FR2801609A1 - Collection of nucleic acids from environmental samples, useful for identifying e.g. genes encoding polyketide synthases and derived antibiotics - Google Patents

Abstract

Preparation of a collection of nucleic acids (I) from organisms in a soil sample, is new. Preparation of a collection of nucleic acids (I) from organisms in a soil sample comprising: (1) milling a dried sample to produce microparticles (MP); (2) suspending these in liquid buffer; (3) extraction of (I) from MP; (4) passing (I)-containing solution through a molecular sieve; (5) passing (I)-enriched fractions through an anion exchange chromatography material; and (6) recovering fractions containing purified (I), is new. Independent claims are also included for the following: (a) processing an environmental sample by: (i) dispersing in liquid; (ii) homogenization of the resulting suspension by gentle mixing; (iii) separation of organisms from other components by density-gradient centrifugation; (iv) lysing separated organisms; (v) extraction of (I); and (vi) purification on a cesium chloride gradient; (b) preparing a collection of recombinant vectors by inserting (I) into a cloning and/or expression vector; (c) new methods for preparing the cloning and expression vectors; (d) a collection of (I) prepared by the new methods; (e) (I) present in the collections of (d); (f) recombinant vectors containing specific (I), prepared by methods (c); (g) collection of recombinant vectors prepared by methods (b) or (c); (h) recombinant vectors present in the collection of (g); (i) recombinant host cells containing (I) of (e) or the vector of (h); (j) collection of cells of (i); (k) detecting a specific (I), or related sequence, in the collection of (j); (l) identifying production of a selected compound (II) in one or more cells of the collection of (j); (m) selected recombinant host cells producing (II) in the collection of (j); (n) producing (II); (o) (II) produced by method (m); (p) polyketide (PK) produced by expressing specified sequences (Ia); (q) determining diversity of (I) in a collection; (r) nucleic acid (Ib) containing a 16S rDNA sequence at least 99% identical with any of 47 specified sequences as given in the specification; (s) producing a type I PK synthase (PKS-I); (t) PKS having any of about 20 specified amino acid sequences as given in the specification; (u) antibodies (Ab) directed against the PKS of (t); (v) detecting PKS-I or its fragments; and (w) kit for method (v).

Description

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 The present invention relates to a method for preparing nucleic acids from a sample of the environment, more particularly to a method of obtaining a nucleic acid collection from a sample. The invention also relates to the nucleic acids or collections of nucleic acids obtained according to the process and their application to the synthesis of new compounds, in particular new compounds of therapeutic interest.

 The subject of the invention is also the novel means used in the above process for obtaining nucleic acids, such as novel vectors and methods for preparing such vectors, or recombinant host cells comprising an acid. nucleic acid of the invention.

 The invention also relates to methods for detecting a nucleic acid of interest within a collection of nucleic acids obtained according to the above method, as well as the nucleic acids detected by such a method and the polypeptides encoded by such methods. nucleic acids.

 The invention also relates to nucleic acids obtained and detected according to the above methods, in particular nucleic acids encoding an enzyme participating in the biosynthetic pathway of antibiotics such as ß-lactams, aminoglycosides, nucleotides heterocyclic or polyketides as well as the enzyme encoded by these nucleic acids, polyketides produced through the expression of these nucleic acids and finally pharmaceutical compositions comprising a pharmacologically active amount of a polyketide produced through the expression of such nucleic acids.

 Since the discovery of streptomycin production by actinomycetes, the search for new compounds of therapeutic interest, and especially new antibiotics, has made greater use of methods for screening the metabolites produced by the microorganisms of the ground.

 Such methods consist mainly of isolating the organisms from the soil microflora, cultivating them on specially adapted nutrient media and then detecting pharmacological activity in the products found in the supernatants of the soil.

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 culture or in cell lysates having, if necessary, previously undergone one or more separation and / or purification steps.

 Thus, the methods of isolation and in vitro culture of the organisms constituting the telluric microflora have made it possible, as of today, to characterize about 40,000 molecules, of which about half have a biological activity.

 Major products have been characterized according to such in vitro culture methods, such as antibiotics (penicillin, erythromycin, actinomycin, tetracycline, cephalosporin), anticancer agents, antichololesterolemic agents or even pesticides.

 The products of therapeutic interest of microbial origin known to date come mainly (about 70%) from the group of actinomycetes and more particularly of the genus Streptomyces.

However, other therapeutic compounds, such as teicoplanins, gentamycin and spinosins, have been isolated from more difficult to cultivate microorganisms such as Micromonospora, Actinomadura, Actinoplanes, Nocardia, Streptosporangium, Kitasatosporia or Saccharomonospora.

 But the practice illustrates that the characterization of new natural products synthesized by the organisms of the soil microflora has remained limited, partly because the in vitro culture step most often results in a selection of organisms already known previously.

 The methods of separation and in vitro culture of telluric organisms to identify new compounds of interest therefore have many limitations.

 In actinomycetes, for example, the antibiotic rediscovery rate already known previously is about 99%. Indeed, fluorescence microscopy techniques made it possible to count more than 1010 bacterial cells in 1 g of soil, whereas only 0.1 to 1% of these bacteria can be isolated after seeding on culture media.

 Using DNA reassociation kinetics techniques, it has been shown that between 12,000 and 18,000 bacterial species can be contained in 1g of soil, whereas to date only 5,000 microorganisms eukaryotes have been described, all habitat confused.

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 Molecular ecology studies have amplified and cloned many new 16S rDNA sequences from environmental DNA.

 The results of these studies led to a tripling of the number of bacterial divisions previously characterized.

 As of today, the bacteria are subdivided into 40 divisions, some of which consist only of bacteria that can not be cultivated. These latest results demonstrate the extent of microbial biodiversity that has remained untapped to date.

 Recent work has attempted to overcome the many obstacles to access to soil microflora biodiversity, including the in vitro culture step prior to isolation and the characterization of compounds of industrial interest, especially of therapeutic interest.

 Methods have thus been developed which include a step of extracting the DNA from telluric organisms, if appropriate after prior isolation of the organisms contained in the soil samples.

 The DNA thus extracted, after lysis of the bacterial cells without prior in vitro culture step, is cloned into vectors used to transfect host organisms, to form DNA libraries from soil bacteria.

 These libraries of recombinant clones are used to detect the presence of genes coding for compounds of therapeutic interest or alternatively to detect the production of compounds of therapeutic interest by these recombinant clones.

 However, the methods for direct access to the DNA of the soil microflora described in the state of the art have drawbacks in the implementation of each of the steps described above, which can considerably affect the quantity and quality of genetic material obtained and exploitable.

 The state of the art relating to each of the steps of constructing DNA libraries from soil samples is detailed below, as well as the technical drawbacks identified by the applicant and which have been overcome according to the present invention.

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 1. Step of extracting DNA from a soil sample.

 1. 1 Direct DNA extraction from the environment.

 It is essentially a process using DNA extraction techniques performed directly on the sample in the environment, usually after a prior in situ lysis of the sample organisms.

 Such techniques have been implemented on samples from aquatic environments, whether freshwater or marine. They comprise a first stage of prior concentration of the cells present freely or in the form of particles, generally consisting in a filtration of large volumes of water on various filtration devices, for example conventional membrane filtration, tangential or rotational filtration or else ultrafiltration .

 The pore size is between 0.22 and 0.45 mm and often requires prefiltration in order to avoid clogging due to the treatment of large volumes.

 In a second step, the harvested cells are lysed directly on the filters in small volumes of solutions, by enzymatic and / or chemical treatment.

 This technique is for example illustrated by the work of STEIN et al. , 1996, Journal of Bacteriology, Vol.178 (3): 591-599 which describes the cloning of genes coding for ribosomal DNA and for a transcriptional elongation factor (EF 2) from Archaebacteria marine plankton.

 Techniques for the direct extraction of DNA from soil or sediment samples have also been described, based on physical, chemical or enzymatic lysis protocols performed in situ.

 For example, US Patent No. 5,824,485 (Chromaxome Corporation) discloses chemical lysis of bacteria directly to the sample taken by addition of a hot lysis buffer based on guanidium isothiocyanate.

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 International Application No. WO 99/20799 (WISCONSIN ALUMNI RESEARCH FOUNDATION) describes a step of lysing bacteria in situ using an extraction buffer containing a protease and SDS.

 Other techniques have also been used, such as carrying out several cycles of freezing-thawing of the sample and then pressing the thawed sample at high pressure.

Bacterial lysis techniques have also been used by means of a succession of sonication steps, microwave heating and thermal shocks (PICARD et al (1992).

 However, the techniques of direct DNA extraction of the state of the art described above have a very variable efficiency in terms of quantity and quality.

 Thus, the chemical or enzymatic treatments in situ of the sample have the disadvantage of only lysing certain categories of microorganisms because of the selective resistance of the various indigenous microorganisms to the lysis stage because of their heterogeneous morphology.

 Thus, Gram-positive bacteria are resistant to SDS detergent heat treatment while almost all Gram-negative cells are lysed.

 In addition, some of the direct extraction protocols described above promote the adsorption of the extracted nucleic acids on the mineral particles of the sample, thus significantly reducing the amount of accessible DNA.

 Furthermore, if certain protocols of the state of the art disclose a mechanical treatment step for lysing the microorganisms of the sample taken, such a mechanical lysis step is systematically performed in a liquid medium in an extraction buffer, which does not allow a good homogenization of the starting sample in the form of fine particles allowing maximum accessibility to the diversity of organisms present in the sample. Grinding tests were also performed on a raw soil sample using glass beads, but the amount of DNA extracted was small.

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 It has been observed according to the invention that a first step of in situ mechanical lysis in a liquid medium has negative effects on the amount of DNA that can be extracted.

 The amount of directly usable DNA for cloning into recombinant vectors is also dependent on the purification steps subsequent to its extraction.

 In the state of the art, the extracted DNA is then purified, for example by the use of polyvinylpolypyrrolidone, by precipitation in the presence of ammonium or potassium acetate, by cesium chloride gradient centrifugations, or else chromatographic techniques, in particular on hydroxyapatite support, ion exchange column or molecular sieving or by agarose gel electrophoresis techniques.

 Previously described DNA purification techniques, especially when these are combined with the above-mentioned DNA extraction techniques from the environment, are likely to lead to co-purification of DNA with inhibitory compounds from of the initial sample that are difficult to eliminate.

 The coextraction of inhibitory compounds with the DNA requires the multiplication of the number of purification steps which leads to significant losses of the DNA initially extracted and simultaneously reduces the diversity of the genetic material initially contained in the sample, as well as than its quantity.

 Another object of the invention has been to overcome the disadvantages of previous purification protocols and to develop a DNA purification step to optimally maintain the DNA diversity of the original sample, from on the one hand, and, quantitatively, to favor its obtaining, on the other hand.

 In particular, the qualitative and quantitative improvements in DNA purification are maximum when they involve a combination of a direct extraction method of the DNA according to the invention and a subsequent purification process, such as this will be described below.

 1. 2. Indirect DNA extraction from the environment.

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 Such techniques use a first step of separating the different organisms from the telluric microflora from the other constituents of the starting sample, prior to the step of extracting the DNA proper.

 In the state of the art, the prior separation of a microbial fraction from a soil sample most often comprises a physical dispersion of the sample by grinding the latter in a liquid medium, for example using devices of the type Waring Blender or a mortar.

 It has also been described chemical dispersions, for example on ion exchange resins or dispersions with non-specific detergents such as sodium deoxycholate or polyethylene glycol. Whatever the mode of dispersion, the solid sample should be suspended in water, phosphate buffer or saline solution.

 The physical or chemical dispersion step may be followed by a density gradient centrifugation to separate the cells in the sample and particles from the sample, it being understood that the bacteria have densities lower than those of most soil particles.

 The physical dispersion step can also be alternately followed by a low speed centrifugation step or a cell elutriation step.

 The DNA can then be extracted from the cells separated by all available lysis methods and purified by many methods, including the purification methods described in the previous paragraph. In particular, the inclusion of the cells in low-melting agarose can be carried out in order to preserve the lysis.

 However, the methods described in the state of the art known to the applicant are unsatisfactory because of the presence, in the fractions containing the extracted DNA, of undesirable constituents of the starting sample having a significant influence on the quality. and the amount of final DNA.

 The present invention proposes to solve the technical difficulties encountered in the methods of the prior art as will be described below.

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 2. Molecular characterization of the extracted DNA.

 When it is desired to construct a DNA library from a sample of the environment, in particular from a soil sample, it is advantageous to check the quality and diversity of the extracted DNA source. and purified prior to insertion into appropriate vectors.

 The objective of such a molecular characterization of the extracted and purified DNA is to obtain profiles representing the proportions of the different bacterial taxons present in this DNA extract. The molecular characterization of the extracted and purified DNA makes it possible to determine whether artifacts have been introduced during the implementation of the various steps of extraction and purification and, if appropriate, whether the original diversity of the DNA extracted and purified is representative of the microbial diversity initially present in the sample, especially in the soil sample.

 To the applicant's knowledge, quantitative hybridization methods using oligonucleotide probes specific for different bacterial groups applied directly to the DNA extracted from the environment are used in the state of the art.

 Unfortunately, such an approach is insensitive and can not detect taxonomic genera or groups present in low abundance.

 The state of the art also describes quantitative PCR methods, such as MPN-PCR or competitive quantitative PCR. However, these techniques have significant disadvantages.

 Thus, the MPN-PCR is of a complex use because of the multiplication of the dilutions and the repetitions which makes it inappropriate for a large number of samples or couples of primers.

 Moreover, the quantitative PCR by competition is difficult to implement due to the need to build a competitor specific to the target DNA which, moreover, does not induce any bias or artifacts in the competition itself. called.

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 It is thus proposed according to the invention a precranking method of a DNA library from a sample of the environment which is at once fast, simple and reliable and makes it possible to test the quality of the DNA extracted beforehand. and purified and thereby determine the interest of constructing a library of clones prepared from this purified starting DNA.

 3. Vectors for cloning extracted and purified DNA from a sample of the environment.

 Many vectors have already been described in the state of the art in order to clone DNA previously extracted from a sample of the environment.

 Thus, according to the description of International Application No. WO 99/20799, it is possible to use viral vectors, phages, plasmids, phagemids, cosmids, phosmides, vectors of the BAC (artificial bacterial chromosome) type, or the bacteriophage P1, PAC-type vectors (artificial chromosome based on bacteriophage P1), YAC (artificial chromosome yeast) vectors, yeast plasmids or any other vector capable of stably maintaining and expressing a DNA genomics.

 Example 1 of PCT Application No. WO 99/20799 describes the construction of a genomic DNA library by cloning into a BAC vector.

 To the applicant's knowledge, no DNA library from a sample of the environment had actually been made with conjugative vectors, such a technique being made accessible for the first time and reproducible by humans. of the art through the teaching of the present invention.

4. Cellular hosts
In the state of the art, many host cells have been described as being able to be used to harbor vectors containing DNA inserts from extracted and purified DNA from a sample of the environment.

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 Thus, PCT application WO 99 / 20.799 cites many suitable cell hosts, such as Escherichia coli, in particular strain DH 10B or strain 294 (ATCC 31446, strain E. coli B, E.

Col. X. 1776 (ATCC N 31,537), E. coli DH5α and E. coli W3110 (ATCC No. 27,325).

 This PCT application also cites other appropriate host cells such as Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, Serratia, Schigella or Bacillus-type strains such as B. subtilis and B. licheniformis as well as bacteria of the genus Pseudomonas. Streptomyces or Actinomyces.

 US Patent No. 5,824,485 particularly cites the Streptomyces lividans strain TK66 or yeast cells such as those of Saccharomyces pombe.

 5. Characterization of genes of interest in DNA libraries from a sample of the environment.

 PCT application WO 99 / 20,799 describes an identification of the phenotype of different clones belonging to the B. cereus DNA library, respectively a clone producing hemolysin, an esculin-hydrolyzing clone or a clone producing a pigment. orange.

 Mutagenesis techniques based on the use of a transposon coding for the pho A enzyme have subsequently made it possible to isolate mutated clones and to characterize the sequences responsible for the observed phenotypes.

 The article by STEIN et al. (1996) supra discloses the use of ribosomal specific DNA primers to amplify DNA inserted into vectors harbored by certain clones of a marine plankton archaebacteria genomic DNA library and identification. of several coding sequences in the DNA thus amplified.

 The article by BORSCHERT S. et al. (1992) describes the screening of a Bacillus subtilis genomic DNA library using pairs of primers hybridizing with conserved regions of known peptide synthetases in order to identify one or more corresponding genes in the genome of Bacillus subtilis.

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 This technique detected a chromosomal DNA fragment of about 26 kb carrying part of the surfactin biosynthesis operon.

 The article by KAH-TONG S. et al (1997) describes the screening of a DNA library originating from the soil using primers hybridizing with conserved sequences of the operon responsible for the biosynthetic pathway. type II polyketides and shows the identification, within this DNA library, of sequences related to the PKS-p gene. This article also describes the construction of hybrid expression cassettes in which the sequence of the PKS- subunit, found naturally in the operon responsible for the biosynthesis of polyketides, has been replaced by different related sequences found in the library. DNA.

 Similarly, the article by HONG-FU et al. , (1995) describes the construction of expression cassettes containing the different open reading phases of the operon responsible for the biosynthesis of polyketides, the different expression cassettes having been artificially constructed by combining the open reading phases which are not not found together naturally in the genome of Streptomyces coelicolor. This article shows that the combination, in artificial expression cassettes, of open reading frames originating from different bacterial strains allows the production of polyketides with different structural characteristics and more or less large antibiotic activities with respect to Bacillus subtilis. and Bacillus cereus.

 Polyketides are part of a large family of natural products of variable structure and with a great diversity of biological activities. Examples of polyketides include tetracyclines and erythromycin (antibiotics), FK506 (immunosuppressant), doxorubicin (anti-cancer agent), monensin (a coccidiostatic agent) and avermectin (a pest control agent).

 These molecules are synthesized through multifunctional enzymes called polyketide synthases, which catalyze repeated condensation cycles between acyl thioesters (usually acetyl, propionyl, malonyl or methylmalonyl thioesters). Each cycle of condensation results in the formation, on a growing carbon chain,

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 of a ss-keto group which can then undergo, if necessary, one or more series of reducing steps.

 In view of the important clinical interest of polyketides, their common mechanism of biosynthesis, and the high degree of conservation observed between gene clusters encoding polyketide synthases, there has been increased interest in the development of new polyketides. by genetic engineering.

 New artificial polyketides have thus been produced by genetic engineering, such as medronhodin A or dihydrogranatirhodine. The vast majority of new molecules of genetically engineered polyketides are structurally very different from the corresponding natural polyketides.

 From the state of the art, it thus emerges that there is a need to obtain new polyketides of interest and more particularly polyketides of therapeutic interest having in particular, compared with their natural counterparts, an increased level of antibiotic activity or a spectrum of different antibiotic activity, either wider than that of known polyketides, or rather more selective.

 This need is, as will be described hereinafter, partly filled according to the present invention.

DESCRIPTION OF THE INVENTION
The invention firstly relates to a method for the construction of DNA libraries from a sample of the environment, such a sample can be indifferently an aquatic medium (freshwater or marine), a soil sample (layer superficial soil, subsoil or sediment), or a sample of eukaryotic organisms containing an associated microflora, such as for example a sample from plants, insects or marine organisms and having an associated microflora.

 The development of a method of constructing a DNA library of a sample of the environment, and particularly of a soil sample, comprises critical steps whose implementation must necessarily be optimized for obtaining a DNA bank

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 whose content in nucleic acids of interest meets the objectives initially set.

 A first critical step consists in the extraction and subsequent purification of the nucleic acids initially contained in the sample, that is to say mainly nucleic acids contained in the various organisms composing the microflora of this sample.

 The quality of purification of the extracted DNA is decisive on the result obtained.

 A second important step in a process for constructing a nucleic acid library from a sample of the environment is the evaluation of the genetic diversity of extracted and purified nucleic acids. The development of a simple and reliable step of pre-screening the extracted and purified DNA to verify that it accounts, at least partially, for the phylogenetic diversity of the organisms initially present in the sample. departure, makes it possible to determine the interest or not to use the initial source of DNA extracted and purified for the construction of the nucleic acid library proper or otherwise not to continue the construction of the bank of nucleic acids because of excessive artifacts introduced at the time of extraction and purification of nucleic acids. It has furthermore been identified according to the invention that the quality of the inserts introduced into the vectors to build the bank is decisive. It has thus been determined that the use of restriction enzymes to cleave the DNA extracted and purified from the sample of the environment was likely to introduce artifacts or "biases" into the structure of the inserts obtained.

Indeed, DNA extracted from soil or other environments, mostly from non-culturable organisms, is composed of molecules whose G and C bases are by definition unknown and more variable depending on the origin of these organisms
A third critical step is the insertion of extracted and purified nucleic acids into vectors capable of integrating nucleic acids of selected length, on the one hand, and, on the other hand, to allow transfection thereof or else integration into the genome in specific cellular hosts and, if appropriate, to allow expression in such cellular hosts.

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 Vectors of interest are vectors capable of integrating nucleic acids of large size, that is to say larger than 100 kb when the objective pursued consists in cloning and in identifying an operon. A compound capable of directing a complete biosynthetic pathway of a compound of industrial interest, particularly a compound of pharmaceutical or agronomic interest.

DEFINITIONS
Within the meaning of the present invention, "nucleic acids", "polynucleotides" and "oligonucleotides" are understood to mean both DNA and RNA sequences, and RNA / DNA hybrid sequences of more than 2 nucleotides, regardless of whether single-stranded or double-stranded form.

 The term "library" or "collection" is used in the present description with reference equally to a set of nucleic acids extracted and, where appropriate purified, from a sample of the environment, to a set of recombinant vectors, each of the recombinant vectors of the set comprising a nucleic acid from the set of nucleic acids extracted and, where appropriate purified above, and to a set of recombinant host cells comprising one or more nucleic acids from the all nucleic acids extracted and, where appropriate, purified above, said nucleic acids being carried by one or more recombinant vectors, or integrated into the genome of said recombinant host cells.

 The term "environmental sample" refers to a sample of aquatic origin, for example of fresh or saline water, or a telluric sample originating from the superficial layer of a soil, sediments or even lower layers of the soil. (subsoil), as well as samples of eukaryotic organisms, possibly multicellular, of plant origin, from marine organisms or insects and having an associated microflora, this associated microflora constituting organisms of interest .

 The term "operon" according to the invention means a set of open reading frames whose transcription and / or translation is co-

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 regulated by a unique set of transcriptional and / or translational regulatory signals. According to the invention, an operon can also comprise said signals for regulating transcription and / or translation.

 By "metabolic route" for purposes of the invention or "biosynthetic pathway" is meant a set of anabolic or catabolic biochemical reactions effecting the conversion of a first chemical species into a second chemical species.

 For example, an antibiotic biosynthesis pathway consists of all biochemical reactions converting primary metabolites into intermediates of antibiotics, and subsequently into antibiotics.

 By regulatory sequence placed "in phase" (in English operably linked) with respect to a nucleotide sequence whose expression is sought, it is meant that the transcription regulation sequence (s) are localized, with respect to the nucleotide sequence of the nucleotide sequence. interest whose expression is sought, so as to allow the expression of said sequence of interest, the regulation of said expression being dependent on factors interacting with the regulatory nucleotide sequences.

 According to another terminology, it may also be said that the nucleotide sequence of interest whose expression is sought is placed "under the control" of the nucleotide transcription regulatory sequences.

 The term "isolated" in the sense of the present invention refers to biological material that has been removed from its original environment (the environment in which it is naturally located).

 For example, a polynucleotide or a polypeptide naturally occurring in an organism (virus, bacteria, fungus, yeast, plant or animal) is not isolated. The same polypeptide separated from its natural environment or the same polynucleotide separated from the adjacent nucleic acids in which it is naturally inserted into the genome of the organism, is isolated.

 Such a polynucleotide may be included in a vector and / or such a polynucleotide may be included in a composition and remains

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 nevertheless in the isolated state, because the vector or composition does not constitute its natural environment.

 The term "purified" does not require that the material be present in a form of absolute purity, exclusive of the presence of other compounds. It is rather a relative definition.

 A polypeptide or a polynucleotide is in the purified state after purification of the starting material of at least one order of magnitude, preferably 2 or 3 and preferably 4 or 5 orders of magnitude.

 The "percent identity" between two nucleotide or amino acid sequences, within the meaning of the present invention, can be determined by comparing two optimally aligned sequences, through a comparison window.

 The part of the nucleotide sequence or polypeptide in the comparison window may thus comprise additions or deletions (for example "gaps") with respect to the reference sequence (which does not include these additions or deletions) so as to obtain an optimal alignment of the two sequences.

 The percentage is calculated by determining the number of positions at which an identical nucleobase or amino acid residue is observed for the two (nucleic or peptide) sequences compared and then dividing the number of positions at which there is identity between the two bases. or amino acid residues by the total number of positions in the comparison window, then multiplying the result by 100 to obtain the percentage of sequence identity.

 The optimal alignment of the sequences for the comparison can be performed in a computer manner using known algorithms contained in the package of the company WISCONSIN GENETICS SOFTWARE PACKAGE, GENETICS COMPUTER GROUP (GCG), 575 Science Doctor, Madison, WISCONSIN.

 By way of illustration, the percentage of sequence identity can be carried out using the BLAST software (BLAST versions 1. 4.9 of March 1996, BLAST 2. 0.4 of February 1998 and BLAST 2. 0.6 of September 1998. ), using only the default parameters (SF Altschul et al., J. Mol Biol., 1990 215: 403-410, SF Altschul et al., Nucleic Acids Res 1997 25: 3389-3402) .Blast searches for similar sequences / homologues to a reference "query" sequence, using

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 Altschul et al. The query sequence and the databases used may be peptide or nucleic, any combination being possible.

EXTRACTION AND PURIFICATION OF NUCLEIC ACIDS FROM AN ENVIRONMENTAL SAMPLE.

1. Direct extraction of nucleic acids
It has been shown according to the present invention that, in order to obtain a nucleic acid library from organisms contained in a soil sample, it was important to create conditions in which, on the one hand, the different organisms in the sample are made accessible for subsequent nucleic acid extraction steps and that the initial stage of soil sample processing allows maximum mechanical lysis of the organisms in the sample of nature to make the nucleic acids of these organisms, mainly genomic and plasmid DNA, directly accessible to the buffers used for the subsequent extraction steps.

 It has thus been demonstrated according to the invention that a maximum accessibility of the nucleic acids originating from the microorganisms of a soil sample is reached by a thorough and dry grinding of the previously dried soil sample in order to obtain microparticles. The Applicant has thus determined that the drying of the soil sample prior to any subsequent treatment causes a significant decrease in the cohesion of the raw soil sample and consequently favors its subsequent disintegration in the form of microparticles, when a suitable milling treatment is operated.

 Surprisingly, the applicant has shown that microparticles of dry soil samples have physicochemical properties favorable to the extraction of an optimal quantity of nucleic acids which, in their nature, could be representative of the genetic diversity of the organisms. initially present in the starting soil sample. It has been shown in particular that the method of direct extraction of nucleic acids according to the invention

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 the extraction of DNA from rare microorganisms, such as some rare Streptomyces or sporulated microorganisms.

 By "microparticles" of the soil sample for the purpose of the present invention is meant particles derived from the sample having an average size of about 50 m, that is to say between 45 and 55 on average. m /.

 According to the invention, the microparticles are obtained from previously dried or desiccated soil samples then crushed to obtain micro-particles of average size between 2 m and 50 m, before resuspension in a liquid buffer medium of the microparticles obtained.

 Such a liquid buffer medium may consist of a nucleic acid extraction buffer, in particular a conventional DNA extraction buffer well known to those skilled in the art.

 Grinding of the soil sample into microparticles has the dual function of mechanically lysing most of the organisms present in the initial soil sample and making non-lysed organisms accessible by this mechanical treatment to optional later stages of lysis. chemical and / or enzymatic.

 Thus, a first object of the invention is a method of preparing a nucleic acid collection from a soil sample containing organisms, said method comprising a first step (I- (a)) of obtaining microparticles by grinding the previously dried or desiccated soil sample, and then suspending the microparticles in a liquid buffer medium.

 Most preferably, the grinding step is carried out using an agate ball or tungsten device or using a tungsten ring device. These devices are preferred because the hardness of materials such as agate or tungsten significantly facilitates obtaining microparticles of the size specified above. For this reason, it will not be preferable, or even avoided, recourse to a glass ball grinding device, which has proved much less effective.

 Drying or classification of the soil sample can be carried out by any method known to those skilled in the art. For example,

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 the raw soil sample can be dried at room temperature for a period of 24 to 48 hours.

 As indicated above, the liquid buffer medium may consist of an extraction medium for the DNA present in the microparticles. An extraction buffer designated TENP, containing 50 mM tris, 20 mM EDTA, 100 mM NaCl and 1% (w / v) polyvinylpolypyrrolidone, at pH 9.0, will be used most preferably.

 The process for preparing a nucleic acid collection from a soil sample is further characterized in that the step of obtaining microparticles by grinding the previously dried or desiccated soil sample is followed by a step I- (b) of extraction of the nucleic acids present in the microparticles.

 It is common ground that nucleic acid extraction is accompanied by co-extraction of undesirable compounds and / or soil constituents necessitating subsequent purification of the extracted nucleic acids, such a subsequent purification step having to be both selective enough. to allow the removal of undesirable compounds and / or soil components and a yield sufficient to cause a small loss in the amount of DNA previously extracted.

 It has been shown according to the invention that a purification step of the DNA extracted from the microparticles of the soil sample meeting the selectivity and yield criteria defined above, comprises a treatment of the extracted DNA by a combination of two successive chromatography steps, respectively molecular sieve chromatography and anion exchange chromatography.

 According to another characteristic of the above process, the nucleic acid extraction step 1- (b) is followed by a step I- (c) of purification of the nucleic acids extracted using the two chromatography steps. following: - passage of the solution containing the nucleic acids on a molecular sieve, then recovery of elution fractions enriched in nucleic acids;

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 passage of elution fractions enriched in nucleic acids on an anion exchange chromatography support, then recovery of the elution fractions containing the nucleic acids.

 The nature and order of the chromatography steps above are essential to good selectivity and excellent yield of the DNA purification step previously extracted from the microparticles of the previously dried or desiccated soil sample.

 Very advantageously, the chromatographic support of the "molecular sieve" type of the nucleic acid purification step above consists of a Sephacryl # S400 HR type chromatographic support or a chromatographic support of equivalent characteristics.

 Most preferably, the anion exchange chromatographic support used in the second step of purification of the extracted DNA is a support of the type Elutip d, or a chromatographic support of equivalent characteristics.

 By combining steps I- (a) of obtaining microparticles from the dry soil sample, I- (b) extraction of the nucleic acids present in the microparticles and I- (c) purification by the chromatographic steps described above, it was possible according to the invention to directly extract the DNA from the soil without prior purification of the cells of the organisms initially contained in the sample, while avoiding the co-extraction of soil contaminants such as for example humic acids which is observed with the methods of the state of the art.

 Contaminants, such as humic acids, severely affect the subsequent analyzes and uses of the nucleic acids whose purification is sought.

 According to the above method, it is furthermore possible to access the nucleic acids contained in the organisms which have not been mechanically lysed during step I- (a) of obtaining microparticles of the sample of the soil, with the aim of obtaining an almost exhaustive collection of the genetic diversity of the nucleic acids initially present in the soil sample. Thus, the microparticles of the soil sample may be the subject of subsequent chemical, enzymatic or physical lysis treatment steps, or a combination of chemical, enzymatic or physical treatments.

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 According to a first aspect, the method for preparing a nucleic acid collection from a soil sample according to the invention may be further characterized in that step I- (a) is followed by steps following: # treatment of the soil suspension in a liquid buffer medium by sonication; # extraction and recovery of nucleic acids.

 Preferably, for a sonication treatment, a titanium microtip type device, such as the 600 W Vibracell Ultrasonicator device marketed by the Bioblock Company or a Cup Horn type of sonicator, will be used.

 Most preferably, the sonication step is carried out at a power of 15 W for a period of 7 to 10 min and comprises successive cycles of sonication, the actual sonication being performed for 50% of the duration of each cycle.

 According to a second aspect, the above method can be further characterized in that step I- (a) is followed by the following steps: # treatment of the soil suspension in a liquid buffer medium by sonication; incubation of the suspension at 37 ° C. after sonication in the presence of lysozyme and of achromopeptidase; # addition of SDS before centrifugation and precipitation of nucleic acids; # recovery of precipitated nucleic acids.

 Preferably, the incubation step in the presence of lysozyme and achromopeptidase will be carried out at a final concentration of 0.3 mg / ml of each of the two enzymes, preferably for 30 minutes at 37 ° C.

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 Preferably, the SDS will be used at a final concentration of 1% and for an incubation time of 1 hour at a temperature of 60 ° C. before centrifugation and precipitation.

 According to a third aspect, the method for preparing a nucleic acid collection from a soil sample above is further characterized in that step I- (a) is followed by the following steps: homogenization of the soil suspension with a violent mixing step (vortex) followed by a simple stirring step; freezing of the homogeneous suspension followed by thawing; treatment by sonication of the suspension after thawing; incubation of the suspension at 37 ° C. after sonication in the presence of lysozyme and of achromopeptidase; addition of SDS before centrifugation and precipitation of the nucleic acids; - recovery of nucleic acids.

 Preferably, the suspensions of soil microparticles are vortexed and then homogenized by gentle stirring on a circular rotation stirrer for a period of two hours before being frozen at -20.degree.

 Preferably, the suspensions are again violently vortexed for 10 minutes, after thawing and before the sonication step.

 It goes without saying that the nucleic acids extracted by the embodiments of the direct nucleic acid extraction method described above are preferentially purified according to the purification step consisting of a first pass on molecular sieve and then a subsequent passage of the fractions. of elution obtained after the chromatography on molecular sieve on an anion exchange chromatographic support.

 2. Indirect extraction of nucleic acids

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According to a second embodiment of the method for preparing a nucleic acid collection from a sample of the environment, according to the invention, said sample of the environment undergoes a first treatment of such a nature as to allow separation. organisms, contained in this sample, of the other macro-constituents of the sample.

 This second embodiment of the process for preparing a nucleic acid collection according to the invention promotes the production of nucleic acids of large size, which are practically impossible to obtain according to the first embodiment of the method according to the invention. invention described above, the step of mechanical lysis performed to obtain microparticles also having the effect of physically breaking the nucleic acids of the soil sample or nucleic acids contained in the organisms of the sample of ground.

 The obtaining of large nucleic acids has been sought by the applicant in order to isolate and characterize the nucleic acids comprising, at least partially, all of the coding sequences belonging to the same operon capable of directing the biosynthesis of a compound of industrial interest.

 Preferably, using the second embodiment of the process for preparing a nucleic acid collection from a soil sample according to the invention, nucleic acids having a size greater than 100 are obtained. kb, preferably greater than 200,250 or 300 kb, and most preferably nucleic acids larger than 400,500 or even 600 kb.

 This second embodiment of a method for preparing a nucleic acid collection from a sample of the environment according to the invention consists of a combination of four successive stages intended to obtain the acids nucleic acids having the characteristics described above.

 When the sample of the environment is a soil sample, it has been shown according to the invention that a first step of obtaining a suspension by dispersion of the soil sample in a liquid medium facilitates the accessibility of the samples. organisms contained in the sample without causing significant mechanical lysis of the cells.

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 The first step of obtaining a dispersion of the above soil sample makes the organisms in the sample accessible to the external environment and also allows partial dissociation of the sample organisms and the macro-constituents. It thus makes possible a subsequent separation of the organisms initially contained in the sample of the other constituents of the latter.

 When the environmental sample comes for example from plants, marine organisms or insects, a pre-treatment by grinding is necessary in order to make the organisms of the associated microflora accessible at the later stages of the process.

 Thus, the present process comprises a step of separating the organisms from the other inorganic and / or organic constituents obtained previously by centrifugation on a density gradient. The organisms thus separated are then subjected to a step of lysis and extraction of the nucleic acids.

 The step of centrifugation on a density gradient has, surprisingly, allowed the cells of organisms to be separated from the soil particles contained in the suspension of the sample. It would indeed be expected that a proportion of the cells are driven with the macro-particles within the gradient phase. In addition, it has never before been demonstrated that a density gradient centrifugation of a soil sample allows the discovery at the aqueous phase / gradient interface of a representative population of organisms. organisms present in the starting sample, because these organisms are of extremely variable volume, density and shape. It was reasonable to assume that they would be found indifferently within the aqueous phase, at the aqueous phase / density gradient interface and also within the density gradient itself.

 Thus, one skilled in the art could expect organisms with densities lower or greater than the density density density used (density gradient density between 1.2 and 1.5 g / ml). preferentially 1.3 g / ml) could not be recovered, which would have had the effect of introducing a bias in the representativity of the organisms actually separated and, consequently, also in the diversity of the nucleic acids extracted.

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 In addition, in a particular embodiment of the method, a step of germinating the spores, in particular actinomycetes, is performed, which has the effect of significantly increasing the amount of actinomycetes DNA recovered.

 The last step consists of a purification step of the nucleic acids thus extracted on a cesium chloride gradient.

 Surprisingly, the purification of the nucleic acids on the cesium chloride gradient allows a substantial or complete elimination of the substances composing the density gradient. This characteristic is decisive with regard to the subsequent use of the purified nucleic acids because the density gradient is known as a potent enzyme inhibitor, capable of inhibiting the catalytic activity of the enzymes used to prepare the insertion of the acids, where appropriate. nucleic extracts in vectors.

 According to this second embodiment, the method for preparing a nucleic acid collection from a sample of the environment containing organisms according to the invention comprises the following succession of steps: (i) obtaining a suspension by dispersion of the sample of the environment in a liquid medium and then homogenization of the suspension obtained by gentle stirring; (ii) separating the organisms from the other inorganic and / or organic constituents of the homogeneous suspension obtained in step (i) by centrifugation on a density gradient; (iii) lysing the separated microorganisms in step (ii) and extracting the nucleic acids; (iv) purification of nucleic acids on a cesium chloride gradient.

 Preferably, the suspension of the soil sample is obtained by dispersing this sample by grinding with the aid of a Waring Blender type device or a device of characteristics

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 equivalent. Most preferably, the sample suspension is obtained after three successive grindings lasting one minute each in a Waring Blender type device. Preferably, the milled sample will be cooled in ice between each of the grindings.

 Preferably, the organisms are then separated from the soil particles by centrifugation on a "Nycodenz" type density cushion marketed by the company Nycomed Pharma AS. (Oslo, Norway). The preferred conditions for centrifugation are 10,000 g for 40 minutes at 4 ° C., advantageously in a rotor with moving cups of the "rotor TST 28.38" type marketed by KONTRON.

 The ring of organisms located, after centrifugation, at the interphase of the aqueous upper phase and the lower phase of Nycodenz is then removed and washed by centrifugation before taking up the cell pellet in an appropriate buffer.

 The step (iii) of lysing the organisms separated in step (ii) described above can be carried out in any manner known to those skilled in the art.

 Advantageously, the cells are lysed in a 100 mM Tris 10 mM-EDTA solution at pH 8. 0 in the presence of lysozyme and of achromopeptidase, advantageously for one hour at 37 ° C.

 The actual extraction of the DNA can be advantageously carried out by adding a solution of lauryl sarcosyl (1% of the final weight of the solution) in the presence of proteinase K and incubation of the final solution at 37 ° C. for 30 minutes.

 The nucleic acids extracted in step (iii) are then purified on a cesium chloride gradient. Preferably, the step of purifying the nucleic acids on a cesium chloride gradient is carried out by centrifugation at 35,000 rpm for 36 hours, for example on a Kontron type rotor 65.13.

 According to a particular aspect of the process for preparing a nucleic acid collection from a soil sample containing organisms according to the invention, said nucleic acids consist mainly, if not exclusively, of DNA molecules.

 In another aspect, the nucleic acids can be recovered after inclusion of the organisms, separated on a gradient of

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 density, in a block of agarose and lysis, for example chemical and / or enzymatic, organisms included in the agarose block.

 Another subject of the invention consists in a nucleic acid collection constituted by the nucleic acids obtained in stage II- (iv) of the process for preparing a nucleic acid collection according to the invention or obtained at the same time. step (c) or a subsequent step of the process for preparing a nucleic acid collection according to the invention.

 The invention also relates to a nucleic acid characterized in that it is contained in a nucleic acid collection as defined above.

 According to a first aspect, such a nucleic acid constituting a nucleic acid collection according to the invention is characterized in that it comprises a nucleotide sequence encoding at least one operon, or part of an operon.

 Most preferably, such an operon encodes all or part of a metabolic pathway.

 Example 9 describes the construction of a genomic DNA library from a strain of Streptomyces alboniger and its cloning respectively in shuttle cosmos pOS7001 and pOS700R. It has been shown according to the invention that in the DNA library made in the integrative vector pOS7001 nine clones contain nucleotide sequences belonging to the operon responsible for the puromycin biosynthetic pathway. Similarly, twelve clones containing nucleotide sequences of the operon responsible for the puromycin biosynthetic pathway could be identified within the DNA library made in the replicative vector pOS 700R.

 In particular, some integrative and replicative cosmids of the libraries produced have, after digestion with ClaI and EcoRV restriction endonucleases, a fragment of a size of 12 kb capable of containing all the sequences of the operon responsible for the biosynthetic pathway. puromycin.

 Thus, according to another aspect, a nucleic acid according to the invention contains, at least in part, nucleotide sequences of the operon responsible for the puromicyne biosynthetic pathway.

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 Example 2 below describes the construction of a DNA library according to a method according to the present invention in a pBluescript SK- vector from a soil contaminated with lindane.

 The recombinant vectors were transfected into Escherichia coli DH10B cells and the transformed cells were cultured in a suitable culture medium in the presence of lindane. Screening the transformed cell clones of the library showed that out of 10,000 clones screened, 35 had a lindane degradation phenotype. The presence of the linA gene in these clones could be confirmed by PCR amplification using primers specific for this gene.

 Thus, according to another aspect, the invention also relates to a nucleic acid containing a nucleotide sequence of the metabolic pathway causing the biodegradation of lindane.

 It is thus clearly demonstrated, as described above, a method for preparing a nucleic acid collection from a soil sample containing organisms according to the invention as well as a method for preparing a The collection of recombinant vectors containing the nucleic acids constituting the above-mentioned nucleic acid collection was entirely suitable for the isolation and characterization of nucleotide sequences included in an operon.

 A further demonstration of the ability of a method according to the invention to identify coding nucleotide sequences involved in a regulated biosynthetic pathway in the form of an operon is further described below: this is cloning and the characterization of sequences coding for polyketide synthases involved in the polyketide biosynthetic pathway, which belong to a family of molecules, some of whose representatives are of major therapeutic interest, in particular antibiotics.

 The present invention therefore also relates to a nucleic acid constituting a nucleic acid collection according to the invention, characterized in that it comprises all of a nucleotide sequence encoding a polypeptide.

 According to a first aspect, a nucleic acid constituting a collection of nucleic acids according to the invention is of procaryotic origin.

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 According to a second aspect, a nucleic acid constituting a collection of nucleic acids according to the invention comes from a bacterium or a virus.

 According to a third aspect, a nucleic acid constituting a collection of nucleic acids according to the invention is of eukaryotic origin.

 In particular, such a nucleic acid is characterized in that it comes from a fungus, a yeast, a plant or an animal.

MOLECULAR CHARACTERIZATION OF THE NUCLEIC ACID COLLECTION EXTRACTED FROM THE SOIL.

