, 31, 28, and 22 kDa were observed. As in all cases where the molecular mass was determined by electrophoretic mobility analysis, these molecular masses were subject to error effects, which were associated with differences in the ionic strength of the buffers, different powers of electrophoresis, etc. One of ordinary skill in the art will understand that the final values of the molecular weights cannot be determined by these standard methods and that

GF) each molecular weight underwent variations. It was hypothesized that proteins of these sizes are degradation products of large protein molecules (about 200 kDa in size) that were observed in the larger primary complex of the toxin. Finally, some preparations contained a protein having the M-terminal sequence shown in ZEO IU Mo10. This sequence had strong homology to known "mentor" proteins, auxiliary proteins known to be involved in the assembly of large protein assemblies.

Although the applicants could not attribute such an assembly function to the protein identified in 5HEO IU Mo10, this agrees with the existence of the described protein complex of the toxin, so that such a "mentor" protein could participate in its assembly. Furthermore, although such proteins have not been directly suggested to have toxic activity, this protein could be important in determining the overall structural nature of the protein toxin and therefore could contribute to the toxic activity or the persistence of this ip mimo complex after oral delivery.

The following analysis of the stability of the protein complex of the toxin to proteinase K was used. It was shown that after 24 hours of incubation of the complex in the presence of a 10-fold molar excess of proteinase K, the activity essentially disappeared (mortality on oral administration was reduced to about 595). These data confirm the protein nature of the toxin.

The toxic activity was also retained by the dialysis membrane, which also confirms the large size of the native complex of the toxin.

Example 7 70 Isolation, characterization and partial amino acid sequencing of Rpoijugparadiv toxins

Isolation and sequencing of M-type amino acids:

In a number of experiments, which were carried out in parallel with Examples 5 and 6, precipitation of proteins with ammonium sulfate

Riioiugpardyz was carried out by bringing the broth of Riioiugpardy, as a rule, from 2 to 3 liters, to a final concentration of 1095 or 2095 by slowly adding ammonium sulfate crystals. After stirring for 1 7/5 hours at 49, the material was centrifuged at 12,000x9 for 30 minutes. The supernatant was adjusted to 8090 ammonium sulfate, stirred at 49C for 1 hour and centrifuged at 12,000 x9 for 60 minutes. The precipitate was resuspended in 1/10 volume of 10 mM Ma»RBO, pH 7.0 and dialyzed against the same phosphate buffer overnight at 42C. The material that was dialyzed was centrifuged at 12,000 g for 1 hour before ion exchange chromatography.

Anion exchange column NK 16/50 0 Zerpagose (Ripagtasia) was equilibrated with 10 mM of MaoRBO»X, pH, 0. The precipitate that was centrifuged and dialyzed, obtained by precipitation with ammonium sulfate, was applied to the column C

Zerpagose at a speed of 1.5 ml/min. and washed thoroughly at 3.Oml/min. buffer for equilibration until the optical density (0.0. 280) did not reach a value of less than 0.100. The column was then eluted either with a 600-minute mass gradient (from 0 to 0.5M at 3 ml/min.), or a stepwise elution was performed using 001M, 04M and finally 1.0M Mass! for 60 minutes (each). Fractions were pooled and concentrated using a Sepigirger 100. Alternatively, the bilyuates can be eluted with a single wash of 0.4M Mass without i) pre-elution with 0.1M Mass.

Aliquots of 2 ml of concentrated samples from C) Zerpagose were applied at a speed of 0.5 ml/min. on a gel filtration column NK 16.50 Biregose 12 (Rpagtasia), balanced with 10 mM Ma»PO,), pH7.O. This column was washed with the same buffer for 240 minutes at 0.5 ml/min. and collected 2-minute samples. Free volume material was collected and concentrated using a Sepigirger, 100. Aliquots of 2 ml of samples that were concentrated were applied with Zuregose 12 at a speed of 0.bml/min. on a gel filtration column NK 16/50 chI

Zerpagose 48-SI (Rpaptasia), balanced with 10 mM MaoRO, pH 7.0. The column was washed with the same buffer for 240 minutes at 0.5 ml/min. and collected 2-minute samples. with

The protein peak that was isolated was subjected to a second fractionation by applying to a gel filtration column with Zerpagose SI-48, which separates proteins in the range from "ZOkDa to 1000 kDa. This fraction was separated on the column into two peaks: a minor peak at free vol. emi (21000OkDa) and a major peak eluting at an average molar mass of about 8bOkDa.Over a period of one week, subsequent gel-filtered samples revealed the gradual appearance of a third peak (about 325kDa), which probably arose from " 470 of the main peak, possibly due to limited proteolysis Bioassays performed on these three peaks showed that the free volume peak had no activity, whereas the 8b0kDa fraction of the toxin complex was highly active and the 325kDa peak was less active, although quite active. Analysis of peaks of the toxin complex from "» different fermentation processes, obtained using Zerpagose SI -48, by means of electrophoresis in

PAAG-DSN revealed two different distributions of peptides, which are designated as "P" and "5". These two distributions of peptides had marked differences in molecular weights and concentrations of peptide components in their fractions. Distribution "5", (se), which is obtained most often, had 4 high-molecular-weight peptides (»150kDa), while distribution "P" had 3 high-molecular-weight peptides. In addition, it was found that the peptide fraction "5" has 2-3 times greater activity against the grain borer. This bias may be associated with modifications in protein expression associated with the age of the inoculum and/or with other factors based on the growth parameters of aging cultures. The amount of about mg fractions of the complex of the toxin, which were determined as the distribution of peptides "P" or "5", were subjected to preparative electrophoresis in PAAG-DSN and trans-blotting with Tris-glycine (Zergaritm RMOEG tetrgapase (RgoVioetm, Arriei Viozuvietv)) for 3 - 4 hours. Blots were sent for amino acid analysis and M-terminal amino acid sequencing to Nagmag MystoSpet and Satrbgidde RgoSpet, respectively. Three peptides 22 in distribution "Z" had unique M-terminal amino acid sequences compared to the sequences identified in the previous example. Peptide 201 kDa (TsaA;), which is given in the form of ЭЭО IЯ Mo13 has i) 33905 identical amino acids and 5095 similar amino acids from ZES IЭЯ Moi (TsrА;) (Table 10, where vertical

Dashed lines indicate amino acid identity, and colons indicate substitutions of conservative amino acids). The second peptide of 197 kDa, ZEO IU Mo14 (Tsav), had 4295 identity and 5895 homology with ZEO IU Mo2 (TsasC). The third peptide of 205 kDa was designated as TsaA;. In addition, the restricted M-terminal amino acid sequence, ZEO IU Mo16 (TeBA), of a peptide of at least 235 kDa was identical in homology to the amino acid sequence of 5EO IU Mo12, which was decoded from the cloned gene (sBA), ZEO IU Mo11, and which contained the transcribed amino acid sequence that corresponded to 5ES I Moi (TsrA;). This indicates that the larger 235kDa peptide was proteolytically converted to the 201kDa peptide, (TsBA 5), (ZEBO IJOO Moi) during fermentation, possibly 65 causing activation of the molecule. In the case of yet another sequence, the sequence originally disclosed as BEO IU Mo5 (TsavB;) in Example 5 above, was found to contain an aspartic acid residue (Avr)

in position C rather than glycine (SiIu) and two additional amino acids Siu and Azr in positions 8 and 9, respectively. In the case of two other sequences, ZEBEO IU Mo2 (Tsas) and 5EO IU Moz3 (Tsav), additional amino acids were obtained. Densitometric determinations were performed using a sample that was identical to preparation "5" that was sent for M-terminal sequence analysis. This analysis revealed that the 201kDa and 197kDa peptides accounted for 7.095 and 7.295, respectively, of the total protein in the Coomassie-stained "5" distribution (Sootazwzziee

Bhiyapi Bice), and are present in amounts similar to other peptides that form larger amounts.

It is discussed that these peptides may be protein homologues, similar to the situation found for other bacterial toxins, such as the various Sgu toxins! You These proteins show variation from 4095 to 9090 7/0 homology in their M-terminal amino acid sequence, which includes their toxic fragment.

Sequencing of the internal amino acid sequence:

To facilitate gene cloning of toxin peptides, the internal amino acid sequences of the selected peptides were obtained as follows. An amount of about a few milligrams of peak 2A fractions, identified as peptide distributions "P" or "5", was subjected to preparative electrophoresis in PAAG-DSN and trans-blotting with Tris-glycine 7/5 (Zergaritm on RMORB-membranes (RgoViotm, Arrielli Viozuvietv) for 3 - 4 hours. Blots were sent for amino acid analysis and M-terminal amino acid sequencing to Naguaga MisgoSpet and Satgidde PgooSNet, respectively. Three peptides, called TebA;, (containing ZEO IUO Mo), TsaA;, and Tsavi ( containing ZEO IU Mo3) was subjected to trypsin digestion in Nagmag MisgoSpet followed by HPLC to separate individual peptides.

Analysis of M-terminal amino acids was performed on selected peptide fragments obtained by trypsin digestion. Two internal peptides were sequenced for the TsaB peptide; (peptide of 205kDa), which are called Tsav RII (ZEO IO Mo17) and Tsav/-P179 (ZEO IO Mo18). Two internal peptides were sequenced for the TsaB peptide; (bacDa peptide), which are named Tsav-PT158 (5EBO IUO Mo19) and Tsav/-PT108 (ZE IO Mo20). Four internal peptides were sequenced for the TebA | peptide (peptide 201kDa), which are called TerA-RT103 (ZEO IO Me21), TsrA-RTZb (ZEOI su

Mo22), TerA;-PT81 (a) (ZEO IU Mo23) and TerA-RTV81 (B) (ZEO IU Mo24). at

Table 10)

M-terminal amino acid sequences of 201 kDa (335 identity and 507 similarity to BEO IU De 1) h s i I s U n Kk o; E 5 Ge Z A BEO TO Me 13 s : I 1 |: «

KE 0 000yai 089 EE sn 0 - A 5BEOtTO La sho s "with 197 kDa (d2" identity and 5385 similarity with BEO TO Lo 2)

M 0 M 8 0 t EE 5 U b EE her 5EOTOYU/Le14 so yuyu | 1 : 1 | 1 :

FT"

M o r 5 ? E U 5 1 T t i. 5EO to Mo 2 so (l

Example 8

Construction of a cosmid library of genomic DNA of РЙойогпардиз Ишптипезсепе MU-14 and its screening for

Isolation of genes encoding peptides contained in the toxic protein preparation

A prerequisite for the production of Rhypoiogpardis insect-toxic proteins in heterologous hosts and (F; for other applications is the isolation and characterization of the genes encoding these peptides. This task was pursued in parallel. One approach, which will be described below, is based on the use of monoclonal and of polyclonal antibodies induced against purified toxin, which were then used to isolate clones from a bo expression library.Another approach, as described in this example, is based on the use of M-terminal and internal amino acid sequence data to design degenerate oligonucleotides for use in PCR -amplification. Each method can be used to identify DNA clones containing peptide-encoding genes, in such a way as to make it possible to isolate the corresponding genes and determine the sequence of their DNA bases. 65 Isolation of genomic DNA:

Strain MU-14 Rpoogpardyz Iyutipezsepz (ATSS assevviop pitreg 55397) was grown on 295 Rgoieovo agar

Reriop Moz (Oyiso I arogaiyugieev, Yueigoi, MI) and insecticidal toxin competence was maintained by repeated post-passage bioassays using the medium described in Example 1 and above. 5 ml of the shaken culture was obtained in 175 ml stoppered flasks in a medium with 295 Rgoieose Reriop

Mo3, cultivation was carried out at 289C and 150 rpm. within about 24 hours. 15 ml of this culture was precipitated and frozen in its medium at -202C until the time when it was thawed for DNA isolation.

The culture, which was thawed, was centrifuged (700 x 9, 30 min.) and the floating orange mucopolysaccharide material was removed. The remaining cellular material was centrifuged (25,000 rpm, 15 min.) to pellet the bacterial cells, and the medium was removed and discarded.

Genomic DNA was isolated by adapting the CTAB method, which is described in section 2.4.1.

Vioiodu I(A!yzyvei ei ai., edz doip UMPeu apa bopz, 1994), which was modified to include salt shock in it and with an increase of all volumes by 10 times. The precipitated bacterial cells were resuspended in

TE-buffer (10mM Tris-HCI, 1mM EDTC, pHe, 0) to a final volume of 10ml, then 12ml of 5M Mass was added; this mixture was centrifuged for 20 minutes at 15,000 rpm. The sediment was resuspended in 5./ml of TE and ZbOml 1095 DSN and boOml Protechnase K 2Omg/ml (Sirso VK. Rgodisiv, Sgapa Iziapa, MM; in sterile distilled water) were added to the suspension. This mixture was incubated at 372C for 1 hour; after that, about 1 µg of lysozyme was added (UMogipdiop Viospetisa! Sogr., Egeepoid, MU). In another 45 minutes, ml of 5M Mass and 800 ml of solution were added

TSAB/Massi (1095 wt./vol. TSAB, 0.7M Mass). This preparation was incubated for 10 minutes at 652C, then gently mixed and further incubated and mixed for approximately 20 minutes to remove cellular material. An equal volume of chloroform/isoamyl alcohol solution (241, v/v) was added, mixed gently and centrifuged. After two extractions with an equal volume of FCI (phenol/chloroform/isoamyl alcohol; 50:49:1, v/v/v, equilibrated with 1 M Tris-HCl, pHaB,O; Ipiegtoipiaip Zsiepiijs Sogrogayop, KauzmShe, OT), the DNA was precipitated 0.6 volumes of isopropanol. The DNA precipitate was carefully removed with a glass rod, washed twice with 70905 ethanol, dried and dissolved in 2 ml of STE (10 mM Tris-HCl, pH 8.0, 10 mM Mass, 1 mM EDTC). This preparation contained 29 2.Bmg/ml of DNA, as determined by optical density at 2b0Onm (ie, 0.05 µg). at

The range of molecular sizes of the isolated genomic DNA was evaluated for suitability for library construction. SNER analysis on the gel was performed in 1.595 agarose (Zeaket? GE, EM5 VioRgodisiv, CosKiapa, ME) gels with 0.5x TBE buffer (44.5 mM Tris-HCl, pHNa, 0, 44.5 mM NVO, 1 mM EDTC) on the Viokad SNER-OK 11 device

Ryizezhmame 760 Zm/yspeg (VioKkaai I arogaigiev, Ips., Kisptopa, SA). Parameters of movement in the gel: initial A yuyu time, Ssec.; final A time, 12 sec.; 200 volts; temperature 4 - 1892C; phoresis time, 16.5 hours. Staining with ethidium bromide and examining the gel under UV light showed DNA in the size range of 30-25O0t.bp.

Construction of the library: ch

A partial hydrolyzate of ZaizA 1 was obtained from this preparation of the genomic DNA of Rpoiiogpardyv. The method of urine is based on section 3.1.3 Аывибре! (Zirga). Adaptations involved running smaller-scale reactions under different conditions until near-optimal results were achieved. Several large-scale reactions with different conditions were carried out, the results were analyzed in SNERB gels, and only the best large-scale reaction method was selected. In such an optimal case, 200 μg of genomic DNA

The cells were incubated with 1.5 units of ZaizA I (Mem Epdiapa Wioyarv, "MEV", Wemepu, MA) for 15 minutes at 372°C in 2 ml of a total volume of their MEV 4-buffer (supplied in the form of 10x by the manufacturer). The reaction with C was stopped by adding 2 ml of FHI and centrifugation at 8000 rpm for 10 minutes. 200 μl of 5M Masi and 1 ml of ethanol cooled on ice were added to the supernatant. This preparation was cooled for 30 minutes at -202C, then centrifuged at 12,000x9 for 15 minutes. The supernatant was removed and the precipitate was dried in a vacuum oven at 402C, after which it was resuspended in 400 μl of STE. Spectrophotometric analysis showed approximately 4095 recoveries of the injected DNA. The cleaved DNA was fractionated by size on a sucrose gradient in accordance with section 5.3.2 of the SRMV (cited above). A linear sucrose gradient 10905 - 4095 was prepared with a device for preparing gradients in ShKga-Sivag tubes (VesKtap Ipzigitepiv, Ips., Rayo Le A, SA) and the DNA sample was layered on the gradient. After centrifugation (26,000 rpm, 17 hours, ZMU4A1 rotor, VesKtap, 2022), fractions (approximately 75 Ωcl) were withdrawn from the upper part of the gradient and analyzed using SNER gel electrophoresis (as described earlier). Fractions containing zaizZA 1-fragments in the size range of 20 - 40 kb were selected and DNA was precipitated using a modification (amounts of all solutions were increased by approximately 6.3 times) of the method in section 5.3.3 of A!yzibei! (Zirga). After precipitation overnight, DNA was collected by centrifugation (17,000 x 9, 15 min.), dried, re-dissolved in TE, pooled in a final volume of 8 µl and re-precipitated by adding 3 µl 3M sodium acetate and 220 µl ethanol. The pellet, which was collected by centrifugation as described above, was resuspended in 12 μl of TE. The concentration of DNA was determined using fluorometry with the dye Noespivi 33258 (Roiuzsiepsev, Ips., MMagipdiop, RA) in a fluorometer (F, Noeieg TK 100 (Noeteg Zsiepiyis Ipvigitepiv, Zap Egapsizso, SA). About 2.5 μg of fractionated DNA was obtained

Go) by DNA size.

Thirty μg DNA of cosmid ruUUE15 (Zigaiadepe, a doMa, CA) was cleaved completely with 100 units of 60 restrictionase WatH I (MEV) in the manufacturer's buffer (final volume 20Omkl, 372C, 1 hour). The reaction mixture was extracted with 1000 μl of FKI and the DNA was precipitated from the aqueous phase by adding 20 μl of 3M sodium acetate and 5500 μl of absolute ethanol cooled to 209. After 20 minutes at -70°C, the DNA was collected by centrifugation (17,000xX9, 15 minutes), dried under vacuum and dissolved in 180 μl of 10 mM Tris-HCl, pHe, 0. 20 μl of 10x CIP buffer (100 mM Tris-HCII, pH8.3, 10 mM 7psIi, 10 mM Massi") and 1 μl (0.25 units) of calf intestinal alkaline phosphatase diluted 1:4 were added to the solution (Voegipdeg Mappeit Sogrogayop, Ipaiaparoiiv, IM). After 30 minutes at

372C the following additions were made: 2 μl of 0.5 M EDTC, pHe, 0; 1 Omkl 1095 DSN; 0.5 µl 2Omg/ml proteinase K (as above) followed by incubation at 55922 for 30 minutes. After successive extractions of 1000 μl of FCI and 100 μl of phenol (Ipiegtoishpiaip Zsiepiyis Sogrogayop), equilibrated with 1M Tris-HCl, pH8B.0, dephosphorylated

DNA was precipitated by adding 72 µL of 7.5 M ammonium acetate and 55 µL of ethanol (-20 C), incubation on ice for 30 minutes, and centrifugation as described above. Precipitated DNA was washed once with 500 μl of 7090 ethanol (-702C), dried under vacuum and dissolved in 20 μl of TE buffer.

Lytation of size-fractionated ZaizA1 fragments from cleaved Vash1 and phosphatase-treated ruUUE15 vector was performed using T4 ligase (MEV) by modifying (ie, using 70% premixed 10x lithiation buffer supplied by the manufacturer) the protocol in Sec. 3.3.3 Aisibei!. Ligation was performed overnight in a total volume of 20 μl at 15 C, followed by storage at -2026. 4 μl of the reaction mixture for ligation of cosmid DNA, containing approximately 1 kg of DNA, was packaged in bacteriophage lambda using a commercial extract for packaging (Sidarask Z | Soid Raskadipa

Ehigasuzigaiadepe) according to the manufacturer's instructions. The drug, which was packed, was stored at 49C until use. The cosmid preparation that was packaged was used to infect cells of Ezspegispia soya

HIT Vice MK (Zigaiadepe) in accordance with the protocols of Sidarask PP (zoii ("Tiegipd (She Sovztiy I irgagu") yes.