 In order to overcome the many technical disadvantages of the methods of characterizing the extracted and purified DNA libraries from a sample of the environment that have been described in the prior art portion of the description, the applicant has developed a simple and reliable method for characterizing qualitatively and semi-quantitatively the nucleic acids obtained at the end of the process described above.

 The method according to the invention thus consists in universally amplifying a 700 bp fragment located inside a 16S-type ribosomal DNA sequence, and then hybridizing the amplified DNA with an oligonucleotide probe of variable specificity and finally, to compare the intensity of hybridization of the sample with respect to an external standard range of DNA of sequence or of known origin.

 The amplification prior to hybridization with the oligonucleotide probe makes it possible to quantify genera or species of scant microorganisms. In addition, the amplification by universal primers makes it possible, during hybridization, to use a large series of oligonucleotide probes.

 Thus, the invention furthermore relates to a method for determining the diversity of nucleic acids contained in a collection of nucleic acids, and more particularly to a collection of nucleic acids from a sample of the environment, preferably a soil sample, said method comprising the following steps:

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 contacting the nucleic acids of the collection of nucleic acids to be tested with a pair of oligonucleotide primers hybridizing with any bacterial 16S ribosomal DNA sequence; performing at least three amplification cycles; detection of the amplified nucleic acids using an oligonucleotide probe or a plurality of oligonucleotide probes, each probe specifically hybridizing with a 16S ribosomal DNA sequence common to a kingdom, order, subclass or a bacterial genus; where appropriate, comparing the results of the preceding detection step with the detection results, using the probe or the plurality of nucleic acid probes of known sequence constituting a standard range.

 Preferably, a first pair of primers hybridizing with universally conserved regions of the 16S ribosomal RNA gene consists respectively of primers FGPS 612 (SEQ ID No. 12) and FGPS 669 (SEQ ID No. 13).

 A second embodiment of a preferred pair of primers according to the invention consists of the pair of universal primers 63 (SEQ ID No. 22) and 1387r (SEQ ID No. 23).

 According to a particular embodiment of a method for determining the nucleic acid diversity of a nucleic acid collection, the amplification step using a pair of universal primers may be performed on a collection of recombinant vectors in each of which has been inserted a nucleic acid of the relevant nucleic acid collection, prior to the step of hybridization with the oligonucleotide probes specific for a kingdom, an order, a sub- class or a particular bacterial genus.

 Such a method for determining the diversity of nucleic acids contained in a collection is particularly applicable to nucleic acid collections obtained according to the teaching of the present description.

 Thus, Example 3 details a process for preparing a collection of nucleic acids from a soil sample containing organisms comprising an indirect DNA extraction step by dispersing a soil sample prior to the separation of

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 cells on a Nycodenz gradient, lysis of the cells and then purification of the DNA on a cesium chloride gradient.

 The nucleic acid collection thus obtained was used as such or in the form of inserts in cosmid type vectors in an amplification process using the above-mentioned universal primers of the 16S rDNA, and then the DNAs. amplified were subjected to a detection step using oligonucleotide probes of SEQ ID N 14 to SEQ ID N 21 which are presented in Table 4.

 The results show that a method of preparing a nucleic acid collection from a soil sample containing organisms according to the invention provides access to DNA of more than 14% of the total telluric microflora. that is 2 x 108 cells per gram of soil, whereas the total cultivable microflora represents only 2% of the total microbial population.

 In order to determine the phylogenetic diversity of a collection of nucleic acids prepared in accordance with the invention, 47 sequences of the 16S rRNA gene were isolated and sequenced. These sequences correspond respectively to the nucleotide sequences SEQ ID N 60 to SEQ ID N 106.

 The nucleic acids comprising the sequences SEQ ID N 60 to SEQ ID N 106 are also part of the invention, as well as the nucleic acids having at least 99%, preferentially 99.5% or 99.8% nucleic acid identity. with the nucleic acids comprising the sequences SEQ ID N 60 to SEQ ID N 106. Such sequences can be used in particular as probes to screen clones of a DNA library and thus identify those among the clones of the library , which contain such sequences, these sequences being likely to be close to coding sequences of interest, such as sequences coding for enzymes involved in the biosynthetic pathway of antibiotic metabolites, for example polyketides.

 The comparison of the 16S rRNA sequences from a DNA library made according to the invention with the sequences listed in the RDP data base (Maidak BL, Cole JR, Parker CT, Garrity GM, Larsen N., Li B., Lilburn TG, McCaughey, MJ, Olsen GJ, R. Overbeek, S. Pramanik, Schmidt TM, Tiedje JM, Woese CR

(1999) "A new project of the RDP (Ribosomal Database Project)" Nucleic

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 Acids Research Vol. 27: 171-173) have made it possible to determine that the nucleic acids contained in a collection of nucleic acids according to the invention come from a-proteobacteria, -proteobacteria, 8proteobacteria, γ-proteobacteria, actinomycetes as well as of a genus related to acidobacterium. These results, presented in Table 7 as well as by the phylogenetic tree of FIG. 7, account for the great phylogenetic diversity of the nucleic acids contained in a DNA library prepared according to the process according to the invention.

CLONING AND / OR EXPRESSION VECTORS
Each of the nucleic acids contained in a collection of nucleic acids prepared in accordance with the invention may be inserted into a cloning and / or expression vector.

 To this end, all types of vectors known in the state of the art can be used, such as viral vectors, phages, plasmids, phagemids, cosmids, phosmides, BAC type vectors, P1 bacteriophages. , BAC type vectors, YAC type vectors, yeast plasmids or any other vector known to the state of the art by the skilled person.

 Advantageously, according to the invention, vectors will be used which allow stable expression of the nucleic acids of a DNA library. For this purpose, such vectors preferentially include transcriptional control sequences which are located in phase ("operably linked") with the genomic insert so as to allow the initiation and / or regulation of the expression of at least a portion of said DNA insert.

 As a result of the foregoing, the invention also relates to a process for preparing a recombinant vector collection characterized in that the nucleic acids obtained in step II- (iv) or in step I- (c) or any further step of a method of preparing a nucleic acid collection from a soil sample containing organisms according to the invention are inserted into a cloning and / or expression vector.

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 Prior to their insertion into a cloning and / or expression vector, the nucleic acids constituting a nucleic acid collection according to the invention can be separated according to their size, for example by electrophoresis on a gel of agarose, if appropriate after digestion with a restriction endonuclease.

 In another aspect, the average size of the nucleic acids constituting a nucleic acid collection according to the invention can be made of a substantially uniform size by the implementation of a physical disruption step prior to their insertion into the cloning and / or expression vector.

 Such a step of physical or mechanical breaking of the nucleic acids may consist of successive passages of the latter, in solution, in a metal channel of about 0.4 mm in diameter, for example the channel of a syringe needle having a such diameter.

 The average size of the nucleic acids can in this case be between 30 and 40 kb in length.

 The construction of the preferred vectors according to the invention is shematized in FIGS. 25 (conjugative integrative cosmid) and 26 (integrative BAC).

 Cloning and / or expression vectors that can be advantageously used for the purpose of inserting the nucleic acids contained in a collection or DNA library according to the invention are, in particular, the vectors described in European Patent No. EP-0 350 341. and in US Patent No. 5,688,689, such vectors being especially adapted for the transformation of actinomycete strains. Such vectors contain, in addition to a DNA sequence of the insert, an att attachment sequence as well as a DNA sequence encoding an integrase (int sequence) functional in the actinomycete strains.

 However, it has been observed according to the invention that certain cloning and / or expression vectors have disadvantages and that their theoretical functional capacity has not been reached in practice.

 Thus, it has been found that the integration system contained in vectors of the state of the art, and in particular in the vectors described in European Patent No. EP 0 350 41, did not make it possible

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 reality a good integration of the DNA insert of the bank within the bacterial chromosome.

 Assuming that the functional deficits of integration of such vectors within the bacterial chromosome were due to a defect in the expression of the integrase gene present in these vectors, the applicant first sought to increase expression of the integrase gene by substituting for the promoter of the initial transcription a transcription promoter capable of significantly increasing the number of transcripts of the integrase.

 The results have been disappointing and the chromosome integration function of these vectors has not been improved.

 Surprisingly, it has been shown according to the invention that the difficulties of expression of the integrase contained in this integrative vector family was not at the level of the expression quantity of the transcripts, but at the level of their stability. .

 According to a second hypothesis, the applicant could show that the lack of stability of integrase transcripts was caused by deficits in the transcription termination of the corresponding messenger RNA.

 The applicant then inserted a terminator site placed downstream of the sequence encoding the vector integrase so as to obtain a messenger RNA of determined size. The insertion of an additional termination signal downstream of the nucleotide sequence coding for the vector integrase made it possible to obtain a family of integrative vectors of the cosmid type and of the BAC type.

 Preferably, the terminator site is placed downstream of the att attachment site.

 In addition, the Applicant has developed novel conjugative vectors and novel cosmid-type replicative vectors and novel BAC-type conjugative vectors which can advantageously be used for the insertion of the nucleic acids constituting a nucleic acid collection. prepared according to the process of the invention.

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 When the insertion of medium-sized DNA fragments is desired, cosmid-type vectors are preferably used, capable of receiving inserts having a maximum size of about 50 kb.

 Such cosmidic vectors are particularly suitable for the insertion of nucleic acids constituting a collection of nucleic acids obtained according to the method of the invention comprising a first step of direct extraction of DNA by mechanical lysis of the organisms contained in the initial soil sample.

 When the insertion of large nucleic acids, in particular nucleic acids of a size greater than 100 kb, or even greater than 200, 300, 400,500 or 600 kb, is sought, preference will then be given to vectors of the nucleic acid. BAC type capable of receiving DNA inserts of such size.

 Such BAC type vectors are particularly suitable for the insertion of the nucleic acids constituting a collection of nucleic acids obtained according to the process according to the invention in which the first step consists of an indirect extraction of the DNA. by first separating the organisms contained in the initial soil sample and removing the macromolecules from said soil sample.

 In particular, BAC-type vectors are advantageously used for the insertion of large nucleic acids containing, at least partially, the nucleotide sequence of an operon.

 Thus, the process for preparing a collection of recombinant cloning and / or expression vectors according to the invention is further characterized in that the cloning and / or expression vector is of the plasmid type.

 In another aspect, such a method is characterized in that the cloning and / or expression vector is of the cosmid type.

 According to a first aspect, it may be a replicative cosmid in E. coli and integrative in Streptomyces. A most preferred cosmid vector responding to such a definition is the cosmos pOS7001 described in Example 3.

 In yet another aspect, the cosmid vector is conjugative and integrative in Streptomyces.

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 In general, conjugative vectors of cosmid type or BAC type, which comprise in their nucleotide sequences a pattern recognized by the cellular enzyme machinery called "origin of conjugation" are used whenever it is desired to avoid recourse to techniques of heavy transformation and little automation.

 For example, the transfection of vectors initially harbored by E. coli cells in Streptomyces cells conventionally requires a step of recovery of the recombinant vector contained in Escherichia coli cells, and its prior purification at the protoplast transformation stage. of Streptomyces. It is commonly accepted that transfection of a set of 1000 clones of Escherichia coli into Streptomyces requires the production of about 8000 clones for each clone of E. coli to have a chance of being represented.

 In contrast, a step of transfection by conjugation of an E. coli-hosted vector to Streptomyces cells requires the same number of clones of each of the microorganisms, the conjugation step taking place "clone to clone" and furthermore not including the technical difficulties associated with the step of transferring genetic material by protoplast transformation, for example in the presence of polyethylene glycol.

 In order to optimize the DNA library construction in Streptomyces, new conjugative vectors of cosmid type and BAC type have been developed according to the invention so as to allow maximum efficiency of the conjugation step.

 In particular, the novel conjugative vectors according to the invention have been constructed by placing a selection marker gene at the end of the vector DNA which is transferred to the recipient bacterium in the last place. This enhancement to the conjugative vectors of the state of the technique makes it possible to positively select only the receptor bacteria which have received all the DNA of the vector and, consequently, all the DNA of the insert of interest.

 A conjugative and integrative cosmid in Streptomyces preferred according to the invention is the cosmid pOSV303 described in Example 5.

 In another aspect, a method of preparing a recombinant vector collection according to the invention is carried out using a replicative cosmid in both E. coli and Streptomyces.

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Such a cosmid is advantageously the cosmos pOS 700R described in Example 6.

 In yet another aspect, the above method can be carried out with a replicative cosmid in E. coli and Streptomyces and conjugative in Streptomyces.

 Such a replicative and conjugative cosmid can be obtained from a replicative cosmid according to the invention, by inserting an appropriate transfer origin, such as RK2, as described in Example 5 for the construction of the vector. pOSV303.

 According to another advantageous embodiment of the process for preparing a recombinant vector collection according to the invention, a BAC-type cloning and / or expression vector is used.

According to a first aspect, the BAC-type vector is integrative and conjugative in Streptomyces.

 Most preferably, such an integrative and conjugative BAC vector in Streptomyces is the BAC vector pOSV 403 described in Example 8.

 The invention further relates to a recombinant vector characterized in that it is chosen from the following recombinant vectors: a) a vector comprising a nucleic acid constituting a nucleic acid collection according to the invention; b) a vector as obtained by a method eliminating any recourse to the action of a restriction endonuclease on the DNA fragment to be inserted, as described above.

Most preferably, the invention also relates to a vector chosen from the following vectors: cosmid pOS7001; the cosmid pOSV303; the cosmid pOS700R; the BAC vector pOSV403;
The invention further relates to a collection of recombinant vectors as obtained according to any of the methods according to the invention.

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Process for the preparation of a recombinant cloning and / or expression vector according to the invention

 Conventional techniques for inserting DNA into a vector to prepare a recombinant cloning and / or expression vector typically use a first step in which a restriction endonuclease is incubated with both the DNA to be inserted and with the receptor vector thus creating compatible ends between the DNA to be inserted and the DNA of the vector allowing the assembly of the two DNAs before a final ligation step making it possible to obtain the recombinant vector.

 However, such a conventional technique has notable drawbacks, particularly when the insertion of large nucleic acids into a cloning and / or expression vector is desired.

 Indeed, the prior action of a restriction enzyme on the DNA fragments to be inserted into a vector is likely to significantly reduce the size of this DNA prior to its insertion into the vector. It goes without saying that a significant reduction in the size of the DNA prior to its insertion on a vector is a particularly unfavorable situation when cloning of large DNA fragments that may contain all the sequences is sought. coding and, where appropriate, also regulatory sequences, of an operon whose expression constitutes a complete biosynthetic pathway of a metabolite of industrial interest, and more particularly of a compound of therapeutic interest.

 In order to overcome the drawbacks of the prior art techniques, two methods of preparing a recombinant cloning and / or expression vector which do not require the use of a restriction endonuclease have been developed according to the invention. on the DNA to insert prior to its introduction into the vector. Such methods are therefore quite suitable for cloning long DNA fragments which may contain, at least partially, all the coding sequences and, where appropriate, also

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 regulatory sequences, of a complete operon responsible for a biosynthetic pathway.

 According to a first aspect, a process for preparing a recombinant cloning and / or expression vector according to the invention is characterized in that the insertion of a nucleic acid into the cloning and / or expression vector , comprises the steps of: - opening the cloning and / or expression vector at a selected cloning site, using an appropriate restriction endonuclease; adding a first homopolymeric nucleic acid to the free 3 'end of the open vector; adding a second homopolymeric nucleic acid, of complementary sequence to the first homopolymeric nucleic acid, to the free 3 'end of the nucleic acid to be inserted into the vector; assembling the nucleic acid of the vector and the nucleic acid by hybridization of the first and second homopolymeric nucleic acid with sequences complementary to one another; - close the vector by ligation.

 Such a method is described in Examples 10 and 13 below.

 Advantageously, the above method may comprise the following characteristics, alone or in combination: the first homopolymeric nucleic acid is of poly (A) or poly (T) sequence; the second homopolymeric nucleic acid is of poly (T) or poly (A) sequence.

 Most preferably, the homopolymeric nucleic acids have a length of between 25 and 100 nucleotide bases, preferably between 25 and 70 nucleotide bases.

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 The process for preparing a recombinant cloning and / or expression vector described above is particularly suitable for the construction of DNA libraries in BAC type vectors. Thus, according to an advantageous embodiment of the process for preparing a recombinant vector described above, said method is further characterized in that the size of the nucleic acid to be inserted is at least 100 kb, and preferentially at least 200,300, 400,500 or 600 kb.

 Such a preparation method is therefore particularly suitable for the insertion of the nucleic acids contained in a collection of nucleic acids obtained according to the method of the invention.

 In order to allow the insertion of large DNA fragments into cloning and / or expression vectors, it has been developed according to the invention, a second method having made it possible to eliminate any recourse to the action of a restriction endonuclease on the DNA to be inserted into the vector.

 Such a process for preparing a recombinant cloning and / or expression vector according to the invention is characterized in that the step of inserting a nucleic acid into said cloning and / or expression vector comprises the following steps: - creation of blunt ends on the ends of the nucleic acid of the collection by elimination of the 3 'outgoing sequences and filling the 5' outgoing sequences; opening of the cloning and / or expression vector to a cloning site chosen with the aid of an appropriate restriction endonuclease; addition of complementary oligonucleotide adapters; creation of blunt ends at the ends of the nucleic acid of the vector by elimination of the outgoing 3 'sequences and filling of the 5' outgoing sequences, followed by dephosphorylation of the 5 'ends in order to prevent recircularization of the vector; - Insertion of the nucleic acid of the collection into the vector by ligation.

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 Preferably, the removal of the 3 'outgoing sequences is carried out using an exonuclease, such as the Klenow enzyme.

 Preferably, the filling of the 5 'outgoing sequences is carried out using a polymerase, and very preferably T4 polymerase, in the presence of the four nucleotide triphosphates.

 A method for preparing a recombinant cloning and / or expression vector by removing the outgoing 3 'sequences and filling the outgoing 5' sequences as described above is particularly suitable for the construction of DNA libraries from of vectors of cosmid type.

 Such a process for obtaining recombinant vectors is described in Example 12.

 In a particular mode of preparation of a recombinant vector according to the invention, oligonucleotides comprising one or more rare restriction sites are added to the vector at the cloning site of the DNA to be inserted, in accordance with the teaching of Example 10. This addition of oligonucleotides facilitates the subsequent recovery of the inserts without cleavage of the latter.

HOST CELLS
Although any type of host cell may be used for transfection or transformation with a nucleic acid or a recombinant vector according to the invention, in particular a prokaryotic or eukaryotic host cell, it will be preferable to use host cells whose physiological, biochemical characteristics and genetic are well characterized, easily cultivable on a large scale and whose culture conditions for the production of metabolites are well known.

 Preferably, the host cell receiving a nucleic acid or a recombinant vector according to the invention is phylogenetically close to the donor organisms initially contained in the sample of the environment from which the nucleic acids originate.

 Most preferably, a host cell according to the invention must have a similar or at least a similar codon usage.

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 donor organisms initially present in the environmental sample, especially the soil sample.

 The size of the DNA fragments likely to carry the desired nucleotide sequences of interest may be variable. Thus, enzymes encoded by 1 kb medium size genes can be expressed from small inserts while expression of secondary metabolites will require maintenance of much larger fragments in the host organism, for example from 40 kb to more than 100 kb, 200 kb, 300 kb, 400 kb or 600 kb.

 Thus, Escherichia coli host cells are a preferred choice for cloning large DNA fragments.

 Most preferably, use will be made of the strain of Escherichia coli designated DH10B and described by Shizuya et al; (1992) for which cloning protocols in BAC vectors have been optimized.

 However, other Escherichia coli strains can be advantageously used for the construction of a DNA library according to the invention, such as E. coli Sure strains, E.coli DH5α, or E.coli 294 ( ATCC No. 31446).

 In addition, the construction of a DNA library by transfection of E. coli cells with recombinant vectors according to the invention is also possible, the expression of genes of various prokaryotes such as Bacillus, Thermotoga, Corynebacterium, Lactobacillus or Clostridium having been described in PCT application WO 99/20799.

 In general, E. coli host cells may in all cases be transient hosts in which recombinant vectors according to the invention can be maintained with high efficiency, the genetic material can be easily handled and archived and stable manner.

 In order to express the greatest possible molecular diversity, other cellular hosts may also be advantageously used, such as Bacillus, Pseudomonas, Streptomyces, Myxococcus, Aspergillus nidulans or Neurospora crassa cells.

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 It has furthermore been shown according to the present invention that Streptomyces lividans cells can be used successfully and constitute expression systems complementary to Escherichia coli.

Streptomyces lividans is a model for studying the genetics of Streptomyces and has also been used as a heterologous expression host for many secondary metabolites. Streptomyces lividans, together with other actinomycetes such as Streptomyces coelicolor, Streptomyces griseus, Streptomyces fradiae, as well as Streptomyces griseochromogenes, the precursor molecules and regulatory systems necessary for the expression of all or part of complex biosynthetic pathways, such as, for example, the polyketide biosynthetic pathway or the non-ribosomal polypeptide biosynthesis pathway representing classes of molecules of very diverse structures.

 Streptomyces lividans also has the advantage of accepting foreign DNA with high transformation efficiencies.

 Thus, the invention also relates to a recombinant host cell comprising a nucleic acid according to the invention, constituting a nucleic acid collection prepared according to a method according to the invention, or a recombinant host cell comprising a recombinant vector such as than previously defined.

 According to a first aspect, it may be a recombinant host cell of prokaryotic or eukaryotic origin.

 Advantageously, a recombinant cell according to the invention is a bacterium, and most preferably a bacterium selected from E. coli and Streptomyces.

 In another aspect, a recombinant host cell according to the invention is characterized in that it is a yeast or a filamentous fungus.

 The invention also relates to a collection of recombinant host cells, each of the host cells constituting the collection comprising a nucleic acid derived from a collection of nucleic acids made according to a process for preparing a nucleic acid collection. from a soil sample containing organisms as described above.

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 The invention also relates to a collection of recombinant host cells, each of the host cells constituting the collection comprising a recombinant vector according to the invention.

 Due to the large size of the inserts it is necessary to have maximum efficiency of transformation. For this purpose, a Streptomyces lividans receptor strain constitutively expressing the integrase of pSAM2 to promote site-specific vector integration is preferred. For this, the int gene under the control of a strong promoter is integrated into the chromosome. Overproduction of integrase does not induce excision phenomena (Raynal et al., 1998).

 The production of a new metabolite from the insert could be toxic to Streptomyces if the insert does not contain genes for resistance to the antibiotic produced or if this gene is little or not expressed. The capacity of the different genes allowing Streptomyces ambofaciens to resist the antibiotic it produces is studied (Gourmelen et al., 1998, Pernodet et al., 1999). Some of these genes encode ABC-type transporters that may confer a broad spectrum of resistance. These genes can be introduced and overexpressed in the host strain of Streptomyces lividans.

 Conversely, a strain that is hypersensitive to antibiotics can be used (Pernodet et al., 1996) to detect the presence of resistance genes in the library. Indeed, in antibiotic-producing microorganisms, these resistance genes are often associated with genes in the biosynthetic pathway of the antibiotic. The selection of resistant clones can make it possible to simply perform a first sort before the more complex tests for detecting a new metabolite produced by the clone.

ISOLATION AND CHARACTERIZATION OF NEW NUCLEOTIDE SEQUENCES ENCODING SYNTHASE POLYKETIDES.

 According to the invention, a collection of recombinant host cells has been obtained after transfection of the host cells by a collection of recombinant vectors each containing a nucleic acid insert from a collection of nucleic acids prepared in accordance with the method according to the invention. .

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 More specifically, the DNA fragments obtained according to the process of the invention in which it is implemented a step of indirect extraction of DNA from the organisms contained in the soil sample were first cloned into the cosmid integrative pOS7001.

 The step of inserting the DNA fragments into the integrative cosmid pOS7001 was carried out according to the process of the invention in which poly (A) and poly (T) homopolymeric polynucleotide tails were added at the 3-end. respectively, the nucleic acid of the vector and the DNA fragments to be inserted.

 The recombinant vectors thus constructed were encapsidated in lambda phage heads and the resulting phages were used to infect E. coli cells according to techniques well known to those skilled in the art.

 A library of approximately 5000 Escherichia coli clones was obtained.

 This library of clones was screened with pairs of primers specific for a nucleotide sequence coding for an enzyme involved in the polyketide biosynthetic pathway, the type I PKS enzyme, also referred to as β-ketoacyl synthase.

 It is recalled here that polyketides constitute a chemical class of great structural diversity comprising a large number of molecules of pharmaceutical interest such as tylosin, monensin, vermectin, erythromycin, doxorubicin or FK506.

 Polyketides are synthesized by condensation of acetate molecules by the action of enzymes called polyketide synthases (PKSs).

There are two types of polyketide synthases. Type II polyketide synthases are generally involved in the synthesis of polycyclic aromatic antibiotics and catalyze the condensation of acetate units iteratively.

 Type I polyketide synthases are involved in the synthesis of macrocyclic or macrolide polyketides and are multifunctional modular enzymes.

 In view of their therapeutic interest, there is a need in the state of the art to isolate and characterize novel polyketide synthases that can be used for the production of

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 new pharmaceutical compounds, especially new pharmaceutical compounds with antibiotic activity.

 Screening of the recombinant clone library described above with PCR primers selectively amplifying nucleotide sequences encoding # type polyketide synthases has identified recombinant clones containing DNA inserts comprising a nucleotide sequence encoding for new polyketide synthases. The nucleotide sequences coding for these novel polyketide synthases are referenced as the sequences SEQ ID N 33 to SEQ ID N 44.

 Another subject of the invention consists of a nucleic acid coding for a novel polyketide synthase I, characterized in that it comprises one of the nucleotide sequences SEQ ID N 34 to SEQ ID N 44.

 Preferably, such nucleic acid is in an isolated and / or purified form.

 The invention also relates to a recombinant vector comprising a polynucleotide comprising one of the sequences SEQ ID N 34 to SEQ ID N 44.

 The invention also relates to a recombinant host cell comprising a nucleic acid selected from polynucleotides comprising one of the nucleotide sequences SEQ ID N 34 to SEQ ID N 44 as well as to a recombinant host cell comprising a recombinant vector in which is inserted a polynucleotide comprising one of the nucleotide sequences SEQ ID N 34 to SEQ ID N 44.

 Advantageously, the recombinant vectors containing a DNA insert coding for a new type 1 polyketide synthase according to the invention are cloning and expression vectors.

 Preferably, a recombinant host cell as described above is a bacterium, a yeast or a filamentous fungus.

 The amino acid sequences of novel polyketide synthases from organisms in a soil sample were deduced from the nucleotide sequences SEQ ID N 34 to SEQ ID N 44 above. These are polypeptides comprising one of the amino acid sequences SEQ ID N 48 to SEQ ID N 59.

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 The invention also relates to novel polyketide synthases comprising an amino acid sequence selected from the sequences SEQ ID N 48 to SEQ ID N 59.

 Genomic DNA from pure bacterial strains, such as Streptomyces coelicolor (ATCC N 101.478), Streptomyces ambofaciens (NRRL N 2.420), Streptomyces lactamandurans (ATCC N 27.382), Streptomyces rimosus ( ATCC N 109.610), Bacillus subtilis (ATCC N 6633) or Bacillus lichenifornis and Saccharopolyspora erythrea.

 PCR amplification of the DNA of each of the bacterial strains described above was carried out using the primer pairs specific for the type 1 polyketide synthase nucleic acid sequences.

 New bacterial-type polyketide synthase genes have thus been isolated and characterized. These are the nucleic sequences of sequences SEQ ID N 30 to SEQ ID N 32.

 The invention therefore also relates to nucleotide sequences coding for novel polyketide synthases # selected from polynucleotides comprising one of the nucleotide sequences SEQ ID N 30 to SEQ ID N 32.

 Also included in the invention are recombinant vectors comprising the nucleotide sequences coding for new type # polyketide synthases defined above.

 The invention also relates to recombinant host cells characterized in that they contain a nucleic acid encoding a novel type 1 polyketide synthase comprising a nucleotide sequence chosen from the sequences SEQ ID N 30 to SEQ ID N 32 as well as host cells recombinant comprising a recombinant vector as defined above.

 The subject of the invention is also polypeptides encoded by sequences comprising the nucleic acids SEQ ID N 30 to 32, and more specifically polypeptides comprising the amino acid sequences SEQ ID N 47 to SEQ ID N 50.

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 The subject of the invention is furthermore a process for producing a type 1 polyketide synthase according to the invention, said production process comprising the following steps: obtaining a recombinant host cell comprising a nucleic acid coding for a polyketide type 1 synthase comprising a nucleotide sequence selected from the sequences SEQ ID N 33 to SEQ ID N 44 and SEQ ID N 30 to SEQ ID N 32; - culture of recombinant host cells in a suitable culture medium; recovery and, where appropriate, purification of the type I polyketide synthase from the culture supernatant or the cell lysate.

 The new type 1 polyketide synthases obtained according to the method described above can be characterized by attachment to an immunoaffinity chromatography column on which antibodies recognizing these polyketide synthases have been immobilized beforehand.

 The polyketide synthases of the type # according to the invention, and more particularly the recombinant polyketide synthases described above can also be purified by high performance liquid chromatography (HPLC) techniques, such as, for example, reverse phase chromatography techniques or chromatography of anion exchanges or cations, well known to those skilled in the art.

 The polyketide synthases, recombinant or non-recombinant, according to the invention can be used for the preparation of antibodies.

 According to another aspect, the subject of the invention is therefore an antibody specifically recognizing a # type polyketide synthase according to the invention or a peptide fragment of such a polyketide synthase.

 The antibodies according to the invention may be monoclonal or polyclonal. Monoclonal antibodies can be prepared from

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 of hybridoma cells according to the technique described by KOHLER and MILSTEIN C. (1975), Nature, Vol.256: 495.

 The polyclonal antibodies can be prepared by immunization of a mammal, in particular mice, rats or rabbits with a polyketide synthase type 1 according to the invention, if appropriate in the presence of an adjuvant compound of immunity, such as Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum hydroxide or a compound of the muramyl peptide family.

 Also "antibodies" within the meaning of the present invention include antibody fragments such as Fab, Fab ', F (ab') 2 fragments, or even single chain antibody fragments containing the variable part (ScFv). described by MARTINEAU et al. (1998) J. Mol. Biol., Vol.280 (1): 117-127 or in US Pat. No. 4,946,778, as well as the humanized antibodies described by REINMANN KA et al. (1997), AIDS Res.

Hmm. Retroviruses, vol. 13 (11): 933-943 or by LEGER O. J et al. (1997), Hum. Antibodies, vol.8 (1): 3-16.

 The antibody preparations according to the invention are useful in particular in qualitative or quantitative immunological tests aimed either at simply detecting the presence of a polyketide synthase of type 1 according to the invention, or at quantifying the amount of this polyketide synthase, for example in the culture supernatant or the cell lysate of a bacterial strain capable of producing such an enzyme.

 Another subject of the invention consists in a method for detecting a polyketide synthase of the type # according to the invention or a peptide fragment of this enzyme, in a sample, said process comprising the steps of: a) contacting a antibody according to the invention with the test sample; b) detecting the antigen / antibody complex that may be formed.

 The invention also relates to a kit or kit for detecting a type I polyketide synthase according to the invention in a sample, comprising:

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 a) an antibody according to the invention; b) if necessary, reagents necessary for the detection of the antigen / antibody complex that may be formed.

 An antibody directed against a polyketide synthase of the type # according to the invention may be labeled using an isotopic or non-isotopic detectable label, according to methods well known to those skilled in the art.

 Screening of a DNA library according to the invention using a pair of primers hybridizing with target sequences whose presence is sought, such as sequences of the puromycin biosynthetic pathway, sequences of the linA gene involved in the biodegradation of lindane or sequences encoding # type polyketide synthases have been detailed above.

 The subject of the invention is therefore a method for detecting a nucleic acid with a determined nucleotide sequence, or a nucleotide sequence structurally related to a determined nucleotide sequence, in a collection of recombinant host cells according to the invention, characterized in that it comprises the following steps: bringing the collection of recombinant host cells into contact with a pair of primers hybridizing with the determined nucleotide sequence or hybridizing with the nucleotide sequence structurally related to a determined nucleotide sequence; - perform at least three amplification cycles; detect the optionally amplified nucleic acid.

 For the appropriate amplification conditions depending on the desired target sequences, those skilled in the art can advantageously refer to the examples below.

 According to another aspect, the invention also relates to a method for detecting a nucleic acid, specific nucleotide sequences, or nucleotide sequences structurally related to a determined nucleotide sequence, in a collection of recombinant host cells according to the invention, characterized in that it comprises the following steps: - bringing the collection of recombinant host cells into contact with a probe hybridizing with the determined nucleotide sequence or

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 hybridizing with a nucleotide sequence structurally related to the determined nucleotide sequence; detect the hybrid possibly formed between the probe and the nucleic acids included in the vectors of the collection.

 To screen a DNA library according to the invention for the presence of a nucleotide sequence coding for a polypeptide capable of degrading lindane, the recombinant clones of interest were detected by their phenotype corresponding to their ability to degrade lindane. For this purpose, the isolated clones and / or sets of clones of the prepared DNA library were cultured in a culture medium in the presence of lindane and the degradation of lindane was observed by the formation of a halo trouble in the immediate environment of the cells.

 The invention also relates to a method for identifying the production of a compound of interest by one or more recombinant host cells in a collection of recombinant host cells according to the invention, characterized in that it comprises the following steps: - culture recombinant host cells of the collection in a suitable culture medium; detection of the compound of interest in the culture supernatant or in the cell lysate of one or more of the recombinant cells cultured.

 The invention further relates to a method for selecting a recombinant host cell producing a compound of interest in a collection of recombinant host cells according to the invention, characterized in that it comprises the following steps: - culture of the host cells recombinants from the collection in a suitable culture medium; detecting the compound of interest in the culture supernatant or in the cell lysate of one or more of the recombinant cultured host cells; selection of the recombinant host cells producing the compound of interest.

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 The invention also relates to a method for the production of a compound of interest characterized in that it comprises the following steps: cultivating a recombinant host cell selected according to the method described above; recovering and, if necessary, purifying, the compound produced by said recombinant host cell.

 The invention also relates to a compound of interest characterized in that it is obtained according to the method described above.

 A compound of interest according to the invention may consist of a polyketide produced by the expression of at least one nucleotide sequence comprising a sequence chosen from the sequences SEQ ID N 33 to 44 and SEQ ID N 30 to 32.

 The invention also relates to a composition comprising a polyketide produced by the expression of at least one nucleotide sequence comprising a sequence chosen from the sequences SEQ ID N 33 to SEQ ID N 44 and SEQ ID N 30 to SEQ ID N 32.

 A polyketide produced by the expression of at least one nucleotide sequence above is preferably the product of the activity of several coding sequences included within a functional operon whose translation products are the various enzymes necessary for the synthesis of a polyketide, one of the above sequences being included and expressed in said operon. Such an operon comprising a nucleic acid sequence according to the invention encoding a polyketide synthase can be constructed for example according to the teaching of Borchert et al. (1992).

 The invention also relates to a pharmaceutical composition comprising a pharmacologically active amount of a polyketide according to the invention, where appropriate in combination with a pharmaceutically compatible vehicle.

 Such pharmaceutical compositions will advantageously be suitable for the administration, for example parenterally, of an amount of a polyketide synthesized by a polyketide synthase type 1 according to the invention ranging from 1 μg / kg per day to 10 mg / kg per preferably at least 0.01 mg / kg per day and most preferably between 0.01 and 1 mg / kg per day.

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 The pharmaceutical compositions according to the invention can be indifferently administered orally, rectally, parenterally, intravenously, subcutaneously or intradermally.

 The invention also relates to the use of a polyketide obtained by the expression of a polyketide synthase type 1 according to the invention for the manufacture of a medicament, in particular a drug with antibiotic activity.

 The invention will be further illustrated, without being limited, by the figures and examples below.

 FIG. 1 illustrates the diagram of the various lysis steps carried out according to the protocols 1,2, 3n 4a, 4b, 5a, and 5b described in Example 1.

Figure 2 illustrates a. agarose gel electrophoresis 0.8% of the DNAs extracted from 300 mg of soil n 3 (Côte St André) after various lysis treatments (protocols 1 to 5, see Fig. 1). M: lambda phage molecular weight marker
Figure 3 illustrates the proportion of different kinds of actinomycetes grown as a result of treatments 1 to 5 (see Fig. 1). The number of cfu (colony-forming unit) was determined on a selective medium for this group of bacteria. A total of about 400 colonies were analyzed.

 Figure 4 illustrates the. recovery of lambda digested lambda phage DNA added to the soil at different concentrations before (G) or after (G *) grinding. T (heat shock) and S (sonication) treatments are additional lysis treatments. Quantification was performed by phosphorimager analysis after dotblot hybridization. One sample of each soil was used for each added lambda phage concentration. The soil characteristics are reproduced in Table 1. Samples corresponding to 10 and 15 g of added DNA were not processed.

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 Figure 5 illustrates the PCR amplification of DNAs extracted from soil n 3 according to the protocols 1,2, 3, 5a and 5b. FGPS 122 and FGPS 350 primers (Table 2) were used to target Streptosporangium spp. native. The DNA extracts were used undiluted or diluted to 1 / 10th and 1 / 100th. M: 123 bp molecular weight marker (Gibco BRL), C: amplification without DNA.

 Figure 6 illustrates the amounts of DNA extracted after inoculation of S. lividans OS48 spores (a) or mycelium (b). 3 inoculated in soils at different concentrations. The amount of mycelium added to the soil corresponds to the number of spores inoculated in the germination medium. About 50% of the spores germinated, the number of cells or genomes contained in the hyphae of germinated spores was not determined. The quantities of inoculated spores and mycelium are therefore not directly comparable. The extraction protocol was carried out according to protocol 6 (see section material and methods). The symbol (') indicates that RNA has been included in the extraction buffer. The target DNA was amplified by PCR with the primers FGPS 516 and FGPS 517, the quantification was carried out by phosphoimageur after dot blot hybridization using the FGPS 518 probe. A sample of each soil was used for each concentration of hyphae or spores. Soil characteristics are described in Table 1.

 Figure 7 shows the phylogenetic tree obtained by the Neighbor Joining algorithm, positioning the 16S rDNA sequences contained in the soil DNA library, relative to cultured reference bacteria.

In gray: .the sequences from clone pools of the bank.

 Bootstrap values are indicated at the node level, after resampling 100 repetitions. The scale bar indicates the

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 number of substitutions per site. Sequence access numbers in the Genbank database are indicated in parentheses.

 FIG. 8 represents a diagram of the vector pOSint 1.

 Figure 9 shows a diagram of the vector pWED1.

 Figure 10 is a diagram of the vector pWE15 (ATCC N 37503).

 Figure 11 shows a diagram of the vector pOS 7001.

 Figure 12 shows a diagram of the vector pOSV010.

 Figure 13 shows the fragment containing a cos site inserted into the plasmid pOSV010 during the construction of the pOSV 303 vector.

 Figure 14 is a diagram of the vector pOSV 303.

 Figure 15 shows a diagram of the vector pE116.

 FIG. 16 represents a diagram of the vector pOS 700 R.

 Figure 17 shows a diagram of the vector pOSV 001.

 Figure 18 shows the scheme of the vector pOSV 002.

 Figure 19 is a diagram of the vector pOSV 014.

 Figure 20 shows a diagram of the vector pBAC 11.

 Figure 21 is a diagram of the vector pOSV 403.

 Figure 22 shows the DNA electrophoresis gels of the library after digestion with BamHI and Oral enzymes of the positive clones of the library screened with PKS-I oligonucleotides.

 Figure 23 illustrates the production of puromycin by S. lividans recombinants compared to the production of the S. alboniger wild strain.

 Figure 24 illustrates Alignment of soil PKSs with conserved active sites of other PKSs. References for each peptide are indicated. The beta-ketoacyl synthase domains have been aligned using GCG's PILEUP program (Wisconsin Package Version 9.1, Genetics Computer Group, Madison, Wisc).

 Figure 25 illustrates the construction of a conjugative integrative cosmid.

 Figure 26 illustrates the construction of a conjugative integrative BAC.

EXAMPLES

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 EXAMPLE 1: Preparation of a nucleic acid collection from a soil sample containing organisms, comprising a step of directly extracting DNA from the soil sample.

1. MATERIAL AND METHODS
1. 1 SOILS: The characteristics of the six soils used in this study are listed in Table 1.

 The clay and organic matter content ranges from 9 to 47% and from 1.7 to 4.7% respectively, the pH ranging from 4.3 to 5.8.

 Soil samples were collected from the top layer 5 to 10 cm deep. All visible roots were removed and the soils were kept at 4 ° C for a few days if necessary, after which they were dried for 24 hours at room temperature and sieved (average mesh size 2 mm) before being stored. up to several months at 4 C.

1. 2 BACTERIAL STRAINS AND CULTURE CONDITIONS: Extracellular DNA as well as bacterial strains providing vegetative cells, spores or hyphae, used to innoculate soil samples, were chosen so that their presence could be monitored. specifically.

 In order to obtain large amounts of extracellular DNA, the lysogenic strain of E. coli 1192 Hfr P4X (metB), containing lambda phage C1857 Sam7, was cultured on Luria-Bertani (LB) medium for two hours at 30 ° C. then 30 minutes at 40 ° C. and then 3 hours at 37 ° C. The phage lambda DNA was extracted according to the technique described by SAMBROOK J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd, ed. Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y.

 The avirulent strain of Bacillus anthracis (STERNE 7700) was used as an inoculum of bacterial cells. Bacillus anthracis was propagated on broth culture type "trypticase soy broth" (TSB) (Biomérieux, Lyon, France) for about 6 hours, verifying that OD600 is kept below 0.6. These conditions allow the development of vegetative cells without spore formation (Patra et al., (1996), FEMS Immunol, Medical Microbiology, 15: 223-231). The

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 spores of Streptomyces lividans OS48. 3 (CLERC-BARDIN et al., Unpublished) were mechanically removed from organism cultures on R2YE medium (HOPWOOD et al., (1985), Genetic Manipulation of Streptomyces-A Laboratory Manual, The John Innes Foundation, Norwich , United Kingdom). The hyphae of S.lividans OS48. 3 were obtained from pre-germinated spores, as the use of short hyphae was expected to minimize breakage and subsequent loss of DNA. The spores were suspended in TES buffer (N-Tris [hydroxymethyl] methyl-2-aminoethanesulfonic acid, SigmaAldrich Chemistry, France) (0.05M, pH 8) (Holben WE et al., (1988), APPL. Microbiol, vol.54: 703-711, were then heat-shocked (50 ° C for 10 minutes followed by cooling under a stream of cold water and then added to an equal volume of pregermination medium (extract). yeast 1%, casamino acids 1% CaCl 2 0.01 M).

 The solution was incubated at 37 ° C. on a shaker. The proportion of germinated spores was estimated at about 50%, in agreement with the results of HOPWOOD et al. (1985). After centrifugation, the pellets were resuspended in TES buffer, added to 3% of TSB medium, and incubated at 37 ° C. until a OD 45 0 of 0.15 (HOPWOOD et al., (1985)) was obtained. . Streptomyces hygroscopicus SWN 736 and Streptosporangium fragile AC1296 (Pushino Institute, Moscow) were grown according to techniques described by HICKEY and TRESNER (1952).

 Spore and hyphae DNA from S. Lividans was extracted from the pure cultures according to the lysis protocol 6 described below (except that no grinding was performed), whereas S. hygroscopicus and fragile S. were extracted by chemical / enzymatic lysis (Hintermann et al., 1981).

 1. CHOICE OF EXTRACTION BUFFER: TENP buffer (50 mM Tris, 20 mM EDTA, 100 mM NaCl, 1% wt / vol polyvinylpolypyrrolidone developed by PICARD (1992) was used, similar buffers were subsequently used by other authors (CLEGG et al., 1997, KUSKE et al., 1998, ZHOU et al., 1996).

 Tris and EDTA protect the DNA from nuclease activity, NaCl provides a dispersing effect and PVPP absorbs humic acids and

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 other phenolic compounds (HOLBEN et al (1988), PICARD et al., (1992).

 In this study, the extraction efficiency of this buffer was evaluated at different pH (6.0 - 10.0) using 20 different soils with a pH range of 5.8 to 8.3 and a organic matter between 0.2 and 6.3%. These twenty soils (the other characteristics are not indicated) were used only in this experiment. The amount of DNA was determined colorimetrically as described by RICHARD (1974), and detailed below.

1. 4 IN SITU LYSE AND DNA EXTRACTION PROTOCOL: Several protocols using an increasing number of steps have been tested to evaluate the effectiveness of different techniques for lysing soil microbes in situ. For these experiments, native soil microflora was targeted in six soils. Additional experiments were conducted to investigate the effects of lysis treatments on the released DNA by analyzing the amounts and quality of recovered DNA from lambda phage DNA previously added to the soils.

 Once an optimized protocol (referred to as protocol 6) was developed, this protocol was used to quantify DNA from native Actinomycetes and DNA from Grampositive bacteria inoculated into selected soils. In all cases, the soil samples were dried and sieved as described above.

 After grinding, 0.5 ml of TENP buffer was added at 200 mg dry weight of sol except for protocol 1 in which the buffer was added to unmilled soil).

 For the various lysis treatments (see below), the soil suspensions were vortexed for ten minutes and centrifuged (4000 g for five minutes), after which an aliquot (25 l) of the supernatant was analyzed by gel electrophoresis (0.8% agarose).

 Another aliquot of the supernatant representing a known volume, typically 350 l, was precipitated with isopropanol.

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 Five aliquots (representing DNA derived from 1 g of sol) were pooled and resuspended in 100 μl of sterile TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) prior to purification (Protocol D, see below) and quantification, either by hybridization (Dot Blot) of the total DNA, or by hybridization (Dot Blot) PCR amplification products (see below).

 The hybridization signals were quantified by phosphorescence imaging ("phospho-imaging" technique see below).

1. EVALUATION OF IN SITU CELL LYSE METHODS: The quality and quantity of the DNA extracted after an increasing number of lysis treatment steps (protocol 2-5b) were compared with those of the extracellular DNA obtained. after washing the soil with an extraction buffer (protocol 1, see also Figure 1).

 Protocol 1: No lysis treatment.

 TENP buffer was added to unmilled soil, a DNA extraction step was performed as described above.

 Protocol 2. Soil grinding followed by DNA extraction.

 Two different types of devices were used for ground grinding.

 In order to compare their respective efficiency, 5g of dry soil was milled for 30 seconds in a mill containing tungsten rings, or for various times up to 60 minutes in a soil grinder containing a mortar and agate balls ( 20 mm in diameter).

 The TENP buffer is then added and the DNA is extracted as described above.

 Gel electrophoresis results showed that grinding for 40 minutes using agate beads was necessary in order to obtain extracted amounts of DNA equivalent to those obtained after 30 seconds of grinding using tungsten rings.

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 The size distribution of the DNA fragments is similar regardless of the method used.

 Thus, these treatments have been considered equivalent and the one that will be used in the protocols described below will therefore not be specified.

 In Protocols 3 to 5, the effectiveness of several other lysis treatments subsequent to ground grinding was tested, either separately or in different combinations.

Protocol 3:
This protocol is identical to protocol 2 except that it comprises a homogenization step using an Ultraturrax type mixer (Janker and Kunkel, IKA Labortechnik, Germany) set at half the maximum speed for 5 minutes. .

PROTOCOLS 4a and 4b:
These protocols are identical to protocol 3 with the exception of an additional sonication step.

 Two types of sonicator devices were compared: a titanium microtip sonicator (600W Vibracell Ultrasonicator, Bioblock, Illkirch, France) (Protocol 4a) and a Cup Horn-type sonicator (Protocol 4b).

 The ultrasonic Vibracell microtip is in direct contact with the soil solution.

 With regard to the Cup Horn device, the soil solution is stored in tubes which are placed in a water bath through which the ultrasound passes.

 Preliminary experiments were conducted to determine the optimal conditions for both sonicators (results not shown).

 The best compromise, in terms of amount of extracted DNA and fragment size, consists of sonication with the titanium micro-tip and the Horn-type sonicator respectively for 7 and 10 minutes, setting the power at 15 W and with active cycles at 50%.

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Protocols 5a and 5b:
After sonication with a titanium microtip or a Cup Horn device (respectively Protocols 4a and 4b) lysozyme and achromopeptidase were added, each of the enzymes at a final concentration of 0.3 mg / ml.

 The soil suspensions were incubated for 30 minutes at 37 ° C., after which lauryl sulfate at a final concentration of 1% was added, then suspensions were incubated for 1 hour at 60 ° C. before centrifugation and precipitation as described below. above.

 In addition to the protocols described above, the effect of sonication (Cup Horn, see Protocol 4b) and thermal shock (30 seconds in liquid nitrogen followed by three minutes in boiling water, the treatments being repeated three on Hindlll digested lambda phage DNA previously added to the soil were examined (see below).

 Thermal shocks have been suggested in the state of the art as means of cell lysis in situ (PICARD et al. (1992)). However, because such a treatment has a detrimental effect on free DNA (see results section) it was not included in the protocols described above.

OPTIMIZED PROTOCOL
After evaluation of the different lysis treatments, an optimized protocol was defined, designated protocol 6. Protocol 6 is identical to protocol 5b except that, before sonication, the soil suspensions are subjected to a Vortex treatment and then agitated by rotation on a wheel for two hours before being frozen at - 20 C.

 After thawing, the soil suspensions are vortexed for 10 minutes before sonication. Protocol 6 was used in experiments in which soils were seeded with bacterial cells as well as in experiments in which native actinomycetes were quantified (see below).

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 1. 6 MICROSCOPE COUNTING: The efficiency of soil crushing as a method for lysing bacterial cells was examined under a microscope.

 5 g of dried crude sol were mixed in a Waring Blender device with 50 ml of ultrapure sterilized water for 1.5 minutes; simultaneously, 1 g (dry weight) of ground ground (Protocol No. 2) was suspended in 10 ml by stirring for 10 minutes. Salt suspensions are serial diluted and orange acridine added to a final concentration of 0.001%.

 After 2 minutes, the suspensions were filtered through a membrane brand NUCLEOPORE type 0.2 m black. Each filter was rinsed with lysed sterile water, treated with 1 ml of isopropanol for 1 minute to fix the bacterial cells, and then rinsed again.

 The bacterial cells were counted using an epifluorescence microscope of the Zeiss Universal type with a 100x objective. For each soil type, three filters were counted, and at least 200 cells were counted on each of the filters.

1. 7 NUMBER OF CULTIVATED ACTINOMYCETS AND TOTAL NUMBER OF COLONY-FORMING UNITS (CFU): Actinomycetes surviving lysis treatments (Protocols 1-5) were examined specifically with soil No. 3 (Côte Saint André, see Table 1). ).

 After a 10-fold dilution of a yeast extract (6% w / v) and SDS (0.05%) solution to induce germination (Hayakawa et al (1988)), suspensions of The sol were serially diluted, incubated at 40 ° C. for 20 minutes and seeded on HV medium (HAYAKAWA et al., 1987).

 The HV medium was supplemented with actidione (50 mg / l) and nystatin (50 mg / ml).

 Actinomycete colonies were counted after incubation for 15 days at 28 ° C.

 In total, about 400 colonies were examined. The identification was carried out on the basis of macro-and microscopic morphological characteristics as well as on the analysis of the acid content

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 diaminopimelic isolates (SHIRLING et al., 1966); STANECK et al., 1974; WILLIAMS state., 1993).

 The total amount of cultivable bacteria (total CFU) was also determined for each of lysis protocols 1 to 5. The soil suspensions were serially diluted and seeded in triplicate on Bennett agar medium (WAKSMAN et al., 1961). added with nystatin and actidione (each at 50 mg / l).

 Each petri dish was covered with a cellulose nitrate filter (Millipore) and incubated for three days at 28 C. After counting the colonies on the membranes, the filters were removed and the Petri dishes were again incubated for 7 days at 28 C then counted again.

1. 8 RECOVERY OF LAMBDA PHAGE DNA ADDED TO THE SOIL: The lambda phage DNA was digested with HindIII, extracted with a mixture of phenol-chloroform, precipitated and then resuspended in sterile ultrapure water according to standard protocols. (SAMBROOK et al., 1989).

 Dilutions corresponding respectively to 0.2.5, 5.7.5, 10 and 15 g of DNA / g dry weight of sol were prepared in volumes of 60 μl. These DNA dilutions were added to 5 g batches of dry sol which were subsequently vigorously vortexed for 5 minutes before grinding.

 Lambda phage DNA was also added to soil before grinding at concentrations corresponding to 0, 10 and 15 g DNA / g dry weight of the soil.

 After grinding, the extraction buffer is added and the DNA is extracted according to protocol 2 (see above).

1. 9 SATURATION OF ADSORPTION SITES WITH RNA: to determine whether the saturation of the soil colloid nucleic acid adsorption sites could increase the recovery rate of DNA, the sandy loam (soil n 4) and clay soil (soil 5) were incubated with an RNA solution before further treatment.

 Commercial Saccharomyces cerevisiae RNA (BOHRINGER MANNHEIM, MEYLAN, France) was diluted in phosphate buffer (pH 7.1) and added to the dry soil samples.

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 sieved (2 ml / g soil) at final concentrations of 20.50 and 100 mg RNA / g dry weight of soil.

 The tubes containing the soil suspensions were rotated for two hours at room temperature. After centrifugation, the soil pellets were oven dried (50 ° C.) overnight. Lambda phage DNA was then added to the soils (0.20 or 50 g / g dry weight of soil) to simulate the fate of the DNA released after cell lysis.

 The DNA was extracted according to protocol No. 2. It was subsequently determined that an identical effect of RNA addition on DNA recovery could be achieved by adding RNA directly to the DNA buffer. extraction.

 This simplified procedure was used for # 5 clay soil in experiments in which microorganisms were inoculated into soils.

 The RNA was then added at a concentration corresponding to 50 mg RNA / g dry weight of the soil.

1. 10 QUALITATIVE AND QUANTITATIVE DETERMINATION OF THE EFFECTIVENESS OF EXTRACTION PROTOCOLS: DNA quality (no degradation) was estimated on the basis of the size of the DNA fragments or the relative position of the migration bands of DNA after electrophoresis of an aliquot of a DNA solution on a 0.8% agarose gel.

 The fluorescence intensity allowed a semiquantitative estimation of the extraction yields.

 Another aliquot fraction was used for quantitative determination of DNA content by dot blotting and phospho-imager analysis. The stain hybridization protocol has been described by SIMONET et al. (1990).

 Hybridization membranes (GeneScreen plus, Life Science Products, Boston, USA) were prehybridized for at least 2 hours in 20 ml of a solution containing 6 ml of 20 x SSC, 1 ml of DENHARDT's, 1 ml of 10% SDS and 5 mg of salmon sperm DNA.

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 Hybridization was carried out overnight in the same solution in the presence of a labeled probe prior to two membrane washes in 2 x SSC buffer for 5 minutes at room temperature, followed by a third wash in 2 x SSC buffer, 0.1% SDS and a fourth wash in 1x SSC buffer, 0.1% SDS for 30 minutes at the hybridization temperature.

 The hybridization signals were quantified with a BIORAD radioanalytical imaging system (Molecular Analyst Software, BIORAD, Ivry S / Seine, France).

 In order to quantify the total amount of DNA derived from the native microflora, the different soils were extracted according to the protocols n 1 to 5. The non-amplified DNA was applied on the Dot Blot membranes and hybridized using the universal probe. FGPS431 (Table 2).

 This probe, which hybridizes to positions 1392-1406 of the E. coli 16S rDNA gene (Amann et al (1995)) was labeled at its ends with ATP [alpha] 32P using a T4 polynucleotide kinase ( BOEHRINGER MANNHEIM, Melan, France).

 A calibration curve was prepared from E. coli DH5a DNA. The conversion of stones to soil bacteria necessitated a simplification, assuming that the average number of copies (rrn) was 7, as for E. coli.

 Hindlll digested lambda phage DNA was used to quantify the recovery of extracellular DNA. Unamplified extracts from soils, to which lambda phage DNA had been added, were hybridized with randomly-tagged Hindlll digested phage lambda DNA using the Klenow fragment (Boehringer Mannheim, Melan, France). ).

 The amounts of DNA were calculated by interpolation from a calibration curve prepared with the purified DNA.

 The total amount of DNA extracted from sols 1, 2, 3, 4 and 6 according to protocol No. 2 (grinding) was also quantified in a colorimetric manner according to the technique described by RICHARD (1974).

 Briefly, DNA was mixed with concentrated HClO4 (the final concentration of HC104 was 1.5 N). 2.5 volumes of this solution were mixed with 1.5 volumes of DPA (diphenylamine, Sigma-Aldrich, France) and incubated at room temperature.

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 room temperature for 18 hours, prior to determining the OD at 600 nn. The soil DNA extracts were quantified against a standard curve made by DNA extracted from E. coli DH5 [alpha] according to standard protocols (SAMBROOK et al., (1989)).

1. 11 DEVELOPMENT OF A DNA QUANTIFICATION TECHNIQUE USING PCR AMPLIFICATION AND HYBRIDIZATION: For PCR amplifications, Taq DNA polymerase (Appligene Oncor, France) was used according to the manufacturer's instructions. .

 The PCR program used for all the amplifications is as follows: initial denaturation for 3 minutes at 95 ° C., then 35 cycles consisting of 1 minute at 95 ° C., 1 minute at 55 ° C. and 1 minute at 72 ° C., followed by a final extension to 72 C for 3 minutes.

 DNA isolated and purified from brittle Streptosporangium was used as a control at concentrations ranging from 100 fg to 100 ng.

In order to specifically amplify the DNA of this bacterial genus, primers FGPS122 and FGPS350 (Table 2), complementary to a portion of the 16S rDNA, were chosen after alignment of the actinomycete 16S rDNA sequences. Their specificity was tested on a collection of actinomycete strains (Streptomyces, Streptosporangium and other closely related genera)
The PCR products were hybridized with the oligonucleotide probe FGPS643 (Table 2). In order to simulate the level of purity obtained routinely with DNA extracted from the soil, pure S. fragile DNA controls were mixed with the soil extracts obtained after treatments according to the lysis protocols 4b and 5b and then purified according to protocol D.

 Before use, the soil extracts were treated with DNase (one unit of DNase / ml, GIBCO BRL) for 30 minutes at room temperature. The DNase was then inactivated by heating at 65 ° C. for 10 minutes. An inactivation check was performed by PCR. Humic acid concentrations were measured spectrophotometrically (D028onm) against a standard curve of commercial humic acids (Sigma).

 Undiluted, diluted 10x and diluted x 100 Dnase-treated soil solutions were mixed with 100 μg to 100 μg of S DNA.

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 fragile before amplification by PCR. In another series of experiments, increasing concentrations of Streptomyces hygroscopicus DNA from (100 μg to 1 g) were added to the fragile S. DNA to simulate the presence of non-target DNA and its influence. on the PCR process.

 1. 12 PURIFICATION OF GROSS DNA EXTRACTS: DNA purification methods were compared. The DNA was extracted from 1 g (dry weight of sol according to protocol 4a and resuspended in 100 l of TE8 buffer (50 mM Tris, 20 mM (EDTA, pH 8.0).

Protocol A
Elution through two successive Elutip columns (SCHLEICHER and SCHUELL, Dassel, Germany) (PICARD et al., (1992)).

Protocol B:
Elution through a SEPHACRYL S200 column (Pharmacia Biotech, Uppsala, Sweden) followed by elution through an Elutip column (NESME et al (1995)).

Protocol C:
Separation using a two-phase aqueous system with 17.9% (w / w) of PEG 8000 (Merck, Darmstadt, Germany) and 14.3% (w / w) of (NH4) 2SO4 (ZASLAVSKY) (1995)).

 After vigorous vortex mixing, both phases were left at room temperature for separation.

 1 ml of each of the phases was transferred to another tube, mixed with 100 l of the sample and left at 4 ° C overnight to allow separation.

 The lower phase was dialyzed for one hour through a Millipore membrane in the presence of an excess of a TE 7.5 buffer (10 mM Tris, 1 mM EDTA pH 7.5 and 1 M Mg Cl 2) in order to remove excess salts.

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Protocol D:
Elution through a Microspin Sephacryl S400 HR column (Pharmacia Biotech, Uppsala, Sweden), followed by elution through an Elutip d column.

 Each protocol is terminated by an ethanol precipitation step, and the DNA is resuspended in 10 l of TE 7.5 buffer.

The efficiency of the purification protocols was verified by PCR amplification of undiluted aliquots of diluted DNA solutions and aliquots 10 and x 100-fold, using standard protocols (see below).

1. 13 RECOVERY OF DNA FROM INNOCULATED MICROORGANISMS: The cells, spores and hyphae were washed twice and counted by plate counting or direct microscopic counting. Lots of 5g of dry, sieved soil (soil types 2, 3 and 5) were inoculated with 100 L of spore and hyphae suspension of S. lividans at concentrations corresponding to 0.103, 105, 107 and 109 spores / g dry weight of soil, or with vegetative cells of B. anthracis at concentrations corresponding to 0.107 and 109 cells per gram of dry soil weight.

 The quantities of S. lividans hyphae were calculated on the basis of the number of spores from which they originate. After addition of the bacterial suspensions, the soil samples are vortexed vigorously for 5 minutes before grinding.

DNA is extracted according to Protocol No. 6 (see below).

 PCR amplification followed by dot blotting and phosphorescence imaging was used to quantify the amounts of DNA recovered from the cells, spores and bacterial mycelium inoculated into the cells. soils.

 DNA extraction was performed according to lysis protocol n 6.

PCR amplification and hybridization were performed as described above. The primers and probes are targeted to chromosomal regions located outside the 16S region, and are highly specific to the respective organisms, so as to avoid background signals.

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 For soils seeded with B. anthracis, primers R499 and R500 were used (Patra et al. (1996)) and the amplification products were hybridized with oligonucleotide probe C501 (Table 2).

 For soils seeded with S. lividans, PCR reactions were performed using primers FGPS516 and FGPS517, and the amplification products were hybridized with oligonucleotide probe FGPS518 (Table 2).

 The amplified region is a portion of the cassette specifically constructed to obtain strain OS48.3 (CLERC-BARDIN et al., Unpublished).

 The calibration counts were in all cases obtained using the purified DNA of the target organism.

 2. RESULTS 2. 1 CHOICE OF THE EXTRACTION STAMP: 20 different soils were used to determine the optimal pH of the DNA extraction buffer.

For all soils, the yield of DNA increases with the increasing pH of the buffer. The yield for each pH (+/- sd), calculated as the percentage of the highest value for each soil, is as follows: pH 6.0: 31 +/- 13; pH 7.0: 43 +/- 16; pH 8.0: 60 +/- 14; pH 9.0: +/- 12; pH 10.0: 98 +/- 3.

 For 16 of the 20 soils, the highest yield was obtained at pH 10.0, while for the other four soils the highest yield was obtained at pH 9.0. However, at pH 10.0, larger amounts of humic material were released, compared to pH 9.0 (results not shown). As a result, pH 9.0 was chosen for all the experiments presented below.

2. EFFECTIVENESS OF DNA EXTRACTION PROTOCOLS: The total native soil DNA was extracted and quantified to evaluate the efficacy of many in situ cell lysis protocols. Samples of soils 1-6 (Table 1) were processed according to Protocols 1-5 described in the Materials and Methods section (Figure 1).

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 After the DNA extraction, the soil suspensions were precipitated with isopropanol, and aliquots of the resuspended pellets were analyzed by gel electrophoresis, in a first step, to estimate the quality and the amount of DNA released.

 However, the color of the DNA extract became increasingly dark as the number of lysis steps increased, due to the coextraction of compounds, such as humic acids, with the DNA.

 Some of these dark-colored crude extracts do not migrate in the expected manner in the agarose gels.

 As a result, the crude DNA solutions were purified (protocol B) before quantification. The gel electrophoresis of the purified solutions obtained after the various lysis treatments are exemplified on the soil n 3 (Figure 2).

 A visual comparison with ultraviolet radiation of the intensities of the colored DNA allowed a semi-quantitative estimation of the effectiveness of the treatments. In addition, the presence of multiple size DNA fragment (discrete band) migration profiles and the disappearance of the long fragments indicates that DNA degradation has occurred.

 No DNA could be extracted from # 5 clay soil.

A more precise quantification of the DNA of all the sols, extracted according to the protocols n 1 to 5, was carried out by hybridization on stain (Dot Blot) without step of preliminary PCR amplification and by using an oligonucleotide probe complementary of a highly conserved sequence of the 16S rDNA region (FGPS 431 probe, Table 2).

 DNA was detected in the extracts of all soils after each of the different lysis steps except for # 5 clay soil.

 The results are consistent with estimates made after electrophoresis gel.

 In order to compare with an independent method for quantification, the DNA extracted according to protocol n 2 (all soils except soil n 5) was also quantified using a colorimetric method of DNA detection (RICHARD, 1974) .

 A good correlation (r = 0.88) was found between the quantified DNA using this colorimetric technique and the results obtained by Dot Blot / radioimaging hybridization, confirming

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 the assumption that the average copy number of soil bacteria (rrn) is 7.

 Hybridization (Dot Blot) showed that the amounts of extracellular DNA, as determined by extraction without lysis treatment (Protocol No. 1), ranged from 4 μg / g for the acidic soil (No. 6) to 36 μg / g for soil no. 3 (Table 3).

 Soil crushing (Protocol 2) increased the amounts of DNA extracted from all soils (eg 26 g / g soil) for soil 6 and 59 g / g soil (for soil 3) (Table 3, Figure 2).

 For both grinding treatments (see Materials and Methods section) the discrete migration of DNA was detected on the agarose gels, indicating that the DNA molecules were partially degraded (Figure 2).

 The size of the DNA fragments is between 20 and 0.2 kb.

The band intensity of the smaller fragments is very small, indicating that most of the fragments are much larger than 1 kb.

 Protocol No. 3 comprises a homogenization step in an Ultraturax type mixing device after the addition of the extraction buffer to the soil samples. This step leads to an increase in the amount of DNA extracted, as determined by dot blotting for two of the soils (sandy soil # 3 and acid soil # 6), while both soils rich in organic matter. (# 1 and # 2 sols) led to lower amounts of DNA.

 Protocols # 4a and # 4b evaluated the influence of two types of sonication on DNA yields from previously ground and homogenized soils.

 Sonication did not have a positive effect on DNA yield, compared to protocol # 3, except for soil # 6. However, the lysis efficiency of both types of sonicator differed. For soils # 2, # 3 and # 4, the largest extracted DNA amounts were obtained using titanium microtip (Table 3; 2), whereas for # 1 and # 6 sols, DNA yield was was superior using the Cup Horn device.

 Conflicting results were also obtained when an enzymatic / chemical lysis step was added (Protocols 5a and 5a).

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 5b) after the sonication step: in some cases, the quantities of DNA extracted were larger than those recovered according to protocols 4a and 4b, while in other cases the yields were lower (Table 3).

2. 3 DIRECT COUNTING OF MICROORGANISMS: Microscopic counts of the total number of bacterial cells after staining with acridine orange were performed for all soils, before and after grinding.

 Before grinding, the number of bacteria per gram of soil dry weight ranged from 1.4 x 109 (+/- 0.4) in tropical soil n 5 to 10 x 109 (+/- 0.7) in soil from Côte Saint-André (soil 3) (Table 1).

 After grinding, the cell numbers were respectively 45, 74, 75, 54, 34 and 75% of the initial values for soils 1 to 6.

2. 4 NUMBER OF CULTURABLE ACTINOMYCETES BELONGING TO DIFFERENT GENRES: modification in the populations of actinomycetes in the soil n 3 was noticed after the various lysis treatments (figure 3).

 For example, colonies of Streptomyces sp. dominated the viable actinomycete flora when no lysis treatment was applied (Protocol No. 1), and accounted for 65% of the total number of colonies identified. After milling, the percentage of Streptomyces colonies decreased to 51%, while the proportion of colonies belonging to the genus Micromonospora increased from 14% to 41%.

 Chemical / enzymatic lysis (Protocols 5a and 5b) appeared to be particularly effective for lysis of streptomycetes. When all lysis treatments were applied, including chemical / enzymatic lysis (Protocols 5a and 5b), the actinomycete microflora, which still contained more than 106 CFU / g soil, was dominated by species belonging to the genus. Micromonospora, while none or very few Streptomyces colonies were recovered.

 Organisms belonging to genera such as Streptosporangium, Actinomadura, Microbispora, Dactilosporangium and Actinoplanes appeared on the plates in low numbers (2-8% of the total number of colonies identified) after grinding, homogenization

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 with the Ultraturrax device, and sonication, but were generally absent when these treatments were combined with chemical / enzymatic lysis.

 The total number of cultivable bacteria remaining after each lysis treatment (protocols 2 to 5) was also searched for soil n 4. The results indicate that the number of culturable bacteria does not decrease with the intensity of the lysis treatments (approximately 2 x 106 CFU / g soil in all cases, and also when treatment is applied, as in protocol 1).

The achievement of these low CFU values is probably due to the fact that dry soil was used and only the most resistant bacteria multiplied on the plates. The number of colony-forming actinomycetes was generally greater than that of total CFUs (all bacteria) because a spore germination step, included in the actinomycete detection protocol, was missing in the control of total bacteria 2. RECOVERY OF LAMBDA ADDED PHAGE DNA:
The purpose of these experiments was to estimate how successive lysis treatments could affect the recovery of naked DNA, and whether these successive lysis treatments contributed to its degradation.

 DNA could be either a fraction of extracellular DNA released from already dead organisms, which can persist in the soil for months (WARD et al., 1990), or DNA released from organisms lysed easily during the first stages of treatment. In order to simulate this situation, HindIII digested lambda phage DNA was added at various concentrations to the soil before and after grinding. In addition to grinding, a combination of other lysis treatments was tested, including sonication (Cup Horn device, see Protocol No. 4b) and thermal shock (see Materials and Methods section).

 After extraction, aliquots which should theoretically contain 25 to 150 ng of lambda phage DNA were analyzed by gel electrophoresis. No DNA fragment specific for lambda phage could be observed when the DNA was inoculated into the

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 soil samples prior to grinding, regardless of the rate or type of soil.

 When the DNA was added after grinding, and extracted without additional lysis treatment step, specific lambda phage DNA profiles were detected in the extracts of four of the five soils tested.

 In all these cases, a direct cause-and-effect relationship was obtained between the amount of DNA added and the intensity of the signals on the agarose gels. The signal intensities were, however, less than the expected signal strengths when compared to those of the molecular standards.

 In addition, the 23 kb band was absent in several cases, indicating that long fragments were preferentially adsorbed to soil particles, or were more susceptible to degradation, compared to short fragments.

 No bands were detected in samples of tropical soil n 5 which is characterized by a very high clay content (Table 1).

 For more precise quantification, DNA recovery was determined on a phosphorescence (phosphoimager) imaging device after dot blotting. According to this technique, DNA was detected in all samples, including those that had been inoculated before grinding, except for sol No. 5 in which no DNA could be detected.

 In all other soils, the amount of DNA extracted increases with increasing size of the inoculum (Figures 4a-d).

 However, lambda phage DNA recoveries were low. When grinding was the only lysis treatment applied, the recoveries ranged from 0.6 to 5.9% of the DNA added when it was added before grinding, and from 3.6 to 24% of the DNA. added when the latter was added after grinding. The highest levels of recovery were obtained from soil n 2.

 Gel electrophoresis of aliquots of heat shock and sonication samples failed to observe DNA bands in any of the samples, including the assay in which the DNA was added after grinding. Spot hybridization experiments (Dot Blot) confirmed these results.

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 Hybridization signals obtained from soil suspensions that have been treated by thermal shock and sonication have been, at most, low.

 The sample with the highest amount of DNA (15 g DNA / g dry weight of soil) was the only one for which the signal obtained was significantly different from the level of the background noise.

 No differences (or small differences) were observed between heat shock treated samples and those treated with thermal shock and sonication, indicating that thermal shocks have a detrimental effect on DNA. The best recoveries were observed for soil # 2, which has the highest organic matter content (Table 1), while no DNA was recovered from # 5 clay soil.

 Additional experiments were performed with unmilled samples of # 4 and # 5 sols, which were inoculated with 20 and 50 g of lambda phage DNA per gram of soil.

 The samples were extracted immediately or after an incubation period of one hour at 28 ° C, then the DNA extracts were purified and analyzed by gel electrophoresis.

 Incubation of soil No. 4 for one hour after inoculation did not lead to qualitatively or quantitatively different profiles from those obtained without incubation or from those previously observed when DNA was added after milling.

 These results indicate that enzymatic degradation by soil nucleases would not be involved in the low recovery rate of DNA. In addition, the absence of grinding step does not allow an increase in the recovery of DNA from soil n 5, indicating that the changes in soil structure due to grinding do not significantly increase adsorption of the soil. nucleic acids on colloids.

 2. 6 SATURATION OF ADSORPTION SITES WITH RNA: Most of the profiles obtained on the agarose gels do not differ significantly from the previous profiles in which the RNA treatment was not performed.

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 For example, no band was detected from soil rich in # 5 clay, regardless of the RNA concentrations and lambda phage DNA concentrations used.

 In addition, the Hindlll-digested lambda phage specific DNA bands remained undetectable in RNA-treated sandy loam (sol 4) when RNA was added prior to grinding.

 The intensity of the bands obtained from samples seeded with the DNA after grinding increases with the concentration of RNA, indicating that the treatment could have a positive effect.

 However, the results after hybridization and phosphorescence imaging analysis did not confirm the results of electrophoresis. For example, the positive effect of RNA treatment on DNA recovery from clay loam, when DNA has been added after milling, does not appear clearly.

 On the other hand, a positive effect of RNA was found for clay-rich soil (# 5) when DNA was added after milling.

 Although the hybridization signals for the control samples do not differ from the background levels, significant amounts of DNA were released from the RNA-treated samples, and the signals increased with the amount of DNA added as well as the RNA concentration.

 However, even for the highest RNA concentration (100 mg / g dry weight) the recovery rate never exceeded 3%.

2. 7 PURIFICATION OF DRAFT DNA EXTRACTS: Of the four protocols tested, the best amplification of the undiluted DNA extracts (1 l of extract in 50 l of PCR mixture) was observed after elution through columns Microspin S400 followed by elution through a column of Elutip d type, as shown by gel electrophoresis of PCR products.

 DNA purified by the aqueous double-phase system (protocol C) gave smaller amounts of PCR products after amplification from undiluted DNA extract.

 No amplification product could be obtained from undiluted extracts after amplification following the implementation of Protocols A or B. Therefore, Protocol B (see Materials section

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 and Methods) was used for all experiments in which PCR amplifications and / or dot blots were performed.

2. 8 PCR QUANTIFICATION AND HYBRIDIZATION: The first step was to determine if the amounts of PCR product were proportional to the number of target DNA molecules initially present in the reaction tube. Fragile Streptosporangium DNA was used as a target (see Materials and Methods section).

 The primers used were primers FGPS122 and FGPS350 (Table 2). Gel electrophoresis of PCR products showed that band intensity increases with increasing target concentration. The PCR products were hybridized with the oligonucleotide probe FGPS643 (Table 2), and the signals were quantified by phosphorescence (phospho-imaging) imaging.

 A good correlation (r2 = 0.98) was found between log [number of targets] and log [intensity of hybridization signal].

 It was then investigated whether the efficiency of PCR amplification was affected by humic acids and non-target DNA. When analyzed by gel electrophoresis, the increased intensity of the PCR product bands, corresponding to the different amounts of target DNA, was retained when the amplification was performed with DNA solutions to which extracts had been added. of DNase treated soil, containing wet acids at concentrations up to 8 ng in the PCR mixture of 50 l volume.

 With 20 ng of humic acid in the PCR mixture, the bands corresponding to low levels of target DNA disappeared, and at humic acid concentrations of 80 ng and at higher concentrations no band was visible.

 The varied amounts of S.fragile target DNA provided the expected amounts of PCR product when, prior to amplification, the fragile S.sub.S DNA was mixed with Streptomyces hygroscopicus DNA and added to the PCR mixture of 50 l in a range of 100 μg to 1 g to simulate the non-target DNA released from the soil microflora.

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2. 9 QUANTIFICATION OF INDIGENE ACTINOMYCETES OF SOIL AFTER DIFFERENT LYSE TREATMENTS: The purification protocol D followed by a PCR amplification as described above was used to quantify the actinomycetes belonging to the genus Streptosporangium in soil n 3 after extraction. according to Protocols 1, 2,3, 5a and 5b (Figure 5).

 After grinding, (Protocol No. 2) the amount of target DNA from this actinomycete was estimated by hybridization (Dot Blot) and radioimaging as 2.5 +/- 1.3 ng / g dry soil weight.

 Assuming that the DNA content is 10 fg per cell, as for Streptomyces (Gladek et al., 1984), this value corresponds to approximately 2.5 x 105 genomes. Similar values were obtained after the other lysis treatments (respectively 2.6 +/- 1.1 and 1.8 +/- 1.3 ng of DNA / g of dry soil using respectively protocols 3 and 4b ).

2. 10 EFFECTIVENESS OF DNA RECOVERY FROM SOILS PREVIOUSLY INOCULATED WITH BACTERIA: Three soils (n 2, 3 and 5) were inoculated with spores or hyphae of Streptomyces lividans at different concentrations (see section Material and Methods).

The amounts of mycelium added to the soil (FIG. 6b) correspond to the number of spores inoculated in the germination medium. Approximately 50% of these spores germinated. The exact number of cells in the hyphae of germ spores has not been determined. As a result, the amounts of spores and mycelia seeded in soils are not directly comparable.

 For each soil sample, extraction protocol No. 6, purification method D, and PCR amplification combined with dot blotting and phosphorescence imaging were used. to count the specific target DNAs that had been released. The extracted DNA can be clearly distinguished from the background only when the number of spores added exceeds 105 for the n 3 and n 5 soils and 107 for the n 2 sol (Figure 6a).

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 When the mycelium is added, the extracted DNA can be detected beyond an amount corresponding to 103 spores / g of soil for soils n 2 and n 3, and more than 107 spores / g for soil n 5 (Figure b).

 Above the detection level, the hybridization signal increases with increasing amounts of the inoculated cells.

 For the spore inoculum, a 100-fold increase in the number of seeded cells leads to an almost 100-fold increase in the yield of DNA. This increase is clearly lower when hyphae are inoculated, particularly in n 2 and n 3 soils (Figure 6).

 In contrast, the results obtained when lambda phage DNA was used as an inoculum, DNA was also recovered from clay-rich soil (# 5) when bacterial cells were used as inoculum. However, for the latter as well, RNA treatment increased the recovery of Streptomyces DNA from this soil for both spores and mycelium (Figure 6).

 Seeding of soils with vegetative cells of Bacillus anthracis provided similar recovery rates to those obtained for Streptomyces.

 In addition, DNA recovery rates from soil no. 5 increased after RNA treatment also for this inoculum.

Example 2 Construction of a Low Molecular Weight DNA Library (<10 kb) from a Lindane Contaminated Soil: Cloning and Expression of the LinA Gene This example describes the construction of a DNA library of soil in E. coli. It demonstrates the cloning and expression of small genes from a non-culturable microflora.

 Lindane is an organochlorine pesticide, recalcitrant to degradation and persistent in the environment. Aerobically, its biodegradation is catalyzed by a dehydrochlorinase, coded by the

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 LinA gene, which transforms lindane into 1,2,4-trichlorobenzene.

The linA gene has only been identified from two strains isolated from the soil: Sphingomonas paucimobilis, isolated in Japan (Seeno and Wada 1989, Imai et al 1991, Nagata et al 1993) and Rhodanobacter lindaniclasticus isolated in France (Thomas et al 1996, Nalin et al 1999).

 However, the potential for degradation of lindane, demonstrated by assaying chloride ions released and PCR amplification of the linA gene from soils that have been in contact with lindane or not, seems to be spread more widely in the environment (BiesiekierskaGalguen, 1997).

1. Direct extraction of soil DNA
Dry soils are milled for 10 minutes in a Restch centrifugal force mill equipped with 6 tungsten balls. 10 grams of ground sol are suspended in 50 ml of TENP buffer pH 9 (50 mM Tris, 20 mM EDTA, 100 mM NaCl, 1% w / v polyvinylpolypyrrolidone) and vortexed for 10 min.

After centrifugation for 5 minutes, 4000 g at 4 ° C., the supernatant is precipitated with sodium acetate (3M, pH 5. 2) and isopropanol, to be taken up in sterile TE buffer (10 mM Tris, EDTA 1mM, pH 8.0).

The extracted DNA is then purified on a S400 molecular sieve column (Pharmacia) and on an Elutip d ion exchange column (Schleicher and Schuell), according to the manufacturers' instructions, and then stored in TE.

2. Construction of the soil-extracted DNA library in the vector pBluescript SK-
The pBluescript SK- vector and the DNA extracted from the soil are each digested with the enzymes HindIII and BamHI (Roche), at the rate of 10 units of enzymes per 1 g of DNA (incubation for 2 hours at 37 ° C.). The DNAs are

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 then ligated by the action of T4 DNA ligase (Roche) overnight at 15 C, one unit of enzyme per 300 ng of DNA (about 200 ng of DNA insert and 100 ng of digested vector). The electrocompetent Escherichia coli cells, ElectroMAX DH10B TM (Gibco BRL) are transformed by the ligation mixture (2 NI) by electroporation (25 F, 200 and 500 #, 2.5 kV) (Biorad Gene Pulser).

 After one hour of incubation in LB medium, the transformed cells are diluted to obtain about 100 colonies per dish and are then plated on LB medium (10 g / l Tryptone, 5 g / l yeast extract, 5 g / NaCl ) supplemented with Ampiciline (100 mg / l), γ-HCH (500 mg / l), X-gal 60 mg / l (5-bromo-4-chloro-3-indolyl-α-D-galactoside), and of IPTG 40 mg / l (isopropylthio-s-D-galactoside), and incubated overnight at 37 C. The y-hexachlorocyclohexane (Merck-Schuchardt) being insoluble in water, a solution of 50 g / l is prepared in DMSO (dimethyl sulfoxide) (Sigma).

 A library of 10,000 clones has thus been obtained.

3. Cloning and expression of the linA gene
The library is screened by visualizing a halo of degradation of lindane around the colony (lindane precipitating in the culture media). Of 10,000 clones screened, thus exhibited lindane degrading activity. The presence of the linA gene in these clones could be confirmed by PCR using specific primers described by Thomas et al (1996). Digestions performed on the inserts as well as on the amplification products showed identical profiles between all the clones screened and the reference control, R. lindaniclasticus. Clones carrying the linA gene also had an insert of the same size (about 4 kb).

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 It could thus be demonstrated that the soil DNA could be cloned and expressed in a heterologous host: E. coli, and that genes from a poorly cultivable microflora could be expressed. Banks made from partial digestion of DNA extracted from the soil by restriction enzymes such as Sau3AI are also possible.

EXAMPLE 3: Process for preparing a nucleic acid collection from a soil sample, comprising a step of indirect extraction of the DNA.

 1. MATERIAL AND METHODS.

1. 1 Extraction of the bacterial fraction of the soil.

 5 g of sol are dispersed in 50 ml of sterile 0. 8% NaCl, by grinding with Waring Blender for 3 x 1 minute, with cooling in ice between each grinding. the bacterial cells are then separated from the soil particles by centrifugation on a Nycodenz density cushion (Nycomed Pharma AS, Oslo, Norway). In a centrifuge tube, 11.6 ml of a solution of Nycodenz with a density of 1. 3 g.ml-1 (8 g of Nycodenz suspended in 10 ml of sterile water) are placed under 25 ml of the suspension. ground previously obtained. After centrifugation at 10,000 g in a rotor with moving cups (TST rotor 28. 38, Kontron) for 40 minutes at 4 ° C., the cell ring lying at the interphase of the aqueous phase and of the Nycodenz phase, is removed, washed in 25 ml of sterile water and centrifuged at 10,000 g for 20 minutes. The cell pellet is then taken up in a 10 mM Tris solution; EDTA 100 mMn pH 8.0.

 Prior to soil dispersion with Waring Blender, a soil enrichment step in a yeast extract solution can be included to allow germination of soil bacterial spores. 5 g of sol are then incubated in 50 ml of a sterile solution of NaCl 0.8% - yeast extract 6%, for 30 minutes at

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 C. Yeast extract is removed by centrifugation at 5000 rpm for 10 minutes to prevent foaming during grinding.

1. 2 Lyse bacterial cells of the soil.

 Lysis of the cells in a liquid medium and purification on a gradient of cesium chloride.

 The cells are lysed in a 10 mM Tris solution, 100 mM EDTA, pH 8.0 containing 5 mg / ml-1 of lysozyme and 0.5 mg / ml-1 of achromopeptidase for 1 hour at 37 ° C. A solution of lauryl sarcosyl (1% final) and Proteinase K (2 mg / ml-1) is then added and incubated at 37 ° C. for 30 minutes. The DNA solution is then purified on a cesium chloride density gradient by centrifugation at 35,000 rpm for 36 hours on a Kontron 65 rotor. 13. The cesium chloride gradient employed is a 1 g / ml gradient of CsCl having a refractive index of 1.3860 (Sambrook et al., 1989).

 Lysis of the cells after inclusion in an agarose block.

 The cells are mixed with an equal volume of 1.5% (weight / volume) agarose Seaplaque (Agarose Seaplaque FMC Products, TEBU, Perray en Yvelines, France). low melting point and cast in a 100 l block. The blocks are then incubated in a lysis solution: 250 mM EDTA, 10.3% sucrose, 5 mg / ml-1 lysozyme and 0.5 mg / ml-1 achromopeptidase at 37 ° C. for 3 hours. The blocks are then washed in a solution of 10 mM Tris-500 mM EDTA and incubated overnight at 37 ° C. in 500 mM EDTA containing 1 mg.ml-1 of proteinase K and 1% lauryl sarcosyl. After several washes in Tris-EDTA, the blocks are stored in 500 mM EDTA.

 The quality of the DNAs thus extracted is controlled by pulsed field electrophoresis.

 The amount of extracted DNA was evaluated on electrophoresis gel against a standard range of calf thymus DNA.

1. 3 Molecular characterization of the DNA extracted from the soil.

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 The DNAs extracted from the soil are characterized by PCR hybridization, which method consists in firstly amplifying the DNAs using primers located on universally conserved regions of the 16S rRNA gene, and then hybridizing the amplified DNAs with different Oligonucleotide probes of known specificity (Table 4), in order to quantify the intensity of the hybridization signal relative to an external standard range of genomic DNA.