Cells XII! Vice MK was grown in IV medium (g/l: Bactotrypton, 10; Bacto-yeast extract, 5;

Baktoagar, 15; Masi, 5; (Oiijso I arogajugieez, Oiijgoi, MII) containing 0.295 (m/v) maltose plus 10 mM Mo5O, at 37. After 5 hours of growth, cells were pelleted at 700 x (15 min.) and resuspended in bml 10MM

Mao). The density of the culture was adjusted to ОЮОво0-0.5 10MmM MaoaBzoO). The packaged cosmid library was diluted 1:10 or 1:20 with sterile 3M medium (0.1 M Mass, 10 mM Mo95O, 50 mM Tris-HCl, pH7.5, 0.01965 (w/v) gelatin) and 25 μl the diluted drug was mixed with 25 μl of diluted XII Vide MK cells. The mixture was incubated at 252C for 30 minutes (without shaking), after which 200 μl of IB-broth was added and incubation was continued for about 1 hour with gentle gentle shaking. Aliquots (20 - 4 µm) of this culture were distributed on plates with I B-agar containing 10 µg/l ampicillin (ie I B-Atr 4100) and incubated overnight at 372C. To store the library without amplification, individual colonies were scraped and inoculated into individual wells of sterile 96b-well microtiter plates; each well contained 75 μl Tegptys yu

Vgoiy (TV - medium: 12g/l Bactotryptone, 24g/l Bacto-yeast extract, 0.495 (v/v) glycerin, 17mMM

KNoRO,, 72 mM KoNRO,) plus 10 ug/l ampicillin (ie TV-Atr.oo) and incubated (without rocking) overnight at 372°C. After the replication of the 9b-well plate in the copy plate, 75 μl/well of TV: glycerol (1:11, v/v; with or without 100 mg/l ampicillin) mixture sterilized using a sterilizing filter was added to the plate, the plate was shaken for a short time at 1000 rpm, 372 and then covered with parafilm (Ragait?, Ategisap Mayopa! Sap, Sgeepzhisp, ST) and placed in a refrigerator at 702C for storage. with

Duplicate tablets were grown and processed in the same way as the master tablets. A total of 40 such basic tablets (and their copies) were received.

Screening of the library with radioactively labeled DNA probes

To prepare filters with colonies for probing with radioactively labeled probes, 10 9b-well plates of the library were thawed at 259C (on the table at room temperature). A 96-tooth c-print method tool (replica) was used to inoculate a fresh 96-well replica plate containing 75 μl per well of TV-Atrioo. The replica tablet was grown overnight (stationary) at 37 2C, after which it was shaken for approximately 30 minutes at 100 rpm. at 372C. A total of 800 colonies were represented in these duplicate plates as some isolates did not grow. The tool for obtaining replicas was used to inoculate duplicate prints of 9b-well rows on nylon membranes Madpa MT (M5I, UUevirogo, MA) o (0.45 micron, 220Xx25Omm), which were placed on solid media I B-Atr 400 (1OOml/cup) in plastic cups t VioAzzau (Mips, 243x243x18mm; Sipip Maiipizop Zsiepiyis, Ips., M/osa Oaie, I). Colonies were grown on these membranes at 372C for about 3 hours. t. A positive control colony (a bacterial clone containing the 324 sequence insert, see below) was grown for 50 h on a separate Madpa MT membrane (Mips, 0.45 micron, 82 mM in diameter) on IV medium supplemented with

ZbBmg/l chloramphenicol (i.e. I B-Satuv) and treated together with membranes with library colonies. c Bacterial colonies on the membranes were lysed and DNA was denatured and neutralized according to the protocol that was taken from the Sepiiz tm Zuviet user manual version 2.0 (Voephipdeg Mappeit, Ipaiaparois,

IM). Membranes were placed on filter paper impregnated with 0.5N Mahon plus 1.5M Massey for 15 minutes for denaturation and neutralized on filter paper impregnated with 1M Tris-HCl, pHNa,O,

F! 1.5M Masi, within 15 minutes. After UV-crosslinking using Zigaiadepe OM zigaia|ypKeg vei op atio sgozziipkK membranes were stored dry at 252C until use. Membranes were cut into strips containing exactly reproduced imprints of one 9b-well plate, after which they were washed thoroughly according to the method in section 6.4.1 in SRMV (cited earlier): From hour at 259C in 3x35C, 0.195 (m/v ) SDN, after that for 1 hour at 6523 in the same solution, after that washed 2x at 55C in preparation for the hybridization stage (20x

ZOS-ZM Masi, 0.3M sodium citrate, pH7.0).

Amplification of a specific genomic fragment of the isas gene

On the basis of the M-terminal amino acid sequence determined for the purified peptide fraction Tsas (which is given here as ZEO IUO Mo2), a pool of degenerate oligonucleotides (pool 54Pziy) was synthesized in a standard chemical way using B-cyanoethylation on a DNA/RNA synthesizer Arried Viozuviet AV1394 (Regk

EITeg, Rovieg Spyu, SA). The protective groups of the oligonucleotides were removed for 8 hours at 55 C, after which the oligonucleotides were dissolved in water, determined by spectrophotometric measurement and diluted for use. This pool corresponds to the determined M-terminal amino acid sequence of the Tsas peptide. The determined amino acid sequence and the corresponding degenerate DNA sequence are given below, where A, C, o and

T represents standard DNA bases and I stands for inositol: inosine:

Amino acid Mes Sip Agr Beh Rko Sich Chai

BAR IN ATOo SA(A/b) sSA(T/S) (TIA)(S/)(T/A) SSI SA(A/s) ST!

Another set of degenerate oligonucleotides (pool P2.3.5K) was synthesized, which was the complement of the coding chain for the determined amino acid sequence of ZEO IU Mo17:

Amino acid A1a Rie Avi Pe Avr Avr Chai

Spring codons TI(T/b) AA(T/C) AT(A/T/C) cSA(T/C) cSA(T/C) st

P2.3.5K ZO) AA(A) TT(A/b) TA(T/A/E) ST(A/E) ST(A/C) SA o

These oligonucleotides were used as primers in Polymerase Chain Reactions (RSV 9, Vospe

Moiiesshiag Zuzietvs, Vgapsprigo, MU) for amplification of a DNA fragment from genomic DNA that was obtained from the strain

MU/-14 Riojogpardiz (see above). A typical reaction (5 Ωcl) contained 125 pmol of each pool of primers P2Pzp and yuzo P2.3.5K, 253 ng of genomic template DNA, TONmol of each of ZATR, aSTR, azTR and aTTR, PCR buffer IX

SepeAtr? and 2.5 units of AtriaTad DNA polymerase? (Bospe Moiesshag Zuvietv; 10x SepaTruU buffer contains 100 mM Tris-HCl, pH 8.3, 500 mM KS, 0.0195 m/v gelatin). Amplification was carried out in a thermocycler for hE DNA

Rectkeep EItag Saves (RegKkeep EItag, Rovieg Sioux, CA) using 35 cycles of 949 (Ex), 5592 (2min), 7296 (Ex), followed by a 7min extension period. at 7220. Amplification products were analyzed by electrophoresis at 2965 (m/v) MyZieyeme? 3:1 agarose (EMC VioRgodisov) in TEA buffer (40 mM Tris-acetate, 2 mM 0

EDTC, pHa, 0). A specific product of a certain size of 250 p.n. was observed. among numerous other amplification products when stained with thiidium bromide (0.5 µg/ml) of the gel and examined under UV light.

The zone of the gel containing about 250 p.p. product was excised and a small plug (diameter "

O, b5mm) and used to supply the matrix for PCR amplification (40 cycles). The reaction mixture (5µl) contained the same components as described above, but without genomic template DNA. After amplification, the ends of the fragments were blunted and phosphorylated by incubating at 25 C for 20 minutes with 1 unit of T4 DNA polymerase (MEV), Inmol of ATP, and 2.15 units of T4 kinase (Riagtasia Viogesii Ips., Rizsaiamzhau, MU). DNA fragments were separated from the rest of the primers using electrophoresis at 195 (m/v) STO U adagose (EMC) in TEA. A piece of gel containing fragments with a size of 25 Op.n was cut out, and DNA was extracted using the COiaex kit (Siadep Ips., Spaivmogiy, SA). DNA fragments that were extracted were ligated to the plasmid vector pB5 K(k) (Zigaidepe), which had been cleaved completely with restriction enzyme Zta 1, and extracted in a manner similar to that described for pUUE15 DNA above. A typical reaction mixture for lithiation (16.3 μl) contained 10 ng of cleaved DNA, 7 ng of a 250 bp DNA fragment, tTnmol |Co(MHz)v|Siz and 3.9 units of MUeizv DNA ligase T4 (CoIIarogaime Vioteais ! Rhodisiv, oo 20 Vedgoga, MA) in IX buffer for lithiation (50 mM Tris-HCl, pH7.4; 10 mM MacIi", 1 mM dithiothreitol; 1 mM spermidine, 1 mM ATP, 100 mg/ml bovine serum albumin). After overnight incubation at 14 2, the ligated products were transformed into frozen, competent ОН5BA EvzspPegispia soybean cells (Birso VKI) according to the manufacturer's recommendations and plated on plates with II B-Sat»zv containing IPTH (119 μg/ml) and X- gal (5 Ohmkg/ml).

Independent white colonies were scraped off and plasmid DNA was obtained in a modified manner using alkaline 59 lysis of PEG (RKIZM Keaidu Keasiop JuOueMeohu tm Tegtipaig Susie Zedcepsipd Ki Rgoosoi!v; (FI AViI/Regkkip EIteg). The nucleotide sequence of both strands of the inserted DNA was determined. using primers 17 (bases 601 - 623 rVS kK5 (8): TAAAASOASOOSsSASTODOSOSO) and I as7 primers base 792 - o 816 rV kK5 (i: ATOASSATOATTASOSSADOSOSOS) and protocols supplied with the kit for sequencing RKIZM" m (AVi/Regkip EItag ).Dye-terminating dideoxyribonucleotide complexes, which were not included, were removed by passing through Sepigu-Zer 100 columns (Rhipsep 5eragaiopv, Ips., AdeiIrpia,

M) in accordance with the manufacturer's instructions. The DNA sequence was obtained by analyzing samples on a DNA sequencer

AVI Moae! 37ZA (AVI/Regkeep EITeg). It was found that the DNA sequences of the two isolates, 5724 and HB14, were as shown in Fig.1.

This sequence illustrates the following features: b5 , y y y y) bases 1-20 represent one of 64 possible sequences of degenerate oligonucleotides 54Rzp,

i) the sequence of amino acids 1 - 3 and 6 - 12 corresponds exactly to the sequence determined for the M-end of TsasC (which is presented in the form of ZEO IU Mo2), i) the fourth amino acid encoding it is a cysteine residue rather than a serine residue; this difference is encoded in the degeneracy for serine codons (see above), i.e.) the fifth amino acid encoded is proline, which corresponds to the M-terminal sequence of TsasC, which is represented as ZEO IU Mo2,

M) bases 257 - 276 encode one of the 192 possible sequences that were constructed in the degenerate pool, 70 m) the terminating TOA codon introduced at bases 268 - 270 is the result of complementarity of the degenerate part embedded in the pool of oligonucleotides in the corresponding position, and not indicates a shortening of the reading frame for the corresponding gene.

Labeling of a probe specific for the Tsas peptide gene.

DNA fragments corresponding to the above 276 bases were amplified (35 cycles) by 7/5 PCR in a reaction volume of 100 μl using 1000 pmol of each of the primers P2Pzp and P2.3.5K, 1 µg of plasmid 03724 or HB14 as a template, 2 µmol of each of ATP, STR, aztTR and attTR, 5 units

DNA polymerases of AtriaTtad? and SepeAtr buffer in their concentration under the temperature regimes described above.

Amplification products were extracted from 196 ZTO-agarose gel using the Oiaex kit and determined by fluorometry.

Extracted amplification products from HB14 plasmid as a matrix (approximately 400 ng) were divided into five aliquots and labeled with Z2P-DSTR using a mixture of Nidp Rgite Iabreipud Mic (Voyipdeg Mapppeit) according to the manufacturer's instructions. Unincorporated radioisotopes were removed by passing through MysTgar probe purification columns (Zigaiadepe) according to the supplier's instructions. Specific activity of the marked

The DNA product was determined using a scintillation counter, and it was equal to 3.11x109 decays per SM minute per μg. This labeled DNA was used to probe membranes that were prepared from 800 members of a genomic library.

Screening with a probe specific for the Tsas peptide gene.

The radioactively labeled HB14 probe was boiled for approximately 10 minutes, after which "minimum gib" was added to the solution. (Note: "minimum gib" method Taken from protocol SEKE5; "Kezigisyop Egadtepi Io)

Iepdius Roiutogrpizt And aroghaiyugu Mapia! megwiop 4.0", chapters 4 - 40 and 4 - 47; SECEB/MRI, Zak ake Spu, CE. co

Now the MRI does not function, its successors act as I ipkade Sepeiisv|)|. The solution of "minimum gib." contains 1090 m/rev. PEG (polyethylene glycol, molar mass approximately 8000), 795 m/rev. DSN; 0.6x55C, 10 mM sodium phosphate buffer (from 1 M stock solution containing 95 g/l ManoRO,u.1H50O and 84.5g/l ManoRO,.7NuO), 5 mM EDTC and with 10O0mg/ml denatured salmon sperm DNA. The membranes were dried by short-term soaking, and after 32 This, without prior hybridization, 5 membrane strips were placed in each of 2 plastic boxes, which (he) contained 75 ml of "minimum hyb" solution. and 2.6 ng/ml of radioactively labeled HB14 probe. They were incubated overnight with slow shaking (50 rpm) at 602C. Filters were washed three times for about 10 minutes. (each) at 259C in "minimum gib washing solution" (02555550, 0.296 DSN) with further two "

With 0-minute washings with slow shaking at 602C in the same solution. Filters accommodated 70 on paper coated Zagap Uugar? (Om Vgapaz, Ipadiaparoiykh, IM) in a light-proof autoradiographic cassette was exposed with X-Otay X-ray film (Kodak, Kospevieg, MU) with two intensifying screens Yui:z" Ropi Stopeh I idpipipo-RiIiz SI (Zbidta Spetis! So. , 91. I otsiv, Moscow State) for 4 hours at -702С. When developed (standard photography procedures), significant signals were visible in both copies amid a high background of weaker, more patchy signals. The filters were washed again for about 4 hours at 682C in "minimum gib washing solution." and then placed again in these cassettes and the film was exposed overnight at -7090. 12 possible positive prints were identified due to strong signals on both duplicate prints of the 96-well colonies. No signals were detected with the membranes of the negative control (XI1 Vice y" MK cell colonies containing ruUE15), and a very strong signal was observed with the membranes of the positive control (50.ONFBA cells containing isolate 224 of the PCR product), which were processed in parallel with the experimental samples Twelve possible hybridization-positive colonies were obtained from frozen 96-well plates with sp library and grown overnight at 372C on solid medium I B-Atr.ioro. Then they were placed on a solid medium of I B-Atr.100 (3 per plate plus 3 negative controls: XI/1 Vice MK cells containing the pUUE15 vector). Two sets of membranes (Madpa MT puiop 0.45 micron) were prepared for hybridization. The first set was prepared by placing the filter directly on the colony on the patch plate and extracting it with the attached bacterial cells, followed by processing as described below. Filters of the second (F) set were placed on cups containing medium I B-Atr.oo, which was then inoculated by transferring cells from cups with "patches" onto the filters. After overnight growth at 37°C, the filters were removed from the plates and processed. 60 Bacterial cells on filters were lysed and DNA denatured by placing each filter (colony side up) on a pool (1.0ml) of 0.5n Mahon in a plastic cup for 3 minutes. The filters were dried by blotting on a paper towel, then the process was repeated with fresh 0.5N Mahon solution. After soaking, the filters were neutralized by placing each in a pool of 1.0 ml of 1M Tris-HCl, pH 7.5, for three minutes, blotted to dry, and re-neutralized with fresh buffer. After that, two similar seepages (5 minutes each) were carried out in puddles with 0.5M Tris-HCl, pH 7.5, plus 1.5M Masi. After blotting for drying, the DNA was cross-linked with UV light to the filter (as described above) and the filters were washed (252C, 100 rpm) in approximately 100 ml of Cx C5C plus 0.195 (m/v) DSN (4 times for 30 minutes with fresh solution for each wash) . Then they were placed in the minimum volume of the pre-hybridization solution

Ibh 55C plus 190 (m/v) (each) RisoiI400 (Rnagtasia), polyvinylpyrrolidone (average mol. mass 360,000; Bidta) and bovine serum albumin (fraction M) (Zidta)) for 2 hours at 65 2C, 5b0rpm.

The prehybridization solution was removed and replaced with P-labeled NVI14 probe, which was saved from pre-hybridization of the membranes with the library and which was denatured at 9529 for 5 minutes.

Hybridization was carried out at 602C for 16 hours with rocking at 5 bob/min.

After removing the solution of the labeled probe, the membranes were washed three times at 252 (5 bbl/min, 15 minutes) in Zx 70.88C (approximately 150 ml for each wash). They were then washed for 3 hours at 682 (B5bob/min) in 0.25x 55C plus 0.295 DSN (minimum gib washing solution) and exposed on X-ray film as described above for 1.5 hours at 252C (without intensifying screens). . This exposure revealed very strong hybridization signals with cosmid isolates 22012, 25A10, 26A5, and 26810 and a very weak signal with cosmid isolate 8810. No signal was observed with negative control colonies (pUUE15), and a very strong signal was observed with positive control membranes (ON5BA cells, containing isolate 5724 of the PCR product), which were processed in parallel with the experimental samples.

Amplification of a specific genomic fragment of the Iysav gene

Based on the M-terminal amino acid sequence that was determined for the purified fraction of the Tsav peptide; (which is described here as zEO IU Mo3), a pool of degenerate oligonucleotides (PVE pool) was synthesized as described for the Tsas peptide. The determined amino acid sequence and the corresponding degenerate DNA sequence are shown below, where A, C, o, and T are the standard DNA bases and I stands for inosine:

Amino acid Tec Re Tg o cSip Tvzh tei puye sich Aia AgE (o)

RVE vi TIT ASI SA(A/Ss) ASI (S/TUGUI lAH SAA SsI Ky z iv)

Another set of degenerate oligonucleotides (pool P8.108.3K) was synthesized, which was the complement of the coding chain for the determined amino acid sequence of the internal peptide Tsav y-PT108 (which is described here as BEO IU Mo20): chE c

Aminoki- Mayes Tug Tug Iie beep A1a beep Sip so slots

Codons AtTe TA(T/S) tTA(T/S) AT(T/SIA) sA(A/sSu OAKBL sSA(A/S) sSAxA/s) «

RUZ. 108.3B. Z'TAS AtT(A/C) AT(A/C) TAT) stT(T/C) ss st(t/se) st - s and "» These oligonucleotides were used as primers for PCR using Noiseiag 50 Tyres tubes ( MoiIesshiag Viorgodisiv, Ips., Zap Oiedo, CA) for amplification of a specific DNA fragment from genomic DNA obtained from strain MU-14 RIpoiogparaiz (see above). A typical reaction mixture (5µl) contained (lower layer) (se) 25 pmol of each pool primers, REE and P8.108.3K with 2 pmol each of dZATP, asStR, aSTR and atTPR in PCR buffer with 1x bepeAtr? and (upper layer) 23 ng of genomic template DNA, vNmol each of aATP, asStR, aSTR and attR and 2.5 units of AtriaTtad DNA polymerase in their ZepeAtrU buffer. Amplifications were carried out by 35 cycles as

T- is described for the TsasC peptide. Amplification products were analyzed by electrophoresis at 0.795 (m/v)

Go! 20 Weaket?biE agarose (PEM) in TEA-buffer. A specific product with a determined size of 1600 p.n. was observed.