 The soil-extracted DNAs as well as genomic DNAs extracted from pure cultures are amplified with FGPS primers 612-669 (Table 1) under standard PCR amplification conditions. The amplification products are then denatured with an equal volume of 1N NaOH, deposited on a nylon membrane (GeneScreen Plus, Life Science Products) and hybridized with an oligonucleotide probe end-labeled with g32P ATP by action of T4. polynucleotide kinase. After prehybridization of the membrane in a 20 ml solution containing 6 ml of 20X SSC, 1 ml of Denhardt's solution, 1 ml of 10% SDS and 5 mg of heterologous salmon sperm DNA, the hybridizations are conducted overnight. at the temperature defined by the probe. The membranes are washed twice in 2X SSC for 5 minutes at room temperature, then once in 2% SDS SDS and a second time in 1X SSC, 0.1% SDS for 30 minutes at room temperature. 'hybridization. The hybridization signals are quantified using the Molecular Analyst software (Biorad, Ivry sur Seine, France) and the quantities of DNA are estimated by interpolation of the standard curves obtained from the genomic DNAs.

2. RESULTS AND DISCUSSION 2. 1 Extraction and lysis of the bacterial fraction of the soil.

 The separation of microbial cells from soil particles, prior to DNA extraction, is an alternative with many advantages over direct DNA extraction methods in the soil. Indeed, the extraction of the microbial fraction limits the contamination of the DNA extract by extracellular DNA present

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 freely in the soil or by DNA of eukaryotic origin. Most importantly, the DNA extracted from the microbial fraction of the soil has longer fragments and better integrity than directly extracted DNA JACOBSON and RASMUSSEN (1992). In addition, the separation of the soil particles makes it possible to avoid contamination of the DNA extract by humic and phenolic compounds, compounds which can subsequently seriously affect the cloning efficiencies.

 One of the decisive steps for the extraction of soil cells is the dispersion of the soil sample in order to dissociate the cells adhering to the surface or inside the aggregates of soil particles.

Three successive milling cycles of one minute each make it possible to obtain a better extraction efficiency of the cells as well as a larger quantity of recovered DNA, compared with a single milling cycle of one minute 30.

 Table 5 reports the extraction efficiencies obtained after centrifugation on a Nycodenz gradient, on the total viable microflora (counted by microscopy after staining with acridine orange), on the total cultivable microflora (counted on Trypticase-Soy solid medium 10% ), and on the microflora of actinomycetes cultivable on HV agar medium (after incubation at 40 C in a solution of yeast extract 6% -SDS 0.05% in order to cause sprouting sprouts). On the other hand, the extracted DNA was quantified either after lysis of the cells in a liquid medium (without purification on a cesium chloride gradient) or after lysis of the cells included in an agarose block (after digestion of the agarose with a b-agarase).

 The results show that more than 14% of the total soil microflora is recovered by this method (ie 2 108 cells per gram of soil), and that the total cultivable microflora represents only 2% of the total microbial population.

 On the other hand, the amount of DNA extracted from the cells is 330 ng per gram of dry soil. By estimating the DNA content per microbial cell of the soil between 1. 6 and 2. 4 fg, and taking into account the quantity of cells extracted (2 108 cells per gram of soil), it can be estimated that almost all cells have been lysed and thus the lysis does not bring significant bias to this approach.

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 Pulsed-field electrophoresis showed that the soil DNA extracted after the Nycodenz and CsCl gradient could reach a size of 150 kb and that the agarose block lysis allowed fragments larger than 600 kb to be extracted.

 These results confirm the interest of this culture-independent approach for the construction of environmental DNA libraries as an alternative to direct DNA extraction methods.

2. 2 Molecular characterization of the DNA extracted from the soil.

 The purpose of the molecular characterization of the DNA extracted from the soil is to obtain profiles representing the proportions of the different bacterial taxons present in the DNA extract. It was also a question of knowing the extraction biases induced by the preliminary separation of the cellular reaction of the soil, in comparison with a direct extraction method for lack of direct visualization of the microbial diversity present in the soils. Indeed, little information has been collected on the extraction of Nycodenz gradient cells according to their morphological structure (cell diameter, filamentous or sporulated forms).

 The methods hitherto in place were based on: - quantitative hybridizations using oligonucleotide probes specific to different bacterial groups, applied directly to DNA extracted from the environment Unfortunately, this approach is not very sensitive and does not allow detect genera or taxonomic groups present in low abundance AMANN (1995).

 - Quantitative PCRs such as the MPN-PCR (Most Probable Number) SYKES et al. (1992) or quantitative PCR by competition DIVIACCO et al. (1993). The respective drawbacks of each of these approaches are (i) the heaviness of use due to the multiplication of dilutions and repetitions which makes the technique inappropriate for a large number of samples or primer pairs, and (ii) the need to build a competitor specific to the target DNA and not inducing bias in the competition.

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 The method implemented according to the present invention consists in universally amplifying a fragment of 700 bp inside the 16S rDNA sequence, to hybridize this amplifiat with an oligonucleotide probe of variable specificity (at the level of the kingdom, of the order, subclass or genus) and to compare the intensity of hybridization of the sample with respect to an external standard range. The amplification prior to hybridization makes it possible to quantify genera or species of scant microorganisms. In addition, the amplification by universal primers makes it possible, during hybridization, to use a large series of oligonucleotide probes. It makes it possible to compare different modes of lysis (direct or indirect extraction) with well-defined taxonomic groups.

 The results are summarized in Table 6.

 They show similar profiles between the two extraction methods (direct and indirect). Thus, it appears that the prior extraction of the telluric microbial fraction does not introduce any real bias among the taxa tested. The only significant difference between the two extraction approaches would seem to be the greater abundance of rDNA sequences belonging to the proteobacteria in the extract by the indirect extraction method.

 In addition, a significant effect of incubation of the soil sample in a yeast extract solution was observed on sporulated soil populations (Gram +, low percent GC and Actinomycetes).

This step causes germination of the spores, and on the one hand certainly allows a better recovery of this type of cells and, on the other hand, a greater efficiency of the lysis on germinating cells.

 This approach allows a semi-quantitative analysis, targeted at the main taxa defined from cultivated micro-organisms and usually found in soils. Only molecular tools make it possible to estimate the importance of the different taxa, the methods of cultivation being too restrictive and dependent on the specificity of the medium used.

 The results show that a large part of the microbial population is not represented in the phylogenetic groups described, thus highlighting the existence of new groups

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 composed of micro-organisms not cultivated until now, or non-cultivable.

 Thus, new probes can be defined from sequences determined from DNA extracted from the soil (new phyla composed of non-cultured microorganisms, LUDWIG et al., (1997) to obtain a more accurate image of the composition of the DNA extract.

Example 4: - CONSTRUCTION OF COSMIDE POS 7001 Features of POS 7001:
Replicative in E. coli
Integrative with Streptomyces
Selectable from E. coli AmpR, HygroR and Streptomyces HygroR
The properties of the cosmid allow to insert large DNA fragments between 30 and 40kb.

It comprises 1 - The inducible promoter tipA of Streptomyces lividans 2 - The specific integration system of the element pSAM2 3 - The hygromycin resistance gene 4- the cosmid pWED1, derived from pWED15 1) - The inducible promoter of the gene tip A from S. lividans
The tipA gene encodes a 19 KD protein whose transcription is induced by the antibiotic thiostrepton or nosiheptide. The tipA promoter is well regulated: exponential and stationary phase induction (200X) Murakami T, Holt TG, Thompson CJ. J. Bacteriol 1989; 171: 1459-66 2) - The hygromycin resistance gene - Hygromycin: produced by S. hygroscopicus - The resistance gene codes a phosphotransferase (hph) - The gene used comes from a cassette built by Blondelet et al in which the hyg gene is under the control of its own promoter

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 and IPTG-inducible promoter Blondelet-Rouault et al; .

Gene 1997; 190: 315-7 3) - The site-specific integration system
The pSAM2 element integrates into the chromosome through a site-specific integration mechanism. Recombination takes place between two identical 58 bp sequences present on the plasmid (attp) and on the chromosome (attB).

 The int gene, located near the attP site, is involved in the site-specific integration of pSAM2, and its product has similarities to the integrases of bacteriophages tempered with Enterobacteriaceae. It has been shown that a fragment of pSAM2 containing only the attP attachment site as well as the int gene was able to integrate in the same way as the whole element. See French Patent No. 88 06638 of May 18, 1988, and Raynal A et al. Mol Microbiol 1998 28: 333-42).

 4) Construction of Cosmos pOS7001 Step 1 The TipA promoter was isolated from plasmid pPM927 (Smokvina et al., Gene 1990; 94: 53-9) on a 700 base pair HindIII-BamHI fragment and cloned into the pUC18 vector (Yannish-Perron et al., 1985) HindIII / BamHI digested Step 2 / This HindIII-BamHI fragment was subsequently transferred from pUC18 to pUC19 (Yannish-Perron et al., 1985).

Step 3: A 1500 base pair BamHI-BamHI insert carrying the int gene and the attP site of pSAM2 was isolated from plasmid pOSint1, shown in Figure 8 (Raynal A et al., Mol Microbiol 1998 28: 333-42 and cloned at the BamHI site of the previous vector (pUC19 / TipA), in the orientation to bring the int gene under control of the TipA promoter.

Step 4 / The BamHl site located 5 'of the int gene was deleted by BamHl partial digestion and treatment with Klenow enzyme. A

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 HindIII-BamHI fragment carrying TipA-int-attP was thus isolated from pUC19 and transferred into pBR322 HindIII / BamHI.

Step 5 / Hygromycin cassette isolated from pHP45Qhyg (BlondeletRouault et al., 1997) on a HindIII-HindIII fragment was cloned at the HindIII site located upstream of the TipA promoter.

Step 6 / The HindIII site located between the QHyg cassette and the TipA promoter was removed by Klenow treatment after HindIII partial digestion.

Step 7 / The plasmid obtained at the end of the preceding step makes it possible to isolate a single HindIII-BamHI fragment bearing all the QHyg / TipA / int attP elements, which was cloned after Klenow treatment at the EcoRV site of the cosmid pWED1. The cosmid pWED1, represented in FIG. 9, derives from the cosmid pWE15, represented in FIG. 10 (Wahl GM, et al., Proc Natl Acad Sci USA 1987 84: 2160-4) by the deletion of a Hpal-Hpal fragment bearing the Neomycin gene and SV40 origin.

A map of the vector pOS 7001 is shown in Figure 11.

Example 5 Construction of Conjugative and Integrative Cosmid in Streptomyces, Vector pOSV 303

 Since the packaging selects clones larger than 30kb, only 10-15% of the clones do not contain an insert, so there is no real need for a recombinant selection system, which allows to build a smaller vector.

Construction: Step 1: the vector pOSV001
Cloning of a 800 base pair PstI-PstI fragment carrying the OriT transfer origin of the RK2 replicon (Guiney et al., 1983), in the plasmid pUC19 opened by PstI. This cloning step makes it possible to obtain a transferable vector of E. coli to Streptomyces by conjugation.

 The map of the vector pOSV 001 is shown in Figure 17.

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Step 2: the vector pOSV002
Insertion of Hygromycin marker (hyg hygienic), and selectable in Streptomyces, so that the gene conferring resistance to hygromycin is transferred last which makes it possible to ensure complete transfer of the BAC with the DNA insert of the ground.

 Cloning of the hygromycin cassette isolated from pHP45Qhyg on a HindIII-HindIII fragment carrying the hygromycin resistance gene. This fragment is cloned at the PstI site (position 201) of the vector pOSV001. This Pstl site was chosen, given the direction of the transfer, so that the Hygro marker is the last transferred during the conjugation. The PstI and HindIII ends are made compatible after treatment with the Klenow fragment of the DNA polymerase making it possible to generate "free ends". The orientation of the Qhyg fragment is determined at the end of construction.

 The map of the vector pOSV002 is shown in Figure 18.

Step 3: the vector pOSV010
The XbaI-HindIII fragment isolated from the plasmid pOSV002 and containing the hygromycin resistance marker and the transfer origin is cloned into the plasmid pOSint1 digested with XbaI and HindIII. The orientaion of the sites is such that the hygromycin marker will always be transferred last.

Plasmid pOSint1, shown in Figure 8, has been described in the article by Raynal et al (Raynal A et al Mol Microbiol 1998 28: 333-42).

 This construct allows the expression of integrase in E. coli and Streptomyces.

Step 4: inserting the site "cos
The principle is to insert a "cos" site in the plasmid pOSV010 allowing the packaging in the plasmid pOSV010, represented in FIG. 12.

 Obtaining the "cos" fragment is shown in Figure 13.

 This fragment is obtained by PCR. From a fragment bearing the cohesive ends (cos) of # (bacteriophage lambda or cosmid pHC79), a PCR amplification is carried out using the

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 oligonucleotides corresponding to the -50 / + 130 sequences relative to the cos site. These oligonucleotides additionally contain the Nsil cloning sites, compatible with PstI, XhoI, SalI compatible, EcoRV, "free end".

 The addition of the rare sites Swal and Pacl makes it possible to isolate and / or map the cloned insert.

 The PCR fragment is bounded by a PstI site at the 5 'end and by a Hindi site at the 3' end, allowing cloning into the vector pOSV010 (FIG. 12), which is first digested with the enzymes Nsil and EcoRV, causing the deletion laclq repressor.

 The map of the vector pOSV303 is shown in Figure 14.

The vector pOSV303 contains cloning sites such as the Nsil site, PstI compatible, the XhoI site, Sali compatible or the EcoRV site for obtaining "free ends".

Example 6: Construction of the replicative cosmid shuttle E. coli Streptomyces pOS700R.

 Fragments of plasmid pEI16 (Volff et al., 1996) shown in Figure 15 were isolated and treated with Klenow. These fragments contain the sequences necessary for replication and stability from the SCP2 plasmid.

 These two fragments are inserted separately into the EcoRV site of cosmid pWED1 leading to 2 different clones.

 The hygromycin cassette isolated from pHP45Qhyg on a HindIII-HindIII fragment was cloned at the HindIII site of cosmids pWED1 containing the ScP2 insert as PstI-EcoRI or XbaI fragments. It confers resistance to selectable Hygromycin in both E. coli and Streptomyces.

 Transformation of S. lividans and determination of transformation efficiency.

 It was found that the cosmid containing the XbaI insert was less stable than that containing the EcoRI PstI fragment. It is therefore the latter which has been retained under the name of pOS700R.

 The map of the vector pOS 700R is shown in Figure 16.

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Example 7: Transformative Efficiency of Integrative Vectors (pOS7001) and Replicative Possibilities
Make the S. lividans strain resistant to thiostrepton by integrating the plasmid pT01 carrying the thiostrepton resistance marker
Preparation of protoplasts from S. lividans grown in the presence of thiostrepton
With the vector pOS7001, the transformation efficiency is about 3000 transformants per g of DNA.

 With the vector pOS700R, the transformation efficiency is about 30,000 transformants per g of DNA.

Example 8: Construction of an Integrative BAC Vector in Streptomyces and Conjugate Characteristics: Replicative in E.coli Transferable by Conjugation of E. coli to Streptomyces Integrative in Streptomyces Selectable in E. coli and Streptomyces Capable of Inserting Large DNA Fragments ; It must be emphasized that it is necessary to have soil DNAs ranging in size from 100 to 300 kb and not contaminated with small fragments. Indeed the small fragments are very preferentially integrated.

 With a screen for selecting the plasmids carrying an insert. This screen makes it possible by eliminating the closed and undigested vectors to work with a higher ratio between vector and DNA to be inserted, which makes it possible to have a better cloning efficiency for forming banks.

Construction :

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Step 1: the vector pOSV001
Cloning of a 800 base pair PstI-PstI fragment carrying the OriT transfer origin of the RK2 replicon (Guiney et al., 1983), in the plasmid pUC19 opened by PstI. This cloning step makes it possible to obtain a transferable vector of E. coli to Streptomyces by conjugation.

 The map of the vector pOSV 001 is shown in Figure 17.

Step 2: the vector pOSV002
Insertion of Hygromycin marker (Qhyg cassette), and selectable in Streptomyces, so that the gene conferring resistance to hygromycin is transferred last, which ensures the complete transfer of the BAC with the DNA insert of the ground.

 Cloning of the hygromycin cassette isolated from pHP45Qhyg on a HindIII-HindIII fragment carrying the hygromycin resistance gene. This fragment is cloned at the PstI site (position 201) of the vector pOSV001. This Pstl site was chosen, given the direction of the transfer, so that the Hygro marker is the last transferred during the conjugation. The PstI and HindIII ends are made compatible after treatment with the Klenow fragment of the DNA polymerase making it possible to generate "free ends". The orientation of the Qhyg fragment is determined at the end of construction.

 The map of the vector pOSV002 is shown in Figure 18.

Step 3: the vector pOSV010
The XbaI-HindIII fragment isolated from the plasmid pOSV002 and containing the hygromycin resistance marker and the transfer origin is cloned into the plasmid pOSint1 digested with XbaI and HindIII. The orientation of the sites is such that the hygromycin marker will always be transferred last.

Plasmid pOSint1, shown in Figure 8, has been described in the article by Raynal et al (Raynal A et al Mol Microbiol 1998 28: 333-42).

 This construct allows the expression of integrase in E. coli and Streptomyces.

Step 4: the vector pOSV014

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Addition of a "cassette" allowing ultimately to select in the final construct the plasmids having inserted foreign DNA.

This "cassette" carries the gene encoding the phage # repressor IC and the tetracycline resistance conferring gene. This gene carries in its 5 'non-coding region the target sequence of the repressor. The insertion of DNA into the HindIII site located in the coding sequence of CI leads to the non-production of the repressor and therefore to the expression of the resistance to tetracycline.

It is carried by the plasmid pUN99 described in the article: Nilsson et al.

(Nucleic Acids Res 1983, 11: 8019-30) A Pvull-HindIII fragment isolated from pOSV010 and containing the Int, attP, Hygro and oriT sequences is cloned at the Mscl site of pUN99.

The map of the vector pOSV014 is shown in Figure 19.

Step 5: the vector pOSV 403, integrative and conjugative BAC factor
This last step of cloning into pBAC11 (shown in Figure 20) provides the final plasmid with BAC (Bacterial Artificial Chromosome) characteristics, particularly the ability to accept very large DNA inserts.

 The PstI-PstI fragment of the vector pOSV014 carrying all of the elements and functions described above is cloned into the pasmide pBAC11 (pBeloBAC11) digested with NotI. The ends are made compatible with the Klenow enzyme treatment.

The map of the vector pOSV403 is shown in Figure 21. The diagram of Figure 21 indicates the selected orientation.

Step 6:
The vector pOSV403 contains the HindIII and Nsil sites. The Nsil site is quite rare in Streptomyces and has the advantage of being compatible with Pstl. On the other hand, the Pstl site is frequent in Streptomyces and can be used to carry out partial digestions.

 The recombinant clones carrying an insert cloned in the repressor CI, and thus inactivating this repressor, become resistant to the tetracycline. Since BACs are only present at one copy per cell, recombinant clones should be selected with a lower dose of tetracycline than the usual dose of 20 g / ml per day.

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 example with a dose of 5 g / ml. Under these conditions there is no background noise.

 It is also possible to use a system developed and marketed by InVitrogen, in which the insertion of DNA into the vector inactivates a gyrase inhibitor whose expression is toxic to E. coli. The fragment is preferably isolated from the vector pZErO-2 (http://www.invitrogen.com/).

Example 9 Construction of an S. alboniger library in the two cosmids integrative (pOS7001) and replicative (pOS700R) 1) - Construction of the Bank
To evaluate the efficiency of the cloning system, the puromycin biosynthetic pathway of Streptomyces alboniger was cloned into both shuttle cosmos pOS7001 and pOS700R. The genes of the puromycin biosynthetic pathway are carried by a BamHI DNA fragment of about 15 Kb.

Genomic DNA from Streptomyces alboniger was isolated. 90% of this DNA has a molecular weight between 20 and 150 Kb, determined by pulsed field electrophoresis.

 Both cosmids were digested with the BamHI enzyme (unique cloning site).

 The BamHl partial digestion conditions of the genomic DNA were determined (50 g of DNA and 12 units of enzyme, 5 minutes of digestion). After checking the size by agarose gel electrophoresis, the partially digested DNA was introduced into the vectors. In the ligation, 15 g of genomic DNA + 2 g of the integrative vector or 5 g of the replicative vector were used.

 Each ligation mixture was used for in vitro encapsidation of DNA in bacteriophage lambda heads. The encapsidation mixtures (0.5 ml) were titrated (integrative vector pOS7001 = 7.5 x 105 cosmids / ml, replicative vector = 5 x 104 cosmids / ml).

 Cosmids were used to transfect E. coli and thereby generate two libraries of approximately 25,000 ampicillin resistant clones. The DNA of all these clones was isolated and quantified.

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 To test the libraries, several clones were chosen, the DNA purified and digested with BamHI to verify the presence and size of the inserts. The clones tested contain between 20 and 35 Kb of S. alboniger insert.

2) - Identification of Clones Containing the Puromycin Biosynthetic Pathway Clones capable of containing the complete puromycin biosynthetic pathway were identified by hybridization with a probe corresponding to the puromycin resistance gene, the pac gene of 1, 1 kb. (Lacalle et al., Gene 1989, 79, 375-80) Bank made in the Integrative Vector pOS 7001:
Among 2000 clones analyzed, 9 clones hybridized with the probe and they contain inserts of about 40 kb.

Bank made in the replicative vector pOS 700R: Among 2000 clones analyzed, 12 clones hybridized with the probe; they contain inserts of about 40 kb.

 Using data published by Tercero et al. (J Biol Chem.

1996; 271,1579-90), clones containing the entire biosynthetic pathway were identified after hybridization with appropriate probes. Some integrative and replicative cosmids have a fragment of 12360 base pairs after Clal-EcoRV digestion, suggesting an insert containing the entire puromycin biosynthesis pathway.

4) - Verification of puromycin production by resistant clones (Rhône-Poulenc). a) Materials and Methods

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 Strains and culture conditions: Three resistant clones were selected to verify the production of puromycin. They correspond to the recombinants of S. lividans containing an insert in the integrative vector pOS7001 (G20) or an insert in the replicative vector (G21 and G22).

Reference strains were used to ensure that the culture media used allowed this production. It is the wild type S. alboniger ATCC 12461 strain producing puromycin and the S. lividans recombinant strain containing the complete puromycin cluster cloned in the plasmid pRCP11 (Lacalle et al., 1992, the EMBO journal, 11). 785-792) (G23).

The strains are seeded in a culture medium whose composition is as follows: Bacteriological peptone Organotechnie 5 g / l of final medium Springer yeast extract 5 Liebig meat extract 5 Prolabo 15 glucose CaC03 (1) Prolabo 3 NaCI Prolabo 5 Agar ( 2) Difco 1 (1) The 3 g of carbonate are mixed with 200 ml of distilled water and sterilized separately. The addition is done after sterilization.

(2) The agar is premixed in 100ml of distilled water before being added to the other ingredients of pH medium adjusted to 7.2 before sterilization sterilization 25 minutes at 121 ° C

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 50 g / l of hygromycin and 5 g / l of thiostrepton are added to the medium after sterilization so as to maintain a selection pressure of the clones containing an insert by virtue of the marker gene present on the vector (the thiostrepton resistance gene being worn by the plasmid pRCP11).

50 ml of liquid culture medium, divided into 250 ml Erlenmeyer flasks, are inoculated with 2 ml of aqueous suspension of spores and mycelium of each of the strains. The cultures are incubated for 4 days at 28 ° C. with stirring of 220 rpm. 50 ml of production media, divided into 250 ml Erlenmeyer flasks, are then inoculated with 2 ml of these pre-cultures. The production medium used is an industrial environment optimized for the production of pristinamycin (RPR 201 medium). The cultures are incubated at 28 ° C. with stirring of 220 rpm. After different incubation times, an Erlenmeyer flask of each culture is brought to pH 11 and then extracted with 2 times 1 volume of dichloromethane. The organic phase is concentrated to dryness under reduced pressure and the extract is taken up in 10 l of methanol. 100 l of the methanolic solution are analyzed by HPLC equipped with a diode array detector in a 0.05% TFA VA / C18 column water-acetonitrile gradient system for the detection of puromycin. b) Results
Comparative HPLC analyzes from the cultures of the different strains show the production of puromycin in the culture of the wild strain from 24 hours of incubation. Production, although lower, is also clearly detected from 48 h in the G20 clone culture containing cosmid pOS7001 (FIG. 23). Puromycin was also detected in trace amounts in clone G23 containing the complete operon encoding the compound in plasmid pRCP11. Nevertheless, no production was observed in

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clones G21 and G22 containing the cosmid pOS700R. The results are shown in Figure 23. c) Conclusions
The results obtained make it possible to demonstrate the effectiveness of the cloning system developed in the cosmid pOS7001 to express in a heterologous host such as S. lividans a complete biosynthetic pathway under the control of its own regulatory sequences. On the other hand, these data also validate the screening of the libraries obtained on the basis of the resistance of the clones to puromycin since it led to identify among a small number of clones, a recombinant capable of expressing the associated biosynthetic pathway. to the resistance gene. The absence of puromycin production in the other clones can probably be explained by the cloning of only part of the operon containing the resistance gene but lacking certain regulatory, transduction or transcription sequences necessary for the synthesis of the compound. .

EXAMPLE 10: CLONING DNA OF THE VECTOR SOLDANS 1) - Preparation of the DNA of the soil to be cloned
The different DNA fragments must be purified according to their destination:
cosmids
The size of the molecules must be between 30 and 40kb. However, the DNA extracted from the soil is heterogeneous in size and comprises molecules up to 200 or 300kb. In order to homogenize the sizes, the DNA is broken mechanically by passing the solution through a needle of 0.4 mm in diameter. Fragments of a size close to 30kb are not affected by these repeated passages through a needle and it is not

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 therefore not necessary to make a separation by the size especially that the packaging in the particles automatically eliminates the short inserts.

BACs
DNA preparation
The soil DNA is separated by pulsed field electrophoresis (type
CHEF) in such conditions as fragments between
100 and 300kb are concentrated in a band of about 5mm. This is achieved by running in a 0.7% normal agarose or 1% low melting agarose gel with a 100 second pulse time for 20 hours and at a temperature of 10 C.

DNA recovery
Two methods are used, their choice depends on the size of the molecules that we want to isolate, up to 150kb or above.

- Up to 150kb
The porosity of a 0.7% agarose gel allows the release of the DNA by electroelution provided complete absence of ethidium bromide. This
DNA is then manipulated with enlarged and hydrophobic orifice pipetting instruments to avoid mechanical fragmentation of the molecules.

- Between 100 and 300kb
The band containing the fragments of a size between 100 and 300 kb is cut. For migration, a 1% agarose gel with a low melting point is used. This property makes it possible to melt the gel at a tolerable temperature for the 65 C DNA and then to digest it with the agarase (Agarase sold by the company Boehringer) at a temperature of 45 ° C. according to the supplier's instructions.

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 2) Use of integrative cosmos pOS7001 and replicative pOS700R PolyA polyT tails construction Principle A cosmid vector, open at any cloning site, is modified at the 3 'ends by adding a monotonous polynucleotide. On the other hand, the DNA to be cloned is modified at the 3 'ends by adding a monotonous polynucleotide that can be paired with the preceding one.

The vector-fragment association to be cloned is by these polynucleotides and the cos sequence of the vector allows the in vitro packaging of the DNA in Lamda phage capsids.

Vector Preparation The vector used is a self-replicating vector in E. coli and integrative in Streptomyces.

For E. coli, the selection is on ampicillin resistance and for Streptomyces, it is on resistance to hygromycin.

The cosmid is opened at one of the two possible sites (BamHI or HindIII) and the 3 'ends are lengthened by polyA with terminal transferase under the conditions where the supplier of the enzyme provides the addition of 50 to 100 nucleotides.

Preparation of the DNA to be inserted

The 3 'ends of the DNA are extended by polyT with terminal transferase under the conditions providing an elongation comparable to that of the vector. Under the experimental conditions described by the manufacturer, polyA polyT tails are 30 to 70 bases long.

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 Assembly of molecules and encapsidation in vitro.

For assembly of the molecules, a vector molecule is mixed for an inserted DNA molecule. The concentration of the DNA in mass is 500 g.ml-1.

The mixture is packaged and the transfection efficiency depends on the strain used as the recipient and the inserted DNA: zero with the test DNA and the strain DH5a, the efficiency is comparable for the SURE and DH10B strains; at extraction, however, the yield of DNA is higher with strain DH10B.

Construction by dephosphorylation The soil DNA is blunt ended by removing the outgoing 3 'sequences and filling the outgoing 5' sequences. This operation is done with: Klenow enzyme, T4 polymerase, the 4 nucleotides triphosphates. The cosmid vector is digested with BamHI, then treated with the Klenow enzyme to make it free and then dephosphorylated to prevent it from closing on itself. After ligation, the mixture is packaged and transfected as previously described.

3) - Use of pBAC Principle.

The conjugative and integrative pBAC plasmid possesses the HindIII and Nsil sites as cloning sites. The insertion of a DNA sequence at these sites inactivates the phage Lambda repressor IC which controls the expression of the tetracycline resistance gene. Inactivation of the repressor therefore makes the cell resistant to this antibiotic (5 g.ml-1). Cloning at these sites is facilitated by vector modification and preparation of the DNA to be cloned.

Preparation of the vector Hindlll example

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 In order for the vector not to close on itself, the Hind III site is modified: the first base (A) is put back in place to form a 5 'outgoing sequence, which can not be paired with its peers.

The operation is performed by the Klenow enzyme in the presence of dATP.

The success of the operation is verified by carrying out a ligation of the vector on itself before and after treatment with the Klenow enzyme. At the same amount of tested DNA, 3000 clones are obtained before treatment and 60 after treatment.

Preparation of the DNA (size between 100 and 300 kb).

 Blunt-ended DNA.

 The DNA is blunt ended by removing the outgoing 3 'sequences and filling the outgoing 5' sequences. This operation is done with: Klenow enzyme, T4 polymerase, the 4 nucleotides triphosphates.

Preparation of the extremities.Example Hindlll
The addition of the DNA to the vector is by means of oligonucleotides recognizing the modified HindIII sequence of the vector. They contain rare restriction sites to allow subsequent cloning (Swal, Notl). this technique is derived from that of: Elledge SJ,
Mulligan JT, SW Ramer, Spottswood M, Davis RW. Proc Natl Acad
Sci USA 1991 Mar 1; 88 (5): 1731-5
Two complementary oligonucleotides are used:
Oligo 1: 5'-GCTTATTTAAATATTAATGCGGCCGCCCGGG-3 '(SEQ ID N 25)
Oligo 2: 5'-CCCGGGCGGCCGCATTAATATTTAAATA-3 '(SEQ ID
N 26)
They are phosphorylated in 5 'by the T4 polynucleotide kinase in the presence of ATP, after their hybridization. This phosphorylation step can be eliminated using the already phosphorylated oligonucleotides.

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 The ligation of this double-stranded adapter with the DNA to be inserted into a vector is made by T4 ligase in the presence of a very large excess of adapter (1000 adapter molecules for a DNA molecule to be inserted), in 15 hours at 14 C.

 The excess adapter is removed by electrophoresis on an agarose gel and the molecules of interest are recovered from the gel by hydrolysis thereof with agarase or electroelution.

 Vector-DNA Ligation.

 Ligation is at 14 C over 15 hours with 10 carrier molecules for one insert molecule.

 Transformation.

 The recipient strain is strain DH10B. The transformation is done by electroporation. To express the resistance to tetracycline, the transformants are incubated at 37 ° C. for 1 hour in an antibiotic-free medium. The clones are selected by culturing overnight on LB agar medium supplemented with tetracycline at 5 g.ml-1.

Example 11 CLONE CLONE CONJUGATION BETWEEN E. COLI AND STREPTOMYCES CONJUGATION BETWEEN E STAINLESS STEEL S17.1 CONTAINING PPM803 AND STREPTOMYCES LIVIDANS TK 21 Introduction It is possible to carry out conjugations between E. coli and Streptomyces (Mazodier et al, 1989) . Adaptation of this method by developing a so-called droplet technique in which 10 l of a culture of E. coli containing a recombinant vector is mixed with a drop of S. lividans receptor consists in carrying out a cloned clone transformation into ensuring that at the end of the operation the entire bank built in E. coli is introduced into S. lividans. A bulk transformation would necessarily lead to a multiplication of clones of Streptomyces transformants in order to be

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 practically sure that the library in E. coli is completely represented in S. lividans.

Moreover this method is easily automatable.

Preliminary tests Conjugation between E. coli strain S17. 1 containing the vector pOSV303 and S. lividans TK21.

Under these conditions, 6 × 10 6 cells of E. coli are mixed with 2 × 10 6 pre-germinated S. lividans spores in a final volume of 20 l.

Development of the method It is known that the DNA extracted from certain actinomycetes is modified and therefore can not be introduced into certain strains of E. coli without being restricted. The strain of E. coli DH10B that accepts these DNAs is not capable of transferring to Streptomyces a plasmid containing only oriT, and it is therefore necessary to construct one. It should be introduced by integration into the chromosome a derivative of RP4 capable of providing in trans all the functions necessary to ensure the transfer of recombinant clones containing the oriT transfer origin.

Example 12 Construction of a cosmid library in E. coli and Streptomyces lividans: Cloning of soil DNA
The objective is the construction of a large library of DNA from the environment, without prior step of culture.of microorganisms, in order to access the metabolic genes of bacteria (or any other organism) that it is not known to grow under standard laboratory conditions.

 The described procedure was used to generate a DNA library in Escherichia coli using E. coli-S shuttle cosmid. lividans pOS7001 and DNA extracted and purified from the bacterial fraction of a soil. This last method makes it possible to obtain DNA of high purity and

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 an average size of 40 kb. Also, in order to avoid for cloning a partial digestion of the extracted DNA, has been adopted an alternative strategy based on the use of the terminal tranferase enzyme which makes it possible to add tails of polynucleotides to the 3 'ends of the DNA and the vector.

 5 g of DNA were extracted from 60 mg of "Côte Saint André" soil according to the protocol described in Example 3 and treated with terminal transferase (Pharmacia) to lengthen the 3 'ends with a monotonous polynucleotide ( poly T) (Example 10).

 The integrative cosmid pOS7001 is prepared according to the protocol B1, Orsay. After a conventional phenol / chloroform purification step, the DNA and vector are assembled by mixing a vector molecule and an inserted DNA molecule. The mixture is then packaged in lambda bacteriophage heads (Amersham kit) which serve to transfect E. coli DH10B. The transfected cells are then seeded on LB agar medium in the presence of ampicillin for selection of recombinants resistant to this antibiotic.

 A library of about 5000 E clones. Each clone was seeded in LB or TB + ampicillin medium in a microplate well (96 wells) and stored at -80 ° C.

The sequence at the site of insertions of the soil fragments in the vector, pOS7001, generated during the construction of the library was analyzed. For this purpose, 17 cosmids of the library were purified and sequenced with a primer, seq. 5 'CCGCGAATTCTCATGTTTGACCG 3', which hybridizes between the BamHI site and the HindIII cloning site present in the vector.

The sequences obtained made it possible to estimate that the length of the homopolymeric tails at the junction points is very variable, between 13 and 60 poly-dA / dT. Beyond the tails, the sequences of the soil fragments thus generated have a percentage in G + C between 53 and 70%. Such high percentages were unexpected, but results

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 similar have already been reported on crude preparation of DNA from soil (Chatzinotas A. et al., 1998).

 A pooling strategy of 48 or 96 clones was used for the analysis of microbial and metabolic richness. The cosmid DNA extracted from these "pools" of clones was then used to perform PCR or hybridization experiments.

Example 13: Diversity of 16S ribosomal DNA within the cloned DNA. a) Materials and methods
The cosmids of the library are extracted from pools of clones by alkaline lysis and are then purified on cesium chloride gradient, in order to collect the cosmid DNA band in supercoiled form and in order to eliminate any chromosomal DNA from Escherichia coli may interfere in the study.

 After linearization of cosmids by action of S1 nuclease (50 units, 30 minutes at 37 ° C.), the 16S rDNA sequences contained in the pools of clones are amplified under the standard amplification conditions, starting from the universal primers 63f ( 5'CAGGCCTAACACATGCAAGTC-3 ') and 1387r (5'GGGCGGWGTGTACAAGGC-3') defined by MARCHESI et al (1998). The amplification products of about 1.5 kilobases are purified from the Qiaquik gel extraction kit (Qiagen) and then directly cloned into the vector pCR II (Invitrogen) in Escherichia coli TOP10, according to the manufacturer's instructions. The insert is then amplified using the M13 Forward and M13 reverse primers specific to the pCR II vector cloning site. Amplification products of expected size (about 1.7 kb) are analyzed by RFLP (Restriction Fragment Length Polymorphism) using the enzymes Cfol, Mspl and BstUI (0.1 units) to select the

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 clones to sequence. The restriction profiles obtained are separated on agarose gel Metaphore 2.5% (FMC Products) containing 0.4 mg of ethidium bromide among.

The 16S rDNA sequences are then determined directly using the PCR products purified by the kit "Qiaquick gel extraction" using the sequencing primers defined by Normand (1995). Phylogenetic analyzes are obtained by comparing the sequences with the prokaryotic 16S rDNA sequences collected in the Ribosomal Database Project (RDP) database, version 7.0 MAIDAK et al (1999) using the SIMILARITY MATCH program, allowing obtain the similarity values with respect to the sequences of the database. b) Results
To determine the phylogenetic diversity represented in the library, 47 sequences of the 16S rRNA gene were isolated from pools of 288 clones and were almost completely sequenced. The results are reported in Table 7.

 Analysis of the sequences by interrogation of the databases reveals that the majority of the sequences (> 61%) have percentages of similarity that are less than or equal to 95% with identified bacterial species (Table 7). Of the 47 sequences analyzed, 28 sequences have, for closer neighbors, non-cultured bacteria whose sequences have been derived directly from DNA extracted from the environment. The majority of these sequences also have very low percentages of similarity (88-95%), so 17 out of 28 sequences differ by more than 5% from their nearest neighbors.

 Of the sequences that can be classified in a phyletic group, a majority of sequences belong to the subclass a proteobacteria (18 sequences with a percentage of similarity

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 between 89 and 99%). A second group of sequences is represented by the subclass g of proteobacteria, comprising 9 sequences whose similarity percentages vary between 84 and 99%). The groups of b-proteobacteria, d-proteobacteria, firmicutes at low G + C% and at high G + C% respectively comprise 1.4, 3 and 5 sequences. Only one sequence could not be classified within the large defined bacterial taxonomic groups: the sequence a22.1 (19), its nearest neighbor Aerothermobacter marianas (with a similarity of 89%) being itself an isolated strain of the Currently, 6 sequences can be classified in the group Acidobacteriuml Holophaga. This group has the particularity to be represented by only two bacteria cultivated Acidobacterium capsulatum and Holophaga foetida, the whole of this group being composed of bacteria of which only the rRNA16S gene was detected by amplification and cloning from extracted DNA. sample of the environment (mainly soil), Ludwig et al (1997). The low similarity values between the different sequences in this group suggest a great heterogeneity and diversity within this group.

* The overall results are shown in Table 7.

 These results show that the sequences contained in the cosmid bank come from microorganisms not only phylogenetically diversified but especially microorganisms that have never been isolated to date.

 The results of the sequencing of the amplified DNAs made it possible to establish a phylogenetic tree of the organisms present in the soil sample from which the characterized sequences originate.

 The phylogenetic tree shown in Figure 7 was made from the sequence alignment by the MASE software (Faulner and Jurak, 1988) and corrected by the Kimura 2 parameter method (1980), and

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 using the Neighbor Joining algorithm (Saitou and Nei 1987). Phylogenetic analysis compared the 16S rDNA sequences cloned in the soil DNA library with the prokaryotic 16S rDNA sequences collected in the Ribosomal Database Project (RDP) databases (version 7. 0, program SIMILARITY-MATCH, Maidak et al 1999), and in the GenBank database thanks to the BLAST 2.0 software (Atschul et al, 1997).

Example 14: Genetic preselection of the bank for the evaluation of metabolic richness
To characterize the bank obtained in terms of metabolic diversity and to identify clones containing inserts carrying genes that may be involved in biosynthetic pathways, PCR-based genetic screening techniques have been developed according to the invention to detect and identify PKS type I genes.

1 Bacterial strains, plasmids and culture conditions
S. coelicolor ATCC101478, S. ambofaciens NRRL2420, S. lactamandurans ATCC27382, S. rimosus ATCC109610, B. Subtilis ATCC6633 and B. licheniformis THE1856 (RPR collection) were used as sources of DNA for PCR experiments. S. lividans TK24 is the host strain used for the cosmic POS1700 shuttle.

 For the preparation of genomic DNA, spore suspensions, protoplasts and for the transformation of S. lividans, the standard protocols described in Hopwood et al (1986) were followed.

Escherichia coli Top10 (INVITROGEN) was used as a host for cloning PCR products and E. coli Sure (STRATAGENE) was used as a host for the cosmos shuttle pOS7001. Culture conditions

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 E. coli, plasmid preparation, DNA digestion, agarose gel electrophoresis were performed according to standard procedures (Sambroock et al., 1996).

2. PCR primers:
The pairs of primers a1-a2 and b1-b2 were defined by the team of N. Bamas-Jacques and their use was optimized for the screening of the DNA of the pure strains and the soil bank for research. of genes encoding PKSI)
Table 8: PCR primers homologous to the PKSI genes used for the screening of the library.

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<Tb>
<tb> a1 <SEP> (+) <SEP> 5 '<SEP> CCSCAGSAGCGCSTSTTSCTSGA <SEP>3'
<tb> a2 <SEP> (-) <SEP> 5 '<SEP> GTSCCSGTSCCGTGSGTSTCSA <SEP>3'
<tb> b1 <SEP> 5 '<SEP> CCSCAGSAGCGCSTSCTSCTSGA <SEP>3'
<tb> b2 <SEP> 5 '<SEP> GTSCCSGTSCCGTGSGCCTCSA <SEP>3'
<Tb>
Amplification conditions:
In the search for PKS I from DNA of pure strains, the amplification mixture contained: in a final volume of 50 l, between 50 and 150 ng of genomic DNA, 200 M of dNTP, 5 mM of final MgCl 2 , 7% DMSO, 1x Appligene buffer, 0.4 M each primer and 2.5 U of Appligene Taq Polymerase. The amplification conditions used are: denaturation at 95 ° C. for 2 minutes, hybridization at 65 ° C. for 1 minute, elongation at 72 ° C. for 1 minute, for the first cycle, followed by 30 cycles where the temperature is decreased to 58 ° C. C as described in K. Seow et al., 1997. The final extension step is at 72 ° C for 10 minutes.

 For the search for PKS I from the library DNA, the PCR conditions are the same as above for the α1-α2 pair using between 100 and 500 ng of cosmid extracted from pools of 48 clones.

For the primer pair b1-b2, 500 ng of cosmids from pools of 96 clones were used. The amplification mixture contained 200 M dNTPs, 2.5 mM final MgCl 2, 7% DMSO, 1x Quiagen buffer, 0.4 M each primer and 2.5 U Taq polymerase Hot-start (Qiagen). The amplification conditions used are: denaturation 15 'to 95 C followed

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 by 30 cycles: 1 'of denaturation at 95 ° C + 1' of hybridization at 65 ° C. for the first cycle and 62 ° C. for the other cycles, elongation at 72 ° C., final extension step of 10 ° to 72 C.

 Identification of positive clones from pools of 48 or 96 clones is performed from replicates of the corresponding parent microplates on solid media or any other standard replication method.