Four such reactions were combined and the DNA that was amplified was extracted from a 1.096 ZeaKet GE gel with sl using the Oiaex kit as described for the TsasC peptide. The DNA that was extracted was used directly as a matrix for sequencing (RKIZM sequencing kit) using RZE and P8.108.3K primer pools. Each reaction contained approximately 10 ng of template DNA and 22 2 bpmole of one of the primer pools and was performed according to the standard protocol as described for

Ge) peptide Tsas. Analysis of the sequence obtained from the extension of the PVE primers revealed a short DNA sequence (and the amino acid sequence that is encoded):

SAT ssA tto nTtT sst 60

Aer A1a Hech (Ua1) A1a which corresponds to part of the M-terminal peptide sequence described in the form of BZEO IU Moz (TsaB;).

Labeling of a probe specific for the TsaB peptide gene; 65 Approximately 5 μg of gel-purified TsavB DNA fragment; ZgR-YAOSTR markers, as described above, and radioisotopes,

that was not incorporated was removed by passing through a MISC column (RPagtasia). It was determined that the specific activity of the labeled DNA was equal to 6bX10 decays per minute per μg. This labeled DNA was used to probe membranes with colonies derived from genomic library members that hybridized with a probe specific for the Tsas peptide.

Membranes containing these 12 colonies identified in the TsaC probe library screening (see above) were freed from the radioactive, TsaC-specific label by boiling 2 times for 30 minutes in 1 liter

Oh 550 plus .195 DSN. The removal of the radioactive label was checked with a single exposure of the film. Then the membranes freed from the label were incubated with the probe specific for the 70 TsaV peptide obtained as described above. The labeled DNA was denatured by boiling for 10 minutes and then added to the filters, which were then incubated for 1 hour in 100 ml of "minimum gib" solution. at 602. After overnight hybridization at this temperature, the probe solution was removed and the filters were washed as follows (all in 0.3x 55C0.1956 SDS): once for 5 minutes at 252C, once for 1 hour at 602C in fresh solution, and once for 1 hour in a fresh solution. After a 1.5-hour exposure on X-ray film, according to standard 75 procedures, 4 strongly hybridizing colonies were observed. These were, as with the Tsas-specific probe, isolates 22012, 25A10, 26A5, and 26810.

The same solution of the probe was diluted with an equal volume (approximately 10 Oml) of the "minimum gib" solution. and then used to screen membranes containing 800 members of a genomic library. After hybridization, washing, and exposure to X-ray film as described above, only four cosmid clones 22012, 25A10, 26A5, and 26810 were found to hybridize strongly with this probe.

Isolation of subclones. containing genes that encode peptides Tsas and TsavB;. and determining the sequence of their DNA bases.

Three hybridization-positive cosmids in strain XII! Vide MK was grown with rocking overnight (200 rpm) at 302 in 100 ml of TV-Atr.oo medium. After collecting cells by centrifugation, cosmid Ha

DNA was obtained using a commercially available kit (VISrgertm, 5 Rgite Z Rgite, Ips., Votsideg, SO) according to the manufacturer's protocols. Only one cosmid, 26A5, has been successfully isolated in this manner. Fragments with approximate sizes of 14, 10, 8 (vector), 5, 3, 3, 2, 9 and 1 bp were detected when digested with restriction enzyme Esok 1 (MEV) and analyzed by gel electrophoresis. In the second attempt to isolate cosmid DNA from the same three strains (culture in ml; TV-Atr.ioo, 302) the tipirger method with boiling (Emape M) was used. Apa village Umani, 1987, "Sovtiy mesioge yog depotis maikipud apa hariy gevigisbyop tarripd" ip Scide Yuu s

Moiesshiag Siopipd Thesppidcez Mei. Epgmtoiiodu, moI.152, 5. Vegdeg apa Kittei, eaz., dis. 604-610). Only one cosmid was successfully isolated in this way. When cleaved by the restriction enzyme EsobB. 1 and analyzed by Х gel - electrophoresis, this cosmid revealed a distribution of fragments that is identical to the distribution that was detected with cosmid 26bА5.

About 0.15 μg of cosmid 26A5 DNA sample was used to transform 5 ml of ONBA E. Soyia cells (Sirso VK) according to the manufacturer's protocols. An isolate of one colony of this strain was inoculated into 4 ml of TV-Atr1oo medium and grown for 8 hours at 372C. Chloramphenicol was added up to the final concentration of 225 μg/ml, the incubation was continued for another 24 hours, then the cells were collected by centrifugation and frozen at -2020. The DNA of cosmid 26A5 was isolated by standard alkaline lysis (tipirger) (Mapiaiis ei ai., cit. above, 70 r.382| with the modification consisting in increasing all volumes by 5095, and with mixing or careful mixing, rather than by vortexing, at each stage. After washing the DNA pellet in 7095 ethanol, it was "» dissolved in TE containing 25 µg/ml ribonuclease A (Voengipdeg MappNeit). " Identification of Esok 1 fragments that hybridize to those obtained from 05724 and TsaV ;-probes.

Approximately 0.4 μg of cosmid 25A10 (from XI 1 VICHE MK cells) and approximately 0.5 μg of cosmid 26A5 (from 49 chloramphenicol-amplified OH5BA cells) were cleaved separately with approximately 15 units of Esok I! (MEV) for 85 so minutes, frozen overnight, then heated at 6592С for 5 minutes and subjected to electrophoresis in

KkZz 0.790 agarose gel (Zeaket? IE, their TEA, 80 volts, 90 minutes). DNA was stained with ethyl bromide as described above and photographed under UV light. Under these conditions, the Esok 1 cleavage product of cosmid 25A10 was fully cleaved in 50%, but the sample of cosmid 26A5 was only partially cleaved. Agarose gels containing (se) DNA fragments were subjected to depurination, denaturation, and neutralization followed by Southern blotting on a Madpa MT nylon membrane using a protocol with a high salt concentration (20x55), all of which are described in Section 2.9 A! ygibei ei ai. (SRMV, cited above)). The transferred DNA was then crosslinked with UV light to a nylon membrane as previously described.

A DNA fragment specific for the TsasC peptide, which corresponded to the insert of plasmid isolate 74, was amplified by PCR in a reaction volume of 100 ml, as described above. Amplification products of three

HF) of such reactions were pooled and extracted from a 196 ZTOZ-agarose gel using the Oiaex kit as described above and quantified by fluorimetry. Gel-purified DNA (10 µg) was labeled with Z2pR-DSTR using Nidp Rgite I abeijpud Mih (Voeyipdeg MappNeit) labeling mixture as described above, up to a specific activity of 6.34x108 decays per minute per μg.

C2p-labeled probe 574 was boiled for 10 minutes, then added to the "minimum gib" buffer. (at ng/ml), a membrane with a Southern blot containing cleaved cosmid DNA fragments was added and incubated for 4 hours at 60 2C with gentle shaking at 50 rpm. Then the membrane was washed three times at 2592; for about 5 minutes in each case (with a washing solution of "minimum gib. "y, followed by two washings for 20 minutes (each) at 60 °C. The blot was exposed on film (with intensifying screens) for about 30 minutes at -702 °C. Probe (324) strongly hybridized with the Esok 1 5,Ot.p.n. (visible size) fragment of both plasmids, 26A5 and 25A10.

The membrane was freed from radioactivity by boiling for 30 minutes in 0.1x 55С--0.195 DSN and the absence of a radioactive label was checked by exposure on the film. Then it was hybridized at 602C for 3.5 hours with a denatured TsavB probe; in the "minimum gib" buffer previously used to screen colony membranes (see above), washed as previously described, and exposed to film for 40 minutes at -70°C with two intensifying screens. In the case of both plasmids, the Tsav probe; hybridized weakly with the fragment Esok 1 5, Ot.p.n. and strongly with a fragment of approximately 2.9 t.b.p.

The previously described cosmid 26A5 DNA sample (from ON5BA cells) was used as a DNA source from which the target bands were subcloned. This DNA (2.5μg) was digested with approximately 3 units of Esok 1 (MEV) in a total volume of ZOmcl for 1.5 hours to obtain a partial cleavage product, which was confirmed by gel electrophoresis. Ten μg DNA of rvs Kk (t) (Zigaiadepe) was digested for 1.5 hours with Esok 1 units in a total volume of 20 μl, causing complete digestion, as confirmed by 75 electrophoresis. Both preparations of cut Esok 1 DNA were diluted to 500 μl with water, an equal volume of FHI was added to each, the suspension was carefully mixed, spun in a microcentrifuge, and the aqueous supernatant was collected. DNA was precipitated with 150 μl of ethanol and the mixture was kept at -202C overnight. After centrifugation and drying, the Esok 1-cleaved plasmid pB5 K5 (w) was dissolved in 10 μm of TE; partially cleaved 26A5 was dissolved in 20 μl TE. DNA extraction was monitored by fluorimetry. 20 In separate reactions, approximately bOng of Esok 1-digested pB5 K5 (F) DNA was ligated with approximately 18Ong or 27Ong of partially cleaved cosmid 26A5 DNA. Lythation was carried out in a volume of 20 μl at 1593 for 5 hours using T4 ligase and buffer from Mem/ Epodiapa Vioi arch. The mixture for lithiation, diluted to 100 μl with sterile TE, was used for the transformation of frozen, competent OH5BA E. Soya cells (birso

VI) in accordance with the manufacturer's instructions. Various amounts (25 - 200Omkl) of cells that were present. / Is transformed, sown on freshly prepared solid medium I B-Satzv with 1MM IPGT and 5mg/l X-gal. at

The dishes were incubated at 372C for 20 hours, then cooled in the dark for about 3 hours to enhance staining for picking inserts. White colonies were scraped and transferred to patch plates of the same composition and incubated overnight at 3726.

Two "lifts" of colonies from each of the selected "patch"-ashoks were prepared as follows. After transfer of the white colonies to fresh plates, Madpa MT round nylon membranes were pressed onto patch plates, the membrane was lifted, and subjected to UV denaturation, neutralization, and crosslinking as described above for library colony membranes.

The cross-linked "elevators" of the colonies were intensively washed, carefully washing off the excess of cell remnants with "I" tissue. One set was hybridized with probe solution 74 (Tsas) described previously, and another set was hybridized with probe solution TsaVi as described above according to the "minimal hyb" protocol, followed by 32 washes and film exposure as described for membranes with colonies of the library. (heh)

Colonies that showed hybridization signals, either only with probe 524, with both probes 074 and TsaV,, or only with the probe Tsav;, were selected for further work and cells were streaked to isolate a single colony on I B-Satzv media with IPTG and X -gal, as described earlier. Approximately 35 individual colonies, from 16 isolates, were transferred to liquid media I B-Sat z5 and grown overnight at 372C; cells were collected by centrifugation, and plasmid DNA was isolated using standard alkaline lysis (tipirger) according to Mapiaizzei a!. (cited above, p. 368). The DNA precipitates were dissolved in TEn 25μg/ml of ribonuclease A and the DNA concentration "" was determined by fluorimetry. The distribution of Esok 1 cleavage products was analyzed by gel electrophoresis. The following " isolates were obtained in the usual way. Isolate A17.2 contains only religated RVS K5(s) and was used as a (negative) control. Isolates Y38.3 and C44.1 (each) contain only 2.9 kbp fragment 49 of Esov 1, which hybridizes with TsaV, which is embedded in rVS KB (g). These plasmids, named pOAV2000 and pOrAV2001, respectively, are shown in Fig.2. isolate AZ5.3 contains only an approximately 5 kb fragment of Esok 1 that hybridizes to 574, which is integrated into rvBs Kk (Zh. This plasmid was named ppAV2002 (also Fig. 2). These isolates provided templates for sequencing in dnk.bo 20 Plasmids pOrAV2000 and rraAV2001 were obtained using the ViSrger im kit as described above.

Cultures (ZOml) were grown overnight in TV-Sat c5 medium up to OO co02, then the plasmid was isolated c according to the manufacturer's instructions. The DNA pellet was redissolved in 10µl TE (each) and the integrity of the samples was checked by Esok 1 digestion and analysis by gel electrophoresis.

Sequencing reactions were performed in duplicate, with one replicate using 99 ppAB2000 DNA as a template and another replicate using ppAB2001 DNA as a template. Reactions were carried out using

Ge) method of cyclic sequencing with a dideoxy-dye complex as a terminator, which is described above for sequencing DNA 74/HB14. The initial rounds of sequencing used as primers the primers and as7 and ko 77 described above, plus primers based on the determined sequence of the PCR-amplified product of Tsav; 60.

After primary structure analysis and preparation of each sequencing product, each was trimmed to 250-350 bases, depending on the completeness of the chromatographic data interpreted by the software

Regkip EIteg Arriiei Viozuzietz Oimizviop ZedEab75. Further "stages" of sequencing were performed by choosing a suitable sequence for new primers. With few exceptions, the primers (synthesized as described above) were 24 bases long with a composition of 5095 C-C. Sequencing in this way was performed on both strands of the Esok 1 fragment with a size of approximately 2.9 tbp.

For further use as a template for DNA sequencing, plasmid DNA was obtained from the pOAB2002 isolate using the BiICrger kit. Sequencing reactions were performed and analyzed as described above. 7/0 Initially, primer TZ (bases 774-796 rvs Kz (8 СОСОСАATTААССССТСАСТАААОС) and primer 17 (bases 621-643 rVS KB (5). OSOSOTAATASOASTSASTATAS) were used for priming sequencing reactions from flanking vector sequences with reading into DNA insertion. Another set primers

Y (C24E: STATSSATIA SA ASOSTOTSASTTSSS;

TNIZ: SSSAASTOSASASSSTTSTAATOSATAS;

ТНИД:АТСТТСОСТОССТoСОССТААТОСАСАТААС; and

IIM/1-204:. SOBAASTOASASOTTOTAATSOATAS) was used to frame the internal sequences, which were previously determined by sequencing using degenerate oligonucleotides Go) subcloned PCR products of the TsasC peptide. Based on the data obtained during the initial rounds of sequencing, new sets of primers were designed and used to "walk" along the entire length of the "5 kb" fragment. A total of 55 oligo primers were used, which made it possible to identify a total of 4832 bp. continuous sequence. details 3o When combining the DNA sequence of the insert of the Esok 1 fragment of рАВ2002 with a part of the determined sequence of pАВ2О00/рпАВ2001 isolates, a common sequence of contiguous nucleotides with a length of bOO5 bp was obtained. (which is given here in the form of ZEO IU Mo25). When the long open reading frames are translated into the corresponding amino acids, this sequence clearly reveals the M-terminal peptide (which is described as ZEO IUO Mo3), which is encoded by bases 19-75, immediately after the methionine residue (starting point of translation). Further along the transcription, in the same reading frame, at bases 1738 - 1773, there is a sequence encoding the internal peptide Tsav;-1108 (which is described as ZEO IU Mo20). Also, in the same reading frame, at bases 1897-1923, the M-terminal peptide of TsaB is encoded; (which is described here as ZEO IUO Mo5) and the reading frame continues without interruption to the transcription termination codon at nucleotides 3586 - 3588.

Absence of the in-frame stop codon between the end of the Tsav-PT108 coding sequence and the beginning of the TsaV coding region. and the absence of a noticeable ribosome binding site directly upstream of the transcriptional region from the TsaB coding region; indicates that TsaB peptides; and TsaV; are encoded by a single 3567-bp open reading frame starting at base pair 16 in ZEO IU Mo25, and most likely arise from a single primary gene product of 1189 amino acids (131586Da; described here as ZEO IU Mo26) in as a result of post-translational cleavage. If the amino acid that co 15 immediately precedes the M-terminal peptide TsaB; is the C-terminal amino acid of the peptide TsaB;, then the predicted mass of TsaB;, (627 amino acids) is 70814Da (which is described here as XEO IUO Mo28), which is somewhat

Kz is larger than the size observed by electrophoresis in PAAG-DSN (6b8kDa). This peptide would be encoded by a continuous segment from 1881 BC. (which is described here as ZEO IU Mo27). One can think that the C-end of TsaV is native; lies somewhat closer to the C-end of Tsav/--1108. Molecular mass of RT108 |Z, 438 kDa; determined during the analysis of the M-terminal amino acid sequence of this peptide assumes a size of 30 amino acids.

Using this peptide size to determine the C-terminus of the TsavB coding region; |SiIy in position 604 5EO IYU sl Mo281, the obtained size of TsaV; can be determined as 604 amino acids or 68463Da, which is more in agreement with experimental observations.

Translation of the coding region of 1686 base pairs of the TsaB peptide; (which is described here as BEO IU Mo29) gives a protein of 562 amino acids (which is described here as cZEO IU Moz30) with a predicted mass of 60789 Da, which is good

F! agrees with the observed b1kDa size.

A potential ribosome binding site (bases 3633 - 3638) was found at a distance of 48 bp. in the course of transcription from the stop codon for the open reading frame of isavB. At bases 3645 - 3677, the sequence encoding the M-end of the TsasC peptide (which is described here as ES IU Mo21) was found. Open reading frame 60, beginning with this M-terminal peptide, continues uninterrupted to base 6005 (2361 bp, represented here as the first 2361 bp of ZEO IUO Mo31). The gene (IsaC) encoding the entire Tsas peptide (average size "165 tbp; 71,500 amino acids) should contain approximately 4,500 bp.

Another isolate containing cloned EsokK 1 fragments of cosmids 26A5, E20.6 was also identified by its homology with the previously mentioned probes 074 and TsaB;. Analysis in an agarose gel of the cleavage products of Esok 1 and the DNA of this plasmid, which is held by this strain (ppАВ2004, Fig. 2), revealed inserted fragments of specified sizes

2,9, 5 and Z,Zt.p.n. Analysis of the DNA sequence initiated from the primers, which were constructed from the sequence of the plasmid ppAV2002, revealed that the fragment of Esok 1 Z,Zt.p.n. ROAV2004 lies next to the fragment

Esok 1 5t.p.n., presented in rrAV2002. The open reading frame from 2361 bp, which was detected in pOAV2002, continues without interruption for the next 2094 bp. in pOAV2004 | which is described here in the form of base pairs 2362 - 4458 5EO IYUO Mo31). DNA sequence analysis using parental cosmid 26A5 DNA as a template confirmed the continuity of this open reading frame. In general, this open reading frame (TsasS 5EO IU

MoZ31) contains 4455 p.n. and encodes a protein (Tsas) of 1485 amino acids (which is presented here as ZEO IU Moz21.

The calculated molecular size of 166214Da agrees with the determined size of the Tsas peptide (165kDa) and the 70 amino acid sequence produced matches exactly the sequence described for the M-terminal sequence of Tsas IZEO IUO Mo21).

The absence of an amino acid sequence corresponding to the BEO IUO Mo17, which was used to construct a pool of degenerate oligonucleotide primers, in the detected sequence indicates that the formation of products

PCRs detected in isolates 574 and HB14, which were used as probes in the initial library screening, 7/5 Performed by randomly priming the reverse strand with one of the primers in the degenerate pool.

In addition, the resulting protein sequence does not contain an internal fragment, which is described here as a ZEO

IO Mo18. These sequences show that the plasmid ppAB2004 contains the complete coding region for the Tsas peptide.

Example 9

Screening of the genomic library of Rpoyugparaitsz for genes encoding the TeBA peptide;,

This example describes a method used to identify DNA clones that contain genes encoding the TebA peptide, isolating this gene and determining its partial DNA base sequence.

Primers and PCR reactions

Polypeptide TsBA.., active against insects of the drug has mol. mass "206 bkDa. Amino acid sequence

The M-end of this peptide is described as 5ESO IJOO Moi. Synthesized 4 pools of degenerate oligonucleotide primers ("Direct primers": TN-4, TN-5, TN-6 and TN-7) to encode this amino acid sequence as described in Example 8. They are shown below. (o) zo Table 11 ju (ge)

Amino- REe Iie sip sSiu tuye Bek Agr TGech Rie « acid s (he) tTn-4 ZLI C) ATI SAdK) sSI TA(T/S) tsI SAT) STI t-U

TN-5 UTIM) ATI SADI) СсСИ 0 TA(T/S) ASK) sa) STI tt-3 « с 70 тТНн-в LO) ATI SA) OSI 0 tTA(T/S) TSI САС; o t(A/s) tt-3 Z z tn-7 9LIYO) ATI SA(DA/) o sSI 0 TA(tT/S); AC) SAT) tt) tt-3

In addition, the primary ("a") and secondary ("B") sequences of the internal peptide (og) preparation (TeA;-PTV81) described here as ZEO IU Mo23 and ZEO IU Mo24, respectively, were determined.

In a similar way, 4 pools of degenerate oligonucleotides ("Reverse primers": ТН-8, ТН-9, ТН-10 and ТН-11) were designed and synthesized to encode the reverse complement of the sequences encoding the t» part peptides ZEO IU Mo23, which is shown below

F) ko 60 b5

Table 12

Amin TE Tut Tep TNti bek o Re Sich bii o Uai AiTa 0 Avp okis lota

TN-8 ZYSI ATAY) SAT TSI Asi AAdDo) SM) sp) sSa1 soi 0 sing tTN-Z Z'SI ATAL) AD) TSI ASI AAfO) STO) sg) sAat o sb1 ps»

TN-10 Z'TSI ATAD) SAT TsI OSA) Or) STO) STIK) SAT si ped"

TN-11 Z'TSI ATaAd) AD) TC TS) Adi) Sp) SGS) SAT ssI lem

Sets of these primers were used in PCR reactions to amplify fragments of the gene encoding TebBA, from the genomic DNA of the strain MU-14 RPoiogpardis Ishtipezsepz, which was obtained in Example 6. All PCR reactions were performed according to the "Noi 5iagi" method using Atrium/akhtm detvs and other Ragkeep reagents and protocols

EITeg. As a rule, a mixture (total volume 11 μl) of MoCI 2, aMTP, 10x buffer II for PCR SepeAtr? and primers were added to tubes containing one wax bead (10x Sepetr? RCE. Solution II consists of 100 mM s 29 Tris-NSII, pHeV,3, and 500 mM KSI). The tubes were heated to 8023 for 2 minutes and allowed to cool. A solution containing 10x ZepeAtrU RCE was added to the (9) upper surface of the wax shutters formed.

DNA template and AtriitadU DNA polymerase. After melting the wax plug and mixing the components by thermocycling, the final reaction conditions (volume 5 Ωcl) were as follows: 10 mM Tris-HCl, pH 8.3; 5 Ohms of KOSI; 2.5 micrograms of Mass", 200 μM of each of ZATR, astR, astR, atteR; 1.25 mM in one pool of Direct primers; 1.25mM in one pool of Reverse primers, 1.25 units of AtriaTtad DNA polymerase and 17Ong of template DNA. The reaction mixture 09 was placed in a thermal cycler (as in Example 8) and PCR was carried out according to the following program: ) using pools of degenerate oligonucleotides. Reactions annealed at 659 did not give amplification products visible after electrophoresis in agarose gels. Reactions annealed at 602C and containing primers TN-5-TN-10 gave an amplification product that had mobility, which corresponded to 2.9 tpp. Less product 2.9 tpp was produced under these conditions with primers TN-7-TN-10. When the reactions were annealed at 5529, these primer pairs produced more product 2 ,9 kb, and this product was also obtained with primer pairs TN-5-TN-8 and i" TN-5-TN-11. Additional very weak bands of 2.9 kb were visible in lanes , which contained amplification products from 20 pairs of primers TN-7-TN-8, TN-9, TN-10 and TN-11. To obtain a sufficient amount of PCR amplification product for clo 10 separate PCR reactions were prepared with primers TN-5-TN-10 (l) and they were carried out under the conditions described above with an annealing temperature of 5520. All reaction mixtures were combined and the product 2.9 t.b.p. purified by Oiaex extraction from agarose gel as described above.

Additional sequences identified for internal TSBA peptides are described here as ZEO IU Mo21 and ZEO 22 |O Mo22. As before, degenerate oligonucleotides (Reverse primers TN-17 and TN-18) were prepared, which

Ge) correspond to the reverse complement of the sequences that encode part of the amino acid sequence of these peptides. ko 60 b5 and Table 14

Z 5EO tre 21 0 Amino-Meie sycy Tikh Siv Avp I1e sip Sich Pko acid tTnH-17 3-TAS cStTtT/C tSI CTT/C tTTA/y TAiYi sttT/C stt/C 6b6-5

15 TGablitsya

From BEO Tr Mo 22

Amino-Dvp RkEo I1e Avp I1e Avp Tak biu She Agr acid cm o tTN-18 3-Ta/)U СсI TAi TAI) TAT LID) TSI ССІ tTAt1 or

Degenerate oligonucleotides TN-18 and TN-17 were used in the amplification experiment with DNA from Rpoyugnaradiz Ishtipezsepe MU-14 as a template and primers TN-4, TN-5, TN-6 or TN-7 as 5-(Direct) primers. These reactions amplified products of approximately 4tbp. and 4, bt.p.n., respectively. These DNAs were transferred from agarose 00 gels to nylon membranes and hybridized with a P-labeled probe (as described above), which was prepared from a 2.9 kb product amplified with primer pair TN-5-TN-10. Both products of amplification - d4t.p.n. and 4, bt.p.n. strongly hybridized with the 2.9t.p.n. probe. These results were used to construct a map that locates the internal peptide sequences of TeBA, as shown in Fig.3.

The sequence of the TeBA coding fragment;; 2.9 t.p.n. co

Approximately 200 ng of the purified fragment of 2.9 t.p.n. (obtained as described above) was precipitated with ethanol and dissolved in 17 ml of water. One half was used as a template with 25 pmol of the TN-5 pool as primers, and the other half was used as a template with the TN-10 primer. Sequencing reactions were performed as described in Example 8. No plausible sequence was obtained with the TN-10 primer pool; однак реакції з пулом З с праймерів ТН-5 продукували описану нижче послідовність: з 1 ААТССТОТТО АТОССТАТОС СОоМОсСсоссТ ТООСТОСАДТ ССАТСТОСТО АссссОссТТ 51 ТАТТМСАСО АНТМСТОСОС ТОАСФССАДА ААМТОСААТО АЛАСААСТТС ААТТТМТТАС 15 121 СТАСАТАЛАС СТОССССООМ ТІТАСАААСМ ТТАМТОМТСА ОССАСАКААТ ТТТОСТТсАС со 121 САААТТОСАС ССМТОСТТСТ СТОТАТТОАТ ТМОССссСТОС ССОССТТОСА АМННААААЯНА 241 ССАЛАТМСАС ААСТТСАССТ САТОСМТТТО TISSMAMSTT MISSTTTASO TSOSSASAAA ko 3101 SSTIMTSAMS ASOMITMTOA AASTOTOSOO SALATOSTOS ATSAMOSTSA MeSASSMTTM 361 SOSSATTOYA

On the basis of this sequence, a sequencing primer (ТН-21, с 5-ССОООСОАСОТТТАТСТАСО-3) was designed for the reverse complement bases 120-139 and initiation of polymerization in the direction of the 5-end (i.e., ТН-5-end) purified on a PCR gel - a fragment of 2.9 t.p.n. TerA;. The determined sequence is shown below and compared with the biochemically determined M-terminal sequence of the TeBA peptide; ZEO IO Mo. 5B Confirmation of the sequence of the PCR fragment of 2.9 t.p.n. ToBA; o (Underlined amino acids: sequences encoded by degenerate oligonucleotides) ko 60 b5 bEO tr l1 y E 1 0 sb Z 5 0 p ' EE sb - - A 2.9 oS АТС САС ССО TAT AST САС STO TTT OST AAT СstТ сстТ t.Pp.n.,

M 0 s 0yai 5 0 EE S M VK A t From the homology of the produced amino acid sequence with the sequence determined biochemically, it follows that

PCR-fragment 2, Ot.p.n. is the TerA coding region. This fragment is 2.9 million years old. was then used as a hybridization probe for screening the genomic library Ripoiogpardyvs MUu-14, obtained in Example 8, for cosmids containing the gene that encodes TebBA;,.

Screening of the Rpoiogparadiv cosmid library 7 Gel-purified PCR fragment of 2.9 Tbp. marked ZR using the Voepyhipdeg marking set

Mappvpeit Nidy Rgite as described in Example 8. Filters containing the remainder of approximately 800 colonies from the cosmid library were screened as previously described (Example 8) and positive clones were streaked to obtain single colonies and rescreened. Three clones (8A11, 25058 and 2601) gave positive results with two during several stages of screening and characterization. Hybridizations of the TsrA ;-specific probe were never obtained with any of the four cosmids identified in Example 8 that contain the genes (caB and isas. (o)

DNA from cosmids 8A11, 25058, and 260 was digested with Wid 2, Esok 1, or Nipa 3 restriction enzymes (alone or in combination with each other), and the fragments were separated on an agarose gel and transferred to a nylon membrane as described in

Example 8. The membrane was hybridized with a Zgr-labeled probe obtained from a 4.5 t.p.n. fragment. (obtained by the amplification of the genomic DNA of Rpoyugparaiz with primers TN-5-TN-17). The distributions (band patterns) obtained from cosmid DNA 8AT11 and 2601 were identical to those obtained from genomic DNA cut in a similar manner on the same membrane. It was concluded that cosmids 8A11 and 2601 are "precise representations of the coding locus of genomic TeBA". However, cosmid 25058 has one Va fragment! 2, which is slightly larger than genomic DNA. This may be the result of the location of the insert within the vector. with

The DNA sequence of the gene encoding TseBA so

Hybridization analysis on the membrane of cosmid 2601 revealed a 4.5 tbp probe that hybridized with one large fragment of Esok 1 (larger than Ot.bp). This fragment was purified on a gel and ligated into the site of Esok 1 rvs

KZ (t), as described in Example 8, to create plasmid pVO-51/K1. The partial DNA sequence of the DNA insert of this plasmid was determined by "primer walking" from the flanking sequence of the vector using "the procedures described in Example 8. The subsequent sequence was generated by extension from new oligonucleotides that were constructed from the previously determined sequence. When compared with the determined DNA sequence for the ISBA gene, which was identified by other methods (which is described here as ZEO IU Mo11, as described in Example 12 below), full homology with nucleotides 1-272, 319-826, 2578-3036 and 3068-3540 (total number of bases - 1712). It was concluded that both approaches can be used to identify DNA fragments that encode the TebA| peptide. co Analysis of the amino acid sequence obtained from the IsBA gene

The sequence of the DNA fragment identified as ZEO IU Mo11 encodes a protein, the amino acid sequence of which is described here as ZEO IUO Mo12. Some features confirm the identity of this gene as a gene encoding their TeBA protein. The M-terminal peptide (ZEO IYUO Mo1: PNe Ie Sip Su Tug Zeg Avr Gei Re Su Avp Aga Aa) encodes amino acids 88-100. Internal peptide TeBA;; ToBA;-RTB81 (a) (ZEO IUO Mo23) encodes amino acids 1065-1077, and TsrA;-PT81 (B) o (ZEO IUO Mo24) encodes amino acids 1571-1592. In addition, the internal peptide TsrA-PT56 (ZEBEO IUO Mo22) encodes amino acids 1474-1488, and the internal peptide TsrA/-PT103 (5EO IUO Mo24) encodes amino acids 1614-1639.

It is obvious that this gene is an authentic clone encoding the TsrA peptide, which was isolated from preparations of the insecticidal protein of the strain MU-14 Rpoiyugpardis Ishtipevzsepz. The protein, which is isolated in the form of the TeBA peptide, was formed as a result of the cleavage of a longer peptide. This is proved by the fact that the nucleotides that code

M-terminal peptide ZEO IYUO My! TsrA;, have 261 bases in front of them (encoding the 87 nearest M-terminal

HF) of amino acids) of a longer open reading frame (ZEO IYUO Mo11). This reading frame begins

Ф nucleotides encoding the amino acid sequence Mei Sip Avzp Zeg Gets, which corresponds to the M-terminal sequence of the large peptide TsBA, and which is described here as ЗБЕБЕО ИХО Мо1б. It can be thought that TsrA is the precursor protein of TerA).

Relatedness of genes (cSBA, isav and isas

The isavB and isasC genes are closely related and can be transcribed as one mRNA (Example 8). Gene

ISA is located on cosmids that apparently do not overlap with cosmids carrying the isaB and isaS cluster, because corresponding genomic library screens identified different cosmids. However, a comparison of the 65 amino acid sequences encoded by the isavB and isasS genes with the IsSbBA gene revealed a significant degree of homology.

The conservation of amino acids (Rgoiveip Aidptepi Mode oi MasMesiogtm bedzuepse Apaiuviz Boimage, zsohipa taiih rat250, pazy maISHe-2; Kodak Zsiepiiis Itadipd Zuvietv, KosPpevieg, MU) is shown in Fig.4. On the score line of each part in Fig.4, the upwardly directed icons (7) indicate homology or changes in conserved amino acids, and the downwardly directed icons (M) indicate the absence of homology.

This analysis shows that the amino acid sequence of the TeBA peptide from residue 1739 to residue 1894 is highly homologous to amino acids 441-603 of the TsavB peptide; (162 of 627 amino acids P8B; ZEO IO Mo28). In addition, the sequence of TerA amino acids 1932-2459 is highly homologous to amino acids 12-531 of the TsaB peptide; (520 of 562 amino acids; 5EO IU Mo30). Considering the fact that the TeBA peptide (ZEO IU Mo12) contains 2505 amino acids, only 684 amino acids (2790) at its C-proximal end are homologous to peptides TsaB, or TsaB;, and 7/0 homologies are collinear with the location of the possible preprotein TsaV ( ZE and Me26). The size of the gap in the TSA homology coincides with the place of connection between the parts of TsaB; and TsaV;; TsaV preprotein. It is clear that the TeBA and TsavB gene products are evolutionarily related, and it is suggested that they share a common function(s) in Riojaugpavradivs.

Example 10

Characterization of zinc-metalloproteases in the broth of RIoioparadiv: Protease inhibition, classification and purification

Protease Inhibition Assay and Classification Assays: Protease assays were performed using

FITZ-casein, which was dissolved in water, as a substrate (final concentration in the test 0.08905). Proteolysis reactions were carried out at 259C for 71 hours in a suitable buffer with 25 μl of Riiogpardivz broth (total reaction volume 150 μl). The samples were also analyzed for the presence and absence of dithiothreitol.

After incubation, an equal volume of 1295 trichloroacetic acid was added to the sediment of the uncleaved protein. After sedimentation for 0.5 hours and subsequent centrifugation, 100 μl of the supernatant was placed in a 9b-well microtiter plate and the pH of the solution was adjusted by adding an equal volume of 4n Mahon. Then proteolysis was determined quantitatively, using a fluorometric tablet reader Ripogozsap I at a wavelength of

Excitation and emission at 485 and 538 nm, respectively. Protease activity was measured in the range of pH5.0 - 10 with increments of 0.5 units. The following buffers were used at a final concentration of 5 MM: sodium acetate (pH5,O - i) 6.5); Tris-HCl (pH7.O - 8.0) and bis-Tris-propane (pH8.5 - 10.0). To identify the class of protease (proteases) observed, the initial broth was treated with various protease inhibitors (final concentration of 0.5μg/μl) and then tested for protease activity at pHa.0 using the substrate described above. The couzo protease inhibitors they used included E-64 (I-trans-epoxysuccinylleucylamido|4-guanidino|-butane), 3,4-dichloroisocoumarin, leupeptin, pepstatin, amastatin, ethylenediaminetetraacetic acid (EDTA), and so 1 ,10-phenanthroline. "Mr

Protease determinations carried out over a range of pH revealed that there was indeed a protease (or proteases) exhibiting maximum activity at pH 8.0 (Table 16). Addition of DTT did not affect the protease activity. with

The stock broth was then treated with various protease inhibitors (Table 17). Treatment of the stock broth with the inhibitors described above revealed that 1,10-phenanthroline caused complete inhibition of all protease activity when added at a final concentration of 5 µg, with ISvo-bmcg in 100 µl of stock stock solution of 2 mg/ml. These data indicate that the most abundant protease(s) in the broth

Rioligpavdis are enzymes of the zinc metalloprotease class. " - "from the activity detected in the products " on the 1st day of Riojognardiv Ishtipezsepe (strain Uu-14)

Activity? so yu pinistliny s: LY N n so sl nit ny NN LY in 01000000 zv in 00100006 ole a Fluor. units - Fluorescence units (Maximum --28000; background --2200). 60 bo Control 13053 9)

vm me pibfenantolnya 10819 vooleyatn 00000116 petiine 116

Kontelzamso 00000000010011000000ю6000000000018

b Percentage of inhibition relative to protease activity at pH 8.0 s Inhibitors were dissolved in methanol 9 Inhibitors were dissolved in DMSO

Isolation of zinc-metalloprotease was carried out by applying the dialyzed precipitate obtained by precipitation with 10 - 8095 ammonium sulfate on a C) Zerpagose column equilibrated with 50 mM MagRO, pH7.0, as described in

Example 5 for the toxin Rioioparadizv. After intensive washing, a gradient of 0 - 0.5 M Mass was used to elute the toxin protein. Most of the biological activity and protein was eluted in the range of 0.15 - 0.45M mass. However, it was found that the main proteolytic activity was present in the 0.25 - 0.35M Mass fraction with some activity in the 0.15 - 0.25M Mass fraction. Analysis using electrophoresis in the PAAG-DSN fraction of 0.25 - 0.35M Mass showed the main peptide band of approximately boOkDa. The 0.15 - 0.25M Mass fraction contained a similar bOkDa band, but at a lower relative protein concentration. Further gel filtration of this fraction using a Ziregose 12 HE 16/50 column gave a major peak migrating at 57.5 kDa, which contained the predominant (29095 of total stained protein) band at 58.5 kDa when analyzed by PAAG-DSN urine electrophoresis. Additional analysis of this fraction using the various protease inhibitors described above indicated that this protease was a zinc metalloprotease. Almost all the protease activity present in the broth of

Ripoiugpavadiz on the 1st day of fermentation corresponded to zinc metalloprotease -58 kDa.

In the second isolation of zinc-metalloproteases (proteases), strain MuU-14 Rpoiiognardiz Ishtipezsepv was taken, which grew for 3 days, and the protease activity was visualized using gel electrophoresis in a polyacrylamide gel with sodium dodecyl sulfate (PAAG-DSN), with the addition of gelatin, as described |Zsptiai, i. t., WieaKieu, V. apa

Meaivop, K. M. 1988). Separating gels used with SDS (5.5 x 8 cm) were prepared with 12.595% polyacrylamide (4095% acrylamide/bisacrylamide stock solution; Zidta SNetisai! Z., Z. Gotziz MO), to which gelatin was added at a final concentration of 195 ( Viogad EIA dgade geadepi; Kisptopa SA) when dissolved in water. Concentrating

DeCH-gels (1.0x8 cm) were prepared with 595 polyacrylamide, also with the inclusion of 0.196 gelatin. As a rule, 2.5 μMkg s of the protein to be tested was diluted in 0.0 ml of loading buffer for electrophoresis in PAAG-DSN s without dithiotrechtol (DTT) and applied to the gel. Proteins were separated in DSN-buffer for separation (I aettii, O.K. 1970.

Maishge 227, 680) at 02С and at 8mMA. After completion of electrophoresis, the gel was washed for 2 hours in 2.590 (vol./06.) Triton X-100. Then the gels were incubated for 1 hour at 37 2C in 0.1M glycine (pH8.0). After incubation, the gels were fixed and stained overnight with 0.195 amido black in a mixture of methanol-acetic acid-water (30:10:60, vol./vol./vol.; Bidta SNetis! So.,). Protease activity was visualized in the form of light zones against a dark, amido black-stained background, caused by proteolysis and subsequent diffusion of included gelatin. At least three distinct bands produced by proteolytic activity were observed at 58, 41, and 90% ZakDa.