Subcloning and Sequencing The PCR products of the identified clones were sequenced according to the following protocol: The fragments are purified on agarose gel (Gel Extraction Kit (Qiagen)) and cloned in E.coli TOP 10 (Invitrogen) using the TOPO TA kit cloning kit (Invitrogen). The plasmid DNA of subclones is extracted by alkaline lysis on a Biorobot (Qiagen) and dialyzed for 2 h on VS 0.025pm membrane (Millipore). The samples are sequenced with primers M13 "Universal" and "Reverse" on the sequencer ABI 377 96 (PERKIN ELMER).

4) Results Definition and validation of PCR primers
Two highly conserved regions of actinomycete type 1 PKS, including the active site of the enzyme, have been targeted for amplification of homologous genes with degenerate primers. These two regions correspond to the sequences PQQR (L) (L) VE (A) HGTGT respectively.

 Primers (Table 8) were tested with the DNA of strains producing or not producing macrolides: Streptomyces coelicolor,

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 Streptomyces ambofaciens, producer of spiramycin, and Saccharopolyspora erythraea, producer of erythromycin. Whatever the primers used, bands representing fragments of about 700 bp and corresponding to the length of the expected fragment, were obtained with all the strains.

 These results demonstrate the specificity of primers a and b for the PKS 1 genes of producer strains or silent genes in S. coelicolor.

 Sequencing of the PCR products obtained with the pair of primers a1-a2 made it possible to identify, from the S. ambofaciens strain, the sequence of a KS gene already described (European Patent Application No. EP0791656) as belonging to the biosynthetic pathway of plantenolide, macrolidic precursor of spiramycin, and two sequences never described, Stramb 9 and Stramb12, (see sequence listing).

 With regard to S. erythraea, the screening method allowed the identification of a sequence of KS (sacery17) identical to that of the KS of module 1 already published in Genebank (access number M63677), coding for the synthetase 1 (DEBS1) of 6-deoxyerythronolide B. Another sequence not correlated to the erythromycin biosynthetic pathway has been identified and it is the sequence SEQ ID N 32.

Conclusion
A method for analyzing the presence of genes encoding PCR-type PKSs from different microorganisms has been developed. The highly conserved structure of the keto-synthetase domain of type # has made it possible to carry out a PCR method based on the use of degenerate GC-biased primers for the choice of codons.

 This approach shows the possibility of identifying genes or clusters involved in the type I polyketide biosynthetic pathway.

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Cloning these genes allows the creation of a collection that can then be used to build polyketide hybrids. The same principle can be applied to other classes of antibiotics.

 The results obtained here also show the presence of genes that can belong to silent clusters (SEQ ID N 30 to 32).

 The presence of silent clusters has already been documented in S. lividans and their expressions are triggered by specific or pleiotropic regulators (Horinouchi et al., Umeyama et al., 1996).

These results suggest that the detection of genes belonging to so-called silent pathways actually encode active enzymes capable of directing, in association with the other pathway-specific enzymes, the enzymatic steps necessary for the synthesis of secondary metabolites.

Screening of the bank
The screening was carried out under the conditions described in the Materials and Methods section using the primer pairs validated from producing strains.

 In the presence of the pair of primers a1-a2, the size of the PCR products obtained from the cosmid DNA extracted from pools of 48 or 96 clones was about 700 bp, thus in agreement with the expected results.

 The intensity of the bands obtained was variable, but only one amplification band was present for each target DNA pool.

 Under these conditions, 8 groups of target DNA were detected, corresponding to 9 positive clones after dereplication.

Screening performed with the second pair of primers, b1-b2, gave less specific amplification results since many satellite bands were observed next to the 700 bp band. Nevertheless, 9 groups of target DNA were detected, corresponding to 14 positive clones after dereplication from these positive clones, the DNA

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 was extracted for the sequencing and transformation steps of .S. lividans.

Cosmid analysis
Digestion of the cosmids identified by PCR with the Dral enzyme, recognizing a site rich in AT, releases a fragment greater than 23 kb (FIG. 22). This suggests that the PCR method preferentially targets soil DNA containing a high percentage of G + C. This result is the consequence of the degeneracy of the primers used, biased in GC for the choice of codons. The inserts, as expected in the case of cosmids, have a size greater than 23 kb, except in one case (a9B12 clone), which could reflect a certain instability of the cosmids.

On the other hand, of all the clones selected, only two of them, GS.F1 and GS.G11, showed the same restriction profile indicating a low redundancy rate in the bank.

 The selected cosmids were transferred to Streptomyces lividans by transformation of protoplasts in the presence of PEG 1000.

The transformation efficiency varies between 30 and 1000 transformants per g of cosmid DNA used.

Sequencing and Phylogenetic Analysis of PKS 1 Soil Genes
The PCR method developed on the pure strains was used as described on the cosmids of the library and 24 clones were thus identified.

PCR products of approximately 700 bp obtained from the DNA of two pools (48 clones) and 8 unique clones were cloned, after purification on agarose gel, and sequenced. This allowed the identification of 11 sequences.

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 The alignment of the PKSs 1 deduced protein sequences of the soil with other PKSs # present in different microorganisms (FIG. 24) shows the presence of a highly conserved region which corresponds to the consensus region of the active site of b-ketoacyl. synthetase.

 Codonpreference sequence analysis (Gribskov et al., 1984, Bibb et al., 1984) revealed a strong bias in the use of G + C rich codons in a single reading phase. The proteins deduced according to this reading phase show a strong similarity with known type 1 KSs (Blast program). In particular, the similarity between the soil KSs and KSs sequences of the erythromycin cluster is about 53%.

 After dereplication of a pool and identification of the single clone, the sequence of the PCR product obtained from this clone is identical to that of the pool which confirms the reliability of the method used.

 The analysis of the sequence of the PCR product of a clone allowed the probable identification of 3 different KSI genes. One of these sequences (SEQ ID N 34) has a similarity of 98.7% with the sequence of another pool, suggesting that they encode the same enzyme. The other two sequences are different but strongly homologous.

 Here, it is described for the first time the cloning and identification in a soil DNA library of biosynthetic pathways of secondary metabolites containing genes encoding type I KS.

 The high G + C percentage of soil sequences suggests that they may be derived from genomes with codon usage similar to those of actinomycetes.

 Although the data available in the literature are reduced, it is known that genes encoding type I PKS are very diverse in their physical organization in the genome, the size and the number of modules contained in each gene.

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 The presence of several domains from a single clone is a confirmation of their membership in asymmetric polyketide clusters. In one case, two clones seem to form a contigue since they share the same sequence for the KS domain.

 The size of the genetic regions involved in PKSI synthesis varies from a few kb for penicillin to about 120 kb for rapamycin. The size of the cosmid inserts may therefore not be sufficient for the expression of the most complex clusters.

 Genes coding for PKSs I, able to iteratively work as PKS II and to control the synthesis of aromatic polyketides, have been described (Jae-Hyuk et al., 1995). The study of soil PKS 1 clusters could bring new innovations in this field.

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<tb> Number <SEP> Origin <SEP> Texture <SEP> Quantity <SEP> (%) <SEP> of <SEP> Material <SEP> pH <SEP> Number <SEP> of <SEP> Number <SEP> of
<tb> sand <SEP> Limon <SEP> Clay <SEP> organic <SEP> cells <SEP> before <SEP> cells <SEP> after
<tb> (g / kg <SEP> of <SEP> soil <SEP> grinding <SEP> grinding '
<tb> sec) <SEP> a (x109 / g <SEP> weight <SEP> (x109 / g <SEP> weight
<Tb>

~~~~~ ~~~~~~~~~~~ ~~~~~~~~~~~ ~~~~~~~~~~~~~ ~~~~~~~~ sol sec sol sec

~~~~~ ~~~~~~~~~~~ ~~~~~~~~~~~ ~~~~~~~~~~~~~ ~~~~~~~~ dry soil dry soil

(1) ation standard

.0 '(1) "C n=3 ; m <tb> 1 <SEP> Australia <SEP> Clay <SEP> sandy <SEP> 62 <SEP> 22 <SEP> 16 <SEP> 49.7 <SEP> 5.8 <SEP> 6.5 <SEP> ( 0.9) <SEP> 2.9 <SEP> (1.3)
<tb> 2 <SEP> Peyrat <SEP> the <SEP> Châ- <SEP> Clay <SEP> sandy <SEP> 61 <SEP> 26 <SEP> 13 <SEP> 48.2 <SEP> 4.9 <SEP> 7.3 <SEP> (0.6)
<tb> 5.4 (0.8) water, France
<tb> 3 <SEP> Coast <SEP> St-André, <SEP> Soil <SEP> sandy <SEP> 50 <SEP> 41 <SEP> 9 <SEP> 40.6 <SE> 5.6 <SEP> 10.0 <SEP> (0.7)
<tb> France
<tb> 4 <SEP> Chazay <SEP> of Azergue, <SEP> Soil <SEP> sandy <SEP> 34 <SEP> 47 <SEP> 19 <SEP> 13.9 <SEP> 5.8 <SEP> 7.8 (1.1) <SEP> 4.2 <SEP> (0.6)
<tb> France <SEP> clay
<tb> 5 <SEP> Guadeloupe, France <SEP> Clay <SEP> 27 <SEP> 26 <SEP> 47 <SEP> 17.0 <SEP> 4,8 <SEP> 1,4 <SEP> (0, 4) <SEP> 0.5 <SEP> (0.1)
<tb> 6 <SEP> Dombes, France <SEP> Soil <SEP> sandy <SEP> 20 <SEP> 67 <SEP> 13 <SEP> 30.3 <SEP> 4.3 <SEP> 7.5 (0 , 5) <SEP> 5.6 (0.9)
<tb> Argileux
<Tb>
<Tb>
re parentheses.

(1) standard item

.0 '(1) "C n = 3; m

<Desc / Clms Page number 121>

!E li a c

- 'C'IS #<s U 2 pour sur

10 ABLI isées datio

= -0 0 m 0 ZT3 so

-< Amorces

PCR and

! E li ac

- 'C'IS # <s U 2 for on

10 ABLI dations datio

= -0 0 m 0 ZT3 so

- <Amorces

1 i fi) M @s, les ont pa on pu

0 .52 m ra c-l

-c" eses. s res st dé

-0) aren nist

en ; es en é <U

ë- "5 #~ -CD U M l'ARNr 16S de E ces chromosomi cassette conten

cr -CD (0 .(D c- 11 Q) 1 1 -I J U (6 <tb> Primer <SEP> or <SEP> probe <SEP> Target <SEP> a) <SEP> Sequence <SEP> (5 '<SEP> to <SEP>3')<SEP> Reference <SEP> n
<tb> FGPS431 <SEP> probe <SEP> Universal <SEP> (1392-1406) <SEP> ACGGGCGGTGTGT (A / G) <SEP> Amann <SEP> and <SEP> al., <SEP> 1995 <SEP >
<tb> FGPS122 <SEP> primer <SEP> Bacteria <SEP> (6-27) <SEP> GGAGAGTTTGATCATGGCTCAG <SEP> Amann <SEP> and <SEP> al <SEP>, <SEP> 1995
<tb> FGPS350 <SEP> Primer <SEP> Streptosporangium <SEP> (616-635) <SEP> CCTGGAGTTAAGCCCCCAAGC <SEP> This <SEP> Study
<tb> FGPS643 <SEP> Probe <SEP> Streptosporangium <SEP> (122-142) <SEP> GTGAGTAACCTGCCCC <SEP> (T / C) GACT <SEP> Study
<tb> R499 <SEP> primer <SEP> Bacillus <SEP> anthracis <SEP> TTAATTCACTTGCAACTGATGGG <SEP> Patra <SEP> and <SEP> al., <SEP> 1996
<tb> R500 <SEP> primer <SEP> Bacillus <SEP> anthracis <SEP> AACGATAGCTCCTACATTTGGAG <SEP> Patra <SEP> and <SEP> al., <SEP> 1996
<tb> C501 <SEP> probe <SEP> Bacillus <SEP> anthracis <SEP> TTGCTGATACGGTATAGAACCTGGC <SEP> Patra <SEP> and <SEP> al., <SEP> 1996
<tb> FGPS516 <SEP> primer <SEP> S. <SEP> lividans <SEP> OS48.3 <SEP> TCCAGATCCTTGACCCGCAG <SEP> This <SEP> study
<tb> FGPS517 <SEP> primer <SEP> S <SEP> lividans <SEP> OS48.3 <SEP> CACGACATTGCACTCCACCG <SEP> This <SEP> study
<tb> FGPS518 <SEP> probe <SEP> S. <SEP> lividans <SEP> Os48.3 <SEP> CCGTGAGCCGGATCAG <SEP> This <SEP> study
<Tb>
norces and ocalised).

1 i) M @ s, have not been able to

0 .52 m ra cl

-c "eses.

-0) aren nist

in ; are in <U

ë- "5 # ~ -CD UM 16S rRNA these chromosomi cassette contained

cr -CD (0. (Dc- 11 Q) 1 1 -IJU (6

<Desc / Clms Page number 122>

(0 '0 emen iation 5b

#> i -0 -f-I -0 ,d) u> ,j " h 0 -0 . 0 15 -≈ ! 1 - II ; n : e n@

#! -0 < Î3 | H (5 " N n - -ro ' x 'o O Bzz <1 Cq .0 CL P V) #4-> O ns </) o (A =' i ! o ! D5 #*#

.0) - E N

(0 0 0ation 5b

## EQU1 ## where: ## STR2 ##

#! -0 <Î3 | H (5 "N n - -ro 'x' o O Bzz <1 Cq .0 CL PV) # 4-> O ns </) o (A = 'i! O! D5 # * #

.0) - EN

.Q"" iver un +

0 1);

x + r tâche avec tion Ultratura (CH); 5a: Cr

CI) idation @ogéné Cup Ho

E 0 ..0 oc -C 0 en '7 L- U C3 c en v. O Q) C'0 en cenc I (G); + H+

C i D..cu::2: 0 Q) 0 0).- fication par imagerie de
2). <tb> 1. <SEP> Australia <SEP> 17 +/- 2 <SEP> 52 +/- <SEP> 2 <SEP> 32 +/- 5 <SEP> 16 +/- 3 <SEP> 33 + / - <SEP> 2 <SEP> 59 +/- 1 <SEP> 27 +/- 0
<tb> 2. <SEP> Peyrat <SEP> 29 +/- 2 <SEP> 58 +/- 1 <SEP> 40 +/- 2 <SEP> 29 +/- 2 <SEP> 18 + / 3 <SEP > 56 +/- 1 <SEP> 15 +/- 1
<tb> 3.Côte <SEP> St-André <SEP> 36 +/- 7 <SEP> 60 +/- 6 <SEP> 148 +/- 10 <SEP> 94 +/- 7 <SEP> 38 + / -6 <SEP> 73 +/- 5 <SEP> 47 +/- 6
<tb> 4. <SEP> Chazay <SEP> 9 <SEP> 16 <SEP> NS <SEP> 32 <SEP> 15 <SEP> 15 <SEP> 70
<tb> 6. <SEP> Dombes <SEP> 4 +/- <SEP> 2 <SEP> 26 +/- 3 <SEP> 43 +/- 1 <SEP> 61 +/- <SEP> 66 +/- 1 <SEP> 160 +/- 7 <SEP> 102 +/- <SEP> 5 <SEP>
<Tb>
GPS43 that /

.Q "" iver a +

0 1);

x + r task with Ultratura (CH); 5a: Cr

CI) idation @ ogen Cup Ho

## STR5 ## in which OQ) C'0 in cenc I (G); + H +

C i D..cu :: 2: 0 Q) 0 0) .- imaging by
2).

:J:J cuZ C CD " + p a Qua@ (table b1: Au 4a: G c ND : In treatment; 2: grinding H + Microtiptic sonication. See also figure Not determined.

: J: J cuZ C CD "+ pa Qua @ (table b1: At 4a: G c ND:

<Desc / Clms Page number 123>

I 0 (J 0 3 s la raits

C A-J qe m 0 bleau lisées s ADN

-0 Amorces et sondes moléculair

tion

I 0 (J 0 3 s the raits

C AJ q e m a lle s s DNA

-0 Molecular primers and probes

(1) -c RNr16S d'Esc

a) c: gè

JU t- :3 a : position s <tb> Target <SEP> Sequence <SEP> (5 '<SEP> - <SEP>3')<SEP> Position <SEP> a
<tb> (primer <SEP> or <SEP> probe)
<tb> FGPS <SEP> 612 <SEP> Eubacteria <SEP> (primer) <SEP> C (C / T) AACT (T / C / A) CGTGCCAGCAGCC <SEP> 506 <SEP> - <SEP> 525 <SEP >
<tb> FGPS <SEP> 669 <SEP> Eubacteria <SEP> (primer) <SEP> GACGTC (A / G) TCCCC (A / C) CCTTCCTC <SEQ> 1174 <SEP> - <SEP> 1194 <SEP>
<tb> FGPS <SEP> 618 <SEP> Eubacteria <SEP> (probe) <SEP> ATGG (T / C) TGTCGTCAGCTCG <SEP> 1056 <SEP> - <SEP> 1073 <SEP>
<tb> FGPS <SEP> 614 <SEP> α-Proteobacteria <SEP> (probe) <SEP> GTGTAGAGGTGAAATTCGTAG <SEP> 683 <SEP> - <SEP> 703 <SEP>
<tb> FGPS <SEP> 615 <SEP> b-Proteobacteria <SEP> (probe) <SEP> CGGTGGATGATGTGGATT <SEP> 939 <SEP> - <SEP> 956 <SEP>
<tb> FGPS <SEP> 616 <SEP> g-Proteobacteria <SEP> (probe) <SEP> AGGTTAAAACTCAAATGA <SEP> 900 <SEP> - <SEP> 917 <SEP>
<tb> FGPS <SEP> 621 <SEP> Gram <SEP> plus <SEP> to <SEP> down <SEP> GC% <SEP> (probe) <SEP> ATACGTAGGTGGCAAGCG <SEP> 532 <SEP> - <SEP> 549
<tb> FGPS <SEP> 617 <SEP> Actinomycetes <SEP> (probe) <SEP> GCCGGGGTCAACTCGGAGG <SEP> 1159 <SEP> - <SEP> 1149
<tb> FGPS <SEP> 680 <SEP> Streptomycetes <SEP> (probe) <SEP> TGAGTCCCCA (A / C / T) C (T / A) CCCCG <SEP> 1132 <SEP> - <SEP> 1149 <SEP >
<tb> FGPS <SEP> 619 <SEP> Streptosporangium <SEP> (probe) <SEP> GCTTGGGGCTTAACTCCAGG <SEP> 609 <SEP> - <SEP> 628 <SEP>
<Tb>
collar

(1) -c RNr16S of Esc

a) c: gè

JU t-: 3 a: position s

<Desc / Clms Page number 124>

'a d Mi-.Q) -<D 3 +> 111 2 +* N ci ent de N ne solut ation su xtrait

= 0) N M M 2: -IIS S 5 g S 3 1*5-=: bactérien chantillon persion et ries extraite

=5 t! 0 **#> C 3 lif CI) s o

(0 Effi

Extra DNA%,

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= 0) NMM 2: -IS S 5 g S 3 1 * 5- =: bacterial sample persion and extracted rays

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z (6 U) #<.2 U N N ô -U E m >(D ombrer DS 0,0@ quantité quantifi@

(D G) m m o # CD ' ' a e <tb> Microflora <SEP> total <SEP> Microflora <SEP> cultivable <SEP> Actinomycetes <SEP> Lyse <SEP> direct <SEP> Lyse <SEP> in <SEP> block
<tb> bacteria / g <SEP> soil <SEP> dry <SEP> cfu <SEP> / g <SEP> soil <SEP> sec <SEP> cultivable <SEP> agarose <SEP> d, e <SEP>
<tb> cfu / g <SEP> soil <SEP> dry <SEP> ng <SEP> DNA / <SEP> g <SEP> soil <SEP> ng <SEP> DNA / <SEP> g <SEP> soil
<tb> dry <SEP> sec
<tb> Without <SEP> incubation
<tb> Suspension <SEP> of <SEP> sol <SEP> 1 <SEP> 3 <SEP> 109 <SEP> (~ <SEP> 0 <SEP> 1) <SEP> 6.9 <SEP> 106 <SEP> ( ~ <SEP> 0 <SEP> 2) <SEP> 8 <SEP> 6 <SEP> 106 <SEP> (~ <SEP> 1 <SEP> 2)
<tb> Extract <SEP> Cellular <SEP> 1.9 <SEP> 108 <SEP> (~ <SEP> 0. <SEP> 2) <SEP> 4.1 <SEP> 106 <SEP> (~ <SEP> 1.5) <SEP> 2.5 <SEP> 106 <SEP> (~ <SEP> 0 <SEP> 7) <SEP> 333 <SEP> (~ <SEP> 35) <SEP> 221 <SEP> (~ <SEP> 70)
<tb> Extraction Efficiency <SEP>
<tb> 15% <SEP> 59% <SEP> 38%
<tb> With <SEP> incubation <SEP> in <SEP> extract <SEP> of <SEP> yeast <SEP> 6%
<tb> Suspension <SEP> of <SEP> sol <SEP> 1 <SEP> 2 <SEP> 109 (<SEP> 0. <SEP> 1) <SEP> 7.6 <SEP> 107 (~ <SEP> 1.1) <SEP> 6.6 <SEP> 107 (~ 0.4)
<tb> Cellular Extract <SEP>
<tb> 1 <SEP> 6 <SEP> 108 (<SEP> 0 <SEP> 3) <SEP> 5.3 <SEP> 106 (~ <SEP> 14) <SEP> 3. <SEP> 7 <SEP> 106 (~ <SEP> 0.7) <SEP> 344 <SEP> (~ <SEP> 30) <SEP> 341 <SEP> (~ <SEP> 67)
<tb> Effectiveness <SEP> of extraction <SEP> 13% <SEP> 7% <SEP> 5%
<Tb>
ure e

> CD "5 U
Z uti

S a II 1 I - (0-CD rra ion

a 1 (0 a5 w (C e - # CL -JE U o. U U7 (0, (D oloratio ase-Soj gar, after te w ge

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z (6 U) # <. 2 UNN ô -UE m> (D shader DS 0,0 @ quantified quantity @

(DG) mmo # CD '' ae

<Desc / Clms Page number 125>

03 "C nal d'hyb

O) , et g nomycètes

" < Se ur proportion bas GC% et
00%.

"C 0 #c E <a <DO <D .9 2 .> 3-505 Me .0.....0)"" J--Q)(/)- .::; 0 ,ft O- -0) 1! des es P prod

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with the

03 "C nal hyb

O), and g nomycetes

"In proportion to low GC% and
00%.

"C 0 #c E <a <DO <D .9 2 .> 3-505 Me .0 ..... 0)""J - Q) (/) -. ::; 0, ft O- -0) 1! Es es prod

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ns

JE vs m LU m 0) >- <tb> a- <SEP> b- <SEP> g- <SEP> Gram +
<tb> Proteobacterium <SEP> Proteobacterium <SEP> Proteobacterium <SEP> Low <SEP> GC% <SEP> Actinomycetes <SEP> Streptomycetes
<tb> s <SEP> s <SEP> s
<tb> Direct <SEP> Extraction <SEP> a <SEP> 7.7 <SEP>% <SEP> (1.4) <SEP> 5.3 <SEP>% <SEP>(<SEP> 0.5) <SEP> 3.3 <SEP> % <SEP>(<SEP> 0.9) <SEP> 3.1 <SEP>% <SEP> (~ <SEP> 1.7) <SEP> 14.7 <SEP>% <SEP> (0.6) <SEP> 0.8 <SEP>% <SEP> (0.1)
<tb> Extraction <SEP> indirect
<tb> Lysis <SEP> + <SEP> CsCI <SEP> 10.9% <SEP> (1.4) <SEP> 6.4% <SEP> (1.4) <SEP> 14.3% <SEP> (1.4) <SEP> 7.9% <SEP> (1.4) <SEP> 8.5% <SEP> (1.4) <SEP> 3.0% <SEP> (1.4) <SEP>
<tb> Lysis <SEP> in <SEP> block <SEP> 2.9% <SEP> (1.4) <SEP> 5.4 <SEP>% <SEP> (~ <SEP> 1.4) <SEP> 11.1 <SEP>% ( 1.4) <SEP> 8.0% <SEP> (1.4) <SEP> 11.3% <SEP> (1.4) <SEP> 2.6% <SEP> (1.4) <SEP>
<tb> Lyse <SEP> in <SEP> block <SEP> 6.3% <SEP> (1.4) <SEP> 7.5% <SEP> (1.4) <SEP> 17.0% <SEP> (1.4) <SEP> 18.1 <SEP>% (1.4) <SEP> 19.4% <SEP> (1.4) <SEP> 4.6% <SEP> (1.4) <SEP>
<tb> + <SEP> incubation <SEP> YE
<Tb>
written in Frostega article

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cn gstene, to force c

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<Desc / Clms Page number 126>

tu c s o
0 Tableau 7 : Nr16S contenu
Q

U) quenc

-0 (0 Q) '0 Diver

cosmic bank

you are
0 Table 7: Nr16S content
Q

U) quenc

-0 (0 Q) '0 Diver

<tb> N <SEP> pool <SEP> Neighbor <SEP> identified <SEP>% <SEP> from <SEP> Neighbor <SEP><SEP> plus <SEP> close <SEP>% <SEP> from
<tb> (clone <SEP> n <SEP> the <SEP> more <SEP> close <SEP> similarity <SEP> (classification, <SEP> reference) <SEP> similarity
<tb> a-Proteobacteria
<tb> a24. <SEP> 1 <SEP> (2) <SEP> Azospirillum <SEP> brasilense <SEP> 97.7%
<tb> a4-a6-a7 <SEP> (7) <SEP> Azospirillum <SEP> brasilense <SEP> 95.4%
<tb> a4-a6-a7 <SEP> (23) <SEP> Azospirillum <SEP> brasilense <SEP> 88. <SEP> 9% <SEP> Str <SEP> L-87 <SEP> (a-proteobacterium) '<SEP> 89. <SEP> 8%
<tb> a52-a53-a5 <SEP> (15) <SEP> Azospirillum <SEP> Lipoferum <SEP> 97.6%
<tb> a49-a50-a51 <SEP> (22) <SEP> Agrobacterium <SEP> tumefaciens <SEP> 95.0% <SEP> Clone <SEP> JN15d <SEP> (no <SEP> published) <SEP> 95.5%
<tb> a49-a50-a51 <SEP> (11) <SEP> Rhizobium <SEP> sp <SEP> 99.7%
<tb> a4-a6-a7 <SEP> (14) <SEP> Rhizobium <SEP> sp <SEP> 99.7%
<tb> a30-a31-a32 <SEP> (7) <SEP> Bradyrhizobium <SEP> japonicum <SEP> 99.4%
<tb> a19-a20-a26 <SEP> (5) <SEP> Bradyrhizobium <SEP> genosp <SEP> 93 <SEP> 3% <SEP> Clone <SEP> DA122 <SEP> (no <SEP> published) <SEP> 95.9%
<tb> a37-a38-a39 <SEP> (6) <SEP> Mesorhizobium <SEP> sp. <SEP> 98.9 <SEP>% <SEP>
<tb> a19-a20-a26 <SEP> (9) <SEP> Bradyrhizobium <SEP> sp <SEP> 90.2% <SEP> CloneS-26 (aProteobacterium) 2 <SEP> 95.9%
<tb> a46-a47-a48 <SEP> (14) <SEP> Phyllobacterium <SEP> rubiacearum <SEP> 97.6 <SEP>% <SEP>
<Tb>

<Desc / Clms Page number 127>

l'a *0 e 1)
16S contenues ique

::s Z 0 0 E LEAU 7 ( @ces d'AI @que cos

mI:l: c4 TA Diversité des séque la b

ns

the * 0 e 1)
16S contained

:: s Z 0 0 E LEAU 7 (@ces of AI @que cos

mI: l: c4 TA Diversity of seques

<tb> N <SEP> pool <SEP> Neighbor <SEP> identified <SEP>% <SEP> from <SEP> Neighbor <SEP><SEP> plus <SEP> close <SEP>% <SEP> from
<tb> (clone <SEP> n) <SEP> the <SEP> plus <SEP> close <SEP> similarity <SEP> (classification, <SEP> reference) <SEP> similarity
<tb> a49-a50-a51 <SEP> (1) <SEP> Caulobacter <SEP> henricii <SEP> 97. <SEP> 0%
<tb> a1-a2-a3 <SEP> (13) <SEP> Caulobacter <SEP> sp <SEP> 96.3%
<tb> a52-a53-a5 <SEP> (8) <SEP> Mesorhyzobium <SEP> mediterranean <SEP> 92.1% <SEP> Clone <SEP> DA122 <SEP> (no <SEP> published) <SEP> 94. <SEP> 8%
<tb> a34-a35-a36 <SEP> (3) <SEP> Rhodobium <SEP> orientis <SEP> 91. <SEP> 8% <SEP> Clone <SEP> (no <SEP> published) <SEP> 95.1 %
<tb> a1-a2-a3 <SEP> (4) <SEP> Sphingomonas <SEP> sp. <SEP> 94. <SEP> 7% <SEP> Clone <SEP> PAD23 <SEP> (no <SEP> released) <SEP> 95.1%
<tb> a8-a9-a10 <SEP> (13) <SEP> Sphingomonas <SEP> sp. <SEP> 94.0%
<tb> [gamma] -Proteobacteria
<tb> a40-a41-a42 <SEP> (13) <SEP> Pseudomonas <SEP> sp <SEP> 98. <SEP> 9% <SEP> clone <SEP> G26 (g-Proteobacterium) 3 <SEP> 99 <SEP> 7%
<tb> a15-a16-a17 <SEP> (12) <SEP> Lysobacter <SEP> antibioticus <SEP> 94. <SEP> 4% <SEP> clone <SEP> vadinHA77 (g-Proteo) <SEP> 93. <SEP> 6%
<tb> a15-a16-a17 <SEP> (5) <SEP> Xanthomonas <SEP> sp <SEP> 93. <SEP> 4% <SEP> clone <SEP> vadinHA77 (g-Proteo) 4 <SEP> 94 <SEP> 6%
<tb> a19-a20-a26 <SEP> (13) <SEP> Luteimonas <SEP> mephitis <SEP> 92. <SEP> 9% <SEP> Strain <SEP> rJ15 <SEP> (no <SEP> released) <SEP> 93. <SEP> 5%
<tb> a46-a47-a48 <SEP> (6) <SEP> Methylobacter <SEP> whittenburyi <SEP> 88. <SEP> 3% <SEP> soil <SEP> clone <SEP> S-43 (g-Proteo ) <SEP> 2 <SEP> 88.9 <SEP>% <SEP>
<tb> a11-a12-a13 <SEP> (11) <SEP> Methylobacterwhittenburyi <SEP> 88. <SEP> 3% <SEP> soil <SEP> clone <SEP> S-43 (g-Proteo) <SEP> 88. <SEP> 9%
<Tb>

<Desc / Clms Page number 128>

ra *0 ) contenues de

C-4 CI) = (1) (t) CX I: T3 ;:] c E 0 rn S < es ue

0 M 03 0 (0 <3 z
T s séq la

-0 Diversité

ns

ra * 0) contained in

C-4 CI) = (1) (t) CX I: T3;:] c E 0 rn S <es ue

0 M 03 0 (0 <3 z
T s seq la

-0 Diversity

<tb> N <SEP> pool <SEP> Neighbor <SEP> identified <SEP>% <SEP> from <SEP> Neighbor <SEP><SEP> plus <SEP> close <SEP>% <SEP> from
<tb> (clone <SEP> n <SEP> the <SEP> more <SEP> close <SEP> similarity <SEP> (classification, <SEP> reference) <SEP> similarity
<tb> a34-a35-a36 <SEP> (5) <SEP> Methylococcus <SEP> capsulatus <SEP> 84.9% <SEP> soil <SEP> clone <SEP> S-12 <SEP> (d-Proteo) <SEP> 85 <SEP> 6% <SEP>
<tb> a43-a44-a45 <SEP> (10) <SEP> Legionella <SEP> birminghamensis <SEP> 88.9%
<tb> a8-a9-a10 <SEP> (2) <SEP> Lamprocystis <SEP> roseopersicina <SEP> 87. <SEP> 5% <SEP> Clone <SEP> 2-100C14 <SEP> (no <SEP> published) <SEP> 95.1%
<tb> p-Proteobacteria
<tb> a27-a28-a29 <SEP> (5) <SEP> Rhodocyclus <SEP> tenuis <SEP> 90. <SEP> 2% <SEP> Clone <SEP> OPB37 <SEP> (b-proteo) 5 <SEP> 91%
<tb> 8-Proteobacteria
<tb> a8-a9-a10 <SEP> (18) <SEP> Nannocystis <SEP> exedens <SEP> 92.0%
<tb> a11-a12-a13 <SEP> (5) <SEP> Geobacter <SEP> sulfurreducens <SEP> 91.5%
<tb> a27-a28-a29 <SEP> (8) <SEP> Desulfoacinum <SEP> infernum <SEP> 88. <SEP> 4% <SEP> Clone <SEP> S-31 <SEP> (d-Proteo) <SEP> 89. <SEP> 1 <SEP>%
<tb> a40-a41-a42 <SEP> (6) <SEP> Desulfivibrio <SEP> aminophilus <SEP> 85. <SEP> 3% <SEP> Clone <SEP> S-34 <SEP> (d-Proteo) <SEP> 2 <SEP> 86.2%
<tb> G + <SEP> low <SEP> GC%
<tb> a23.1 <SEP> Kurthia <SEP> zopfii <SEP> 97. <SEP> 3%
<tb> a25.1 <SEP> Kurthia <SEP> zopfii <SEP> 97.2%
<tb> a18.1 <SEP> (22) <SEP> Kurthia <SEP> gipsonii <SEP> 94.4% <SEP> G + <SEP> low <SEP> GC% <SEP> no <SEP> identified <SEP> RS19 <SEP> (no <SEP> released) <SEP> 94.8%
<Tb>

<Desc / Clms Page number 129>

<0 '0 M M
EAU 7 (suite
6S contenue

bzz u.1 ^ IJI Diversité des séquences

s the cosmidiq bank

<0 '0 MM
WATER 7 (continued
6S contained

bzz u.1 ^ IJI Variety of sequences

en s# . <tb> Actinomycetes
<tb> a33. <SEP> 1 <SEP> Cellulomonas <SEP> sp <SEP> 99.5%
<tb> a <SEP> 14.7 <SEP> Streptosporangium <SEP> longisporum <SEP> 99. <SEP> 8%
<tb> a <SEP> 21.7 <SEP> Arthrobater <SEP> polychromogenes <SEP> 99.2% <SEP> no <SEP> identified <SEP> RSW1 <SEP> (no <SEP> released)
<tb> a8-a9-a10 <SEP> (7) <SEP> Arthrobacter <SEP> oxidans <SEP> 983% <SEP> actinomycete <SEP> no <SEP> identified <SEP> RSW1 <SEP> (no <SEP > published) <SEP> 98. <SEP> 5%
<tb> a27-a28-a29 <SEP> (3) <SEP> Arthrobacter <SEP> oxidans <SEP> 98.9% <SEP> actinomycete <SEP> no <SEP> identified <SEP> RSW1 <SEP> (no <SEP > published) <SEP> 99.3%
<tb> Acidobacterium <SEP>?
<tb> a43-a44-a45 <SEP> (4) <SEP> Holophaga <SEP> Foetida <SEP> 87.3% <SEP> Clone <SEP> 32-10
<tb> (Acidobacterium <SEP> phylum) 6 <SEP> 95.0%
<tb> a27-a28-a29 <SEP> (12) <SEP> Desulfuromonas <SEP> acetexigens <SEP> 88. <SEP> 8% <SEP> Clone <SEP> Sva0515 <SEP> (Acidobacterium <SEP> phylum) 6 <SEP> 91.0%
<tb> a37-a38-a39 <SEP> (12) <SEP> Desulfuromonas <SEP> palmitatis <SEP> 90. <SEP> 3% <SEP> Clone <SEP> Sva0515 <SEP> (Acidobacterium <SEP> phylum) 6 <SEP> 91. <SEP> 5%
<tb> a37-a38-a39 <SEP> (14) <SEP> Halothermothrix <SEP> orenii <SEP> 87.5% <SEP> Clone <SEP> ii3-7 <SEP> 93. <SEP> 3%
<tb> (Acidobacterium <SEP> phylum) 6 <SEP> 93.3%
<tb> a8-a9-a10 (9) <SEP> Pelobacter <SEP> carbinolicus <SEP> 865% <SEP> Clone <SEP> ii3-15 <SEP> 92.6%
<tb> (Acidobacterium <SEP> phylum) 6 <SEP> 92.6%
<tb> a34-a35-a36 (10) <SEP> Nitrococcus <SEP> mobilis <SEP> 90.6% <SEP> clone <SEP> RB43 <SEP> Hum) <SEP> 6 <SEP> 93.7%
<tb> (Acidobacterium <SEP> phylum) 6 <SEP> 93.7%
<tb> No <SEP> classified <SEP>
<tb> a22.1 (19) <SEP> Aerothermobacter <SEP> marianas <SEP> 89. <SEP> 1% <SEP> Eubacteria <SEP> no <SEP> identified <SEP> (no <SEP> published) <SEP> 93.4%
<Tb>
98

in s #.

CL en 0) 1,'c' (6 GONZALEZ et al (1996) - Zhou et N o (D erson et al (1996 - 4 Godon et al (19

CL in 0) 1, 'c' (6 GONZALEZ et al (1996) - Zhou and

<Desc / Clms Page number 130>

TABLE 9:

<Tb>
<tb> SequencesDesignation <SEP> SEQ <SEP> ID <SEP> N <SEP>
<tb> Probes <SEP> and <SEP> primers
<tb> FGPS431 <SEP> 1
<tb> FGPS122 <SEP> 2
<tb> FGPS350 <SEP> 3
<tb> FGPS643 <SEP> (T) <SEP> 4
<tb> FGPS643 <SEP> (C) <SEP> 5
<tb> R499
<tb> R500 <SEP> 7
<tb> C501 <SEP> 8
<tb> FGPS516
<tb> FGPS517 <SEP> 10
<tb> FGPS518 <SEP> 11
<tb> FGPS612 <SEP> 12
<tb> FGPS669 <SEP> 13
<tb> FGPS618 <SEP> 14
<tb> FGPS614 <SEP> 15
<tb> FGPS615 <SEP> 16
<tb> FGPS616 <SEP> 17
<tb> FGPS621 <SEP> 18
<tb> FGPS617 <SEP> 19
<tb> FGPS680 <SEP> 20
<tb> FGPS619 <SEP> 21
<tb> 63f <SEP> 22
<tb> 1387r <SEP> 23
<tb> Oligo-1 <SEP> (Example <SEP> 10) <SEP> 24
<tb> Oligo-2 <SEP> (Example <SEP> 10) <SEP> 25
<tb> A1 <SEP> 26
<tb> A2 <SEP> 27
<tb> B1 <SEP> 28
<tb> B2 <SEP> 29
<tb>SEP> nucleic acids <SEP> PKS-I
<tb> Amb9 <SEP> 30
<tb> Amb12 <SEP> 31
<tb> Ery19 <SEP> 32
<tb> A9b12 <SEP> 33
<tb> A23G1 <SEP> 1-1 <SEP> 34
<tb> A26G1 <SEP> 1-2 <SEP> 35
<tb> A26G1-10 <SEP> 36
<Tb>

<Desc / Clms Page number 131>

TABLE 9 (continued 1): Sequences

<Tb>
<tb> Designation <SEP> SEQ <SEP> ID <SEP> N
<tb> A35 <SEP> E4-16 <SEP> 37
<tb> A49F1-32 <SEP> 38
<tb> A17d2-3 <SEP> 39
<tb> A53F11-13 <SEP> 40
<tb> A53F11-14 <SEP> 41
<tb> A22A <SEP> 2-11 <SEP> 42
<tb> A36E8-1 <SEP> 43
<tb> A52E8-2 <SEP> 44
<tb> Sequences <SEP> of <SEP> amino acids <SEP> PKS-I
<tb> Amb9 <SEP> 45
<tb> Amb12 <SEP> 46
<tb> Ery19 <SEP> 47
<tb> A9b12 <SEP> 48
<tb> A23G1 <SEP> 1-1 <SEP> 49
<tb> A26G1 <SEP> 1-2 <SEP> 50
<tb> A26G1-10 <SEP> 51
<tb> A35 <SEP> E4-16 <SEP> 52
<tb> A49F1-32 <SEP> 53
<tb> A17d2-3 <SEP> 54
<tb> A53F11-13 <SEP> 55
<tb> A53F11-14 <SEP> 56
<tb> A22A <SEP> 2-11 <SEP> 57
<tb> A36E8-1 <SEP> 58
<tb> A52E8-2 <SEP> 59
<tb> Sequences <SEP> RNA <SEP> 16S
<tb> a24.1 (2), <SEP> 60
<tb> a4. <SEP> a6.a7 <SEP> (7) <SEP> 61
<tb> a52.a53.a5 (15) <SEP> 62
<tb> a49.a50.a51 (11) <SEP> 63
<tb> a4.a6.a7 (14) <SEP> 64
<tb> a30.a31.a32 (7) <SEP> 65
<tb> a37.a38.a39 <SEP> 6) <SEP> 66
<tb> a46.a47.a48 <SEP> (14) <SEP> 67
<tb> a49.a50.a51 (1) <SEP> 68
<tb> a52.a53.a5 (8) <SEP> 69
<tb> a8.a9.a10 (13) <SEP> 70
<tb> a1.a2.a3 (13) <SEP> 71
<tb> a43.a44.a45 (10) <SEP> 72
<tb> a27.a28.a29 <SEP> 5) <SEP> 73
<Tb>

<Desc / Clms Page number 132>

TABLE 9 (cont'd 2): Sequences

<Tb>
<tb> Designation <SEP> SEQ <SEP> ID <SEP> N <SEP>
<tb> a23.1 <SEP> 74
<tb> a25. <SEP> 1 <SEP> 75
<tb> a18.1 (22) <SEP> 76
<tb> a33. <SEP> 1 <SEP> 77
<tb> a14.7 <SEP> 78
<tb> a21.7 <SEP> 79
<tb> a8.a9.a10 (7) <SEP> 80
<tb> a8.a9.a10 (18) <SEP> 81
<tb> a27.a28.a29 <SEP> 3 <SEP> 82
<tb> a34.a35.a36 <SEP> 5 <SEP> 83
<tb> a22.1 (19) <SEP> 84
<tb> a11.a12.a13 (5) <SEP> 85
<tb> a19.a20.a26 <SEP> 9 <SEP> 86
<tb> a40.a41.a42 (6) <SEP> 87
<tb> a27.a28.a29 <SEP> 8 <SEP> 88
<tb> a27.a28.a29 (12) <SEP> 89
<tb> a37.a38.a39 <SEP> 12) <SEP> 90
<tb> a46.a47.a48 (6) <SEP> 91
<tb> a11.a12.a13 (11) <SEP> 92
<tb> a15.a16.a17 (12) <SEP> 93
<tb> a15.a16.a17 (5) <SEP> 94
<tb> a19.a20.a26 (13) <SEP> 95
<tb> a37.a38.a39 (14) <SEP> 96
<tb> a8.a9.a10 (9 <SEP> 97
<tb> a19.a20.a26 (5) <SEP> 98
<tb> a43.a44.a45 (4 <SEP> 99
<tb> a1.a2.a3 (4) <SEP> 100
<tb> a4.a6.a7 (23) <SEP> 101
<tb> a49.a50.a51 <SEP> 22 <SEP> 102
<tb> a8.a9.a10 (2) <SEP> 103
<tb> a34.a35.a36 <SEP> 3) <SEP> 104
<tb> a34.a35.a36 <SEP> 10 <SEP> 105
<tb> a40.a41.a42 <SEP> 13 <SEP> 106
<Tb>

<Desc / Clms Page number 133>

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SEQUENCE LIST <110> Rhône Poulenc Rorer S.A.

<120> Method for obtaining nucleic acids from a soil sample, nucleic acids thus obtained and their application to the synthesis of compounds <130> Soil DNA library - RPR S.A.

<140><141><160> 106 <170> PatentIn Ver. 2.1 <210> 1 <211> 15 <212> DNA <213> Artificial sequence <220><223> Description of the artificial sequence: probe
FGPS431 <220><221> variation <222> (14) <223> Base A replaced by G <400> 1 acggcggtg tgtac <210> 2 <211> 22 <212> DNA <213> Artificial sequence <220><223> Description of the artificial sequence: primer
FGPS122 <400> 2 ggagagtttg atcatggctc ag 22 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <220><223> Description of the artificial sequence: primer
FGPS350 <400> 3 cctggagtta agcccacaagc 20

<Desc / Clms Page number 146>

<210> 4 <211> 24 <212> DNA <213> Artificial sequence <220><223> Description of the artificial sequence: probe
FGPS643 <220><221> variation <222> (20) <223> T replaced by C <400> 4 gtgagtnnna acctgcccct gact 24 <210> 5 <211> 21 <212> DNA <213> Artificial sequence <220><223> Description of the artificial sequence: probe
FGPS643-2 <400> 5 gtgagtaacc tgcccccgac t 21 <210> 6 <211> 23 <212> DNA <213> Artificial sequence <220><223> Description of the artificial sequence: primer
R499 <400> 6 ttaattcact tgcaactgat ggg 23 <210> 7 <211> 23 <212> DNA <213> Artificial sequence <220><223> Description of the artificial sequence: primer
R500 <400> 7 aacgatagct cctacatttg gag 23 <210> 8 <211> 25

<Desc / Clms Page number 147>

<212> DNA <213> Artificial sequence <220><223> Description of the artificial sequence: C501 probe <400> 8 ttgctgatac ggtatagaac ctggc <210> 9 <211> 20 <212> DNA <213> Artificial sequence <220 ><223> Description of the artificial sequence: primer
FGPS516 <400> 9 tccagatcct tgacccgcag 20 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <220><223> Description of the artificial sequence: primer
FGPS517 <400> 10 cacgacattg cactccaccg 20 <210> 11 <211> 16 <212> DNA <213> Artificial sequence <220><223> Description of the artificial sequence: probe
FGPS518 <400> 11 ccgtgagccg gatcag 16 <210> 12 <211> 20 <212> DNA <213> Artificial sequence <220><223> Description of the artificial sequence: FGPS612 <220><221> variation <222> (2 )

<Desc / Clms Page number 148>

 <223> Base C replaced by T <220> <221> variation <222> (7) <223> Base T replaced by C <220> <221> variation <222> (7) <223> Base T replaced by A <400> 12 ccaacttcgt gccagcagcc 20 <210> 13 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Description of the artificial sequence: FGPS669 <220> <221> variation <222> (7) <223> Base A replaced by G <220> <221> variation <222> (13) <223> Base A replaced by C <400> 13 gacgtcatcc ccaccttcct c 21 <210> 14 <211> 18 <212> DNA < 213> Artificial sequence <220> <223> Description of the artificial sequence: FGPS618 <220> <221> Variation <222> (5) <223> Base T replaced by C <400> 14 atggttgtcg tcagctcg 18 <210> 15 < 211> 21 <212> DNA <213> Artificial sequence

<Desc / Clms Page number 149>

 <220> <223> Description of the artificial sequence: FGPS614 <400> 15 gtgtagaagt gaaattcgat t 21 <210> 16 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Description of the artificial sequence: FGPS615 <400> 16 cggtggatga tgtggatt 18 <210> 17 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Description of the artificial sequence: FGPS616 <400> 17 aggttaaaac tcaaatga 18 <210> 18 < 211> 18 <212> DNA <213> Artificial sequence <220> <223> Description of the artificial sequence: FGPS621 <400> 18 atacgtaggt ggcaagcg 18 <210> 19 <211> 19 <212> DNA <213> Artificial sequence < 220> <223> Description of the artificial sequence: FGPS617 <400> 19 gccggggtca actcggagg 19 <210> 20 <211> 18 <212> DNA <213> Artificial sequence

<Desc / Clms Page number 150>

<220><223> Description of the artificial sequence: FGPS680 <220><221> variation <222> (11) <223> Base A replaced by C <220><221> variation <222> (11) <223> Base A replaced by T <220><221> variation <222> (13) <223> Base T replaced by A <400> 20 tgagtcccca actccccg 18 <210> 21 <211> 20 <212> DNA <213> Artificial sequence <220><223> Description of the artificial sequence: FGPS619 <400> 21 gcttggggct taactccagg <210> 22 <211> 21 <212> DNA <213> Artificial sequence <220><223> Description of the artificial sequence: primer 63f <400> 22 caggcctaac acatgcaagt c 21 <210> 23 <211> 18 <212> DNA <213> Artificial sequence <220><223> Description of the artificial sequence: primer
1387r <400> 23 gggcggngtg tacaaggc 18 <210> 24

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 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Description of the artificial sequence: oligo-1 <400> 24 gcttatttaa atattaagcg gccgcccggg 30 <210> 25 <211> 28 <212> DNA <213 > Artificial sequence <220> <223> Description of the artificial sequence: oligo-2 <400> 25 cccgggcggc cgcattaata tttaaata 28 <210> 26 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Description of the artificial sequence: primer al <400> 26 ccncagnagc gcntnttnct nga 23 <210> 27 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Description of the artificial sequence: primer a2 <400> 27 gtnccngtnc cgtgngtntc na 22 <210> 28 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Description of the artificial sequence: primer bl <400> 28 ccncagnagc gcntnctnct nga 23 <210> 29

<Desc / Clms Page number 152>

 <211> 22 <212> DNA <213> Artificial sequence <220> <223> Description of the artificial sequence: primer b2 <400> 29 gtnccngtnc cgtgngcctc na 22 <210> 30 <211> 672 <212> DNA <213> Streptomyces ambofaciens <400> 30 ccccagcagc acgtgttcct cgagacggtg tgggagacct tcgaatccgc cggagtggac 60 ccgcgcgcgg tacgcggtcg ttccgtcggg atgttcgtcg gcaccaacgg acaggactac 120 ccggtggtgt tggccggatc cgccgacgag ggcctggacg cccacgcggc caccggtaac 180 gcggcggcgg tgctgtccgg ccgggtctcg tacgccttcg gcctggaagg gccggcggtc 240 accgtcgaca cggcgtgttc gtcgtcgctg gtggcccttc acctggccgc gcaggcgctg 300 cggcgcggcg agtgcgatct ggcactcgcc ggcggtgtgt cggagatgtc caccgaggcg 360 gcgttcaccg agttcgcccg gcagggcggc ctggccgacg acggccgctg caaggccttc 420 tcggccgacg ccgacggcac gggctggggc gagggcgtcg gcgtcctgct ggtggagcgg 480 ctggcggacg cccgccgcaa cgggcaccgg gccctcgcgc tggtacgggg cagcgcggtc 540 aaccaggacg gcgcctccaa cggtctgacg gcacccaacg gcccgtccca gcagcgagtc 600 atccggcagg cactggcgga cgcccggctg tcgccgtcgg aggtcgacgc ggtcgagacc 660 cacggcaccg gc 672 <210> 31 <211> 665 <212> DNA <213> Streptomyces ambofaciens <400> 31 ccccagcagc gcgtgttcct ggaagcgtcc tgggaggcgg tcgagcgggc aggcatcgac 60 atgcgcaccc tgcgcggtgg acgcaccggc gtcttcgccg gcgtgatgta ccacgactac 120 ccgtcggtgg tcgaccccga agcgctcgac ggctacctgg gcacggccaa cgccggcagc 180 gttctctccg gccgcatcgc ctacaccttc gggcttcagg gaccggcggt caccgtggac 240 acggcctgct cctcgtccct ggtggcgctg cacctcgccg cccaggcgct gcccgccggc 300 gagtgcgaac tcgccctggt cggtggggtc acggtcatgt ccggcccgat gatgttcgcg 360 ggcttcggcc tggaagacgg ctctgccgcc gacggccgct gcaaggcgtt cgccgccgcc 420 gccgacggca ccggctgggg cgagggtgtc ggtgtgctgc tggtggagcg gctgtcggac 480 gcccggcgcc acgggcaccg ggtgctggcc gtggtgcgcg gtagcgcggt caaccaggac 540 ggtgcctccg gcggcctcac cgcccccaac ggacctgccc agcagcgcgt catccgtcag 600 gccctggcga gcgcggcact cgtaccggcc gaggtcgacg cggtcgagac ccacggcacc 660 gggac 665 <210> 32 <211> 671 <212> DNA <213> Saccharopolyspora erythraea <400> 32 ccgcaggagc gcgtgttcct ggaactcgct tgggaagcac ttgataacgc gggcatcgca 60 ccgcaca gcc tcagggacag ccggacgggc gtgttcttcg gagctatgtg gcacggctac 120 gcgcagttcg cagccggagc cgtcgaccgc atcacccagc acaccgcgac cgggcacgac 180

<Desc / Clms Page number 153>

 ctgagcatca tcccggccag gatcgcctac ttcctgggct tgcgcggccc ggacatgacc 240 ctgaacaccg cgtgctcatc ggctttggtg gccatgcacc aggcacgcca aagcatcctg 300 ctgggcgaat cctcggtcgc cttggtcggc gggatcagct tgttggtcgc gctggacagc 360 atggtcgcca tgtcgcggtt cggagcgatg gccccggacg gccggtgcaa ggcattcgac 420 tctcgcgcga acggctacgt gcgcggcgaa ggcggcggtg tcgtggtgct caaaccgctg 480 tcgcgcgctc tggccgatgg caacccggtc tactgcgtcc tgcgcggcag cgcggtcaac 540 aacgacggct tcagcaatgg ccttaccgcg ccgagcccgg cggcgcagga gcaggtactg 600 cgcgacgcct acgccaacgc cggggtcgat ccggcacagg tcgactacgt cgagacccac 660 gggaccggca c 671 <210> 33 <211> 686 <212> DNA <213> Unknown organism <220> <223> Origin of the sequence: soil organism <400> 33 ccgcaggagc gcgtgttcct cgagtcgtgc tgggaggcgc tggagcatgc tggatacgat 60 actgcacgct accccggccg catcgggctg tgggccggcg cgggcttcaa cagctacctc 120 ctgaccaatc tcatgaacaa ccgcgccttt ttagagagcg tgggcatgta ccagatcttt 180 ctgagcaacg acaaggactt catcgccacc cgcacggctt acaagttaaa cctgcgcggt 240 ccggcgatgg ccgtcggcac cgcctgt cc acatcgctgg tggcggttca cgaagcttgc 300 caggcgctgc ggctgggcga gtgtgacatg gcactggccg gtgctgcgtc tgtcagcacg 360 cccctccggg agggctacct ctaccaggaa ggcatgatta tgagccgtga cggcgtctgc 420 cgcccgtttg acgccgacgc cgatggcacg gtgctgggca atggcgtggc ggtcgtggtg 480 ctcaagcggc tggacgaagc gctccgggac ggtgacacgg tctacgccgt gattcgtggc 540 acggcggtca acaacgacgg ctctgtcaag atcgggttca cggcgcccag cgccgagggg 600 cagagccggg tcgtgcggga cgccctgcgg gcggccgcgg tcccggcgga gagcgtgacc 660 tacgtcgaca cgcacggcac cggcac 686 <210> 34 <211> 689 <212> DNA <213> Unknown organ <220> <223> Origin of the sequence: soil organism <400> 34 ccccagcagc gcctgttcct cgagtgcgcg tgggaagcga tggagaacgc gggatatgcg 60 gcgcgaagct ataagggttc gatcggcgtt ttcgcgggat gcggcgtcaa tacctacctg 120 ctgaacaacc tcgccaccgc ggagccgttc gatttctcac gcccctccgc gtaccagctg 180 ctgacggcca acgacaagga tttcctggcc acgcgtgtct cttacaagct gaacctccgc 240 gggcccagcc tgacggttca gacggcgtgc tccacctcgc tggtgtcggt ggtgatggca 300 tgcgagagct tgcagcgcgg cgcctcggac attgccttg g ccgggggagt tgccatcaat 360 gttccgcagt ccgtggggta cctgcaccag ccgggcatga tcctgtcgcc cgacgggcgc 420 tgccgcgcct tcgatgagtc cgctcaaggc acggtgccgg gcaacggcgc gggtgtggtc 480 gtcctcaagc gcttgagccg cgctctggcc gatggcgaca cgatctacgc cgtcattcgc 540 ggagcggcta ttaataatga tggcgccgag cgcatggggt ttaccgctcc aggtgtggac 600 ggtcagacgc gattgattcg gcgcactcaa gagatggcgg gcgtgaagcc ggagtccatc 660 ggctacatgg acacccacgg caccggcac 689 <210> 35 <211> 671 <212> DNA

<Desc / Clms Page number 154>

 <213> Organime Unknown <220> <223> Origin of sequence: soil organism <400> 35 ccgcagcagc gcctcttcct cgaggtggca tgggaagctt tggagcgtgc gggtcggccg 60 cccgacagtc tcgcgggcag cgacaccgga gtgttcatcg ggatcagcac cgacgactac 120 agccggctga aacctaccga tccggcgctc attgacgcct ataccggtac cggaaccgcg 180 ttcagcactg ccgccggacg gatctcctat ctgctggggt tgcagggacc gaacttcccc 240 gtcgacacgg cgtgctcttc ctcactcgtg gcggttcatc tggcgtgccg cagcttgcag 300 tcgcgagagt gcagcatggc gctggccggc ggcgtgaacc tgattctggc gccggaaagc 360 acgatctact tctgccgcct gcgggccatg gcggccgatg gccgttgcaa aagtttcgct 420 gcctccgccg acggttacgg ccgcggcgag ggatgcggaa tgctggtgct gaagcggctg 480 tccgatgcga cgcgtgacgg cgatcgtatt ctggcgctga ttcgcggatc ggccgtcaac 540 cacggcggcc gcagcaacgg cctcacggcg ccgaacggtc cggcgcagga agccgtgatt 600 cgggcggcgc tcaagaacgc cggcatggcc cccgccgatg tcgattacgt ggacacccac 660 ggcaccggca c 671 <210> 36 <211> 758 <212> DNA <213> Unknown organ <220> <223> Origin of the sequence: soil organism <400> 36 ccgcaggagc gc gtcttcct cgaacgcatt gacggtttcg atgcggaatt cttcggcatc 60 tccccccgcg aagctctgaa catggatccg cagcagcggc tgctgctgga agtgtgctgg 120 gaagcggcag aggacgccgg catctctccc ggccctctgg cgggcagcgc gaccggcgtc 180 tttgccggct cctgcgccca ggacttcgga ctgtttcagt acgccgaccc tgcccgcatc 240 ggagcttggt cgggttccgg cgtggcgcat agcatgttgg ccaatcgcat ctcctatctg 300 ctcgacctgc gcggtccgag catggcggtc gatacggcct gctcctccgc gctcgtcgcc 360 gtccatctgg cttgccaaag cctgcgccgg cgcgaatgcg atgcggcatt cgccggcgga 420 gtgaacttga tcctgactcc cgagggcatg atcgctttgt cgaaggctcg catgttggcg 480 cccgacggac gctgcaagac gttcgacgcc gcagccgacg gttatgtgcg cggcgagggc 540 tgcggcatcg tgctgctgaa gcggctctcc gatgcgctgg ccgatggcga tgccatctgt 600 gcagtcatcc gcggctcggc aatcaatcag gacggacgga gcaatggcat cacggcgccg 660 aatctgcagg cgcagaaggc ggtcctgcaa gaggcggtgg ccaacgcgca catcgatcca 720 tcccacgtat cgttgatcga cacgcacggc accggcac 758 <210> 37 <211> 704 <212> DNA <213> Unknown organism <220> <223> Origin of the sequence: soil organism <400> 37 ccgcagcagc gc gtgttcct cgagtgcgcc tgggaggcgg tggaaagcgc gggctacgat 60 cccgaaaaat atcccggcct gatcggagtt ttcgccgggg ccagcatcaa cagctatttc 120 ctttataacc tcgcgcacaa ccgggaattc gtcgcccgca tggcggggga gtaccaagtg 180 ggcgagtacc agacgatcct cggaaacgac aaggactacc tccccactcg cgtctcctac 240 aaattgaacc tgcgcggccc cagcctggcc gtgcagtccg cctgctcgac cggcctcgtc 300 gccgtttgtc aggccattca aaatctgcag acttatcagt gcgatatggc cctcgcgggc 360 ggcatctcga tttcgtttcc gcaaaagcgc gactaccgct tcaccgacga aggaatggtc 420

<Desc / Clms Page number 155>

 tctcgcgacg gtcactgccg cccgttcgac gccagcgcgc aaggcacggt cttcggcaac 480 ggggccggcg tcgtcctgat gaaaagattg gccgacgcag tgaccgatcg ggacacgatc 540 ctcgccgtga ttaggggcgc tgccgtgaac aacgacggcg gcgtcaaaat gggttacacg 600 gcgcccagtg ccgaaggtca ggcggaggcc atcaccctgg ccctcgcgct cgctggcgtc 660 agcccggaga ccatcacttg catggacacc cacggcaccg CCGA 704 <210> 38 <211> 680 <212> DNA <213> Organime unknown <220> <223> Origin of sequence: soil organism <400> 38 ccccagcagc gcgtgttcct cgaatgcgcc tgggcggcgc tggagcgccg ccggatatca 60 gggcgacacc ttccacggtg tccatcggcg gtctatgcct caagcggctt taacacctat 120 cttctgaacc tgcatgccaa tgccgcggtg cgccaatcga tcagcccgtt tgaactgttc 180 gtcgccaacg acaaggattt tctggcgacg cgcacggctt acaagctcaa tctgcgcggc 240 ccggccatga cagtgcagac ggcctgctcc tcatcgttgg ttgccgttca tgtcgccgcg 300 caaagcctcc tagcgggcga atgcgatatt gcgctcgcgg gcggcatcac ggtttcccgt 360 tcgcatggat atgtggcgcg cgaaggtgga atattgtctc ctgacgggca ttgccgggcg 420 ttcgatgcgg atgccggcgg aaccgttcca ggcagcggcg tcggcgttgt cgtgctca ag 480 cgtctcgaag atgcgcttgc agacggcgat acgatcgacg ccgtcatcat cggttcggcc 540 atcaacaatg atggcgcgct gaaggcgagc tttaccgcac cgcaggtgga cagccaggcc 600 ttggtcatca gcgaggccca tgcagctgcc ggaatatcgg ccgattccat cggttatatg 660 gacacccacg gcaccgggac 680 <210> 39 <211> 671 <212> DNA <213> Organime Unknown <220> <223> Origin of the sequence: soil organism <400> 39 ccgcagcagc gcctcttcct cgagctcacc tgggaagcgc tggaagatgc cggcatcccg 60 ccgtccacga ttgccggcac gaatgtcggc gttttcatgg gcgcgtcgca ggctgactac 120 ggccacaagt tcttcagcga ccacgccgtc gcggattccc atttcgccac cggcacctcg 180 ctggcggtcg tcgccaatcg catttcctac atctacgacc tgcgcggccc aagcctcact 240 gtagacacgg cgtgctcgtc gtcgctcgtc gcgctgcatc aggcggtgga agcgctccgc 300 tcggggcgga tcgaaacagc cattgtcggc ggcattaacg ttatcgccag cccggcgtcc 360 ttcatcgcct tctcgcaggc ctcgatgctg tcgccgacgg ggttgtgcca ggctttctcc 420 gccaaggccg atggctttgt ccgcggcgag ggcggcacgg ttttcgtcct gcgcaaggcg 480 gcgcatgcgc atggcagccg caacccggtg cgcgggctca ttctcgccac 540 cgacgtcaat gt tccgacgggc aqccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccccbc

<Desc / Clms Page number 156>

 <400> 40 ccgcagcagc gcgtgttcct cgacggcatc gaccggttcg atccgcgtca cttcgcgatc 60 acgccgcgcg aggcgatcag catggacccg cagcagcggc tcctgctcga ggtcacgtgg 120 gaagcgctgg agcgcgccgg cgtggcgccc gatcgcctga ccggatccga caccggcgtc 180 ttcatcggca tcagcaccaa cgactacggc cagatcctgc tgcgcgcctc ggaccagatc 240 gatccgggga tgtacttcgg caccggcaac ctgttgaacg cggcggcggg acgcctctcg 300 tacgtcctcg gccgcaggg tccgagcatg gcggtcgaca ccgcatgtcc gtcgtcgctg 360 gtggcgattc atctcgcgtg tcagagcctg cgcaaccgcg agtgccgcat ggcgctcgcc 420 ggcggcgcca acctggtgct cgtcccggaa gtgacggtca actgctgccg cgccaagatg 480 ctcgcgcctg acgggcgctg caagacgttc gacgccgcgg cggacggcta cgtccgcggc 540 gaaggggccg cggtgatcgt gctgaagcgg ctctccgacg cgctggcgga cggcgatccg 600 atcgtcgcgc tgatccgcgg atccgcggtc aatcaggacg gccgcagcgg cggcttcacc 660 gcgccgaacg aactggcgca gcaggcggtg atccggaccg cgctcgcggc agcgggcgtc 720 gccgcgtccg acatcggcta cgtggacacg cacggcaccg GGAC 764 <210> 41 <211 > 763 <212> DNA <213> Unknown Organ <220> <223> Origin of the sequence: organism u soil <400> 41 ccgcagcagc gcgtgttcct cgacggcatc gaccgcttcg atccgcagtt tttcgggatc 60 gcgccgcgcg aagcggccgg catcgatccg cagcagcggc tgctgctcga gacgacgtgg 120 gaagcgctgg aagacgccgg gacgtcgccg gaaaagctgc agggaacccc ggccggcgtg 180 ttcgtcggca tcaacagcat cgactacgcg acgctgcagc tgcagaactg cgatctggcc 240 agcatcgacg cctattcgct ctccggcagc gcgcacagca tcgcggccgg gcggctcgcc 300 tacgtgctcg gcctgcaggg gccggcgatg gcggtcgaca ccgcctgctc gtcgtcgctg 360 gtcgcgatcc acctggcgtg ccagagcctg cgcaacgacg actgccgcgt cgccgtggcc 420 ggcggcgtgc acgtcacgct gacgccgatc aacatggtcg tgttctcgaa gctgcgcatg 480 ctggcggcgg acggcaagtg caagacgttc gacggccgcg gcgacggatt cgtcgaaggc 540 gagggctgcg cggtcatcgt cctcaagcgg ttgtcgcacg cgcttgccga caaggatcgg 600 atcctcgcgc tggtgcgcgg ttcggcggtc aaccaggacg gcgcgagcag cggtctcacc 660 gcgccgaacg gtccggcgca ggaagcggtc atccgcgcgg cgttgaagcg ggccggcgtg 720 cagccggcgg aggtcggcta cgtggacacc cacggcaccg gca 763 <210> 42 <211> 668 <212> DNA <213> Unknown Organ <220> <223> Origin of the sequence: organ ism soil <400> 42 ccgcaggagc gcgtgctgct ggaatcctcg tggcatgcgc tggaagacgc cggctatgcc 60 ggcgaaagca tcgccggcgc gcgctgcggc gtgtacatgg gcttcaacgg cggcgactac 120 ggcgacctgc tgtacggcca gccgtcgctg ccgccgcacg cgatgtgggg caacgccgcc 180 tcggtgctgt cggcgcgcat cgcctattac ctggacctgc aaggcccggc gatcaccctc 240 gacaccgcct gttcgagctc gttggtcgcg gtgcatctgg cctgccaggg gctgtggacc 300 ggcgagaccg atctggccct ggccggcggc gtgtggatcc agtgcacgcc cggattcctg 360 atctcctcca gccgcgccgg catgctctcg ccgaccggcc agtgccgcgc gttcggcgcc 420 ggcgccgacg gcttcgtgcc gtccgaaggc gtcggcgtgg tcgtgctcaa gcgcctgcag 480 gacgcgctcg acgccggcga ccacatntac ggcgtgatcc gcggcagcgc gatcaaccag 540 gacggcgcca gcaacggcat caccgcgccg agcgccgccg cccaggagcg cttgcagcgc 600

<Desc / Clms Page number 157>

cgtctacg acagcttcgg catcgacgcc tcgcgcctgc agatgatcga ggcccacggc 660 accgcac 668 <210> 43 <211> 671 <212> DNA <213> Unknown Organ <220><223> Origin of the sequence:: soil organism <400> 43 ccgcaggagc gcgtgctgct ggaggtgact tgggaggcac tcgaagacgc cggccaagac 60 gtggaccgtc tggccgggcg gcccgtcggc gtcttcgtcg ggatctcgtc gaacgattac 120 ggccagcttc agaacggcga cccggccgac gtggacgcct acgtcggcac cggtaacgcg 180 ctgagcatcg ccgccaaccg actcagctac acgtttgact ttcgcggccc gagtctggcg 240 gtggacacgg cgtgctcgtc ttcactcgtc gcgatccatc tcgcctgcca gagcgttcgc 300 cgcggtgaag cggaactcgc cgtcgcggcc ggcgtcaact tgattctgac ccccggcctg 360 acggtgaatt tcacccgcgc cggcatgatg gcgcctgacg gccggtgcaa gacgttcgac 420 gcggccgcca acggctacgt gcgcggcgaa ggcgccggcg tcgtcgtgct caagccgctg 480 gcccaggcta tcgccgacgg cgacccgatc tacgcgatcg tccgtggcag cgccgtcaac 540 caggacggcc gttccaacgg cctcaccgcc ccgaaccgac aggcccaaga ggtcgtgctg 600 cgggccgcgt atcgtgacgc gggcatcagc ccggccgatg tcgacgccgt cgaggcccac ggcaccggca c 660 671 <210> 44 <211> 707 <212> DNA <213> Organime Unknown <220><223> Origin of sequence: soil organism <400> 44 ccccagcagc gcgtgttcct cgaggacgcg actgaggtcg acgtggatgc gctttcagac 60 ggcgaagacg tcgtgatcgc cggcatcatg cagcacatcg aggaggccgg catccactcg 120 ggcgattcat cgtgcgtgct tccgccggtc gacatcccgc cgaaggcgct gcagacgatc 180 cgcgatcaca cgttcaagct cgcgcgcgcg ttgaaggtca tcggcctgat gaacgtgcag 240 tacgcgattc agcgcgacaa ggtctacgtg attgaggtaa accctagggc ttctcgaact 300 gtcccgtatg tctcgaaggc gacaggcgtg ccgctggcga aggtcgcgtc acgcttgatg 360 accggacgca aactgcacga gctgttgccg gaaggggtcg agcgcggctg gatcaccacc 420 gcgggcgaga atttctacgt gaagtcgccg gtcttcccgt ggggtaagtt cccgggcgtt 480 gacactgtgc tcgggccgga gatgaaatcg accggcgaag tcatgggcgt cgccgacaac 540 ttcggcgagg ccttcgccaa ggcacagatc gccgccggca catacctgcc gaccgaaggt 600 accgtcttca tctcggtcaa cgaccgtgac aaaggcaacg tcattcagct ggcgcagcgt 660 ttctccgaac tcggtttcgg cattgtcgac acgcacggca ccgggac 707 <210> 45 <211> 225 <212> PRT <213> Streptomyces ambofaciens <400> 45 Pro G ln Gln His Val Phe Glu Thr Thru Glu Thr Phe Glu Ser
1 5 10 15 Ala Gly Val Asp Pro Arg Ala Val Arg Gly Arg Ser Val Gly Met Phe

<Desc / Clms Page number 158>

20 25 30 Val Gly Thrn Asn Gly Asp Gln Asp Val Al Val Val Val Ala Gly Ser Ala
35 40 45 Asp Glu Gly Leu Ala Asp Ala Ala Thr Gly Asn Ala Ala Ala Val
50 55 60 Leu Ser Gly Arg Val Ser Tyr Ala Phe Gly Leu Glu Gly Pro Ala Val
65 70 "75 80 Thr Asp Asp Val Ala Cys Ser Ser Leu Leu Val Ala Leu His Leu Ala
85 90 95 Ala Gln Ala Leu Arg Arg Gly Glu Cys Asp Leu Al Leu Ala Gly Gly
100 105 110 Val Ser Glu Met Ser Thr Glu Ala Ala Phe Thr Glu Phe Ala Arg Gln
115 120 125 Gly Gly Ala Asp Asp Gly Arg Cys Ala Phe Ser Lys Ala Asp Ala
130 135 140 Asp Gly Thr Gly Trp Gly Glu Gly Val Gly Val Leu Leu Val Glu Arg 145 150 155 160 Leu Ala Asp Ala Arg Arg Asn Gly His Arg Ala Leu Ala Leu Val Arg
165 170 175 Gly Ser Ala Val Asn Asp Gln Gly Ala Asn Ser Gly Leu Thr Ala Pro
180 185 190 Asn Gly Pro Ser Gln Gln Arg Val Arg Arg Gln Ala Leu Ala Asp Ala
195 200 205 Arg Leu Ser Ser Ser Glu Val Asp Ala Glu Gl Thr Thr Gly Thr Gly
210 215 220 Thr 225 <210> 46 <211> 207 <212> PRT <213> Streptomyces ambofaciens <400> 46 Ala Ser Trp Glu Ala Val Glu Arg Ala Gly Ile Asp Met Arg Thr Leu 1 5 5 10 15 Arg Gly Gly Arg Thr Gly Val Phe Ala Gly Val Met Tyr Tyr Asp Tyr
20 25 30 Pro Val Val Val Pro Asp Glu Ala Leu Asp Gly Tyr Leu Gly Thr Ala
35 40 45 Asn Ala Gly Ser Val Leu Ser Gly Arg Ala Tyr Thr Phe Gly Leu
50 55 60

<Desc / Clms Page number 159>

Gln Gly Pro Ala Thr Thr Val Asp Thr Ser Ala Ser Cys Ser Ser Leu Val
65 70 75 80 Ala Leu Ala Ala Gla Ala Leu Leu Ala Gly Glu Leuk Leu
85 90 95 Ala Leu Val Gly Gly Val Thr Met Met Ser Gly Met Met Phe Ala
100 105 110 Gly Phe Gly Leu Asp Asp Glly Ala Ala Ala Asp Gly Arg Ala Cly Lys
115 120 125 Phe Ala Ala Ala Ala Gly Thr Gly Trly Gly Glly Gly Val Gly Val
130 135 140 Leu Leu Val Glu Arg Leu Ser Asp Ala Arg Arg His Gly His Arg Val 145 150 155 160 Leu Ala Val Val Arg Gly Ser Ala Val Asn Asp Gln Gly Ala Ser Gly
165 170 175 Gly Leu Thr Ala Pro Asn Gly Pro Ala Gln Gln Arg Val Arg Gln Island
180 185 190 Ala Leu Ala Ser Ala Ala Val Leu Pro Ala Glu Asp Val Ala Val
195 200 205 <210> 47 <211> 223 <212> PRT <213> Saccharopolyspora erythraea <400> 47 Pro Gln Glu Arg Val Phe Leu Glu Leu Ala Trp Glu Ala Leu Asp Asn 1 5 10 15 Ala Gly Ile Ala Pro His Ser Arg Arg Ser Ser Arg Thr Gly Val Phe
20 25 30 Phe Gly Ala Met Trp His Gly Ala Gla Phe Ala Ala Gla Ala Val
35 40 45 Asp Arg Thr Thr Thrn His Thr Ala Thr Gly His Asp Leu Ser Ile Ile
50 55 60 Pro Ala Arg Ala Tyr Phe Leu Gly Leu Arg Gly Pro Asp Met Thr
65 70 75 80 Leu Asn Thr Ala Ser Cys Ser Ala Leu Ala Val Met His Gln Ala Arg
85 90 95 Gln Ser Ile Leu Leu Gly Glu Ser Ser Val Ala Leu Val Gly Gly Ile
100 105 110
Ser Leu Leu Ala Val Leu Asp Ser Met Ala Val Met Ser Arg Phe Gly
115 120 125

<Desc / Clms Page number 160>

Ala Met Ala Pro Asp Gly Arg Cys Lilies Ala Phe Asp Ser Arg Ala Asn
130 135 140 Gly Tyr Val Arg Gly Glu Gly Gly Gly Val Val Val Leu Lys Pro Leu 145 150 155 160 Ser Arg Ala Leu Ala Asp Gly Asn Pro Val Tyr Cys Val Leu Arg Gly
165 170 175 Ser Ala Asn Asp Asn Asp Gly Phe Ser Asn Gly Ur Thr Ala Pro Ser
180 185 190 Pro Ala Ala Gln Glu Gln Val Leu Arg Asp Ala Tyr Ala Asn Ala Gly
195 200 205 Val Asp Pro Ala Gln Val Asp Tire Val Glu Thr His Gly Thr Gly
210 215 220 <210> 48 <211> 211 <212> PRT <213> Unknown organism <220><223> Origin of the sequence: soil organism <400> 48 Ser Cys Trp Glu Leu Ala Glu His Ala Gly Tyr Asp Thr Ala Arg Tyr
1 5 10 15 Pro Gly Arg Ile Gly Leu Trp Ala Gly Ala Gly Phe Asn Ser Tyr Leu
Leu Leuk As Leun Asn Asn Arg Ala Phe Leuk Glu Ser Val Gly Met
35 40 45 Tyr Gln Ile Phe Leu Ser Asn Asp Lys Asp Phe Ile Ala Thr Arg Thr
50 55 60 Ala Tyr Lilies Leu Asn Leu Arg Gly Pro Ala Ala Val Gly Thr Ala
65 70 75 80 Cys Ser Thr Ser Leu Leu Val Ala Val His Glu Ala Cys Gln Leu Al Arg
85 90 95 Leu Gly Glu Asp Cys Ala Al Ala Gly Ala Ser Val Ser Thr
100 105 110 Pro Leu Arg Glu Gly Tire Leu Tyr Gln Glu Gly Met Ile Met Ser Arg
115 120 125 Asp Gly Val Cys Arg Pro Phe Asp Ala Asp Ala Asp Gly Thr Val Leu
130 135 140 Gly Asn Gly Val Ala Val Val Val Leu Lys Arg Leu Asp Glu Ala Leu 145 150 155 160 Arg Asp Asp Gly Asp Thr Val Tyr Ala Val Arg Island Gly Thr Ala Val Asn

<Desc / Clms Page number 161>

165 170 175
Asn Asp Gly Ser Val Lys Gly Phe Thr Ala Pro Ser Ala Glu Gly
180 185 190
Gln Ser Arg Val Val Arg Asp Ala Ala Al Arg Ala Ala Val Pro Ala
195 200 205
Glu Ser Val
210 <210> 49 <211> 229 <212> PRT <213> Unknown organism <220><223> Origin of the sequence: soil organism <400> 49
Pro Gln Gln Arg Leu Phe Leu Glu Cys Ala Trp Glu Ala Met Glu Asn 1 5 10 15
Ala Gly Tyr Ala Ala Arg Ser Tyr Lys Gly Ser Gly Val Phe Ala
20 25 30
Gly Cys Gly Val Asn Thr Leu Leu Leu Asn Leu Leu Ala Thr Ala Glu
35 40 45
Pro Phe Asp Phe Ser Arg Pro Ser Ala Tyr Gln Leu Leu Thr Ala Asn
50 55 60
Asp Lys Asp Phe Leu Ala Thr Arg Val Ser Tyr Lys Leu Asn Leu Arg
65 70 75 80
Gly Pro Ser Leu Thr Thrn Gln Thr Ala Ser Cys Ser Ser Ser Val Val Ser
85 90 95
Val Val Met Ala Cys Glu Ser Gln Arg Glly Ala Ser Asp Ala Island
100 105 110
Leu Ala Gly Gly Val Ala Val Asn Val Val Gln Ser Val Gly Tyr Leu
115 120 125
His Gln Pro Gly Met Leu Ser Pro Asp Gly Arg Cys Arg Ala Phe
130 135 140
Asp Glu Ser Ala Gln Gly Thr Pro Val Gly Asn Gly Ala Gly Val Val
145 150 155 160
Val Leu Lys Arg Leu Ser Arg Ala Leu Ala Asp Gly Asp Thr Tyr Island 165 170 175
Ala Val Arg Island Gly Ala Ala Asn Asn Island Asp Gly Ala Glu Arg Met
180 185 190
Gly Phe Thr Ala Pro Gly Val Asp Gly Arg Gln Arg Arg Arg Arg
195 200 205

<Desc / Clms Page number 162>

Thr Gln Glu Met Ala Gly Val Lys Pro Glu Ser Gly Tyr Island Met Asp
210 215 220 Thr His Gly Thr Gly 225 <210> 50 <211> 223 <212> PRT <213> Unknown organism <220><223> Origin of the sequence: soil organism <400> 50 Pro Gln Gln Arg Leu Phe Leu Glu Val Ala Trp Glu Ala Leu Glu Arg 1 5 10 15 Ala Gly Arg Pro Pro Ser Ser Ala Gly Ser Ser Asp Thr Gly Val Phe
20 25 30 Gly Island Ser Ser Asp Asp Asp Tyr Ser Ser Arg Leu Lys Pro Thr Asp Pro
35 40 45 Ala Leu Asp Asp Ala Tyr Thr Gly Thr Gly Thr Ala Phe Ser Thr Ala
50 55 60 Ala Gly Arg Ser Ser Tyr Leu Leu Gly Leu Gln Gly Asn Pro Phe Pro
65 70 75 80 Val Asp Thr Ala Cys Ser Ser Ser Leu Val Ala Val His Leu Ala Cys
85 90 95 Arg Ser Leu Gln Ser Arg Glu Cys Ser Met Ala Leu Ala Gly Gly Val
100 105 110 Asn Leu Leu Leu Ala Pro Glu Ser Thr Ile Tyr Phe Cys Arg Leu Arg
115 120 125 Ala Met Ala Ala Asp Gly Arg Cys Lily Ser Phe Ala Ala Ser Ala Asp
130 135 140 Gly Tyr Gly Arg Gly Glu Gly Cys Gly Met Leu Val Leu Lys Arg Leu 145 150 155 160 Ser Asp Ala Thr Arg Asp Gly Asp Arg Ile Leu Ala Leu Arg Gly Island
165 170 175 Ser Ala Val Asn His Gly Gly Arg Ser Asn Gly Leu Thr Ala Pro Asn
180 185 190 Gly Pro Ala Gln Ala Val Glu Ala Ala Ala Lys Ala Gly
195 200 205 Met Ala Pro Asp Asp Asp Asp Asp Asp Gly Thr Gly
210 215 220

<Desc / Clms Page number 163>

<210> 51 <211> 252 <212> PRT <213> Unknown organ <220><223> Origin of the sequence: soil organism <400> 51 Pro Gln Glu Arg Val Phe Leu Asp Glu Asp Asp Asp Glu 1 5 10 15 Phe Phe Gly Ile Pro Ser Arg Glu Ala Asn Leu Met Asp Pro Gln Gln
Arg Leu Leu Leu Glu Val Cys Trp Ala Glu Ala Glu Asp Ala Gly Ile
35 40 45 Ser Pro Gly Pro Leu Ala Gly Ser Ala Thr Gly Val Phe Ala Gly Ser
50 55 60 Cys Ala Gin Asp Phe Gly Leu Phe Gln Tire Ala Asp Pro Ala Arg Ile
65 70 75 80 Gly Ala Ser Ser Gly Ser Gly Ser Ala His Ser Ser Leu Ala Asn Arg
85 90 95 Ser Ser Tyr Leu Leu Asp Leu Arg Gly Pro Ser Met Ala Val Asp Thr
100 105 110 Ala Cys Ser Ser Ala Leu Val Ala Val His Leu Ala Cys Gln Ser Leu
115 120 Arg Arg Arg Glu Cys Asp Ala Ala Phe Ala Gly Gly Val Asn Leu Ile
130 135 140 Leu Thr Pro Glu Gly Met Ile Ala Leu Ser Lys Ala Arg Met Leu Ala 145 150 155 160 Pro Asp Gly Arg Cys Lys Thr Asp Asp Ala Ala Asp Gly Tyr Val
165 170 175 Arg Gly Glu Gly Cys Gly Ile Val Leu Leu Lys Arg Leu Ser Asp Ala
180 185 190 Leu Ala Asp Gly Asp Ala Island Cys Ala Val Island Arg Gly Ser Ala Island
195 200 205 Asn Gln Asp Gly Arg Ser Asn Gly Thr Island Ala Pro Asn Leu Gln Ala
210 215 220 Gln Lys Ala Val Leu Gln Glu Ala Val Ala Asn Ala His Asp Asp 225 230 235 240 Ser His Val Ser Leu Asp Asp Thr His Gly Thr Gly
245,250

<Desc / Clms Page number 164>

<210> 52 <211> 234 <212> PRT <213> Unknown Organ <220><223> Sequence Origin: Soil Organism <400> 52 Pro Gln Gln Arg Val Phe Leu Glu Cys Ala Trp Glu Ala Val Glu Ser
1 5 10 15 Ala Gly Tyr Asp Pro Glu Lys Tire Pro Gly Leu Isle Gly Val Phe Ala
20 25 30 Gly Ala Ser Asn Ser Ser Tyr Phe Leu Tyr Asn Ala His As Asn Arg
35 40 45 Glu Phe Val Ala Arg Ala Gly Glu Gln Gln Val Gly Glu Tyr Gln
50 55 60 Thr Leu Leuk Asn Asp Asp Asp Tyr Tyr Leu Pro Thr Arg Val Ser Tyr
65 70 75 80 Lys Leu Asn Leu Arg Gly Pro Ser Leu Ala Val Gln Ser Ala Cys Ser
85 90 95 Thr Gly Leu Val Ala Valley Cys Gln Ala Gln Island Asn Leu Gln Thr Tyr
100 105 110 Gln Cys Asp Met Ala Leu Ala Gly Gly Ser Island Ser Phe Pro Gln Island
115 120 "125 Lys Arg Asp Tyr Tyr Arg Phe Thr Asp Glu Gly Met Val Ser Arg Asp Gly
130 135 140 His Cys Arg Pro Phe Asp Ala Ser Ala Gln Gly Thr Val Phe Gly Asn 145 150 155 160 Gly Ala Gly Val Val Leu Met Arg Lys Leu Ala Asp Ala Asp Asp Val
165 170 175 Arg Asp Thr Island Leu Ala Val Island Arg Gly Ala Ala Val Asn Asn Asp
180 185 190 Gly Gly Val Lys Met Gly Tyr Thr Ala Ser Ser Ala Glu Gly Gln Ala
195 200 205 Glu Ala Island Thr Leu Ala Leu Ala Leu Ala Gly Ser Val Pro Glu Thr
210 215 220 Thr Cys Thr Met Asp Thr His Gly Thr Gly 225 230 <210> 53 <211> 226 <212> PRT