Analyzes of the activity of various proteases in the culture broth of strain MUU-14 on the third day of fermentation were performed using FITC-casein dissolved in water as a substrate (final concentration in the test was 0.02906). Experiments on proteolysis were carried out at 372C for 0 - 0.5 g in Tris-HCl (pNaV,0) with different protein fractions in a total volume of 0.15 ml. Reactions were stopped by adding an equal volume of 1295 trichloroacetic acid (TCA) dissolved in water. After incubation at room temperature for 0.25 g, the sample was centrifuged at 10,000 x 9 for 0.25 g, and aliquots of 0.1 Oml were taken and placed in 96-well 20 microtiter plates. Then the solution was neutralized by adding an equal volume of 2N sodium hydroxide and quantitative measurements were performed using a fluorimetric tablet reader with wavelengths of excitation and emission of 485 and 538 nm, respectively. Activity measurements were performed with FITC-casein with different concentrations of protease at 372 for 0 -10 minutes. The unit of activity was arbitrarily set as the amount of enzyme required to produce 1000 fluorescence units per minute, and the specific activity 22 was defined as units/mg of protease. Inhibition studies were performed using two inhibitors

Ge) zinc metalloprotease: 1,10-phenanthroline and M-(a-rhamnopyranosyloxyhydroxyphosphinyl)-I en-Tgtr (phosphoramidon) with initial solutions of inhibitors dissolved in 10095 ethanol and water, respectively. The starting concentrations were typically 100 mg/ml and 5 mg/ml for 1,10-phenanthroline and phosphoramidon, respectively, with final inhibitor concentrations of 0.5-1.00 mg/ml of the reaction mixture. Treatment of the three-day initial broth of UU-14 60 with 1,10-phenanthroline, an inhibitor of all zinc-metalloproteases, led to the complete elimination of all protease activity, while treatment with phosphoramidon, an inhibitor of thermolysin-like proteases |(MUeamg,

G.N., Kevieg, MuU.K., apa Maipemuv, V.M/. 1977, 9U.Moi. Vioi 114, 119-132), led to "5695 reduction of protease activity. The remaining proteolytic activity did not decrease further with the additional addition of phosphoramidon. Proteases of the three-day-old broth of MuU-14 of Riioiiogpardis were purified as follows: 4.0 liters of the broth were concentrated using a spiral ultrafiltration cartridge Artichoke type 51MISO, which is connected to an Artichoke M-12 filtration device. The material passing through, which had native proteins less than 100kDa in size (3.8L), was concentrated to 0.375L using the indicated ultrafiltration cartridge.

The material remaining on the filter contained proteins in the range of 10 - 100 kDa. This material was applied to Column NK16/10 RNaghtasia, which was packed with strong anion exchange material Regzeriime Viovuziet (Egatipdiop, MA) Rogose? HO), which was equilibrated in 10 mM sodium phosphate buffer (pH7.0). Proteins were applied to this column at a flow rate of 5 ml/min., then the unbound protein was washed away with Agovo-0.00 buffer. Then the proteins were eluted using a gradient of 0 - 1.0 M Mass within 40 minutes at a flow rate of 7.5 ml/min.

Fractions were assayed for protease activity as above, and active fractions were pooled. Proteolytically 70 active fractions were diluted 2 times (by volume) with 10 mM phosphate buffer (pH7.0) and applied to a 10/10 Mogo O RNAghtasia column equilibrated in 10 mM sodium phosphate. After washing the column with buffer to A 280-0.0, the proteins were eluted using a gradient of 0 - 0.5M Mass within 1 hour at a flow rate of 2.Oml/min.

Fractions were analyzed for protease activity. The fractions that had the greatest amount of phosphoramidon-sensitive protease activity, which was due to the 41/38 kDa protease, Ipita, were combined. It was found that these 75 Fractions were oiled in the range of 0.15 - 0.25M MassSi. Fractions containing the predominant amount of phosphoramidon-insensitive protease activity, 58 kDa protease, were also combined. It was found that these fractions melt in the range of 0.25 - 0.35M Mass. Then the phosphoramidon-sensitive protease fractions were concentrated to a final volume of 0.75 ml using a Viotakh-5K MMUMI membrane of a centrifugal filter device

Miyiroge Shga-itee?. This material was applied at a flow rate of 0.5 ml/min. to a Rpagtasia HE column. 10/30, which was packed with Zerpadeh 0-50 (Rpagtasia), equilibrated in phosphate buffer (pH7.0) (10mM sodium phosphate-0.1M Mass). Then the fractions that had the maximum phosphoramidon-sensitive activity were combined and centrifuged through the Viotakh-5bOK MMUU membrane of the MiSroghe centrifugal filter device

Wow?9.15, Analysis of proteolytic activity, wheezing, showed that this material had only phosphoramidon-sensitive protease activity. After combining the phosphoramidon-insensitive protease, protein 58 kDa, M, it was concentrated in the same device and further separated on a Zyregaech-75 column (Riagtasia). Fractions containing this protease were combined.

Analysis of the purified 58kDa and 41/38kDa proteases revealed that, while both types of protease were completely inhibited by 1,10-phenanthroline, only the 41/38kDa protease was inhibited by phosphoramidon. Further analysis of the stock broth showed that the protease activity of the one-day MU-14 broth had 2395 total M) of protease activity due to the 41/38kDa protease, increasing to 4495 in the three-day MU-14 broth. co

Standard analysis using electrophoresis in PAAG-DSN to test the purity of the protein and obtain the amino-terminal sequence was performed using 4-2095 gradient gels (MipiRiiz Zergasei!v), "I purchased from Ipigedgaiye Zeragaiop BZuvietv (MayisK, MA). Proteins, which were to be subjected to amino-terminal sequencing, were blotted onto a PMOE membrane after purification, IPTA (Rhobiosystem Metrhapez; Arriea

Zo Viovuzietv, Eovieg Sue, SA), visualized with amido black, excised, and sent to Satbrgidde 89

Rhospet; Satgidde, MA, for sequencing.

The deciphered amino-terminal sequence of proteases of 58kDa (ZEBO and Mo45) and 41/38kDa (5EBEO IYUO Mo44) from the broth of three-day M/-14 was: OM-OZEKAMEKI K (5EO IYU Mo45) and OBSOOSOKMTMTOINK (ZEO I Mo44), respectively. Sequencing of the 41/38 kDa protease revealed multiple amino termini, each of which had an additional amino acid that was removed by proteolysis. Examination of the primary, secondary, tertiary and quaternary sequences for the 38 and 41kDa polypeptides allowed the above sequence to be deciphered and ""revealed that these two proteases are homologous." Example 11, Part A

Screening of the genomic library of Rpoiogpardis using antibodies against genes encoding the TerA peptide

In parallel to the sequencing described above, the relevant probing and sequencing was performed on the co TerA peptide; (5EO IO Mo1). This sequencing was performed by obtaining bacterial culture broths and purifying the toxin as described in Examples 1 and 2 above. Genomic DNA was isolated from the MU-14 strain of Ripoiogpardyv

Ishtipezsepz grown on Grace's medium for insect tissue culture. Bacteria were grown in Bbml of culture medium in a 250 ml Erlenmeyer flask at 282C and 250 rpm for approximately 24 hours and 20 hours. Bacterial cells from 100 ml of culture medium were precipitated at 5000 x 9 for 10 minutes.

The supernatant was discarded and the cell pellet was then used to isolate genomic DNA. sl Genomic DNA was isolated using a modification of the CTAB method, which is described in Section 2.4.3 of Aizibei (Zirga).

The section with the heading ""where Zsaie Sv8vSiI rger oi rasiegiai depotis OMA" was used up to stage 6 (inclusive). At this point, an additional extraction of DNA with a mixture of chloroform/isoamyl alcohol (24:1) was carried out, with the next stage of extraction with a mixture of phenol/ chloroform/isoamyl alcohol (25:24:1) and the final stage

Ge) extraction with a mixture of chloroform/isoamyl alcohol (24:11). DNA was precipitated by adding 0.6 volume of isopropanol.

The precipitated DNA was caught and wound onto the end of a bent glass rod, immersed briefly in 70905 L ethanol as a final wash, and dissolved in 3 mL of TE buffer.

The concentration of DNA, which was determined by optical density at 280/26 Ohm, was approximately 2 mg/ml. 60 A genomic library was obtained using this genomic DNA. Approximately 5 µg of genomic DNA was partially cleaved with the restriction enzyme bacZ At. Then, centrifugation in a MacSi gradient was used for size fractionation of partially cleaved DNA fragments. Fractions containing DNA fragments with an average size of 12 tbp. or more, as determined by agarose gel electrophoresis, ligated into the plasmid Winpezsgiri, Ziga(adepe, Ia doia, Saijogpia, and transformed into strain ONba or ONVOE. soya. bo Separately, purified aliquots of the protein were sent to the center of hybridoma biotechnology at University of Wisconsin

(Madizop) to obtain monoclonal antibodies to these proteins. The material sent was an HPLC-purified fraction containing native bands 1 and 2, denatured at 652C, and 20μg of each injected into each of four mice. Stable cell lines producing monoclonal antibodies were obtained after fusion of spleen cells from non-immunized mice with a stable myeloma cell line. Monoclonal antibodies were obtained with a hybrid.

Separately, polyclonal antibodies were obtained by taking band 1 protein purified on a native agarose gel (see Example 1), which was then used to immunize a New Zealand white rabbit. Protein was obtained by cutting a band from native agarose gels, briefly heating the gel pieces to 65923 to melt the 70 agarose, and immediately emulsifying with adjuvant. Complete Freund's adjuvant was used for primary immunizations and incomplete Freund's adjuvant was used for additional injections at monthly intervals.

For each administration, approximately 0.2 ml of emulsified strip 1, containing 50 - 100 μg of protein, was delivered by multiple subcutaneous injections into the back of the rabbit. Serum was obtained 10 days after the last injection and additional bloodlettings were performed at weekly intervals for 3 weeks. Serum complement 75 was inactivated by heating to 562C for 15 minutes and then stored at -202C. Monoclonal and polyclonal antibodies were then used to screen the genomic library for the expression of antigens that could be detected by the epitope. Positive clones were detected on nitrocellulose filters, which were used for colony lifting. Immunoblotting of positive clones was used.

Analysis of clones determined by both immunoblotting and Southern analysis led to the experimental identification of five classes of clones. In the clone of the first class there was a gene that encodes a peptide, which is called here TebBA and.

The complete DNA sequence of this gene (TebBA) was obtained. it is presented as ZEO IYUO Mo11. Confirmation that this sequence encodes the internal sequence of ZEO IU Mo1 is shown by the presence of ZEO IUO Mo1 at amino acid position 88 of the decoded amino acid sequence, which is created by the open reading frame of 5EO IU

Mo11. This can be confirmed by reference to ZEO IU Mo12, which is the decoded amino acid Ha sequence created by ZEO IU Mo11. The second class of toxin peptides includes the segments referred to above as TsaV;, TsaV; and Tsas. After screening the library with polyclonal antisera, this second class of toxin genes was identified as several clones producing proteins of different sizes, all of which cross-reacted with the polyclonal antibodies on the immunoblot and were found to have DNA homology on the Southern blot. A comparison of the sequences revealed that they belong to a complex of genes that Io) is called above TsavB and Tsas. Three other classes of clones were also identified by polyclonal screening. These classes produced proteins that cross-reacted with polyclonal antibodies and also had DNA homology to these classes as determined by Southern blotting. hE

These classes have been named Class I, Class IM, and Class M. Monoclonal antibodies that cross-react with Classes I!, II, AI, and IM can also be detected. This suggests that all of them have a high homology with protein. Thus, it is obvious that the genes of extracellular proteins of Rpoiogpardis Ishtipezsepz represent a family of genes that are evolutionarily related.

To further develop the concept that there may be evolutionarily related variations in the toxin peptides found in this organism, two approaches were taken to examine other strains of Rpoiogpardis Istipezsepz for the presence of related proteins. This was done by PCR amplification of genomic DNA and immunoblotting using polyclonal and monoclonal antibodies. in c These results show that related proteins are produced by strains of Rpoyugparadiz Ishtipevzsepe MUH-2, M/X-3, c MUH-4, MUH-5, MUH-6, MUH-7, MUH-8, MUH-11, MUH- 12, Mukh-15 and Mukh-14. "» Example 11. Part B

Sequencing and analysis of Class PI-yss toxin clones

Further DNA sequencing was performed on plasmids that were isolated from E. soya Class III clones described (se) in Example 11, Part A. It was shown that the nucleotide sequence was three closely related open reading frames at this gene locus. This locus was named (es), and the three open reading frames were named issA 5EO IUO Mo56, issB ZEO IU Mo58 and issS ZEO IU Mobo (Fig. 6B). The deciphered amino acid c" sequence from the open reading frame of issA shows that this gene encodes a protein of 10545 DA. This protein was named TssA. The first 12 amino acids of this protein match the M-terminal sequence that was obtained from the 108 kDa ZEO IU Mo7 protein, which was previously identified as part of the toxin complex. c Deciphered amino acid sequence from the open frame reading of issB shows that this gene encodes a protein of 175716Da. This protein was named TosB. The first 11 amino acids of this protein match the M-terminal sequence that was obtained from a protein with a determined molecular weight of 185kDa 5EO IU Mov.

The deciphered amino acid sequence of ysC shows that this open reading frame encodes the protein 111694Da and this protein product was named Tess. (F) Example 12. Characteristic of Riojogpardy strains

To confirm that the collection described here consists of Rpoiogparadive strains, these 60 strains were evaluated for microbiological characters that are characteristic of Rioiogpardys and distinguish it from other species of Epiegorasiegiaceae and from species of Hepogpardys (Ragteg, ..). 1984. Wegdeuz Mapia! Oi Zuzietis

Vasiegioodu, my I, years 510-511. MUMShiatwe apa M/UyKipv, Waiitoge.;

AkKkpygvi apa Voetage, 1988, Voetage ei a!., 1993). These characteristic features are: Gram-negative rods, organism size 0.5 - 2 µM wide and 2 - 1 µM long, red/yellow pigmentation of colonies, presence of 65 crystalline inclusion bodies, presence of catalase, inability to reduce nitrates, presence of bioluminescence, ability to absorb dye from media for growth, positive reaction to protease production, temperature limit of growth below 37 C, survival under anaerobic conditions and motility (Table 18). Standard strains of EzspPegispia soii, Hepogpardis and Rpoiogpardis were included in all tests for comparison. All results agree that all strains belong to the family Epiegobacieegiacede and the genus Rpoyugtabraise.

A luminometer was used to detect the bioluminescence of each strain and provide a quantitative and relative measurement of light production. To measure relative light emission units, broths from each strain (cells and media) were measured at three time intervals after inoculation in liquid culture (6, 12, and 24 h) and compared to background light (non-inoculated media and water). Before 7/0 measurement of light emission from different broths, cell density was determined by measuring light absorption (5bonm) in a Siyoga Zuzietwe spectrophotometer (ObBegip, ON) using a zirreg element. Then suitable dilutions were made (to normalize the optical density to 1.0 units) before measuring the illuminance. Then aliquots of the broths that were diluted were placed in cuvettes (30 Ωcl each) and the readings of the Vio-Ogria 1251 lumenometer (Vio-Ogria Su, Tm/iki, Ripiapa4) were taken. The integration period for each sample was equal to 45 7/5 seconds. Samples were continuously agitated (swirled in baffled cuvettes) during readings to ensure access to oxygen. A positive response was defined as "5 times the background luminescence (-5 - 10 units). In addition, the luminosity of the colonies was detected by overlaying on photographic films and visually, after adaptation in a dark chamber. The Gram staining characteristics of each strain were determined with a commercial Gram staining kit (VVI, Soskeuzmiye, MO) used together with control Gram staining slides (Rizspeg Zsiepiyis, Riivrygo, RA). Then a microscopic evaluation was carried out using a Zeiss microscope (Sagi 7eizv, (zeppapu) with a 100x oil immersion objective (with an eyepiece magnification of 10x and a case magnification of 2x). Microscopic examination of the sizes of individual strains of the organism, description of cells and inclusion bodies (the latter - after logarithmic phase of growth) were performed using wet microscopic preparations (uei toipi ziides) (10x eyepiece magnification, 2x housing magnification and 40x objective lens magnification) using oil immersion microscopy and phase-contrast microscopy with a micrometer

IAKpigvi, K.). apa Voetage, M.E. 1990. Epiotoraiiodepis Metaiiodes ip Viioidisa! Sopigo! (ie. Satsdieeg, K. apa o

Kaua, N.). years 75-90. SK5 Rgezz, Vosa Kaiop, OA; Vaodpaiidchciap 5., Voueg-Sidio MN, Tnaieg, 9U.0., Voppoi 0.,

Voetage M. 1993. VioI. SeiI 79, 177-185). Pigmentation of the colonies was observed after inoculation on Bactoagar (Wasio piigiepi adag, Oso Iarogaiogiez, Yuoigoii, MI) which was prepared according to the instructions supplied with it. Incubation took place at 28 2С, and descriptions were made after 5 - 7 days. To test for the presence of the catalase enzyme, a colony of the organism being tested was removed from a plate with nutrient agar on a small plug and placed at the bottom of a glass test tube. One ml of the hydrogen peroxide solution prepared in the CHE laboratory was carefully added along the side wall of the test tube. A positive reaction was registered when bubbles of gas appeared immediately or within 5 seconds (as it was assumed - oxygen). Controls with non-inoculated nutrient agar and hydrogen peroxide solution were also studied. To determine the recovery of nitrates, each culture was inoculated into 1 Oml of Bacto-Nitrate Broth (Oiiso Iarogaiogiez, Ohio, MI). After 24 hours of incubation at 282, the formation of nitrites was determined by adding two drops of sulfanilic acid reagent and two drops of alpha-naphthylamine reagent |see .

Ohio, MI, 1984). The formation of a clear pink or red color indicates the formation of nitrite from nitrate. The ability of each strain to absorb the dye from the growing medium was determined with MasSopkKeu bactoagar containing neutral red dye; with Bactoagar Teguilloi-7 containing the dye bromothymol blue and Bactoagar EMV containing the dye Eosin-U (Agars from Oiso and Arogaigies, Juegoii, MI, all "" were prepared according to the accompanying instructions). After inoculation on these dye uptake media were recorded after incubation at 28 oC for 5 days. Growth on these media is characteristic for members of the Epiegorasiegiaceae family. The motility of each strain was determined using the motility test medium (Vasu Moitsiu Tevzi Meaisht MI), which was prepared according to the accompanying instructions, was stably inoculated with each strain and motility was assessed macroscopically by a diffuse zone of growth extending from the inoculum line.

FT" In many cases, motility was also observed under a microscope from a liquid culture on wet preparations 20 (of which toshpi viiedev). Biochemical nutritional assessment for each strain was performed using VVI. Epiegoishbe I so (Vepyup, OisKipzop, Segtapu). The recommendations accompanying this product were followed, except that the incubation was carried out at 282C for 5 days. The results agreed with previously reported data for

RIioiogpardiv. Protease formation was determined by monitoring the hydrolysis of gelatin using bactogelatin (Oso Iarogaigiez, Ohio, MI), plates were prepared according to the included instructions. 29 Cultures were inoculated and plates were incubated at 282C for 5 days. To assess growth at various temperatures, agar plates (295 µg/ml MoZ with 27 µg Bactoagar (Oysso I Arogayuz, Ohio, MI)) in deionized water were inoculated with streaks from a common inoculum source. 372C for a period of up to three weeks. Cups that did not show growth at 37 C did not show viability after being transferred to a thermostat at 28 C for one week. Oxygen requirement 60 for strains of Riojogpardis were tested as follows. containing thioglycollate (Oso I arogayugiez, YOeigoii MI). The tubes were incubated at room temperature for a week and then the cultures were tested for type and degree of growth. Resazurin indicator shows moderate oxidation level or aerobic zone (Oyso Mapia), 10-2 eaiyop, YOyso Iabrogaiyugiez , YuOeiigoi, Mi).

The results for the growth zone obtained for the strains of R. coli that were tested correspond to the results for the facultative anaerobic microorganism.

Table 18

Taxonomic features of RvobogNardyiiv iatipevsepa

Signs that evaluate them

Strain c b I Iii i 14 x xx / dky| x x x x x x E x x x 5

A- gram-positive strain, B- crystalline inclusion bodies, C- bioluminescence, JO- cell shape, E- motility, E- nitrate reduction, C3- presence of catalase, H- gelatin hydrolysis, I- dye absorption, .)- pigmentation, K - growth on EMV agar, I - growth on MasSopKeu agar, M - growth on TegoyoI-7 agar, M - facultative anaerobe, O - growth at 202C, P - growth at 282C, 0 - growth at 372C; s (8) iv) (ge) "I s so - s;" co

FT" with "sl

F) ko 60 b5 mo LEADING BA PENI BA PENI BA PENI BA PENI NB A NBN NBN NO NBN ; х хи хЗ| and-th their th th Cl J her th-th th x-2 x x | кй х х х х х х ХЕ х

WITH

Vk Y NEON NBN A NYA NOY NO NEO PO Po NYA MO NO NO NO NBN NO m in NO I o Z PI ZH NO NYA NO oo NBN NI NO PNO BI NO M

WITH

On NEO I METY NO M NO SONG oo NO NI BUT NO EN NOT YET 5 ; And i x | x4| х х х VIV хх х х х х и 5 и хх Z frИ4Й| х х и и х х х им и

WITH

ХВ и х Гтх4| 5 х х х х х х х х 5 m a NE PED EI NO NO NI NS PENYA os NO PE NO PES NO BJ 5 th || x x y || Х Х Х Х Х

With I | hehe hehe ||| 111 p. 8 o

PSIO NO PE PE EZIA NO NO NEO PE PE NBN NO NI PENYA NI NI NEON

WITH

Uk kh yah | hee | х х и х|BK| x x x x x x x x x)

M ii PEUGEOT NEO PE PE NO NO NO NEO MEN oo NO NO NO NEO NO NO PE NA 5

VALY NYA EI I NYA NYA NYA I s NYA NYA NYA NYA PE NYA I 5 MY 5 cm x Z r|rIhIIA | x y x x | ХТ| x x Z x il x « so

Ble BD I NO NO NO NO NO Be PEN NEO NI NS NO NBN

WITH

ROAR | хих хх их | ii « 5 zo el - r kiy GG khz hih no) s 5 zam zhi PEV NYA PE I NO NO PE NO NEO I PI NO PBS NO NEO BZH p 5

MIV Хх Х Х Х Х Х Х Х Х Х 8 am Bo El PED NO I PE o NOT PE PE PE PI PES NO PEN NO NO PEN W t 5 ra y h | k4| 5 of the same X X X X in Gt Tat TTN (EE). penya lo lyn oh jo minya, nin nin nya nya. nn --m- min. nni ini i- zh minya oh nnya nnya zh, nn - nn nn chu. «сл ж/- positive or negative in relation to the sign, stick, 5- suitable in size for this genus, КО- red-orange, ЙК- bright red, К- red, О- orange, У- yellow, T- reddish-brown, I U - light yellow, UT - yellow-reddish brown, I O - light orange. вы ДЯ Я Я и и ПЕ НБН ПЕ ПЕН М ЛОЖИ ПЕ ПК MB о В я ж KZ ж я К Бы ж х жк Э - zy Po PI V NEO Pe A PE PE PEJO NI PSY PE PE NEK NEO PEJO TOWERS EY sl io PED VE BY BA PE PE PE PE PE PE PE PE V PES PBS PENYA PEN 5 oh PED SUN NO EI NO I NO NO PES NEO PO PO PO MEN NO NO NO

Z g AT b5 Z

Cell fatty acid analysis is a recognized tool for bacterial characterization at the genus and species level

Pogpabepe T.0. 1985. Iiriy Apaiuziv apa Keiaiopepir 0 SPetoiachopotu ip Meiyodz ip Misgorioiodu. My 1.18, 209-224; Soodeyom, M. apa O'boppei, A... 1993. Kooiv oi Vasieegiai Zuzietaiyysv (ey. SoosePyom, M. apa

O'Vapiei, A.O.) years 3-54. | opdop: Asadetis Rgezz Ts.), which are included here as a reference and used to confirm that our collection is related at the genus level. Cultures were sent to another contract laboratory for analysis of fatty acid methyl esters (FAME) using the Microbial Identification System (MI5) from Misgoriai I (MIOI, Memzhagk, OBA). The MIZ system consists of a NemLei Raskaga NRB5VOOA gas chromatograph with a column of fused 595 methylphenylsilicon capillaries, which are lined with 7/0 silicon dioxide (25mM x 0.2mm). Hydrogen is used as a carrier gas and the detector based on plasma ionization works together with an automatic sampler, an integrator and a computer. The computer compares the fatty acid methyl esters of the samples to a microbial fatty acid library and compares to a calibration mixture of known fatty acids. At the discretion of the contract laboratory, strains were grown for 24 hours at 2823 on trypticase soy agar before analysis. Sample extraction in contract laboratory 75 was carried out according to standard EAME methodology.

There was no direct identification of strains relative to any luminescent bacterial group other than

Rpoyugpabayiiz. When performing a cluster analysis comparing the fatty acid profiles of a group of isolates, the fatty acid profiles of the strains were related at the genus level.

The evolutionary diversity of the strains of Rioiogpardis in our collection was measured by analyzing PCR-mediated genomic fingerprinting using genomic DNA from each strain. This method is based on families of repetitive DNA sequences present throughout the genome of bacterial species that differ |see review Megzaiomis, ..,

Zsppeideg, M., Oe Vgyip, E.U. apa I oreKki, UK. 1994. Meiyodvz Moi. SeiI. VioiI.,, 5, 25-40). It is believed that three of them, the extragenic palindromic repeating sequence (KER), the enterobacterial intergenic si 2rb Kconsensus repeating (EKIS) and the VOX element, play an important role in the organization of the bacterial genome. It is believed that the organization of the genome is created by selection, and the differential dispersion (scattering) of these o elements in the genome of closely related bacterial strains can be used to distinguish these strains (for example, I oshtsmuv, B.9U., Eshrgidny O.MU., e(Ferpepz , ST apa OE Vgyip, R.). 1994. Arri. Epmigop.

Misgo. 60, 2286-2295). Ker-RSK uses oligonucleotide primers complementary to these (U) repetitive sequences to amplify DNA fragments of different sizes that lie between them. The resulting products are separated by electrophoresis to establish a DNA "fingerprint" for each strain.

To isolate genomic DNA from our strains, cell sediments were resuspended in TE buffer (10 mM

Tris-HCl, 1mM EDTC, pHNeB, 0) to a final volume of 10ml and then 5M Mass was added. This mixture was centrifuged for 20 s and 5 minutes at 15,000x9. The obtained sediment was resuspended in 5.7 ml of TE and added ZOOmcl 1095 DSN and c bOmcl 20mg/ml proteinase K (Sirso VKI Rgodisiv, Sgapa IziIapa, MU). The mixture was incubated at 372C for 1 h, then approximately 10 mg l of isozyme was added and the mixture was incubated for another 45 minutes. Then one milliliter of 5M Masi and 800 μl of CTAB/Masi solution (1095 m/v CTAB, 0.7M Masi) were added and the mixture was incubated and mixed for another 20 minutes to improve removal of cellular material. An equal volume of chloroform/isoamyl alcohol solution (24:106w/06) was added, mixed carefully and then centrifuged. Then three extractions were carried out with an equal volume of a mixture of phenol/chloroform/isoamyl alcohol (50:49:11). Genomic DNA was precipitated with 0.6 volume of isopropanol. Precipitated DNA was removed with a glass rod, washed twice with 7095% ethanol, dried and dissolved in 2 ml of STE (10 mM Tris-HCII, pH 8.0, 100 mM Mass, 1 mM EDTC). Then the DNA was quantified by optical density at 2 bOhm. To perform Ker- The following primers were used for the RSC analysis of the genomic DNA of RPoyugpavraiv: KERIK-I: 5-IPISOISSISATSIVOS-3 and KER2-1: 5-ISVISTTTATSIVOSSTAS-3 PCR (se) was performed using a reaction volume of 25 μl: 7.75 μl of water, 2.5 μl 10X buffer GA (Rapmega Sogr., Madizop,

M/I) 16 μl of aMTP mixture (2.5 mM each), 1 μl of each primer at 500 pM/μl, 1 μl of DMSO, 1.5 μl of genomic DNA o (with a concentration in the range of 0.075 - 0.48 Ωkg/μl) and 0.25 μl of TaKaKa EX Tad (RapMega Sogr., Madivop, UM). t» PCR amplification was carried out in a DNA thermocycler Regkip EIteg (MopoikK, ST) using the following conditions: 959077 sec for 50 minutes, then 35 cycles: 942С/1min., 652С/8min., then 1bmin. at 6520. After carrying out the cycles, 25 μl of the reaction mixture was added to 5 μl of 6X buffer for applying to the gel (0.25956 bromophenol blue, 40905 m./v. "(pl sucrose in HO). Then electrophoresis was performed in a 195 agarose gel 15x20 cm in TBE buffer (0.09M Tris-HCl, 0.002M EDTC) using 8 µl of each PCR reaction.Electrophoresis was performed for approximately 16 hours at 458. The gels were then stained in 20 µg/ml ethidium bromide for 1 hour and destained in TBE 59 buffer for approximately 3 hours . The gels were then photographed with a Polaroid under UV light illumination. o The presence or absence of bands at strain-specific sizes was assessed from these photographs and entered as similarity matrices into a digital computer taxonomy program, MTZU5-rs (Ehefeg Zoimage, Zegatskyi, Ko MU) Controls, strain RY101 E. Soyii and Hepogppardyvs ogugaae ru. U.K. 1991. 60 Mysivys Asidz Bevs. 19, 6823-6831; Mega Sgy7, SM., Naida-Aiya, I. Iov, R., ZKippeg, O.7., Seogde, M.Y.,

Meyvop, K.Yu., OEE Vgyip, B.), Kise, S. and Ieasp, U.E. 1995. |p7i Kise Kev. Moiev5, 20, 23-24; Mega Sgy?,

SM., Agaaiez, E.U., ZKippeg, O.7., Taiad, 9U., Meivop, K.U., Gotsmuv, E.), Getspd, N., Mem, T.MU. apa I easi, U.E. 1996. РНуиораипоиоду (ip rzhezv5, gezresiimeIu)). Then, the data for the strains of RIoiigpardis were analyzed with a series of programs in MTZUZ-rs; ZIMOSHAG (Similarity for Quantitative Data) to obtain a matrix of similarity coefficients (using the Jaccard coefficient) and the formation of ZANM-clusters (Zedpepiia), Addiotegaiiime, Neigagspisa!

apa Mezied) |with the use of OROMA (puedpi Raig-Stgotsr Meijod myp AgpAteiis Amegadev)-method)| that group related strains and can be expressed in the form of a phenogram (Fig. 5). The programs SORN (co-phenetic values) and МХСОМР (comparison of matrices) were used to obtain the matrix of co-phenetic values and compare the correlation between it and the original matrix on which the formation of clusters is based. The resulting normalized Mantel statistic (r) was obtained, which is a measure of agreement for cluster analysis (1-0.8-0.9 means very good agreement). In our case, g-0.919. Thus, our collection consists of a diverse group of easily distinguishable strains that are typical of the genus

Repoujnaraitsv 70 Example 13

Suitability as insecticides of the toxin(s) produced by strains of Rpoiogpavrdiv

Source cultures for sowing different strains of Riojopardis were obtained by inoculation of 175 ml of 295 Rgoieose

Rerup Moz (RRZ) (Oiiso I abogaigiez, Yueigoi, MI) of liquid medium primary variant subclone in a 500ml three-wall Delong-necked, Karsha-capped flask. The inoculum for each culture for 7/5 inoculation was obtained from oil-infused cultures on slanted agar or from plate cultures. After inoculation, these flasks were incubated for 16 g at 28°C on a rotary shaker at 150 rpm. These inoculation cultures were then used as homogeneous sources of inoculums for the specific fermentation of each strain. In addition, coating the post-log phase growth culture with sterile mineral oil, adding a sterile magnetic rod for subsequent resuspension, and storing the culture in the dark at room temperature ensured long-term preservation of the inoculum in a toxin-competent state.

Production broths were inoculated by adding 195 actively growing seed culture to fresh 295

RRZ medium (for example, 1.75 ml per 175 ml of fresh medium). Broths were prepared either in flasks with three partitions of 500 ml (see above), or in flasks with a convex bottom of 2800 ml (volume 500 ml), closed with corks made of organosilicon foam. The production flasks were incubated for 24-48 hours under the conditions described above. After incubation, the broths were distributed into sterile polyethylene bottles at Tl, spun at 2600x9 for 1 hour at 102C and decanted from the cell sediment and cell remnants. Then the liquid broth i) was filtered under vacuum through M/paytap SRH/O filters (2.7 μM retention) and SR/B glass filters (1.0 μM retention) to remove cell debris. Further clarification of the broth was achieved using a tangential flow microfiltration device with open channels of 0.5 μm. If necessary, additional lighting can be achieved by cooling the broth (to 42C) and centrifuging for several hours at 26b00x9. After these procedures, the broth was passed through a sterilizing filter using a 0.2 μm nitrocellulose membrane. Then, the sterile broths were used directly for chE biological analysis, biochemical analysis or concentrated (up to 15 times) using a cutting mol. mass 10000 ultrafiltration device M12 (Artichoke, Vemepu MA) or centrifugal concentrators (Mijiroge, Veatoga, MA and Rai! Riigop, Mopyprogotsdy, MA) with a pore size of 10000 (mol. mass). In the case of centrifugal concentrators, the broth was spun at 2000x9 for about 2 hours.

The material that passed through (permeate) from mol. with a mass of 10,000 was added to the corresponding material retained by the filter (retentate) to achieve the desired concentration of components with mol. with a mass greater than 10,000. "

Inactivation by heating samples of the treated broth was achieved by heating the samples at 100 9 in a sand-filled heating block for 10 minutes. - c Broth (broths) and complex (complexes) of toxin from different strains of Rpoiogpardis are applicable for "" reduction of insect populations and have been used in the method of inhibiting the insect population, which involves "applying to the insect cell an amount of the described active component effective for inactivating insects."

A demonstration of the breadth of insecticidal activity observed with broths of a selected group of Riojopardy strains fermented as described above is shown in Table 19. It is possible that additional insecticidal activity could be elicited with these strains by increasing the concentration of the broth or by using different fermentation methods. According to the protein-bound activity, the insecticidal activity of all tested strains was thermolabile (see above). The culture broth(s) of various strains of Riojopardis show differential insecticidal activity (mortality and/or growth inhibition, reduced hatching of adults) against larvae of the long-bearded flea and larvae of the cotton weevil, which are members of the order of insects Soieoriega. Other members of the SoiIeoriega include «sl wireworms, the canola flower borer and the Colorado potato beetle. Activity against aster leafhoppers, which are members of the Notoriega order, was also observed. Other members of the Notoriega include cycads, pear leafhoppers, common honeybees, vine worms and scale insects, whiteflies, shrisse beetles, and numerous 22 host-specific species of thallus. Broths and purified complexes of the toxin are also active against the scoop and the grain grinder, which are members of the series I eridoriega. Other typical members of this order are the small moth, the cabbage moth, the upsilon moth, the cotton moth, the apple moth, the true moth, the southern barn firefly, io) leafhoppers, the panther moth, the American ringed cocoon, the meadow butterfly, and the grass moth. Activity was also observed against fruit fly larvae and mosquito larvae, which are 60 members of the Oiriega family. Other members of the Oiriega family include the pea fly, carrot fly, spring cabbage fly, turnip fly, onion fly, weevil and housefly, and various species of mosquitoes. The activity of the broths and toxin complexes was also observed against the two-spotted spider mite, a member of the Asagipa order, which also includes strawberry mites, mites, red citrus mite, hairy spider mite, pear mite and destructive mite. Because the activity against the larvae of the long-bearded flea was determined as follows. Culture broths of RPIogpardis (concentrated 0 - 15 times, which was passed through a sterilizing filter), 296 Rgoieose Reriop MoZ,

purified toxin complexes |0.2Zmg/ml| or a buffer containing 10 mM sodium phosphate, pH7.0O, was applied directly to the surface (approximately 1.5 cm?7) of artificial feed (Kose, V.I. apa MeSarbe, -.M. (1973). U. Esop.

Epiotoi 66, (398-400)) in the form of aliquots of 400 μl. The toxin complex was diluted in 10 mM phosphate buffer, pH 7.0. Cups with feed were air-dried in a sterile laminar box, and the wells were infected with individual neonates of Oiabgoiis ipdesitripsiaia pouagai (southern long-bearded flea, ZSK) hatched from surface-sterilized eggs. Cups were sealed, placed in a humidified growth chamber and maintained at 272 for a suitable period of time (3 - 5 days). Then mortality and larval mass were assessed. As a rule, 16 insects per variant were used in all studies. Mortality in the control as a rule was less than 5905.

The activity against the cotton weevil (Apitopaz adgapaiz) was tested as follows. Concentrated (1 - 10 times) broths of Riojopardy, control medium (295 Rgoieose Reriop Mo3), purified toxin complexes

IO, 2 Zmg/ml| or 10 mM phosphate buffer, pH 7 0, was applied in the form of aliquots of bomcl to the surface of 0.35 g of artificial fodder (lepidoptera fodder, whitefish with a yellow color) and allowed to dry. One 12- to 24-hour-old larva 75 of the cotton weevil was placed on the feed and the wells were sealed and kept at 259C and 50905 relative humidity for 5 days. Then mortality and larval mass were assessed. Mortality in the control ranged from 0 to 1,395.

Activity against mosquito larvae was tested as follows. Each well contained 20 μl of an aqueous solution (concentrated 10 times) of culture broths of Ryoigpardy, control medium (295 Ryoegose

Reriyuop Mo3), 10MM phosphate buffer, toxin complexes (0.2Zmg/ml) or water and approximately one-day-old larvae (Aedes aeduria). We would use the hole for the option. The results were recorded at 3 - 4 days after infection. Mortality in controls was between 0 and 20,965.

Activity against common fruit fly was tested as follows. Purchased medium for Oyugozorpia theiapodavzieg was prepared using 5095 of dry medium and 50905 of a liquid such as water, control medium (295 µg/ml, 10-fold concentrated culture broth of RPyugparatsv, or purified toxin complex (0.2 µg/ml| or 10 µM phosphate buffer, pH 7.0 This was done by placing 4.Oml of dry medium in each of the 3 vials for growth per variant and adding 4.Oml of the appropriate liquid.

Then 10 Ogozorpia teiIapodazieg larvae of the late age stage were added to each 25 ml bottle. Vials were kept on a laboratory table at room temperature under the light of fluorescent lamps on the ceiling. M

Pupae or adults were counted after 15 days of exposure. Adult hatching was compared with water and control medium (0 - 1695 reduction). co

Activity against adult aster leafhoppers (Masgoziwiez zemegipi) and nymphs of the corn leafhopper (Regedgipis taidis) was tested using an oral challenge test designed to involve ingestion of the active ingredient without other external contact. The reservoir for the solution of the active ingredient/"feed" was prepared by making 2 holes in the center of the lower part of the Petri dish measuring 35x100mm. A square of parafilm SO (Ragate U) (2 inches) was placed on top of the cup and secured with an "0" ring. A 1-ounce plastic cup was then infested with about 7 cicadas, and the tank was placed over the cup with the parafilm down.

Then the test solution was added to the tank through the opening. In assays using culture broth(s) that had been concentrated 10-fold, the broth and control medium (295 Rgoieose Reriop Mo3) were dialyzed against 10 mM sodium phosphate buffer, pH 7.0, and sucrose (up to 595) was added to obtained more) with the solution to reduce mortality in the control. Purified complexes of the toxin (0.2 µg/ml| or 10 mM phosphate buffer, pH7.0) were also tested. Mortality was recorded on the 3rd day. The analysis was carried out in a thermostat at " 282, 7090 relative humidity with a photoperiod of 16/8. Evaluation mortality was recorded at 72 hours Mortality in controls was less than 695.