<Desc / Clms Page number 165>

<213> Unknown organ <220><223> Origin of the sequence: soil organism <400> 53 Pro Gln Gln Arg Val Phe Leu Glu Cys Ala Trp Ala Ala Leu Glu Arg 1 5 10 15 Arg Arg Ile Ser Gly Arg His Leu Pro Arg Ser Cys Ser Ala Val Tyr
Ala Ser Ser Gly Phe Asn Thr Leu Leu Leu Asn Leu His Ala Asn Ala
35 40 45 Ala Val Arg Gln Ser Ser Ser Pro Phe Glu Phe Leu Val Ala Asn Asp
50 55 60 Lys Asp Phe Leu Ala Thr Arg Thr Ala Tyr Lily Leu Asn Leu Arg Gly
65 70 75 80 Pro Ala Met Thr Val Gln Ser Ala Cys Ser Ser Ser Ala Val Val Val
85 90 95 His Val Ala Ala Gln Leu Leu Leu Ala Gly Glu Asp Cys Ala Leu
100 105 110 Ala Gly Gly Thr Island Ser Val Ser Arg Ser His Gly Tyr Val Ala Arg Glu
115 120 125 Gly Gly Ile Leu Pro Ser Asp Gly His Cys Arg Asp Asp Ala Phe Asp
130 135 140 Ala Gly Gly Thr Val Pro Gly Ser Gly Val Gly Val Val Val Leu Lys 145 150 155 160 Arg Leu Glu Asp Ala Leu Asp Ala Asp Gly Asp Asp Asp Asp Ala Val Ile
165 170 175 Island Gly Ser Ala Island Asn Asn Asp Gly Ala Leu Lys Ala Ser Phe Thr
180 185 190 Ala Pro Gln Asp Val Gln Al Gla Val Al Ser Glu Ala His Ala
195 200 205 Ala Ala Gly Ser Island Ala Asp Ser Gly Tyr Island Met Asp Thr His Gly
210 215 220 Thr Gly 225 <210> 54 <211> 223 <212> PRT <213> Unknown organism <220><223> Origin of the sequence: soil organism

<Desc / Clms Page number 166>

<400> 54 Pro Gln Gln Arg Leu Phe Leu Glu Leu Thr Trp Glu Ala Leu Glu Asp 1 5 10 15 Ala Gly Pro Pro Ser Thr Island Ala Gly Thr Asn Val Gly Val Phe
Met Gly Ala Ser Gln Ala Asp Tyr Gly His Lys Phe Phe Ser Asp His
35 40 45 Ala Val Ala Asp His Ser Phe Ala Thr Ser Gly Thr Ser Ala Val Val
50 55 60 Ala Asn Arg Isle Ser Tyr Island Tyr Asp Leu Arg Gly Pro Ser Leu Thr
65 70 75 80 Val Asp Thr Ala Ser Cys Ser Ser Al Leu Leu Leu Leu Gln Ala Val
85 90 95 Glu Ala Leu Arg Ser Gly Arg Glu Thr Island Ala Island Val Gly Gly Island
100 105 110 Asn Val Ala Ser Pro Ala Ser Phe Ala Phe Ser Ala Ser
115 120 125 Met Leu Ser Gly Leu Cys Gln Pro Ala Phe Ser Ala Lys Ala Asp
130 135 140 Gly Phe Val Arg Gly Glu Gly Gly Thr Val Phe Val Leu Arg Lys Ala 145 150 155 160 Ala His Ala His Gly Ser Arg Asn Pro Val Arg Gly Leu Ile LeuAla
165 170 175 Thr Asp Val Asn Ser Asp Gly Arg Thr Asn Gly Ile Ser Leu Pro Ser
180 185 190 Ala Glu Ala Gln Glu Val Leu Leu Gln Arg Val Tyr Ser Arg Ala Ser
195 200 205 Asp Asp Island Asn Arg Leu Ala Phe Asp Val Asp Thr His Gly Thr Gly
210 215 220 <210> 55 <211> 254 <212> PRT <213> Unknown organism <220><223> Origin of the sequence: soil organism <400> 55 Pro Gln Gln Arg Val Phe Leu Asp Gly Asp Asp Arg Phe Asp Pro Arg 1 5 10 15 His Phe Ala Island Thr Pro Arg Glu Ala Island Ser Met Asp Pro Gln Gln
20 25 30

<Desc / Clms Page number 167>

Arg Leu Leu Leu Glu Val Thr Trp Glu Ala Leu Glu Arg Ala Gly Val
35 40 45 Ala Pro Asp Arg Green Thr Gly Asp Asp Thr Gly Val Phe Ile Gly Ile
50 55 60 Ser Thr Asn Asp Tyr Gly Gln Ile Leu Leu Arg Ala Ser Asp Gln Ile
65 70 75 80 Asp Pro Gly Met Tyr Phe Gly Thr Gly Asn Leu Leu Asn Ala Ala Ala
85 90 95 Gly Arg Leu Ser Tyr Leu Val Leu Gly Leu Gln Ser Gly Pro Met Ala Val
100 105 110 Asp Thr Ala Cys Ser Ser Ser Leu Val Ala Island His Leu Ala Cys Gln
115 120 125 Ser Leu Arg Asn Arg Glu Cys Arg Met Ala Leu Ala Gly Gly Ala Asn
130 135 140 Leu Val Leu Val Pro Glu Val Thr Val Asn Cys Cys Arg Ala Lys Met 145 150 155 160 Leu Ala Pro Asp Gly Arg Cys Lily Asp Asp Phe Ala Ala Ala Asp Gly
165 170 175 Tyr Val Arg Gly Glu Gly Ala Ala Val Ile Val Leu Lys Arg Leu Ser
180 185 190 Asp Ala Leu Ala Asp Gly Asp Pro Val Ala Island Leu Arg Gly Ser Island
195 200 205 Ala Val Asn Gln Asp Gly Arg Ser Gly Gly Phe Thr Ala Pro Asn Glu
210 215 220 Leu Ala Gln Gln Ala Val Arg Arg Ala Al Ala Ala Gly Val 225 230 235 240 Ala Ala Ser Asp Gly Tyr Island Val Asp Thr Thr Gly Thr Gly
245 250 <210> 56 <211> 254 <212> PRT <213> Unknown organ <220><223> Origin of the sequence: soil organism <400> 56 Pro Gln Gln Arg Val Phe Leu Asp Gly Ile Asp Arg Phe Asp Pro Gln 1 5 10 15 Phe Phe Gly Ile Ala Pro Arg Glu Ala Ala Gly Ile Asp Pro Gln Gln
20 25 30

<Desc / Clms Page number 168>

Arg Leu Leu Leu Glu Thr Thr Trp Glu Ala Glu Asp Leu Ala Gly Thr
35 40 45 Ser Pro Glu Lilies Leu Gin Gly Thr Pro Ala Gly Val Phe Val Gly Ile
50 55 60 Asn Ser Asp Asp Tyr Ala Thr Leu Gln Gln Leu Asn Cys Asp Leu Ala
65 70 75 80 Ser Asp Ala Ser Ser Ser Ser Ser Ser Gly Ser Ala His Ser Ala Ala Ser
85 90 95 Gly Arg Leu Ala Val Leu Val Leu Gly Leu Gin Val Ala Ala Val
100 105 110 Asp Thr Ala Cys Ser Ser Ser Leu Val Ala Island His Leu Ala Cys Gln
115 120 125 Ser Leu Arg Asp Asn Asp Asp Cys Arg Val Ala Val Ala Gly Gly Val His
130 135 140 Val Thr Leu Thr Pro Ile Asn Met Val Val Phe Ser Lys Leu Arg Met 145 150 155 160 Leu Ala Ala Asp Gly Lys Cys Lys Thr Phe Asp Gly Arg Gly Asp Gly
165 170 175 Phe Val Glu Gly Glu Gly Cys Ala Val Ile Val Leu Lys Arg Leu Ser
180 185 190 His Ala Leu Ala Asp Lys Asp Arg Arg Leu Ala Leu Val Arg Gly Ser
195 200 205 Ala Val Asn Asp Gln Gly Ala Ser Ser Gly Leu Thr Ala Pro Asn Gly
210 215 220 Pro Ala Gln Glu Ala Val Arg Island Ala Ala Leu Lys Arg Ala Gly Val 225 230 235 240 Gln Pro Ala Glu Val Gly Tyr Val Asp Thr His Gly Thr Gly
245 250 <210> 57 <211> 222 <212> PRT <213> Unknown organism <220><223> Origin of the sequence: soil organism <400> 57 Pro Gln Glu Arg Leu Leu Leu Ser Glu Ser Trp His Ala Leu Glu Asp 1 5 10 15 Ala Gly Tyr Ala Gly Glu Ser Ile Ala Gly Ala Arg Cys Gly Val Tyr
20 25 30 Met Gly Phe Asn Gly Gly Asp Tyr Gly Asp Leu Leu Tyr Gly Gin Pro

<Desc / Clms Page number 169>

35 40 45 Ser Leu Pro Pro His Ala Met Trp Gly Asn Ala Ala Ser Val Leu Ser
50 55 '60 Ala Arg Ala Tyr Tyr Leu Leu Asp Leu Gln Gly Pro Ala Ile Thr Leu
65 70 75 80 Asp Thr Ala Ser Cys Ser Ser Led Ala Val Val His Leu Ala Cys Gln
85 90 95 Gly Leu Trp Thr Gly Glu Asp Asp Leu Al Leu Ala Gly Gly Val Trp
100 105 110 Gln Island Cys Thr Pro Gly Phe Leu Ser Ser Ser Ser Arg Ala Gly Met
115 120 125 Leu Ser Pro Gly Gln Cys Arg Ala Phe Gly Ala Gly Ala Asp Gly
130 135 140 Phe Val Pro Ser Glu Gly Val Gly Val Val Val Leu Lys Arg Leu Gln 145 150 155 160 Asp Ala Leu Asp Ala Gly Asp His Xaa Tyr Gly Val Isle Arg Gly Ser
165 170 175 Ala Asn Island Gln Asp Gly Ala Ser Asn Gly Thr Island Ala Pro Ser Ala
180 185 190 Ala Ala Gln Glu Arg Leu Gln Arg His Val Tyr Asp Ser Phe Gly Ile
195 200 205 Asp Ala Ser Arg Leu Gln Met Glu Island Ala His Gly Thr Gly
210 "215 220 <210> 58 <211> 223 <212> PRT <213> Unknown organism <220><223> Origin of the soil organism sequence <400> 58 Pro Gln Glu Arg Leu Leu Leu Glu Val Thr Trp Glu Ala Leu Glu Asp
1 5 10 15 Ala Gly Asp Gln Val Asp Arg Ala Leu Gly Arg Pro Val Gly Val Phe
20 25 30 Val Gly Ser Ser Asn Asp Asp Gly Gln Gln Leu Gln Asn Gly Asp Pro
35 40 45 Ala Asp Val Asp Ala Tire Val Gly Thr Gly Asn Ala Leu Ser Ala Island
50 55 "60 Ala Asn Arg Ser Ser Tyr Thr Phe Asp Arg Phe Gly Pro Ser Leu Ala
65 70 75 80

<Desc / Clms Page number 170>

Val Asp Thr Ala Ser Cys Ser Ser Leu Leu Val Ala Ile His Leu Ala Cys
85 90 95 Gin Ser Arg Arg Val Gly Glu Ala Glu Leu Val Ala Ala Ala Gly Val
100 105 110 Asn Leu Leu Leuk Pro Gly Leu Thr Asn Phe Thr Arg Ala Gly
115 120 125 Met Met Ala Pro Asp Gly Arg Cys Lys Asp Thr Phe Asp Ala Ala Ala Asn
130 135 140 Gly Tyr Val Arg Gly Glu Gly Ala Gly Val Val Val Leu Lys Pro Leu 145 150 155 160 Ala Gin Ala Island Ala Asp Gly Asp Pro Island Tyr Ala Island Val Arg Gly
165 170 175 Ser Ala Val Asn Gin Asp Gly Arg Ser Asn Gly Leu Thr Ala Pro Asn
180 185 190 Arg Gln Ala Gin Glu Val Val Leu Arg Ala Ala Tyr Arg Asp Ala Gly
195 200 205 Pro Ser Island Ala Asp Asp Val Ala Val Glu Ala His Gly Thr Gly
210 215 220 <210> 59 <211> 235 <212> PRT <213> Unknown organism <220><223> Origin of the sequence: soil organism <400> 59 Pro Gln Gin Arg Val Phe Leu Glu Asp Ala Thr Glu Val Asp Val Asp
1 5 10 15 Ala Leu Ser Asp Asp Gly Glu Val Val Val Ala Gly Island Met Gin His
20 25 30 Island Glu Glu Ala Gly Island His Ser Gly Asp Ser Ser Cys Val Leu Pro
35 40 45 Pro Val Asp Pro Island Pro Lys Ala Leu Gln Thr Arg Asp Asp Thr Island
50 55 60 Phe Lys Leu Ala Arg Ala Leu Lys Val Ile Gly Leu Met Asn Val Gin
65 70 75 80 Tyr Ala Island Gin Arg Asp Lys Val Tyr Val Glu Val Asn Pro Arg
85 90 95 Ala Ser Arg Thr Val Pro Tire Val Ser Lys Ala Thr Gly Val Pro Leu
100 105 110

<Desc / Clms Page number 171>

Ala Lys Val Ala Ser Arg Leu Met Thr Gly Arg Lys Leu His Glu Leu
115 120 125 Leu Pro Glu Gly Val Glu Arg Gly Trp Thr Thr Island Ala Gly Glu Asn
130 135 140 Phe Tyr Val Lys Ser Pro Val Phe Pro Trc Gly Lys Phe Pro Gly Val 145 150 155 160 Asp Thr Val Leu Gly Pro Glu Met Lys Ser Thr Gly Glu Val Met Gly
165 170 175 Val Ala

* Asp Asn Phe Gly Glu Ala Phe Ala Lys Ala Gln Ala Ala Island
180 185 190 Gly Thr Tyr Leu Pro Thr Glu Gly Thr Val Phe Val Asn Asp Asp
195 200 205 Arg Asp Lys Gly Asn Val Gln Leu Ala Gln Arg Phe Ser Glu Leu
210 215 220 Gly Phe Gly Asp Asp Thr His Gly Thr Gly 225 230 235 <210> 60 <211> 1269 <212> DNA <213> Unknown Organism <220><223> Origin of the sequence: Soil organism <400 > 60 taacaggaag aagcttgctt ctttgctgac gagtggcgga cgggtgagta acacgtggga 60 acctgcctta tggttcggga taacgtctgg aaacggacgc taacaccgga tgtgcccttc 120 gggggaaagt ttacgccatg agaggggccc gcgtccgatt aggtagttgg tggggtaatg 180 gcccaccaag ccgacgatcg gtagctggtc tgagaggatg atcagccaca ctgggactga 240 gacacggccc agactcctac gggaggcagc agtggggaat attggacaat gggggcaacc 300 ctgatccagc aatgccgcgt gagtgatgaa ggcctaggg ttgtaaagct ctttcgcacg 360 cgacgatgat gacggtagcg tgagaagaag ccccggctaa cttcgtgcca gcagccgcgg 420 taatacgaag ggggcgagcg ttgttcggaa ttactgggcg taaagggcgc gtaggcggcc 480 cgatcagtca gatgtgaaag ccccgggctc aacctgggaa ctgcatttga tactgtcggg 540 cttgagttcc ggagaggatg gtggaattcc cagtgtagag gtgaaattcg tagatattgg 600 gaagaacacc ggtggcgaag gcggccatct ggacggacac tgacgctgag gcgcgaaagc 660 gtggggagca aacaggatta gataccctgg tagtccacgc CGTA aacgat gaatgctaga 720 cgctggggtg catgcacttc ggtgtcgccg ctaacgcatt aagcattccg cctggggagt 780 acggccgcaa ggttaaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg 840 tggtttaatt cgaagcaacg cgcagaacct taccaaccct tgacatgtcc attgccggtc 900 cgagagattg gaccttcagt tcggctggat ggaacacagg tgctgcatgg ctgtcgtcag 960 ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccctac cgccagttgc 1020 catcattcag ttgggcactc tggtggaact gccggtgaca agccggagga aggcggggat 1080 gacgtcaagt cctcatggcc cttatgggtt gggctacaca cgtgctacaa tagcggtgac 1140 agtgggacgc gaagtcgcaa gatggagcaa atccccaaaa gccgtctcag ttcggattgc 1200 actctgcaac tcgggtgcat gaagttggaa tcgctagtaa tcgcggatca gcacgccgcg 1260 gtgaatacg 1269 <210> 61

<Desc / Clms Page number 172>

 <211> 1500 <212> DNA <213> Organime Unknown <220> <223> Origin of sequence: Floor Organism <400> 61 ttttaaaacg acggccagtg aattgtaata cgactcacta tagggcgaat tgggccctct 60 agatgcatgc tcgagcggcc gccagtgtga tggatatctg cagaattcgc ccttcaggcc 120 taacacatgc aagtcgaacg agggcttcgg ccctagtggc gcacgggtga gtaacacgtg 180 ggaacctgcc ttatggttcg ggataacgc tggaaacgga cgctaacacc ggatgtgccc 240 ttcgggggaa agtttacgcc argagagggg cccgcgtccg attaggtagt tggtggggta 300 atggcccacc aagccgacga tcggtagctg gtctgagagg atgatcagcc acactgggac 360 tgagacacgg cccagactcc tacgggaggc agcagtgggg aatattggac aatgggggca 420 accctgatcc agcaatgccg cgtgagtgat gaaggcctta gggttgtaaa gctctttcgc 480 acgcgacgat gatgacggta gcgtgagaag aagccccggc taacttcgtg ccagcagccg 540 cggtaatacg aagggggcga gcgttgttcg gaattactgg gcgtaaaggg cgcgtaggcg 600 gcccgatcag tcagatgtga aagccccggg ctcaacctgg gaactgcatt tgatactgtc 660 gggcttgagt tccggagagg atggtggaat tcccagtgta gaggtgaaat tcgtagatat 720 tgggaagaac accggtggcg aaggcggcca tctggacgga cactgacgct gaggcgcgaa 780 agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccgtaaac gatgaatgct 840 agacgctggg gtgcatgcac ttcggtgtcg ccgctaacgc attaagcatt ccgcctgggg 900 agtacggccg caaggttaaa actcaaagga attgacgggg gcccgcacaa gcggtggagc 960 atgtggttta attcgaagca acgcgcagaa ccttaccaac ccttgacatg tccattgccg 1020 gtccgagaga ttggaccttc agttcggctg gatggaacac aggtgctgca tggctgtcgt 1080 cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaacccc taccgccagt 1140 tgccatcatt cagttgggca ctctggtgga actgccggtg acaagccgga ggaaggcggg 1200 gatgacgtca agtcctcatg gcccttagg gttgggctac acacgtgcta caatggcggt 1260 gacagtggga cgcgaagtcg caagatggag caaatcccca aaagccgtct cagttcggat 1320 tgcactctgc aactcgggtg catgaagtg gaatcgctag taatcgcgga tcagcacgcc 1380 gcggtgaata cgttcccggg ccttgtacac accgcccaag ggcgaattcc agcacactgg 1440 cggccgttac tagtggatcc gagctcggta CCAAGCTTGG cgtaatcatg gtcatagctg 1500 <210> 62 <211> 1366 <212> DNA <213 > Unknown organism <220> <223> Origin of the sequence: Soil organism <400> 62 acgacggcca gtgaattgta atacgactca ctatagggcg aattgggccc tctagatgca 60 tgctcgagcg gccgccagtg tgatggatat ctgcagaatt cgcccttcag gcctaacaca 120 tgcaagtcga acgaaggctt cggccttagt ggcgcacggg tgagtaacac gtgggaacct 180 gcctttcggt tcggaataac gtctggaaac ggacgctaac accggatacg cccttcgggg 240 gaaagttcac gccgagagag gggcccgcgc cggattaggt agttggtgag gtaatggctc 300 accaagcctt cgatccgtag ctggtctgag aggatgatca gccacactgg gactgagaca 360 cggcccagac tcctacggga ggcagcagtg gggaatattg gacaatgggc gcaagcctga 420 tccagcaatg ccgcgtgagt gatgaaggcc ttagggttgt aaagctcttt cgcacgcgac 480 gatgatgacg gtagcgtgag aagaagcccc ggctaacttc gtgccagcag ccgcggtaat 540 acgaaggggg ctagcgttgt tcggaattac tgggcgtaaa gggcgcgtag gcggcctgct 600 tagtcagaag tgaaagcccc gggctcaacc tgggaatagc ttttgatact ggcaggcttg 660 agttccggag aggatggtgg aattcccag gtagaggtga aattcgtaga tattgggaag 720 aacaccggtg gcgaaggcgg ccatctggac ggacactgac gctgaggcgc gaaagcgtgg 780 ggagcaaaca ggattagata ccctggtagt: ccacgccgta aacgatgaat gctagacgtc 840 ggggtgcatg cacttcggtg tcgccgctaa cgcattaagc attccgcctg gggagtacgg 900 ccgcaaggtt aaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt 960

<Desc / Clms Page number 173>

 ttaattcgaa gcaacgcgca gaaccttacc aacccttgac atgtccatta tgggcttcag 1020 agatgaggtc cttcagttcg gctgggtgga acacaggtgc tgcatggctg tcgtcagctc 1080 gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccctaccgt cagttgccat 1140 cattcagttg ggcactctgg tggaaccgcc ggtgacaagc cggaggaagg cggggatgac 1200 gtcaagtcct catggccctt atgggttggg ctacacacgt gctacaatgg cggtgacagt 1260 gggaagcgaa gtcgcgagat ggagcaaatc cccaaaagcc gtctcagttc ggatcgcact 1320 ctgcaactcg agtgcgtgaa gttggaatcg ctagtaatcg cggatc 1366 <210 > 63 <211> 1360 <212> DNA <213> Unknown Organ <220> <223> Origin of the sequence: Soil organism <400> 63 acagctatga ccatgattac gccaagcttg gtaccgagct cggatccact agtaacggcc 60 gccagtgtgc tggaattcgc ccttcaggcc taacacatgc aagtcgaacg ccccgcaagg 120 ggagtggcag acgggtgagt aacgcgtggg aacataccct ttcctgcgga atagctccgg 180 gaaactggaa ttaataccgc atacgcccta cgggggaaag atttatcggg gaaggattgg 240 cccgcgttgg attagctagt tggtggggta aaggcctacc aaggcgacga tccatagctg 300 gtctgagagg atgatcagcc acattgggac tgagacacgg cccaaactc c tacgggaggc 360 agcagtgggg aatattggac aatgggcgca agcctgatcc agccatgccg cgtgagtgat 420 gaaggcctta gggttgtaaa gctctttcac cggagaagat aatgacggta tccggagaag 480 aagccccggc taacttcgtg ccagcagccg cggtaatacg aagggggcta gtgttgttcg 540 gaattactgg gcgtaaagcg cacgtaggcg gatatttaag tcaggggtga aatcccagag 600 ctcaactctg gaactgcctt tgatactggg tatcttgagt atggaagagg taagtggaat 660 tccgagtgta gaggtgaaat tcgtagatat tcggaggaac accagtggcg aaggcggctt 720 actggtccat tactgacgct gaggtgcgaa agcgtgggga gcaaacagga ttagataccc 780 tggtagtcca cgccgtaaac gatgaatgtt agccgtcggg cagtatactg ttcggtggcg 840 cagctaacgc attaaacatt ccgcctgggg agtacggtcg caagattaaa actcaaagga 900 attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgcagaa 960 ccttaccagc tcttgacatt cggggtttgg gcagtggaga cattgtcct cagttaggct 1020 ggccccagaa caggtgctgc atggctgtcg tcagctcgcg tcgtgagag ttgggttaag 1080 tcccgcaacg agcgcaaccc tcgcccttag ttgccagcat ttagttgggc actctaaggg 1140 gactgccggt gataagccga gaggaaggtg gggacgacgt caagtcctca tggcccttac 1200 g ggctgggct acacacgtgc tacaatggtg gtgacagtgg gcagcgagac agcgatgtcg 1260 agctaatctc caaaagccat ctcagttcgg attgcactct gcaaccgag tgcatgaagt 1320 tggaatcgct agtaatcgca gatcagcatg tgcggtgaat 1360 <210> 64 <211> 1288 <212> DNA <213> Unknown Organ <220> <223> Origin of the sequence: Soil organism <400> 64 tccaggaaac agctatgacc atgattacgc caagcttggt accgagctcg gatccactag 60 taacggccgc cagtgtgctg gaattcgccc ttcaggccta acacaLgcaa gtcgagcgcc 120 ccgcaagggg agcggcagac gggtgagtaa cgcgtgggaa tctacccatc cctacggaac 180 aactccggga aactggagct aataccgtat acgccctttg ggggaaagat ttatcgggga 240 tggatgagcc cgcgttggat tagctagttg gtggggtaaa ggcctaccaa ggcgacgatc 300 catagctggt ctgagaggat gatcagccac attgggactg agacacggcc caaactccta 360 cgggaggcag cagtggggaa tattggacaa tgggcgcaag cctgatccag ccatgcccgc 420 gtgagtgatg aaggtcttag gattgtaaag ctctttcacc ggagaagata atgacggtat 480

<Desc / Clms Page number 174>

 ccggagaaga agccccggct aactttcgtg ccagcagccg cggtaatacg aagggggcta 540 gcgttgttcg gaattactgg gcgtaaagcg cacgtaggcç gatatttaag tcaggggtga 600 aatcccagag ctcaactctg gaactgcctt tgatactggg tatcttgagt atggaagagg 660 taagtggaat tgcgagtgta gaggtgaaat tcgtagata tcgcaggaac accagtggcg 720 aaggcggctt actggtccat tactgacgct gaggtgcgaa agcgtgggga gcaaacagga 780 ttagataccc tggtagtcca cgccgtaaac gatgaatgtt agccgtcggc aagtttactt 840 gtcggtggcg cagctaacgc attaaacatt ccgcctgggg agtacggtcg caagattaaa 900 actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca 960 acgcgcagaa ccttaccagc ccttgacatg cccggacagc tacagagatg tagtgttccc 1020 ttcggggacc gggacacagg tgctgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg 1080 gttaagtccc gcaacgagcg caaccctcgc ccttagttgc cagcattcag ttgggcactc 1140 taaggggact gccggtgata agccgagagg aagtgggga-- gacgtcaagt cctnatggcc 1200 cttacgggct gggctacaca cgtgctacaa tgggtggtga cagtgggcag cgaaggaacg 1260 atcccgagct aatctccaaa agccatct 1288 <210> 65 <211> 1386 <212> DNA <213> Organism Inc Onnu <220> <223> Origin of sequence: Soil Organism <400> 65 cgacggccag tgaattgtaa tacgactcac tatagggcga attgggccct ctagatgcat 60 gctcgagcgg ccgccagtgt gatggatatc tgcagaattc gcccttcagg cctaacacat 120 gcaagtcgag cgggcgtagc aatacgtcag cggcagacgg gtgagtaacg cgtgggaaca 180 taccttttgg ttcggaacaa cacagggaaa cttgtgctaa taccggataa gcccttacgg 240 ggaaagattt atcgccgaaa gattggcccg cgtctgata gctagttggt agggtaatgg 300 cctaccaagg cgacgatcag tagctggtct gagaggatga tcagccacat tgggactgag 360 acacggccca aactcctacg ggaggcagca gtggggaata ttggacaatg ggcgcaagcc 420 tgatccagcc atgccgcgtg agtgatgaag gccctaggg ,: tgtaaagctc ttttgtgcgg 480 gaagataatg acggtaccgc aagaataagc cccggctaac ttcgtgccag càgccgcggt 540 aatacgaagg gggctagcgt tgctcggaat cactgggcg-: aaagggtgcg taggcgggtc 600 tttaagtcag gggtgaaatc ctggagctca actccagaac tgcctttgat actgaagatc 660 ttgagttcgg gagaggtgag tggaactgcg agtgtagagg tgaaattcgt agatattcgc 720 aagaacacca gtgggcgaag gcggctcact ggcccgatac tgacgctgag gcacgaaagc 780 gtggggagca aacaggatta gataccctgg tagtccacgc cgtaaacgat gaatgccagc 840 cgttagtggg tttactcact agtggcgcag ctaacgctt aagcattccg cctggggagt 900 acggtcgcaa gattaaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg 960 tggtttaatt cgacgcaacg cgcagaacct taccagccc tgacatgtcc aggaccggtc 1020 gcagagatgt gaccttctct tcggagcctg gagcacaggt gctgcatggc tgtcgtcagc 1080 tcgtgtcg = g agatgttggg ttaagtcccg caacgagcgc aacccccgtc cttagttgct 1140 accatttagt tgagcactct aaggagactg ccggtgataa gccgcgagga aggtggggat 1200 gacgtcaagt cctcatggcc cttacgggct gggctacaca cgtgctacaa tggcggtgac 1260 aatgggacgc taaggggcaa cccttcgcaa atctcaaaaa gcccgtctca gttcggattg 1320 ggctctgcaa ctcgagccca tgaagttgga atcgctagta atcgtggatc agcacgccac 1380 ggtgaa 1386 <210> 66 <211> 1223 <212> DNA <213> Organime Unknown <220> <223> Origin of the sequence: Soil organism <400> 66

<Desc / Clms Page number 175>

 agcggcagag ggtgagtaac gcgtgggaat ctacccatct ctacggaaca actccgggaa 60 actggagcta ataccgtata cgtccttcgg gagaaagatt tatcggagat ggatgagccc 120 gcgttggatt agctagttgg tggggtaatg gcctaccaag gcgacgatcc atagctggtc 180 tgagaggatg atcagccaca ctgggactga gacacggccc agactcctac gggaggcagc 240 agtggggaat attggacaat gggcgaaagc ccgatccagc catgccgcgt gagtgatgaa 300 ggccctaggg ttgtaaagct ctttcaacgg tgaggataat gacggtaacc gtagaagaag 360 ccccggctaa cttcgrgcca gcagccgcgg taatacgaag ggggctagcg ttgttcggaa 420 ttactgggcg taaagcgcac gtaggcggac tattaagtca ggggtgaaat cccggggctc 480 aaccccggaa ctgcctttga tactggtagt ctcgagtccg gaagaggtga gtggaattcc 540 gagtgtagag gtgaaattcg tagatattcg gaggaacacc agtggcgaag gcggctcact 600 ggtccggtac tgacgctgag gtgcgaaagc gtggggagca aacaggatta gataccctgg 660 tagtccacgc cgtaaacgat ggaagctagc cgttggcaag tttacttgtc ggtggcgcag 720 ctaacgcatt aagcttcccg cctggggagt acggtcgcaa gattaaaact caaaggaatt 780 gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg cgcagaacct 840 taccagccct tgacatcccg gtcgcggtta ccagagatgg tatccttcag ttcggctgga 900 ccggtgacag gtgctgcatg gctgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 960 cgcaacgagc gcaaccctcg cccttagttg ccagcattca gttgggcact ctaaggggac 1020 tgccggtgat aagccgagag gaaggtgggg atgacgtcaa gtcctcatgg cccttacggg 1080 ctgggctaca cacgtgctac aatggtggtg acagtgggca gcgagaccgc gaggtcgagc 1140 taatctccaa aagccatctc agttcggatt gcactctgca actcgagtgc atgaagttgg 1200 aatcgctagt aatcgcggat cag 1223 <210> 67 <211 > 1237 <212> DNA <213> Organime Unknown <220> <223> Origin of sequence: floor Organism <400> 67 cccgcagggg agtggcagag ggtgagtaac gcgtgggaat ctaccctttt ctacggaaca 60 actgagggaa acttcagcta ataccgtata cggccgagag gcgaaagatt tatcggagaa 120 ggatgagccc gcgttggatt agctagttgg tggggtaaag gcctaccaag gcgacgatcc 180 atagctggtc tgagaggatg atcagccaca ctgggactga gacacggccc agactcctac 240 gggaggcagc agtggggaat attggacaat gggcgcaagc ctgatccagc catgccgcgt 300 gagtgatgaa ggccctaggg ttgtaaagct ctttcaccgg tgaagataat gacggtaacc 360 ggagaagaag ccccggctaa cttcgtgcc has gcagccgcgg taatacgaag ggggctagcg 420 ttgttcggat ttactgggcg taaagcgcac gtaggcggac tattaagtca ggggtgaaat 480 cccggggctc aaccccggaa ctgcctttga tactggtagt cttgagttcg aaagaggtga 540 gtggaattcc gagtgtagag gtgaaattcg tagatattcg gaggaacacc agtggcgaag 600 gcggctcact ggctcgatac tgacgctgag gtgcgaaagc gtggggagca aacaggatta 660 gataccctgg tagtccacgc cgtaaactat gagagctagg cgtcgggcag tatactgttc 720 ggtggcgcag ctaacgcatt aagctcttcg cccggggagt acggtcgcaa gattaaaact 780 caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg 840 cgcagaacct taccagccct tgacatcccg atcgcggtta ccagagatgg tatccttcag 900 ttaggctgga tcggtgacag gtgctgcatg gctgtcgtca gctcgtgtcg tgagatgttg 960 ggttaagtcc cgcaacgagc gcaaccctcg cccttagttg ccatcattca gttgggcact 1020 ctaaggggac tgccggtgat aagccgagag gaaggtgggg atgacgtcaa gtcctcatgg 1080 cccttacggg ctgggctaca cacgtgctac aatggtggcg acagtgggca gcgagaccgc 1140 gaggtcgagc taatctccaa aagccatctc agttcggatt gcactctgca actcgagtgc 1200 atgaagttgg aatcgctagt aatcgtggat cagaatg 1237 < 210> 68 <211> 1346 <212> DNA <213> Unknown Organism

<Desc / Clms Page number 176>

 <220> <223> Origin of sequence: Floor Organism <400> 68 acgacgggcc agtgaattgt aatacgactc actatagggc gaattgggcc ctctagatgc 60 atgctcgagc ggccgccagt gtgatggata tccgcagaat tcgcccttca ggcctaacac 120 atgcaagtcg aacggatccc ttcggattag tggcggacgg gtgagtaaca cgcgggaacg 180 tgccctttgg ttcggaacaa ctcagggaaa cttgagctaa taccggataa gcctttcgag 240 ggaaagattt atcgccattg gagcggcccg cgtaggatta gctagttggt gaggtaaaag 300 ctcaccaagg cgacgatccc tagctggtct gagaggatga tcagccacat tgggactgag 360 acacggccca aactcctacg ggaggcagca gtggggaatc ttgcgcaatg ggcgaaagcc 420 tgacgcagcc atgccgcgtg aatgatgaag gtcttaggat tgtaaaattc tttcaccggg 480 gacgataatg acggtacccg gagaagaagc cccggctaac ttcgtgccag cagccgcggt 540 aatacgaagg gggctagcgt tgctcggaat tactgggcgt aaagggagcg taggcggata 600 gtttagtcag aggtgaaagc ccagggctca accttggaat tgcctttgat actggctatc 660 ttgagtacgg aagaggtatg tggaactccg agtgtagagg tgaaattcgt agatattcgg 720 aagaacacca gtggcgaagg cgacatactg gtccgtact gacgctgagg ctcgaaagcg 780 tggggagcaa acaggattag ataccc TGGT agtccacgct gtaaacgatg agtgctagtt 840 gtcggcatgc atgcatgtcg gtggcgcagc taacgcatta agcactccgc ctggggagta 900 cggtcgcaag attaaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt 960 ggtttaattc gaagcaacgc gcagaacctt accacctttt gacatgcccg gaccgctcca 1020 gagatggagc tttcccttcg gggactggga cacaggtgct gcatggctgt cgtcagctcg 1080 tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac cctcgctatt agttgccatc 1140 aggtttggct gggcactcta ataggaccgc cggtggtaag ccggaggaag gtggggatga 1200 cgtcaagtcc tcatggccct tacaaggtgg gctacacacg tgctacaatg gcgactacag 1260 agggctgcaa tcccgcgagg gggagccaat ccctaaaagt cgtctcagtt cggattgcac 1320 tctgcaactc gagtgcatga agttgg 1346 <210> 69 <211> 1500 <212> DNA <213> Unknown Organ <220> <223> Origin of the sequence: Soil organism <400> 69 acagctatga ccatgattac gccaagcttg gtaccgagct cggatccact agtaacggcc 60 gccagtgtgc tggaattcgc ccttcaggcc taacacatgc aagtcgaacg ccgtagcaat 120 acgagtggc agacgggtga gtaacacgtg ggaacgtgcc ctttggttcg gaacaacaca 180 gggaaacttg tgctaatacc gaataagccc ttacggggaa agattatcg ccaaaggatc 240 ggcccgcgtc tgattagcta gttggtgggg taacggccca ccaaggctac gatcagtagc 300 tggtctgaga ggatgatcag ccacactggg actgagacac ggcccagact cctacgggag 360 gcagcagtta ggaatcttgg acaatgggcg caagcctgat ccagccatgc cgcgtgagtg 420 atgaaggcct tagggttgta aagctctttc agcggggaag ataatgacgg tacccgcaga 480 agaagccccg gctaacttcg tgccagcagc cgcggraata cgaagggggc tagcgttgct 540 cggaatcact gggcgtaaag cgcacgtagg cggatcttta agtcaggggt gaaatcctgg 600 agctcaactc cagaactgcc tttgatactg gggatctcga gtccggaaga ggtgagtgga 660 actccgagtg tagaggtgaa attcgtagat attcggaaga acaccagtgg cgaaggcggc 720 tcactggtcc ggtactgacg ctgaggtgcg aaagcgtggg gagcaaacag gattagatac 780 cctggtagtc cacgccgtaa acgatggatg ctagccgttg gcgggtttac tcgtcagtgg 840 cgcagctaac gcattaagca tcccgcctgg ggagtacggt cgcaagatta aaactcaaag 900 gaattgacgg gggcccgcac aagcggtgga gcatgtggtt caattcgaag caacgcgcag 960 aaccttacca gcccttgaca tgtcccgtat ggacttcaga gatgaggtcc ttcagttcgg 1020 ctggcgggaa cacaggtgct gcatggctgt cgtcagctcg tgtcgtgag 1080 has tgttgggtta agtcccgcaa cgagcgcaac cctcgccctt agttgccatc atttagttgg gcactctaag 1140 gggactgccg gtgataagcc gcgaggaagg tggggatgac gtcaagtcct catggccctt 1200 acgggctggg ctacacacgt gctacaatgg cggtgacagt gggacgcaat ggagcaatcc 1260 tgcgcaaatc tcaaaaagcc gtctcagttc ggattggggt ctgcaactcg accccatgaa 1320

<Desc / Clms Page number 177>

 gtcggaatcg ctagtaatcg cagatcagca cgctgcggtg aatacgttcc cgggccttgt 1380 acacaccgcc caagggcgaa ttctgcagat atccatcaca ctggcggccg ctcgagcatg 1440 catctagagg gcccaattcg ccctatagtg agtcgtatta caattcactg gccgtcgttt 1500 <210> 70 <211> 1113 <212> DNA <213> Organimè Unknown <220> <223> Origin of sequence: Organization soil <400> 70 gagctaatac cgtataatga cttcggtcca aagatttatc gcctgaggat gagcccgcgt 60 cggattagct agttggtagg gtaaaagcct accaaggcga cgatccgtag ctggtctgag 120 aggatgatca gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagtg 180 gggaatattg gacaatgggc gcaagcctga tccagcaatg ccgcgtgagt gatgaaggcc 240 ttagggttgt aaagctcttt tacccgggaa gataatgact gtaccgggag aataagcccc 300 ggctaactcc gtgccagcag ccgcggtaat acggaggggg ctagcgttgt tcggaattac 360 tgggcgtaaa gcgcacgtag A ccggaattgc ggtggggtgt ggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggfggggggggg ca ggattagata ccctggtagt 600 ccacgccgta aacgatgatg actagctgtc ggggcgctta gcgtttcggt ggcgcagcta 660 acgcgttaag tcatccgcct ggggagtacg gccgcaaggt taaactcaaa gaaattgacg 720 ggggcctgca caagcggtgg agcatgtggt ttaattcgaa gcaacgcgca gaaccttacc 780 agcgtttgac atgccaggac ggtttccaga gatggattcc ttcccttacg ggacctggac 840 acaggtgctg catggctgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac 900 gagcgcaacc ctcgtcttta gttgctacca tttagttgag cactctagag aaactgccgg 960 tgataagccg gaggaaggtg gggatgacgt caagtcctca tcccttac gcgctgggct 1020 acacacgtgc tacaatggcg gtgacaacgg gcagcaaact cgcgagagtg agcaaatccc 1080 gaaaagccgt ctcagttcgg attgttctct gca 1113 <210> 71 <211> 1225 <212> DNA <213> Unknown Organ <220> <223> Origin of the sequence: Soil organism <400> 71 ggagcggcgg acgggtgagt aacgcgtggg aacgtgccct ttggtacgga acaactgagg 60 gaaacttcag ctaataccgt atgtgccctt cgggggaaag atttatcgcc attggagcgg 120 cccgcgttgg attaggtagt tggtggggta aaggcctacc aagcctacga tccatagctg 180 gtctgagagg atgatcagcc acactgggac tgagacacg g cccagactcc tacgggaggc 240 agcagtaggg aatcttgcgc aatgggcgaa agcctgacgc agccatgccg cgtgtatgat 300 gaaggtctta ggattgtaaa atactttcac cggggaagat aatgacggta cccggagaag 360 aagccccggc taacttcgtg ccagcagccg cggtaatacg aagggggcta gcgttgctcg 420 gaattactgg gcgtaaaggg cgcgtaggcg gatatttaag tcgggggtga aagcccaggg 480 ctcaaccctg gaattgcctt cgatactgga tatcttgagt tcgggagagg tgagtggaat 540 gccgagtgta gaggtgaaat tcgtagatat tcggcggaac accagtggcg aaggcgactc 600 actggcccga tactgacgct gaggcgcgaa agcgtgggga gcaaacagga ttagataccc 660 tggtagtcca cgctgtaaac gatgagtgct agttgtcggc atgcatgcat gtcggtgacg 720 cagctaacgc attaagcactccgcctgggg agtacggtcg caagattaaa actcaaagga 780 attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgcagaa 840 ccttaccacc ttttgacatg ccctgatcgc tggagagatc cagttttccc ttcggggaca 900 gggacacagg tgctgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 960