Activity against lepidopteran larvae was tested as follows. Concentrated (10-fold) culture broth (broths) of Rpoiogpardiv, control medium (2956 Rgoieozve Reriop Mo3), purified t toxin complex (0.2 µg/ml| or 10 mM buffer containing sodium phosphate, pH 7.0) were applied directly to surface (71.5 cm) of standard artificial food for lepidoptera (Biopemishe Mayom food) in the form of aliquots of 4 µl. Tablets with food were allowed to air dry in a sterile laminar and each well was infested with one oo 20 newborn larva. pybyaiv) and the weevil (Mapatcysa zwzechia) were obtained from commercial sources and hatched in the laboratory, while the larvae of the scoop were obtained "self. After larval infestation, the food plates were sealed, placed in a humidified rearing chamber and kept in the dark at 272C for an appropriate period . Mortality and mass were assessed on day 5. Typically, all studies used 16 insects per variant. Control mortality was between 4 22 and 1 2.596 for control medium and less than 1095 for phosphate buffer.

GF! The activity against the two-spotted spider mite (Teigapuspyv cigpiisae) was determined as follows. Young pumpkin plants were cut to one cotyledon and sprayed until runoff with a 10-fold concentrated broth, a control medium (295 Rgoieose reriop Mo3), a purified toxin complex (0.2 µg/ml| or 10 mM phosphate buffer, pH 7.0. After drying plants were infested with a mixed population of spider mites and 60 maintained at laboratory temperature and humidity for 72 hours. Live mites were then counted to determine the level of control. bo Strain Rpoiogpardiz Insect species susceptible"

k"-1: scoop; 2: grain borer; C: weevil; 4: long-bearded flea; 5: cotton weevil; b: mosquito; 7: common fruit fly (Drosophila); 8: aster cicada; 9: corn cicada; 10 : two-spotted spider mite, 2 cm wide, 35 cm, 14 cm

Strains of Riojugpavaiv that are not MU-14:

Purification, characterization and spectrum of activity

Cleaning «

The protocol outlined below is similar to that developed for the purification of MU-14 and is based on the 37Z purification of the fractions with the greatest activity against the southern long-bearded flea (SLF) as determined in bioassays (see Example 13). As a rule, 4 - 20 liters of broth were obtained, which was filtered as described in Example 13 and concentrated using a spiral ultrafiltration cartridge of the BIMIOO (Artichoke) type, which was attached to an Artichoke M-12 filtration device. The retained material contained native proteins with mol. with masses greater than 100 kDa, while the material passing through contained native proteins less than 100 kDa in size. Most of the activity against 5SC was contained in the retained 100 kDa material. Then, the retained material was continuously diafiltered with 10 mM phosphate buffer (pH-7.0) until the filtrate reached pH 0.100. Unless otherwise indicated, all procedures up to this point have been performed in a buffer such as 10 mM buffer containing sodium phosphate (pH7.9). The retained material was then concentrated to a final volume of approximately 0.20 L and filtered using a sterilizing filter element of 0.45 mm MaiYidepe (ee) Eiyepmage. The filtered material was applied at a rate of 7.5 ml/min. on the РНаптация сл НЕ16/10 column, which was packed with a strong anion-exchange matrix RegZeriime Viovuviet Rogoze? BUT, equilibrated in the buffer, using the HPLC system RegZeriime Viozuviet Zrgipi?. After the application, the column was washed with buffer until A zvo 0.100 was reached. Then the whites were eluted from the column at a speed of 2.5 ml/min. using a buffer with 0.4M Mass within 20 minutes for a total volume of 5Oml. Then the column was washed with a buffer with 1.0 M mass at the same flow rate for another 20 minutes (final volume - 500 ml). (F; Proteins that were eluted with 0.4M and 1.0M Mass were placed in separate dialysis bags and dialyzed overnight at 42C in 12L of buffer. Most of the activity against ZSK was contained in the 0.4M fraction.

The 0.4M fraction was further purified by applying 20 ml to a 25/100 Rpagtasia column packed with Zerpagose SI 48 bo (Rpaptasia), using a flow rate of 0.75 ml/min. Fractions were combined based on the profile of the Aodo peaks and concentrated to a final volume of 0.75 ml using a Viotach-50K MMUMI membrane. centrifugal filtering device MiPiroge OKgaigtee?f. Protein concentrations were determined using the Viogai Rgoiep kit

Azzau with bovine gamma globulin as standard.

Characterization 65 The native molecular weight of the anti-BSK toxin complex was determined using a RIagtasia 16/50 column pre-packed with Zerpagose SIAV in buffer. Then the column was calibrated using proteins -A3-

of known molecular size, which made it possible to calculate the approximate native molecular size. As shown in Table 20, the molecular size of the toxin complex ranges from 777 kDa for the Hb strain to 1900 kDa for the Mukh-12 strain. The output of the toxin complex also varied, from 0.8mg/l for the MUH-12 strain to 7.0mg/l for the НБ strain.

Proteins detected in the toxin complex were tested for the size of individual polypeptides using electrophoresis analysis in PAGE-DSN. As a rule, 20 mg of toxin complex protein from each strain was applied to a 2-1595 polyacrylamide gel (Ipigedgaged Zeraganop Zuzietv) and subjected to electrophoresis at 20mA in Viogad electrophoresis buffer. At the end of electrophoresis, the gels were stained overnight in Viogaa Sootavvi Bie 7/0 K-250 (0.295 in a mixture of methanol:acetic acid:water; 40:10:40 vol./vol./06.). Then the dye was removed from the gels in a mixture of methanol:acetic acid:water; 40:10:40. The gels were then washed with water for 15 minutes and scanned using a personal laser densitometer Moiesshag Juupatisv. Lanes were quantified and molecular sizes were calculated against high molecular weight Viogad standards ranging from 200 to 45 kDa.

The sizes of the individual polypeptides containing the anti-SC toxin complex from each strain are shown in Table 21. The sizes of the individual polypeptides range from 230 kDa in strain MUK-1 to 16 kDa, as seen in the case of strain MUK-7. Each strain, with the exception of strain HE, had polypeptides in the toxin complex that ranged from 160-230kDa, 100-160kDa, and 50-8OkDa. These data indicate that the toxin complex may vary in peptide composition and components in different strains, however, in all cases the toxin apparently consists of a 2or Large oligomeric protein complex. of toxins from Rpoiognaradiv strains, which are not M/-14 zv s o yu zo so a 1vyu1111011086 her sch zv so a Native molecular weight, which was determined using a 16/50 Rnagtasia NE column packed with Sepharose SI AB « - s Spectrum of activity y As shown in Table 21, the toxin complexes purified from the Ht and HO strains were tested for activity against "" numerous insects, with the toxin complex from the MU-14 strain for comparison. The tests were carried out as described in Example 13. The toxin complex from all three strains showed activity against the scoop, grain grinder and aster leafhopper. In addition, the complex of toxin from strains Ht and MU-14 showed activity against the two-spotted mite

Go! cobweb In addition, the toxin complex with M/-14 showed activity against mosquito larvae. These data indicate that a toxin complex that had similar activities against some insect species may also exhibit different co-activities against other insect species. t so slov o lyu rage mo) led on 05 july 0 molo yu zlyu in mo l6 9 9800 v 0yaa00o 100130 vti vviyayu) lo 1174 76064332 05 vo vovi yav vv vv 60560 zov) v vo o vv iyu. in 83) 85300060 in 0 in60 55 in ovit pf yuyu | I entered 1019 353289 | to viyu 5003101 560 vv vv i i 08118318 5100065 52 BA 45 zo 24 18 37 63 -dA-

li | | with | 128 22111613 1111 tt I o V Po PO PO PON NO PO NOT in 11151111 1 111111

PISHNE PI NN VO I V V fields po VE shi PI PO PO PO PON POLON PON NO NOYA NI o spectrum of the purified toxin complex from the strains of Rpoiogpardyvz, which it is observed and k"-1: scoop; 2: grain grinder; C: southern long-bearded flea ; 4: mosquito; 5: two-spotted spider mite; 6: aster leafhopper; 7: common fruit fly; 8: cotton weevil 720 Example 15

Subfractionation of the complex of protein toxin Rpoiogpardis

The protein toxin complex of Riojagpardis was isolated as described in Example 14. Then approximately 10 mg of the toxin was applied to a Mopos) 5.5 column equilibrated with 20 mM Tris-HCl, pH 7.0, at a flow rate of Tml/min. The column was washed with 20 mM Trxo-HCl, pH 7.0, until the optical density at 280 nm returned to the absorption of 25 psi; baseline. Proteins bound to the column were eluted with a linear gradient of 0 - 1.0M Mass in 20mM Tris-HCII, at pH 7.0, at Tml/min., for 30 minutes. Fractions were collected according to Iml and subjected to their bioanalysis with the southern long-bearded flea (ZSK) (see Example 13). Activity peaks were determined using a series of dilutions of each fraction in bioassays with SC. Two peaks of activity against ZSK were observed, which were named A (eluted at approximately 0.2 - 0.3M Mass) and B (eluted at approximately 0.3 - 0.4M Mass). Activity peaks A and B were pooled separately and both peaks were further purified using the 3-step procedure described below. (heh)

Solid (MH/)»5O) was added to the above-mentioned protein fraction to a final concentration of 1.7M. Then the proteins were applied to a 5/5rpepuI-Zyregose column equilibrated with 1.7M (MNA)»ZO; in 50MM K-phosphate buffer, pH7.0, - at a flow rate of Tml/min. Proteins bound to the column were eluted with a linear gradient of 1.7M se (MNA)»ZO,, 095 ethylene glycol, BHOMM potassium phosphate, pH7.0, - 2595 ethylene glycol, 25MM potassium phosphate, pH7.0 (without ammonium sulfate ) at 0.5 ml/min. Fractions were dialyzed overnight against 10 mM K-phosphate buffer, pH7.0.

The activity in each fraction against ZSK was determined using a bioassay.

Fractions with the highest activity were combined and applied to a 5/5 Mopos column) which was equilibrated with 20MM

Tris-HCI, pH7.0, at Iml/min. Proteins bound to the column were eluted at 1 ml/min with a linear gradient of 20 0O-In Mass in 20 mM Tris-HCl, pH 7.0. WITH

For the final stage of purification, the most active fractions (which were determined using a bioassay with 5SK) were combined and subjected to purification on the second column of rpepuI-Zyregose 5/5. Solid (Mn.)»5O) was added up to :z» final concentration of 1.7M. Then the solution was applied to a column equilibrated with 1.7M (MNA)»ZzO; in 50 mm

K-phosphate buffer, pH7.0O, at Tml/min. Proteins bound to the column were eluted with a linear gradient of 1.7 M (MNA)»ZO»X, 5 mM potassium phosphate, pH 7 0, - 1 mM potassium phosphate, pH 7 0, at 0.5 ml/min. Fractions were dialyzed overnight against 10 mM Ma-phosphate buffer, pH7.0. The activity in each fraction against ZSK was determined using a bioassay. kz The final protein purified using the above-described 3-step procedure from peak A was named toxin A, and the final protein from peak B was named toxin B. Characterization and amino acid sequencing of both toxin A and toxin B. (se) By electrophoresis in PAAG-DSN, toxin A and toxin B contained two main (29095 of all stained

Coomassie protein) peptide: 192 kDa (named AT and VI, respectively) and 58 kDa (named A2 and B2, respectively). Toxin A and toxin B revealed only one major band in native electrophoresis, suggesting that AT and A? were subunits of one protein complex, and B1 and B2 were subunits of one protein complex. Further, the native molecular weight of both toxins (A and B) was determined to be 8 bokDa according to gel filtration chromatography. Relative molar concentration of AT to A2 was estimated as

GF) eq. to Current, as determined by densitometric analysis of PAAG-DSN gels. Similarly, peptides B1 and B2 are present at the same molar concentration. o Toxin A and toxin B were subjected to electrophoresis in 1096 PAAG-DSN and transblotting on RMOBE membranes. Blots were sent for amino acid analysis and M-terminal amino acid sequencing to Nagmag Misgtospet and

Satrgidde Rhospet, respectively. It was determined that the M-terminal sequence of B1 is identical to ZEO IYUO Mo1, district

TerA; ISBA gene (ZEO IYUO Mo12, position 87-99). A unique M-terminal sequence was obtained for peptide B2 (ZEBEO IC Mo40). The M-terminal amino acid sequence of the B2 peptide was identical to the TeBA region, the obtained amino acid sequence for the ISBA gene (ZEO I Mo12, position 1935-1945). Therefore, toxin B contained mostly two peptides, TSOBA;; and ToBA;, which were produced from one gene product, TerA.

The M-terminal sequence of A2 (ZE IU Mo41) was unique compared to the TerA peptide;; and other peptides. Peptide A2 was named TsaA;, (see Example 17). It was determined that ZEO I Mob is a mixture of amino acid sequences 5EO IU Mo40 and 41.

Peptides A1 and A2 were further sequenced by internal amino acid sequence. 1 µg of toxin A was subjected to electrophoresis in 1095 PAAG-DSN-electrophoresis and transblotting on the PMOR membrane. After staining the blot with amido black, peptides A1 and A2, named TsaA;, and TsaA;;, respectively, were excised from the blot and sent to Nagmagi Misgospet and Satrbgidde RgoSpet. Peptides were digested with trypsin followed by HPLC to separate individual peptides. Analysis of the M-terminal amino acid sequence was performed on selected peptide fragments obtained by trypsin cleavage. It was found that two 7/0 External amino acid sequences of AT peptide (TsaA;-RK71, BEO I Moz8 and Tsad-RK44, BEO IU Me39) have significant homologies with the decoded amino acid sequences of the TsdA region;; ISBA gene (ZEO IU Mo12).

Thus, the M-terminal sequence (ZEO IYUO Mo41) and two internal sequences of A2 peptides (TsaA;;-РК57,

ZEO IO Mo42 and TsaA;-RK20, 5EO IU Mo43) also revealed significant homology with deciphered amino acid sequences of the TebBA region;; gene ISBA 5EO IU Mo12).

Summarizing the above results, it can be said that the toxin complex has at least two complexes active against ZSC: toxin A and toxin B. Toxin A and toxin B are similar in terms of native molecular weight and molecular weight of subunits, however, their peptide compositions differ. Toxin A contains peptides TheaA; and TsaA;; as the main peptides, and toxin B contains TebBA;; and TebA;;, as the main peptides.

Example 16

Cleavage and activation of TeBA peptide

In the toxin B complex, TebA peptides; and TerA;; originate from one TSBA gene product (Example 15).

Processing of the TsBA peptide to TerA; and TsrA; hypothesized to be carried out by protease(s)

Rippoyugpardis and, most likely, by metalloproteases, which are described in Example 10. In some cases, it was noted that during the processing of MU-14 Ripopyugpardy broth, TerA peptide is present in the toxin B complex as the main component, in addition to TsBA peptides;; and TerA;;. Identical procedures described for the purification of the toxin B complex (Example 15) were used to enrich the TeBA peptide from the complex fraction (8) of the MU-14 toxin broth. The final purified material was analyzed in 4-2095 gradient electrophoresis in

PAAG-DSN and the main peptides were quantified by densitometry. It was determined that TeBA, TeBA; and TeBA;; contained 5895, 3695 and 695, respectively, of total protein. The identity of these peptides was confirmed by their respective molecular sizes in PAAG-DSN electrophoresis and Western blotting using monospecific antibodies. It was determined that the native molecular weight of this fraction is equal to 8 bOkDa. co

Cleavage of TerA was assessed by treating the material purified as described above with metalloproteases "U

38kDa and 58kDa MU-14 Riioiogpardis (Example 10) and trypsin as a control enzyme (Zidta, Moscow).

The standard reaction contained 17.5 μg of purified fraction, 1.5 units of protease and 0.1 M Tris-buffer, pH 8O, in a total volume of 10 μl. Protease was not added to the control reaction. The reaction mixture was incubated at 372°C for 90 minutes. At the end of the reaction, 200 μl was taken and boiled with sample buffer for electrophoresis in

PAAG-DSN immediately before electrophoresis in a 4 - 2095 gradient gel. It was determined on the basis of electrophoresis in PAGE with SDS that in both versions (with protease 38kDa and protease 58kDa) the number of peptides

TeBA; and ToBA;; increased approximately 3 times, while the amount of TerA peptide decreased proportionally " "Table 23). The relative decrease and increase of selected peptides was confirmed by Western blotting. In addition, gel filtration of the cleaved material revealed that the native molecular size of the complex remained the same. When treated with trypsin, TeBA and TerA peptides; were nonspecifically cleaved into small "" peptides. This indicated that the 38kDa and 58kDa Rpoijupvacase proteases can specifically process the peptide

TeBA into peptides TebBA;, and ToerA;;. Protease-treated and untreated control of the remaining - 8 Омкл - reaction mixture were serially diluted with 10 mM sodium phosphate buffer, pH 7.0, and analyzed using a bioassay with ZSK.

Go! By comparing the activity in several dilutions, it was determined that treatment with 38 kDa protease increased the insecticidal activity in relation to ZSK by approximately 3 - 4 times. Inhibition of growth of insects remaining in the protease treatment was more significant than in the control (Table 23).

FT» o Transformation and activation of ToBA peptide into peptides and 11111111017111zhontyaol | Protavy treatment of building

Example 17

Screening of the library for the gene encoding the TesaA peptide;,

Cloning and characterization of the gene encoding the TsaAu peptide, which is described in ZEO IU Mo17 (M-terminal sequence of the internal peptide TsaA;-RTII), and 5BEO 10 Mo18 (M-terminal sequence of the internal peptide TsaA;-P1T79) was carried out. Two pools of degenerate oligonucleotides designed to encode the amino acid sequences of ZEO IU Mo17 (Table 24) and 5EO IU Mo18 (Table 25) and reverse complements of these sequences were synthesized, as described in Example 8. The DNA sequence of these oligonucleotides is given below:

Table 29

Degenerate oligonucleotide for 5EO TO 17

B M Be DA (TS At (TISTA) SA STO) BA STISU B

Pi podivnn Pino drink NN kN BURIAL OF GUILT from rtasei TT(T/s) IDAST/S) (AT (T/S/A) (SAT) SA(T/SU B (8)

Table 24 oyu so

Degenerate oligonucleotide for 5EO TI De 18 "I lo rep, so binyi Bin wa Biy nni v vka i a vii i i rep eh

P2.79.2 О'ТТХ|ATU|STK|TATI ASU TST| ТК |ССХ |STK|DAT|ISSKIAATIDAT U - s so R2.798.sВ p ATTIATTIHOSI|ATTIMASTASSTSASIVESTISTIST|ATA MASTAATIAAA 3 ko

FT" page 50

Ж According to PSHIRAS-IIV encodes nucleotides: U - Sabo T, NA, C or

Polymerase Chain Reactions (PCRs) were performed essentially as described in Example 8, using as

GF) forward primers P2.3.6.SV or P2.3.5 and as reverse primers P2.79.K.1 or P2.79K.SV in all forward/reverse combinations, using genomic DNA of RPIiogpardis as a template. In another series of reactions, primers P2.79.2 or P2.79.3 were used as forward primers and P2.3.5K, P2.3.5K.1 and P2.ZK.SV br were used as reverse primers in all forward/reverse combinations. Only in reactions that contained

P2.79.K.I or P2.79K.SV as forward primers in combination with P2.79.K.I or P2.79K.SV as reverse primers revealed a non-artifact amplification product with a determined size (by mobility at agarose gels) 2500 p.p. The order of the primers used to obtain this amplification product indicates that the peptide fragment TsdA;-PTII is aminoproximal to the peptide fragment v5 TsdA;-PT79.

PCR products 2500 p.n. ligated with the roti plasmid vector! (Impygodep, Zap Oiedo, CA) according to the manufacturer's instructions, and the DNA sequences crossing the ends of the insertion fragments of the two isolates (NBZ24 and H5Z27) were determined using the primers recommended by the supplier and the sequencing methods described above. The sequence of both isolates was identical. New primers were synthesized on the basis of the determined sequence and used to prime additional sequencing reactions to obtain a total of 2557 bases of insert I(IZEO I Mo36b). Translation of the partial peptide that encodes EC IUO Mo3b gives a sequence of 845 amino acids described in ZE IUO Mo37. Analysis of the protein homology of this part of the TsaA peptide fragment; revealed a significant amino acid homology (6895 similarity; 5390 identity) with residues 542-1390 of the TeBA ISEO IU Mo12 protein). Thus, it is obvious that the gene, which is represented in the part of ZEO IU Mozb, produces a protein of a similar, but not identical, amino acid sequence to the TsBA protein, and this protein, as can be seen, has a similar, but not identical, biological activity compared to the TerA protein .

In another case, a gene was isolated that encodes TsA;-PK4A4 peptides and the M-terminal peptide of 58 kDa TesA;;, which are described as BEO IU Me9 (TsaA;-PKa44 internal peptide sequence), and 52EO IU Mo41 (M-terminal peptide sequence of 58 kDa TsadA;). Two pools of degenerate oligonucleotides designed for 7/5 Coding of amino acid sequences described as ZEO IU Mo39 (Table 27) and ZEO IUO Mo41 (Table 26) and reverse commissments of these sequences as described in Example 8 and their DNA sequences were synthesized .

Table 26

Degenerate oligonucleotide for 5BKO TO Up to 41 2 codons 2 3 and 3 7 10|11, 12 713 14 s amino acid | Ge | Ah A1Ta |Avip Tech AvriTech yeech sip pota iv) om NI NO NO i Boni Boni io Moi Koli Bai BA Bin B God's NIK s d2.3.V pto|TOs| CHA AAA | HAVIKTS|KESsST|HAV | WEST|KTT| OS KsStT|Co. In so

PooinY NIO NI NO i i Bio ri Bon BY B Bi By My Bii « - s z so ko

FT" with "sl

F) ko 60 b5

Table 26 9 Degenerate oligonucleotide for ZEO Ir Le 39 2 ami-| (8) (9) (10) (11) (12y (13) (15) (15) (16) oxygen and codo- 1 2 3 and c) 7

WELL

Amino-s1u Uai sich I1e Avip Teg Aia Ii1e i o iv) so

Polymerase Chain Reactions (PCR) were performed essentially as described in Example 8, using forward primers A1.44.1 or A1.44.2 and reverse primers A2.3BE. or A2.4E in all forward/reverse combinations, with the SAME use of the genomic DNA of the UUUU-14 RIoioparadiva as a template. In another series of reactions, primers A2.1 or A2.2 were used as forward primers, and A1.44.1K and A1.44.2K were used as reverse primers in all

From direct/reverse combinations. A non-artifact amplified product with a size of 1400 bp. (determined by co-mobility on agarose gels) was detected only in reactions containing A 1.44.1 or A 1.44.2 as forward primers in combination with A2.Z3K quality reverse primer. The order of the primers used to obtain this amplified product indicates that the peptide fragment of TsaA /|-RK44 lies "aminoproximal to the peptide fragment of 58 kDa TeadA".

PCR products 1400 p.n. ligated with the plasmid vector pOctmi! according to the supplier's instructions. - c DNA sequences crossing the ends of the insertion fragments of the four isolates were determined using "" primers similar in sequence to the primers recommended by the supplier and using "" sequencing methods that were previously described. The nucleic acid sequences of all isolates differed, as expected, in the regions corresponding to degenerate primer sequences, but the amino acid sequences deduced from these data were the same as the true amino acid sequences of these peptides previously determined (ZEO IU Mo41 and 39). t Screening of the M/-14 genomic cosmid library, as described in Example 8, with a radiolabeled probe consisting of the DNA obtained above (ZEO IU Mo3b) identified five cosmid isolates that hybridized, and

T - namely, 1709, 20810, 2102, 27810 and 26ba1. These cosmids differed from the cosmids previously identified with OO 20 probes that corresponded to genes described as ZESO IUO Mo1i or ZEO IO Mo25. Restriction analysis and blot-hybridization of DNA identified three fragments of EsokK I with an approximate size of 3.7, 3.7, and 1.1 kb, covering the region that includes DNA ZEO IUO Mo3b. Screening of the MU-14 genomic cosmid library using as a probe the radioactively labeled DNA fragment of 1.4 tbp obtained in this example identified the same five cosmids (1709, 20810, 2102, 27810 and 2601). Blot hybridization of DNA with 22 cleaved Esov I cosmid DNAs also showed hybridization to the same subset of Esov fragments observed with the TsaA gene probe; 2.5 kb, which indicates that both fragments are encoded on genomic DNA. Determination of the DNA sequence of cloned EsokK fragments revealed a continuous reading frame from 7551 bp. (ZEO IUO Mo46), which encodes a 282.9kDa protein of 2516 amino acids (BEO IUO Mo47). Analysis of the 60 amino acid sequence of this protein revealed all the expected internal fragments of TsaA peptides; (ZEO IJU Mo17, 18, 37, 38 and 39) and the M-end of the peptide TsaA;; (ZEO IU Mo41) and all internal peptides of TsaA;; (ZEO IU Mo42 and ZEO IU Mo43). Each of the peptides isolated and identified as TsaA;; and TsaA;;, is the product of an open reading frame designated TsaA, described in BEO IU Mo46. Next, 560 IU Mo47 shows, starting at position 89, a sequence described as ZEO IUO Mo13, which is the M-terminal sequence of a peptide of approximately 201 kDa, which 62 indicates that the primary protein that is produced from ZEO IU Mo46 is processed in a similar way as described earlier for ZEO IU Mo12. In addition, this protein is then cleaved to produce a 209.2 kDa product encoding ZEO IO Mo48 and described as ES IU Mo49 (TsaA peptide ;) and a 63.bkDa product encoding ZEO IU Mo50O and described as 5ESO and Mo51 (peptide TsaA ;;)). Thus, it can be assumed that the insecticidal activity identified as toxin A (Example 15) produced from products of ZEO IU Mo46, such as the full-length protein of 282.9 kDa, which is described in BEO IU Mo47, is processed with the formation of peptides described in the form хEO IU Mo49 and 5EO IU Mo51. The insecticidal activity identified as toxin B (Example 15) is believed to be produced from 5ZES IU Mo11 products, such as the 280 bkDa protein described as ZEO IU Mo12. This protein is proteolytically processed with the formation of a 207.bkDa peptide, which is described in ЖЭО 70 ИО Мо53, which is encoded by ЗЕО ИХО Мо52, and a peptide of 62.9 kDa, which has an M-terminal sequence described in the form of ZES

IO Mo40 and further described in the form of 5260) IU Mo55, which is encoded by ZBEO IU Mo54.

A comparison of the amino acid sequences between the proteins described as ZEO IU Mo12 and ZEO IU Mo47 revealed that they share 69905 similarities and 5495 identities. This high degree of evolutionary relatedness is not uniform throughout the amino acid sequence of these peptides, but it is high toward the carboxy terminus of these 7/5 proteins, as peptides described as ZEO IU Mo51 (produced from ZEO IU Mo47) and as BZEO IU Mo55 (made from ZES

IO Mo12) have 7695 similarity and 6495 identity.

Example 18

Protection against grain borer-induced foliar damage to corn plants by spray application of broth

Ripoyugpavdyz (MU-14 strain)

The ability of Rpoiogpardis toxin(s) to reduce leaf damage caused by insect larvae was demonstrated by measuring leaf damage caused by the grain borer (Ozygypia pibiliaii) on corn plants treated with Rpoiogpardis broth. The fermentation broth from the MuU-14 strain of RNIOiogpardis was obtained and concentrated approximately 10 times using ultrafiltration (pore size 10,000 (mol. mass)) as described in Example 13. The resulting concentrated broth was sterilized by passing through nitrocellulose membrane filters 0.2 micron. A sample of non-inoculated 295 proteose-peptone Mo3Z prepared in this way (8) was used as a control. Maize plants (inbred line, Oomeiapso Estate) were grown from seed to vegetative stage 7 or 8 in pots containing the mixture (without soil) in a greenhouse (272C day, 222C night, approximately 5095 relative humidity, 14-hour day length , watering and fertilizers - as necessary). Test plants were placed in randomized, replicated groups (3 replicates/variation (treatment), 6 plants/variation)) in a greenhouse with a temperature of approximately 222 °C during the day, 1892 °C at night, with no artificial light and partial shading, with a relative "g with a humidity of about 5095 and with watering and fertilizing as necessary. The treatment solutions (uninoculated media and concentrated Riojopardy broth) were applied with a syringe sprayer, 2.0 ml each from 3-5 inches above the whorl of the leaves and an additional 2.0 ml was applied in a circular motion from about one foot above the whorl. In addition, one group of plants was not treated. After drying of the applied solutions (after approximately 30 minutes), twelve newborn larvae of the grain grinder (eggs were obtained from commercial sources and hatched in the laboratory) were applied directly to the bobbin. After one week, the plants were evaluated for leaf damage using a modified scale " 470 Guthrie Ikoliei M.O., Veiiapa, B.Y., Vom/tap, S, Sagogli, M.V., Stepepamu, K., Stozviapa, H. . M.7., Megpip, E., and KPpodevz, K., MUagtep, .MU., Mugidni, M. apa Emoia, 5.M. 1993) Vio/Hesppoiodu, II, 194-195) and the evaluation was compared statistically |T-criterion (least significant (probable) difference, I ZO) p«0.05 and Tikeuz 5iidepiigea Kapde (H5O)

Tevi p«0.111. These results are shown in Table 28. For reference, a score of 1 means no damage, a score of 2 means small "window glass" type damage on an uncollapsed sheet with no pinhole penetrations, and a score of 5 means penetration of a sheet with elongated damage and/or midvein eating observed on more than 3 leaves (1 inch damage). These data show that broth or other protein-containing fractions can provide protection against specific insect pests when applied in

Whose" in the form of a sprayed drug or when delivering the gene or its derivative encoding this protein or part of it through a transgenic plant or microbe. co

And Rpoiogpardyz broth for leaf damage by grain grinder on corn z o z vo

Example 19

Genetic engineering of genes for expression in E. Soya

Summary of construction in 5 A series of plasmids were constructed for the expression of the IsBA gene of the Rioiiogparatsv M/-14 in Ezspegispia soybean. A list of these plasmids is given in Table 29. A brief description of each construction follows, as well as a summary of the obtained for -BO0-

expression data of E. soya. ; at

Construction of pRAVbZA

Example 9 describes a large fragment of Esog I that hybridizes with the TsrA probe. This fragment 75 was subcloned in pVS (Zigaiadepe, Ha doPa SA). Sequencing analysis shows that this fragment has 8816 bp.

The fragment encodes the IsSBA gene with the initiation codon ATO at position 571 and the termination codon TAA at position 8086.

So, this fragment carries 570 p.n. The DNA of RIIogpardis against the course of transcription from ATO and 7ZOp.n. during transcription from TAA.

Construction of the plasmid раАСОоРб7ВЛсСА

The ІСІА gene was amplified by PCR using the following primers: 5'-primer (ІІАС51) 5 TT

AAA SSA TO OAA AST SAT TAT SAA OSA STA TS 3 and 3i--primer (5IAs31) 5 TT AAA SO OSS OST TAA

So ATO OTA TAA SOA ATA TO 3. PCR was performed using the TaKakKa GA RSK kit from Rapmega (Madizop,

MUizsopzip) in the following reaction: 57.5 ml of water, 100 ml of 10X I A-buffer, 1 bml of aMTP (2.5 mM of each starting solution), 20 ml of each of the primers at 1Omol/ml, ZOOng of the plasmid rrAVb6Z34, which contained the gene (srA MUuU-14 and one ml of Td TaKaCa Ha polymerase. The cycling conditions were as follows: 98 2 g/20 sec., 682 C/5 min., 722 C/10 min. for 30 cycles. The expected PCR product of about 7526 bp was isolated in a 0.895 agarose gel in TBE- buffer (100 mM Tris, 90 mM boric acid, 1 mM EDTC) and purified using the Oiaex II kit with Oiadep (Spaivzmogii,

Saiyogpia). The purified ISBA gene was cleaved by Mso | and My | and ligated into the baculovirus carrier vector pAsoRbUV (Rpagmipdep (Zap Oiedo, SaiYogpis) and transformed into YONba E. soybean cells. Then the IsBA gene was excised from pAsOoRb7B and transferred into pET27b to create the pPAV 635 plasmid. A missense mutation in the ISBA gene was repaired in rOAVb635. (ge)

The repaired gene (sBA) contains two changes compared to the sequence shown in ZEO IJU Mo11: A instead of O " at position 212, replacing alanine 71 with serine 71, and C instead of A at position 229, replacing alanine 77 with threonine 77. Both of these modifications are upstream of transcription from the predicted M-terminus of TerA; p

Construction of pET15-IsBA co

The coding region of ISBA rrAVbZ35 was transferred into the pET15B vector. This was done using shotgun ligations,

The DNA was excised with the restriction enzymes Mco I and Xo I. The resulting recombinant was named pET15-ІІСА.

TeBA expression in E. soybean from the pET15-YisBA plasmid

The expression of IsBA in E. soybean was obtained by modifying the methods previously described in Fig. 5. " 20 Izishaieg, E.MU., Kozeprego, A., Yuip, U. apa YuOyBrepadogpi, 9., (1990). Ove oi T7/ KMA roiutegase yu digesi -o s ekhrezviop oi sioped depe5z. Meiyodz Eplutoi!., 185: 60-89). Competent cells of E. soy strain VI21 (OEZ) were transformed with the pET15-IsSBA plasmid and sown on I B-agar containing 100 μg/ml of ampicillin and 40 μM of glucose. Transformed cells were grown to a density of several hundred isolated colonies per dish. After overnight incubation at 37°C, the cells were scraped from the dishes and suspended in IV broth containing 00 μg/ml ampicillin. Typical volumes of cultures were 200-5000 ml. At time 0, the density of cultures (ОЮsoo) reached 0.05 - 0.15, depending on the experiment. Cultures were shaken at one of three temperatures (222С, 302С or 3722) until a density of 0.15 - 0.5 was obtained, after which they were induced with 1 mM co isopropylthio-beta-galactoside (IPTH). Cultures were incubated at the selected temperature for 4-5 hours and then transferred to 42C for processing (12-72g).

Purification and characterization of ToeBA. expressed in E. soybean from the pPET15-IsBA plasmid. Cultures of E. soybean expressing TerA peptides were treated as follows. Cells were collected by centrifugation at 17000X9, and the medium was decanted and stored in a separate container.

The medium was concentrated approximately 8 times using a M12 filtration system (Artichoke, Vemepu

MA) and a filter with a molecular weight cutoff of 100 kDa. The concentrated medium was applied to an anion exchange column and the bound proteins were eluted with 1.0M mass. An elution peak of 1.0 M Massi was found to cause mortality when applied against larvae of the southern long-bearded flea (SLB) (Table 30). Fraction 1.0M

F) masses were dialyzed against 10 mM sodium phosphate buffer, pH 7.0, concentrated and subjected to gel filtration on Zerpagose SI-48 (RIagtasia, Rizsaiajau, Mem egveu). The C-48 elution profile zone corresponding to the calculated molecular mass (approximately 900 kDa) of the native MU-14 toxin complex was collected, concentrated to 60, and subjected to a larval bioassay. The pooled 900kDa fraction was found to have insecticidal activity (see Table 30 below), with symptomatology similar to that caused by the native MU-14 toxin complex. This fraction was subjected to the action of Proteinase K and thermal treatment. The activity in both cases disappeared or decreased, which proves the protein nature of the activity carrier. In addition, this active fraction was immunologically positive for ToBA and TebBA peptides;; in immunoblotting when testing 65 with monoclonal antibodies against TebA;; (Table 30).

YOU ARE FLOWS Polon PON 1.0M ion exchange

SI Av TeOBA; 70 iProteinase K shi si Av tnaharivana

Tov superuklimnssyaV 1777-1111 nt 171111svoodaG///- : inhibition of growth in comparison with control samples: 5 - 249r-"", 25 - 4Z9r"", 5 - 1009",

The cell pellet was resuspended in 10 mM phosphate buffer (with sodium phosphate), pH 7.0, and lysed by passing through a Vio-Mertm cell sprayer (|Iav-Soi Ips., Tetpa Natsie, IM). The pellet was treated with DNase to remove DNA and centrifuged at 17,000 x 9 to separate the cell pellet from the supernatant. The supernatant fraction was decanted and filtered through a 0.2 micron filter to remove large particles and subjected to anion exchange chromatography. Bound proteins were eluted at 1.0 M Mass, dialyzed, and concentrated by using Biotachtm concentrators (MyPiroge Sogr, Veatoga, MA) with a molecular weight cutoff of 50,000

Dalton. The concentrated fraction was subjected to gel filtration chromatography using a granular SI-48 matrix. The bioassay data for the material prepared in this way are shown in table 30 and are called from p. 29 "TerA of super cells". Gee)

In another method for handling large amounts of pellet material, the cells were resuspended in 10 mM sodium phosphate buffer, pH 7.0, and gently homogenized using a tissue mill (Kopiez SiIazv

Sotrapu (Mipeiapa, MO)) for 40 ml. Cell remnants were pelleted by centrifugation at 25,000 x, and the cell supernatant was decanted, passed through a 0.2 micron filter, and subjected to anion exchange chromatography using a 10/10 Ppagtasia column packed with Rogov HO 50 granules. Bound proteins were eluted with o) gradient Masses 0.0 - 1.0M. Fractions containing TerA protein were pooled and concentrated using

B5 OkDa concentrator and subjected to gel filtration chromatography using a granular matrix

RPpagtasia SI -4B. Fractions containing TsBA oligomer with a molecular weight of approximately 9O0OkDa were collected and subjected to anion exchange chromatography using a Rpagtasia Mopo O 10/10 column equilibrated to 20MM

Tris buffer, pH7.3. A gradient of 0.0 - 1.0 M Mass was used for elution of the recombinant TsrBA protein. co

Recombinant TerA was eluted from the column at a salt concentration of approximately 0.3 - 0.4M, at the same molarity at which the native TsSBA oligomer was eluted from the Mopo C 10/10 column. It was found that this fraction of recombinant TerA caused the mortality of ZSC in bioassay experiments similar to the experiments that " 20 of them are listed in Table 30. With s h"

Claims (10)

The formula of the invention p 1. A polynucleotide operably linked to a heterologous promoter encoding a protein having 75 toxin activity against an insect pest, wherein the nucleotide molecule encoding said protein retains the ability to hybridize to a complementary nucleic acid sequence after hybridization and co-washing , and the indicated hybridization is carried out at 60 oC in a solution containing 10 905 (m/o) PEG (polyethylene glycol, M.m. approximately 8000 Da), 0.6 x 55C, 7 95 (m/o) ZO5, 10 mM sodium phosphate buffer, 5 mM EDTA and 100 mg/ml of denatured DNA from salmon milk, where the specified nucleic acid sequence is 79. ES IU MO: 46. 2. Polynucleotide according to claim 1, which differs in that the washing is carried out in 0.25 x З5С and 0.2 96 505 at s voes. C. A polynucleotide encoding a protein having toxin activity against an insect pest, wherein said protein contains the amino acid sequence ZEO IJU MO: 47, where said polynucleotide is functionally linked to a heterologous promoter. GF) 4. A polynucleotide containing a nucleotide sequence that encodes a protein having the amino acid sequence ZEO IU MO: 47, functionally linked to a heterologous promoter, and the indicated protein has toxin activity against an insect pest. in 5. A transgenic plant cell that expresses the polynucleotide according to claim 1. 6. Recombinant protein that has toxin activity against an insect pest, where the nucleotide sequence encoding the specified protein retains the ability to hybridize with the nucleic acid sequence upon hybridization and washing, and the specified hybridization is carried out at 60 C in a solution containing 10 90 (w/o) PEG (polyethylene glycol, MW approx. 8000 Da), 0.6 x C5C, 7 90 (w/o) 5O5, 10 mM sodium phosphate buffer, 5 65 mM EDTA, and 100 mg/ml denatured DNA from salmon milk, where the specified nucleic acid sequence is ZEO IU MO: 46. 7. Protein according to claim 6, which differs in that washing is carried out at 0.25 x 35C and 0.2 95 505 at 60C. 8. Recombinant protein having the activity of a toxin against an insect pest, containing the amino acid sequence ZEO IYUO MO: 47. 9. A method of combating an insect pest, which involves oral delivery of the protein according to item b to the specified pest. 10. The method according to claim 9, which is characterized by the fact that the specified protein is produced and present in a transgenic plant that is accessible to the specified pest. s (8) Io) so «I s Zo so - with . and? o ko FT" o "sl F) ko 60 b5