<Desc / Clms Page number 178>

 gcaacgagcg caaccctcgc cattagttgc catcattaag ttgggcactc taatgggacc 1020 gccggtggta agccggagga aggtggggat gacgtcaagt cctcatggcc cttacggggt 1080 gggctacaca cgtgctacaa tggcgactac agagggttgc aaacctgcga aggggagcta 1140 atccctaaaa gtcgtctcag ttcggattgc actctgcaac tcgagtgcat gaagtcggaa 1200 tcgctagtaa tcgcggatca gcatg 1225 <210> 72 <211> 1286 <212> DNA <213> Organime Unknown < 220> <223> sequence Origin: soil Organism <400> 72 atgattagta gcaatactaa tcgatgacga gcggcggacg ggtgagtaat acgtaggaac 60 ctgcccttaa gcgggggata actaagggaa actttagcta ataccgcata aactcgagag 120 agaaaagctg cagcaatgtg gcacttgagg aggggcctgc gtcagattag ctagttggtg 180 aggtaatagc tcaccaaggc gatgatctgt aactggtctg agaggacgac cagtcacact 240 gggactgaga cacggcccag actcctacgg gaggcagcag tggggaatat tgccgcgtg tgccgcgtgg tgccgcgtgg tgccgcgtgg gtgaagaagg ccttcgggtt gtaaagccct 360 ttaggtcggg aagaaggtta gtagaggaaa tgctattaac ttgacggtac cgacagaata 420 agcaccggca aactctgtgc cagcagccgc ggtaatacag agggtgcgag cgttaatcgg 480 atttactg gg cgtaaagggc gcgtaggcgg tgagatgtgt gtgatgtgaa agccccaggc 540 tcaacctggg aagtgcatcg caaactgtct gactggagta tatgagaggg tggcggaatt 600 tccggtgtag cggtgaaatg cgtagagatc ggaaggaacg tcgatggcga aggcagccac 660 ctggcataat actgacgctg aggcgcgaaa gcgtggggat cgaacaggat tagataccct 720 ggtagtccac gctgtaaact atgagtacta gatgttggta ggggaaccta tcggtatcga 780 agctaacgcg ataagtattc cgcctgggaa gtacggccgc aaggttgaaa ctcaaatgaa 840 ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgatgcaa cgcgaagaac 900 cttacctacc cttgacatcc tgagaatctg gcttagtagc tggagtgccg aaaggagctc 960 agagacaggt gctgcatggc tgtcgtcagc tcgtgttgtg agatgttggg ttaagtcccg 1020 taacgagcgc aacccttgcc cttagttgcc atcatttagt tggggactct aaggggaccg 1080 ccagtgatga actggaggaa ggcggggacg acgtcaagtc atcatggcct ttatgggtag 1140 ggccacacac gtgctacaat ggggcgtacg gagggtcgca aacccgcgag ggggagctaa 1200 tctcataaag cgtctcgtag tccggattgg agtctgcaac tcgactccat gaagttggaa 1260 tcgctagtaa tcgcgaatca gcattg 1286 <210> 73 < 211> 1288 <212> DNA <213> Unknown Organism <220> <223> Origin of sequence: Floor Organism <400> 73 cggggcaacc ctggcggcga gcggcgaacg ggtgagtaat gcatcggaac gtgtcctctt 60 gtgggggata accagtcgaa agactggcta ataccgcatg agatcgaaag atgaaagcag 120 gggaccgcaa ggccttgcgc gagaggagca gccgatgccg gattagctag ttggtggggt 180 aaaagcctac caaggcgacg atccgtagct ggtctgagag gacgaccagc cacactggga 240 ctgagacacg gcccagactc ctacgggagg cagcagtggg gaattttgga cagtgggggc 300 aaccctgatc cagccatgcc gcgtgtgtga agaaggcctt cgggttgtaa agcactttcg 360 gacggaacga aatcgcgcga gttaatagtt cgcgtggatg acggtaccgt aagaagaagc 420 accggctaac tacgtgccag cagccgcggt aatacgtagg gtgcgagcgt taatcggaat 480 tactgggcgt aaagtgtgcg caggcggctt cgcaagtcga gtgtgaaatc cccgagctta 540 acttgggaat tgcgctcgaa actacggagc cggagtgtgg cagaggaagg tggaattcca 600 cgtgtagcgg tgaaatgcgt agagatgtgg aggaacaccg atggcgaagg cggccttctg 660

<Desc / Clms Page number 179>

 ggccaacact gacgctcatg cacgaaagcg tggggagcaa acaggattag ataccctggt 720 agtccacgcc ctaaacgatg atgactagtt gttggaggag ttaaatcctt tagtaacgca 780 gctaacgcgt gaagtcatcc gcctggggag tacggtcgca agattaaaac tcaaaggaat 840 tgacgggggc ccgcacaagc ggtggatgat gtggtttaat tcgatgcaac gcgaaaaacc 900 ttacctaccc ttgacatgct aggaacgctg cagaaatgta gcggtgcccg aaagggaacc 960 tagacacagg tgctgcatgg ctgtcgtcag ctcgrgtcgt gagatgttgg gttaagtccc 1020 gcaacgagcg caacccctgc cattagttgc tacattcagt tgagcactct aatgggactg 1080 ccggtgacaa accggaggaa ggtggggatg acgtcaagtc ctcatggccc ttatgggtag 1140 ggctacacac gtcatacaat ggcgcgtaca gagggttgcc aacccgcgag ggggagccaa 1200 tcccagaaag cgcgtcgtag tccggattgg agtctgcaac tcgactccca tgaagtcgga 1260 atcgctagta atcgcggatc agcatgtc 1288 <210> 74 <211> 600 <212> DNA <213> Organime Unknown <220> <223> Origin of sequence: Soil organism <400> 74 cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta ttgggcgtaa 60 agcgcgcgca ggtggtttct taagtctgat gtgaaagccc acggcttaac cgtggagggt 120 cattg gaaac tgggagactt gagtgcagaa gaggaaagtg gaattccaag tgtagcggtg 180 aaatgcgtag agatttggag gaacaccagt ggcgaaggcg actttctggt ctgcaactga 240 cgctgaggcg cgaaagcatg gggagcaaac aggattagat accctggtag tccatgccgt 300 aaacgatgag tgctaagtgt tagggggttt ccgcccctta gtgctgcagc taacgcatta 360 agcactccgc ctggggagta cgaccgcaag gttgaaactc aaaggaattg acgggggccc 420 gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc gaagaacctt accaggtctt 480 gacatcccga tgancgctct agagatagag ttttcccttc ggggacattg gtgacaggtg 540 gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca 600 <210> 75 <211> 601 <212> DNA <213> Organime Unknown <220> <223> Origin of Organized sequence soil <400> 75 cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta ttgggcgtaa 60 agcgcgcgca ggtggtttct taagtctgat gtgaaagccc acggcttaac cgtggagggt 120 cattggaaac tgggagactt gagtgcagaa gaggaaagtg gaattccaag tgtagcggtg 180 aaatgcgtag agatttggag gaacaccagt ggcgaaggcg actttctggt ctgcaactga 240 cgctgaggcg cgaaagcatg gggagcaaac aggattagat accctgg tag tccatgccgt 300 aaacgatgag tgctaagtgt tagggggttt ccgcccctta gtgctgagct aacgcattaa 360 gcactccgcc tggggagtac gaccgcaagg ttgaaactca aaggaattga cgggggcccg 420 cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg aagaacctta ccaggtcttg 480 acatcccgat gacgctctag agatagagtt ttcccttcgg ggacattggt gacaggtggt 540 gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcacc 600 c '601 <210> 76 <211> 1236 < 212> DNA <213> Unknown Organism

<Desc / Clms Page number 180>

 <220> <223> Origin of sequence: Floor Organism <400> 76 tgccctgtag acggggataa cttcgggaaa ccggagctaa taccggataa tcctcttccc 60 cacatgggga agagttgaaa ggcgctttcg cgtcactaca ggatgggccc gcggtgcatt 120 agctagttgg tagggtaacg gcctaccaag gcgacgatgc atagccgacc tgagagggtg 180 atcggccaca ttgggactga gacacggccc aaactcctac gggaggcagc agtagggaat 240 cttccacaat ggacgaaagt ctgatggagc aacgccgcgt gagtgatgaa ggttttcgga 300 tcgtaaaact ctgttgtaag ggaagaacca gtacgtcagg caatggacgt accttgacgg 360 taccttatta gaaagccacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc 420 aagcgttgtc cggaattatt gggcgtaaag cgcgcgcagg tggtttctta agtctgatgt 480 gaaagcccac ggcttaaccg tggagggtca ttggaaactg ggagacttga gtgcagaaga 540 ggaaagtgga attccaagtg tagcggcgaa atgcgtagag atttggagga acaccagtgg 600 cgaaggcgac tttctggtct gcaactgacg ctgaggcgcg aaagcatggg gagcaaacag 660 gattagatac cctggtagtc catgctgtaa acgatgagtg ctaagtgtta gggggtttcc 720 gccccttagt gctgcagcta acgcattaag cactccgcct ggggagtacg accgcaaggt 780 tgaaactcaa aggaattgac gggggc CCGC acaagcggtg gagcatgtgg tttaattcga 840 agcaacgcga agaaccttac caggtcttga catcccgatg atcgctctgg agatagagtt 900 ttcccttcgg ggacattggt gacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga 960 tgttgggtta agtcccgcaa cgagcgcaac ccttaatctt agttgccatc atttagttgg 1020 gcactctaag gtgactgccg gtgataaacc ggaggaaggt ggggatgacg tcaaatcatc 1080 atgcccctta tgacctgggc tacacacgtg ctacaatgga cggtacaaag agtcgctaac 1140 tcgcgagagt atgctaatct catagaaccg ttctcagttc ggattgtagg ctgcaactcg 1200 cctacatgaa gccggaatcg ctagtaatcg cggatc 1236 <210> 77 <211> 815 <212> DNA <213> Unknown Organ <220> <223> Origin of the sequence: Soil organism <400> 77 caagcgttgt ccggaattat tgggcgtaaa gagctcgtag gcggtttgtc gcgtctgctg 60 tgaaaactcg aggctcaacc tcgggcttgc agtgggtacg ggcagactag agtgcggtag 120 gggtgactgg aattcctggt gtagcggtgg aatgcgcaga tatcaggagg aacaccgatg 180 gcgaaggcag gtcactgggc cgcaactgac gctgaggagc gaaagcatgg ggagcgaaca 240 ggattagata ccctggtagt ccatgccgta aacgttgggc actaggtgtg gggctcattc 300 cacgagttcc gtgccgcagc aaa cgcatta agtgccccgc ctggggagta cggccgcaag 360 gcttaaaact caaagaaatt gacgggggcc cgcacaagcg gcggagcatg cggattaatt 420 cgatgcaacg cgaagaacct taccaaggct tgacatacac cggaaacttc cagagatggt 480 tgccccgcaa ggtcggtgta caggtggtgc atggttgtcg tcagctcgtg tcgtgaagat 540 gttgggttaa gtcccgcaac gagcgcaacc ctcgtcctat gttgccagca cgtgatggtg 600 gggactcata ggagactgcc ggggtcaact cggaggaagg tggggatgac gtcaaatcat 660 catgcccctt atgtcttggg cttcacgcat gctacaatgg ccggtacaaa gggctgcgat 720 accgcaaggt ggagcgaatc ccaaaaagcc ggtctcagtt cggattgggg tctgcaactc 780 gaccccatga agtcggagtc gctagtaatc gcaga 815 <210> 78 <211> 826 <212> DNA <213> Unknown Organ <220> <223> Origin of the sequence: Soil organism

<Desc / Clms Page number 181>

 <400> 78 tcgtaggtgg cttgtcacgt cgggtgtgaa agcttggggc ttaactccag gtctgcattc 60 gatacgggct ggctagaggt aggtagggga gaacggaatt cctggtgtag cggtgaaatg 120 cgcagatatc aggaggaaca ccggtggcga aggcggttct ctgggcctta cctgacgctg 180 aggagcgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac gctgtaaacg 240 ttgggcgcta ggtgtgggga ccttccacgg tttccgcgcc gtagctaacg cattaagcgc 300 cccgcctggg gagtacggcc gcaaggctaa aactcaaagg aattgacggg ggcccgcaca 360 agcggcggag catgttgctt aattcgacgc aacgcgaaga accttaccaa ggcttgacat 420 cgcccggaaa gcttcagaga tggagccctc ttcggactgg gtgacaggtg gtgcatggct 480 gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgttc 540 aatgttgcca gcaacatcct tcggggtggt tggggactca ttggagactg ccggggtcaa 600 ctcggaggaa ggtggggacg acgtcaagtc atcatgcccc ttatgtcttg ggctgcaaac 660 atgctacaat ggccggtaca gagggttgcg ataccgcaag gtggagcgaa tccctaaaag 720 ccggtctcag ttcggattgg ggtctgcaac tcgaccccat gaagtcggag tcgctagtaa 780 tcgcagatca gcaacgctgc ggtgaatacg ttcccgggcc ttgtac 826 <210> 79 <211> 799 <212> AD N <213> Organime Unknown <220> <223> Origin of sequence: Floor Organism <400> 79 cgtaggcggt ttgtcgcgtc tgccgtgaaa gtccggggct caactccgga tctgcggtgg 60 gtacgggcag actagagtga tgtaggggag actggaattc ctggtgtagc ggtgaaatgc 120 gcagatatca ggaggaacac cgatggcgaa ggcaggtctc tgggcattaa ctgacgctga 180 ggagcgaaag catggggagc gaacaggatt agataccctg gtagtccatg ccgtaaacgt 240 tgggcactag gtgtggggga cattccacgt tttccgcgcc gtagctaacg cattaagtgc 300 cccgcctggg gagtacggcc gcaaggctaa aactcaaagg aattgacggg ggcccgcaca 360 agcggcggag catgcggatt aattcgatgc aacgcgaaga accttaccaa ggcttgacat 420 gaaccggaaa cacctggaaa caggtgcccc gcttgcggtc ggtttacagg tggtgcatgg 480 ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caaccctcgt 540 tctatgttgc cagcgcgtta tggcggggac tcataggaga ctgccggggt caactcggag 600 gaaggtgggg acgacgtcaa atcatcatgc cccttatgtc ttgggcttca cgcatgctac 660 aatggccggt acaaagggtt gcgatactgt gaggtggagc taatcccaaa aagccggtct 720 cagttcggat tggggtctgc aactcgaccc catgaagtcg gagtcgctag taatcgcaga 780 tca gcaacgc tgcggtgaa 799 <210> 80 <211> 1250 <212> DNA <213> Unknown Organ <220> <223> Origin of the sequence: Soil organism <400> 80 tgccagcttg ctggtggatt agtggcgaac gggtgagtaa cacgtgagta acctgccctt 60 aactctggga taagcctggg aaactgggtc taatgccgga tatgactcct catcgcatgg 120 tggggggtgg aaagcttttt gtggttttgg atggactcgc ggcctatcag cttgttggtg 180 aggtaatggc tcaccaaggc gacgacgggt agccggcctg agagggtgac cggccacact 240 gggactgaga cacggcccag acttctacgg gaggcagcag tggggaatat tgcacaatgg 300 gcgaaagcct gatgcagcga cgccgcgtga gggatgacgg ccttcgggtt gtaaacctct 360 ttcagtaggg aagaagcgaa agtgacggta cctgcagaag aagcgccggc taactacgtg 420 ccagcagccg cggtaatacg tagggcgcaa gcgttatccg gaattattgg gcgtaaagag 480

<Desc / Clms Page number 182>

 ctcgtaggcg gtttgtcgcg tctgccgtga aagtccgggg ctcaactccg gatctgcggt 540 gggtacgggc agactagagt gatgtagggg agactggaat tcctggtgta gcggtgaaat 600 gcgcagatat caggaggaac accgatggcg aaggcaggtc tctgggcatt aactgacgct 660 gaggagcgaa agcatgggga gcgaacagga ttagataccc tggtagtcca tgccgtaaac 720 gttgggcact aggtgtgggg gacattccac gttttccgcg ccgtagctaa cgcattaagt 780 gccccgcctg gggagtacgg ccgcaaggct aaaactcaaa ggaattgacg ggggcccgca 840 caagcggcgg agcatgcgga ttaattcga-- gcaacgcgag gaaccttacc aaggcttgac 900 atgaaccgga aatacctgga aacaggtgcc ccgcttgcgg tcggtttaca ggtggtgcat 960 ggttgccgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctc 1020 gttctatgtt gccagcgcgt tatggcgggg actcatagga gactgccggg gtcaactcgg 1080 aggaaggtgg ggacgacgtc aaatcatcat gccccttatg tcttgggctt cacgcatgct 1140 acaatggccg gtacaaaggg ttgcgatact gtgaggtgga gctaatccca aaaagccggt 1200 ctcagttcgg attggggtct gcaactcgac cccatgaagt cggagtcgct 1250 <210> 81 <211> 1210 <212> DNA <213> Unknown Organ <220> <223> Origin of the sequence.-Org anismus soil <400> 81 cgctaatacc ggatacggcg cgagagtctr cggactttcg cgagaaagat tcgcaaggat 60 cactgaggga cgagcctgcg gcccatcagc tagttggtga ggtaagagct caccaaggct 120 aagacgggta gctggtctga gaggatgatc agccacactg gaactgagac acggtccaga 180 ctcctacggg aggcagcagt ggggaatatt gcgcaatggg cgaaagcctg acgcagccac 240 gccgcgtgag cgatgagggc cttcgggtcg caaagctctg tggggagaga cgaataaggc 300 cggtgaagag tcggccttga cggtatctcc ttagcaagca ccggctaact ccgtgccagc 360 agccgcggta atacggaggg tgcaaacgtt gctcggaatc attgggcgta aagcgcacgt 420 aggcggcgtg ataagttggg tgtgaaagcc ctgggctcaa cccaggaagt gcattcaaaa 480 ctgtcacgct tgaatctcgg agggggtcag agaattcccg gtgtagaggt gaaattcgta 540 gatatcggga ggaataccag tggcgaaggc gctggcctgg acgaagattg acgctgaggt 600 gcgaaagcgc ggggagcaaa caggattaga taccctggta gtccgcgctg taaacgatga 660 gtgctagacg ggggaggtat tgaccccttc gctgccgaag ctaacgcgtt aagcactccg 720 cctggggagt acggtcgcaa gactaaaact caaaggaatt gacgggggcc cgcacaagcg 780 gtggagcatg tggtttaatt cgacgcaacg cgcaaaacct tacctgggtt aaatccgccg 84 0 gaacctggct gaaaggctgg ggtgccctcc ggggaatcgg tgagaaggtg ctgcatggct 900 gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccctatcg 960 tcagttgcca acattaaggt gggaactctg gcgagactgc cggtctaaac cggaggaagg 1020 tggggacgac gtcaagtcct catggccctt atgcccaggg ctacacacgt gctacaatgg 1080 ctggtacaat gagccgcaaa accgcgaggt caagctaatc tcaaaaaacc agtctcagtt 1140 cggatcggag tctgcaactc gactccgtga agctggaatc gctagtaatc gaagatcagc 1200 acgctttcgg 1210 <210> 82 < 211> 1272 <212> DNA <213> Organime Unknown <220> <223> Origin of sequence: soil Organism <400> 82 gatgccagct tgctggtgga ttagtggcga acgggtgagt aacacgtgag taacctgccc 60 ttaactctgg gataagcctg ggaaactggg tctaataccg gatatgactc ctcatcgcat 120 ggtggggggt ggaaagcttt ttgtggtttt ggatggactc gcggcctatc agcttgttgg 180 tgaggtaatg gctcaccaag gcgacgacgg gtagccggcc tgagagggtg accggccaca 240

<Desc / Clms Page number 183>

 ctgggactga gacacggccc agactcctac gggaggcagc agtggggaat attgcacaat 300 gggcgaaagc ctgatgcagc gacgccgcgt gagggatgac ggccttcggg ttgtaaacct 360 ctttcagtag ggaagaagcg aaagtgacgg tacctgcaga agaagcgccg gctaactacg 420 tgccagcagc cgcggtaata cgtagggcgc aagcgttatc cggaattatt gggcgtaaag 480 agctcgtagg cggtttgtcg cgtctgccgt gaaagtccgg ggctcaactc cggatctgcg 540 gtgggtacgg gcagactaga gtgatgtagg ggagactgga attcctggtg tagcggtgaa 600 atgcgcagat atcaggagga acaccgatgg cgaaggcagg tctctgggca ttaactgacg 660 ctgaggaacg aaagcatggg gagcgaacag gattagatac cctggtagtc catgccgtaa 720 acgttgggca ctaggtgtgg gggacattcc acgttttccg cgccgtagct aacgcattaa 780 gtgccccgcc tggggagtac ggccgcaagg ctaaaactca aaggaattga cgggggcccg 840 cacaagcggc ggagcatgcg gattaattcg atgcaacgcg aagaacctta ccaaggcttg 900 acatgaaccg gaaatacctg gaaacaggtg ccccgcttgc ggtcggttta caggtggtgc 960 atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc 1020 tcgttctatg ttgccagcgc gttatggcgg ggactcatag gagactgccg gggtcaactc 1080 ggaggaaggt ggggacg acg tcaaatcatc atgcccctta tgtcttgggc ttcacgcatg 1140 ctacaatggc cggtacaaag ggttgcgata ctgtgaggtg gagctgatcc caaaaagccg 1200 gtcccagttc ggattggggt ctgcaactcg accccatgaa gtcggagtcg ctagtaatcg 1260 cagatcagca ac 1272 <210> 83 <211> 1247 <212> DNA <213> Organime Unknown <220> <223> Origin of the sequence : floor Organism <400> 83 tgtttagtag caatactaaa tgatgacgag cggcggacgg gtgaggaaca cgtaggaacc 60 tgcccaagag agggggacaa ccaagggaaa ctttggctaa taccgcataa tctctacgga 120 gaaaagttgc ccgtaagggt ggcgcttttg gaggggcctg cgtccgatta gttagttggt 180 gaggtaatag ctcaccaaga ctgtgatcgg taactggtct gagaggacga ccagtcacac 240 tgggactgag acacggccca gactcctacg ggaggcagca gtggggaatc ttggacaatg 300 ggggcaaccc tgatccagcg atgccgcgtg ggtgaagaag gccttcgggt tgtaaagccc 360 tttaggcggg gaagaaggat atgggatgaa taagcctgta ttttgacggt acccgcagaa 420 taagcaccgg caaactctgt gccagcagcc gcggtaatac agagggtgcg agcgttaatc 480 ggatttactg ggcgtaaagg gcgcgtaggc ggttgtgtga gtgtgatgtg aaagccccgg 540 gctcaacctg ggaagtgcat cgcaaacgac acaactgg ag tatatgagag ggtggcggaa 600 tttccggtgt agcggtgaaa tgcgtagaga tcggaaggaa cgtcgatggc gaaggcagcc 660 acctggcata atactggcgc tgaggcgcga aagcgtgggg agcgaacagg attagatacc 720 ctggtagtca cgcccgtaaa cgatgagaac tagatgttgg agggggaacc cttcagtatc 780 gaagctaacg cgataagttc tccgcctggg aagtacagtc gcaagactga aactcaaaag 840 aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgatgc aacgcgaaga 900 accttacctg cccttgacat cctgcgaatc ttgccgagag gtgagagtgc cgcagggagc 960 gcagagacag gtgctgcatg gctgtcgtca gctcgtgttg tgagatgttg ggttaagtcc 1020 cgtaacgagc gcaacccttg tccttagttg ccatcattta gttggggact ctaaggagac 1080 cgccggtgat gaaccggagg aaggcgggga cgacgtcaag tcatcatggc ctttatgggt 1140 agggctacac acgtgctaca atggggcgta cagagggtcg ccaacccgcg agggggagcc 1200 aatctcttaa agcgtctcgt agtccggart ggagtctgca actcgac 1247 <210> 84 <211> 1292 <212> DNA <213> Organime Unknown <220> <223> Origin of the sequence: Soil organism

<Desc / Clms Page number 184>

 <400> 84 ggctcgcaag agcaaccggc gaacgggtgc gtaacacgtg aacaacctgc cctcgtgtgg 60 gggatagccg ggctaacgcc cgggtaatac cgcatacgtt ctctctgggg agtcctgggg 120 agaggaaagc tccggcgcac ggggaggggt tcgcggccta tcagctagtt ggcggggtaa 180 tggcccacca aggcgacgac gggtagctgg tctgagagga tggccagcca cattgggact 240 gagagacggc ccagactcct acgggaggca gcagtgggga atcttgcgca atggccgaaa 300 ggctgacgca gcgacgccgc gtgtgggagg acgcctttcg gggtgtaaac cactgttgcc 360 cgggacgaac agcctctttc gagaggtctg acggtaccgg gtgaggaagc accggctaac 420 tccgtgccag cagccgcggt aatacggagg gtgcgagcgt tgtccggaat cattgggcgt 480 aaagggcgcg taggtggccc ggtcagttcg tggtgaaagc gcggggctca accctgcgtc 540 ggccatgaat actgccgcgg ctggagcact gtagaggcag gcggaattcc gggtgtagcg 600 gtggaatgcg tagagatccg gaagaacacc ggtggcgaag gcggcctgct gggcagtagc 660 tgacactgag gcgcgacagc gtggggagca aacaggatta gataccctgg tagtccacgc 720 cgtaaacgat gggcactagg cgcttggggg agcgaccccc cgagggccgg cgctaacgca 780 ttaagtgccc cgcctgggga gtacggccgc aaggctgaaa ctcaaaggaa ttgacggggg 840 cccgcacaag c ggtggagca tgtggtttaa ttcgacgcaa cgcgaagaac cttacctagg 900 cttgacatac acgggaaacc ggtcagaaac ggccggccct cttcggagcc cgtgcacagg 960 tgctgcatgg ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg 1020 caacccctgt ctctagttgc cagcgcgtca tggcggggac tctagagaga ctgccggtgc 1080 caaaccggag gaaggtgggg atgacgtcaa gtcatcatgg tccttacgtc tagggctaca 1140 cacgtgctac aatggcgggg acagagggtc gcgagccggc aacggcaagc caatcccgta 1200 aaccccgcct cagttcggat tgtcgtctgc aactcgacgg catgaagctg gaatcgctag 1260 taatcgtgga tcagctacgc cacggtgaat ac 1292 <210> 85 <211> 1300 <212> DNA <213> Unknown Organ <220> <223> Origin of the sequence:: Soil organism <400> 85 tcccttcggg agcaagtaca gcggcgaacg ggtgagtaac acgtaggtaa cctaccctgg 60 agactgggat aacctgccga aaggcgggct aataccagat aagaccacga gggctgcggc 120 ccttggggca aaaggtggcc tctacttgta agctaccact ccgggatggg cctgcgcgcc 180 attagctagt tggcggggta acggcccacc aaggcagaga tggctagctg gtctgagagg 240 atggccagcc acacagggac tgagacacgg cccagactcc tacgggaggc agcagtgggg 300 aatattgcg c aatgggcgaa agcctgacgc agcgacgccg cgtgggtgat gaaggccttc 360 gggtcgtaaa gccctgtcaa gagggacgaa accttgtcga cctaacacgt cggcaacctg 420 acggtacctc tgaaggaagc accggctaac tccgtgccag cagccgcggt aatacggagg 480 gtgcgagcgt tgttcggaat tactgggcgt aaagcgcgtg taggcggcct cttcagtctg 540 gtgtgaaagc ccggggctca accccggaag tgcattggat actgggaggc tggagtaccg 600 gagaggaggg tggaattcct ggtgtagcgg tgaaatgcgt agatatcagg aggaacacct 660 gtggcgaagg cggccctctg gacggatact gacgctgaga cgcgaaagcg tggggagcaa 720 acaggattag ataccctggt agtccacgct gtaaacgatg ggcactaggt gttcggggta 780 ttgaccccct gagtgccgca gctaacgcat taagtgcccc gcctggggaa tacggccgca 840 aggttaaaac tcaaaggaat gacgggggc ccgcacaagc ggtggagcat gtggtttaat 900 tcgacgcaac gcgaagaacc ttacctgggc tagacaacat cggacagcct cagaaatgag 960 gtctccccgc aaggggccgg tggttcaggt gctgcatggc tgtcgtcagc tcgtgtcgtg 1020 agatgttggg ttaagtcccg caacgagcgc aacccctgtc tctagttgct accattcagt 1080 tgagcactct agagagactg cccngtgtta aacgggagga aggtggggac gacgtcaagt 1140 cctcatggcc cttatgtcca gggct acaca cgtgctacaa tgggcgatac aaagggctgc 1200 gaacccgcga ggggaagcca atcccaaaaa gtcgctctca gttcggattg gagtctgcaa 1260 ctcgactcca tgaaggcgga atcgctagta atcgcggatc 1300 <210> 86 <211> 1186

<Desc / Clms Page number 185>

 <212> DNA <213> Organime Unknown <220> <223> Origin of sequence: Floor Organism <400> 86 caatgggcag cggcggacgg gtgagtaaca cgtgggaatg tacctttcgg tgcggaacaa 60 ctcagggaaa cttgagctaa tgccgcatac gcccttacgg ggaaagattt atcgccgaaa 120 gatcagcccg cgttggatta gctagttggt gaggtaatgg cccaccaagg cgacgatcca 180 tagctggttt gagagaacga ccagcctcac tgggactgag acacggccca gactcctacg 240 ggaggcagca gttgggaatc ttggacaatg ggggaaaccc tgatccagcc atgccgcgtg 300 agtgatgaag gccttcgggt tgtaaaactc tttcgacggg gacgataatg acggtacccg 360 tagaagaagc tccggctaac ttcgtgccag cagccgcggt aatacgaagg gggctagcgt 420 tgttcggaat tactgggcgt aaagcgtgcg caggcggcta tccaagtcag tggtgaaagc 480 ccggagctca actccggaat tgccattgaa actgtttagc ttgagtacga gagaggtgag 540 tggaataccc agtgtagagg tgaaattcgt agatattggg tagaacaccg gtggcgaagg 600 cggctcactg gctcgtaact gacgctcagg CACGACAGCG TAGGAGACCA TAGGAGACTA TAGGAGAGAG TAGGAGAGAG TAGGAGATAG TAGGAGAG TAGGAGAG TAGGAGAG TAGGAGAG TAGGAGAG TAGGAGAG TAGGAGAG TAGGAGAG TAGGAGAG TAGGAGAG TAGGAGAG TAGGAGAG TAGGAGAG TAGGAGAG TAGGAGAG TAGGAGAG 780 agaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc gacgcaacgc 840 gcagaacctt accagggttt gacatcctgt gctcgccggt gaaagccggt tttcccgcaa 900 gggacgcaga gacaggtgct gcatggctgt cgtcagctcg tgtcgtgaga tgttgggtta 960 agtcccgcaa cgagcgcaac cctcgccttt agttgccatc attcagttgg gcactctaga 1020 gggaccgccg gcgacaagcc ggaggaaggt ggggatgacg tcaagtcccc atggccctta 1080 caccctgggc tacacacgtg ctacaatggc ggtgacagtg ggcacgagct cgcgagagtc 1140 agctaatccc aaaaaaccgt cccagttcag attgcactct gcaact 1186 < 210> 87 <211> 1454 <212> DNA <213> Organism Unknown <220> <223> Origin of the sequence: Soil organism <400> 87 cgacggccag tgaattgtaa tacgactcac tatagggcga attgggccct ctagatgcat 60 gctcgagcgg ccgccagtgt gatggatatc tgcagaattc gcccttcagg cctaacacat 120 gcaagtcgag cgagaaaggg cgcttcggcg cctgagtaca gcggcgcacg ggtgcgtaac 180 acgtgggcaa tctgtccttg agatggggat aacccagcga aagttgggct aataccgaat 240 aagactacag gaggcaactc ccgtggttaa agggtgctct ctgcggggag catgcgcttg 300 aggaggagcc cgcggcctat cagctagttg gtagggtcac ggcctac caa ggcgaagacg 360 ggtagctggt ctgagaggat gaccagccac acggggactg agacacggcc ccgactccta 420 cgggaggcag cagtggggaa tattgggcaa tgggggaaac cctgacccag cgacgccgcg 480 tgggtgatga aggccttcgg gtcgtaaagc cctgtcgggc ggaacgaagg ttctcacggc 540 aaatagccgt gagaggtgac ggtaccgccg aaggaagcac cggccaactc cgtgccagca 600 gccgcggtaa gacggagggt gcaagcgttg ctcggaatca ctgggcgtaa agggtgcgta 660 ggcggtctcg caagtctggc gtgaaagccc aaggctcagc cttggaagtg cgctcgaaac 720 tgcgaggctg gagtgccgga ggggagagtg gaattcccgg tgtagcggtg aaatgcgtag 780 agatcgggag gaataccggt ggcgaaagcg actctctgga cggcaactga cgctgaggca 840 cgaaagcgtg gggagcaaac aggattagat accctggtag tccacgccgt aaacgatgga 900 cactaggtgt cgggggtatc cactccctcg gtgccgccgc taacgcagta agtgtcccgc 960 ctgggaagta cggtcgcaag attaaaactc aaaggaattg acgggggccc gcacaagcgg 1020 tggagcatgt ggttcaattc gatgcaacgc gaagaacctt acctgggttt gacatctggc 1080 gaatctctgg gaaaccagag agtgcccgca ggggagcgcc aagacaggtg ctgcatggct 1140 gtcgtcagct cgtgccgtga ggtgttgggt taagtcccgc aacgagcgca acccttaccc 12 00 ttagttgccc ccgggtcaag ccgtggcact ccaagggaac tgcccgtgtt aagcgggagg 1260 aaggtgggga cgacgtcaag tcatcatggc ctttatatcc agggctacac acgtgctaca 1320

<Desc / Clms Page number 186>

 atggctgggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggggg > 88 cccttcgggg agcgagtaca gcggcgaacg ggtgagtaac acgtaggtaa cctaccctgg 60 tgactgggat aacttgccga aaggcgggct aataccagat aagaccacga gggctgcggc 120 ctttggggta aaagatggcc tctgcttgca tgctatcacg ccgggatggg cctgcgcgcc 180 attagctagt tggtgaggta acggctcacc aaggcagaga tggctagctg gtctgagagg 240 atggccagcc acactgggac tgagacacgg cccagactcc tacgggaggc agcagtgggg 300 aatattgcgc aatgggcgaa agcctgacgc agcgacgccg cgtgggtgat gaaggccttc 360 gggtcgtaaa gccctgtcaa gagggacgaa acctcgccga cccaatacgt cggcgacctg 420 acgtacctc tgaaggaagc accggctaac tccgtgccag cagccgcggt aatacggagg 480gtgcaagcgt tgttcggaat cactgggcgt aaagcgcgtg taggcggcct tcttagtctg 540gtgtgaaagc ccggggctca accccggaag agcattgat actggaaggc tggagtaccg 600 gagaggaggg tggaattcct ggtgtagcgg tgaaatgcgt agatatcagg aggaacaccg 660 gtggcgaagg cggccctctg gacggatact gacgctgaga cgcgacagcg tggggagcaa 720 acaggattag ataccctggt agtccacgcc gtaaacgatg ggtactaggt gttcggggta 780 ttgaccccct gagtgccgca gctaacgcat taagtacccc gcctggggac tacggccgca 840 aggctaaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat 900 tcgacgcaac gcgaagaacc ttacctgggc tagacaacac tggacagccc cagaaatggg 960 gtcttcccgc aagggactgg tggttcaggt gctgcatggc tgtcgtcagc tcgtgtcgtg 1020 agatgttggg ttaagtcccg caacgagcgc aacccctgtc tctagttgct accattaagt 1080 tgagcactct agagagactg cccgtgttaa acgggaggaa ggtggggacg acgtcaagtc 1140 ctcatggccc ttatgtccag ggctacacac gtgctacaat ggacagtaca aagggctgcg 1200 aacccgtgag ggggagccaa tcccaaaaag ctgttctcag ttcggattgg agtctgcaac 1260 tcgactccat gaaggcggaa tcgctagtaa tcgcggaca gcatgcc 1307 <210> 89 <211> 1305 <212> DNA <213> Organime Unknown <220 > <223> Origin of the sequence: Soil organism <400> 89 gggagcaatc cccaagtaga gcggcgaacg ggtgagtaac gcgtgggtaa tctgcctccg 60 agtggg GAAC aacatcggga aactggtgct aataccgcat aacatcgttg ggtcttcgga 120 tctgacgatc aaagccgggg accgcaaggc ctggcgcttg gagaggagcc cgcgtccgat 180 tagctagttg gtggggtaat ggcccaccaa ggcttcgatc ggtagccggc ctgagagggc 240 ggacggccac actgggactg agacacggcc cagactccta cgggaggcag cagtggggaa 300 tttttcgcaa tgggcgaaag cctgacgaag caacgccgcg tggaggatga gggccttcgg 360 gtcgtaaact cctgtcgacc gggacgaaag taggatggcc taatacgccg atctattgac 420 tgtaccggtg gaggaagcca cggctaactc tgtgccagca gccgcggtaa tacagaggtg 480 gcaagcgttg ttcggaatta ctgggcgtaa agggcgcgta ggcggcttgg tcagtcccgt 540 gtgaaatccc tcggctcaac tgaggaactg cacgggaaac tgcctggctt gagttcggga 600 gagggaagtg gaattccggg tgtagcggtg aaatgcgtag atatccggag gaacaccggt 660 ggcgaaggcg gcttcctgga ccgacactga cgctgaggcg cgaaagctag gggagcaaac 720gggattagat accccggtag tcctagctgt aaacgatgag tgctgggtgt agggggtatc 780

<Desc / Clms Page number 187>

 aaccccccct gtgccgaagc taacgcatta agcactccgc ctggggagta cggtcgcaag 840 gctgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggttcaattc 900 gacgcaacgc gaagaacctt accggggttt gaactgtacg ggacagctct agagatagag 960 tcttccttcg ggacccgtac agaggtgctg catggctgtc gtcagctcgt gtcgtgagat 1020 gttgggttaa gtcccgcaac gagcgcaacc cttgcctcct gttgccatca ggtaaagctg 1080 ggcactctgg agagactgcc ggtgataaac cggaggaagg tggggatgac gtcaagtcct 1140 catggccttt atgccccggg ctacacacgt gctacaatgg ccggtacaaa gggtcgcaaa 1200 accgcgaggt ggagctaatc ccaaaaagcc ggtcccagtt cggattgcag tctgcaactc 1260 gactgcatga agttggaatc gctagtaatc gcggatcagc atgcc 1305 <210> 90 <211> 1299 <212> DNA <213> Unknown Organ <220> <223> Origin of the sequence: Soil organism <400> 90 gggctttcgg gtcctgagta aagtggcgaa cgggtgagta acgcgtaggt aacctgacct 60 cgagtgtgga ataacctggc gaaagccggg ctaataccgc atgacgtctt cgggtcttcg 120 gacttgagga ccaaaggtgg cgagctttga gcgctgtcgc tcgagaaggg gcctgcgtcc 180 cattagctag ttggtgggg gatggcctac caaggcgacg atgggtagcc g ggctgagag 240 gctgtccggc cacactggaa ccgagacacg gtccagactc ctacgggagg cagcagtggg 300 gaatcttgcg caatggggga aaccctgacg caacgacgcc gcgtgggcga tgaaggcctt 360 cgggtcgtaa agccctgtcg agcgggacga accgtgcgag ctctaacata gctcgtgcct 420 gacggtaccg ctagaggaag ccccggctaa ctccgtgcca gcagccgcgg taatacggag 480 ggggctagcg ttattcggaa ttattgggcg taaagggcgt gtaggcggct ctgtgtgtcc 540 catgtgaaag ccctcggctc aaccggggaa ctgcatggga aactgcggag cttgagtccg 600 ggagaggtga gtggaattcc cagtgtagcg gtgaaatgcg tagatattgg gaggaacacc 660 agtggcgaag gcggctcac ggaccggtac tgacgctgag acgcgaaagc caggggagca 720 aacgggatta gataccccgg tagtcctggc tgtaaacgat gagcacttgg tgtggcgggt 780 atcgacccct gccgtgctga agctaacgca ttaagtgctc cgcctgggga gtacggccgc 840 aaggctgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggttcaa 900 ttcgacgcaa cgcgaagaac cttacctggg tttgaactgc aggtgacagc ccctgaaagg 960 gggtcttcct tcgggacacc tgtagaggtg ccgcatggct gtcgtcagct cgtgtcgtga 1020 gatgttgggt taagtcccgc aacgagcgca acccctactc ctagttgcca gcggctcggc 1080 cggga actct agggggaccg ccggtgataa accggaggaa ggtggggatg acgtcaagtc 1140 ctcatggcct ttatgtccag ggctacacac gtgctacaac ggacggtaca aagggctgcg 1200 aaggcgcgag ccggagccaa tcccaaaaag ccgttctcca gtgcggattg cagtctgcaa 1260 ctcgactgca tgaaggtgga atcgctagta atcgcggat 1299 <210> 91 <211> 1296 <212> DNA <213> Organime Unknown <220> <223> Origin of the sequence: floor Organism <400> 91 atgtctggta gcaataccag atgatggcaa gtggcggacg ggtgagtaat acgtagggat 60 ctgcccagaa gagggggaca acccggggaa actcgggcta ataccgcata ctattctgag 120 gaagaaagct tggcgcaagc caggcgcttt tggaggaacc tacgtccgat tagctagttg 180 gtgaggtaaa ggctcaccaa ggcagagatc ggtagctggt ctgagaggat gaccagccac 240 actgggactg agacacggcc cagactccta cgggaggcag cagtggggaa tattggacaa 300 tgggggcaac cctgatccag cgatgccgcg tgtgtgaaga aggccttcgg gttgtaaagc 360 actttagttg gggaagaagt aatgtttttt aaLagagagc attgttgacg gtacccaaag 420

<Desc / Clms Page number 188>

 aataagcacc ggctaactct gtgccagcag ccgcggtaat acagagggtg caagcgttaa 480 tcggagttac tgggcgtaaa gggcgcgtag gcggtgttgc aagtgagatg tgaaatccct 540 gggcttaacc taggaaccgc attttagact gcaatgctag agtacagtag agggtagtgg 600 aatttccggt gtagcggtga aatgcgtaga gatcggaagg aacaccagtg gcgaaggcga 660 ctacctggac tgacactgac gctgaggcgc gagagcgtgg ggagcaaaca ggattagata 720 ccctggtagt ccacgctgta aacgatgaga actagatgtt ggtgcgcgcg agcgcacaag 780 tatcgaagct aacgcgataa gttctccgcc tggggagtac ggccgcaagg ttaaaactca 840 aaggaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg atgcaacgcg 900 aggaacctta cctacccttg acatccacag aatttgatag agatatcgaa gtgccgaaag 960 gaactgtgag acaggtgctg catggctgtc gtcagctcgt gttgtgagat gttgggttaa 1020 gtcccgtaac gagcgcaacc cttatcctta gttgccaaca cgtaatggtg gggactctaa 1080 ggagactgcc ggtgaagaac cggaggaagg tggggacgac gtcaagtcat catggccttt 1140 atgggtaggg ctacacacgt gctacaatgg ggcgtacaga gggttgccaa cctgcgaagg 1200 ggagccaatc ccggaaagcg cctcgtagtc cagattgaag tctgcaactc gacttcatga 1260 agtcggaatc GCTA gtaatc gcgaatcaga acgtcc 1296 <210> 92 <211> 1250 <212> DNA <213> Unknown Organ <220> <223> Origin of the sequence: Soil organism <400> 92 gtctggtagc aataccagat gatgcaagt ggcggacggg tgagtaatac gtagggatct 60 gcccagaaga gggggacaac ccggggaaac tcgggctaat accgcatact attctgagga 120 aaaaagcttg gcgcaagcca ggcgcttttg gaggaaccta cgtccgatta gctagttggt 180 gaggtaaagg ctcaccaagg cagagatcgg tagctggtct gagaggatga ccagccacac 240 tgggactgag acacggccca gactcctacg ggaggcagca gtggggaata ttggacaatg 300 ggggcaaccc tgatccagcg atgccgcgtg tgtgaagaag gccttcgggt tgtaaagcac 360 tttagttggg gaagaagtaa tgttttttaa tagagagcat tgttgacggt acccaaagaa 420 taagcaccgg ctaactctgt gccagcagcc gcggtaatac agagggtgca agcgttaatc 480 ggagttactg ggcgtaaagg gcgcgtaggc ggtgttgcaa gtgagatgtg aatccctgg 540 gcttaaccta ggaaccgcat tttagactgc aatgctagag tacagtagag ggtagtggaa 600 tttccggtgt agcggtgaaa tgcgtagaga tcggaaggaa caccagtggc gaaggcgact 660 acctggactg acactgacgc tgaggcgcga gagcgtgggg agcaaacagg attagatacc 720 ctggtagtcc acgc tgtaaa cgatgagaac tagatgttgg tgcgcgcgag cgcacaagta 780 tcgaagctaa cgcgataagt tctccgcctg gggagtacgg ccgcaaggtt aaaactcaaa 840 ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgat gcaacgcgaa 900 gaaccttacc tacccttgac atccacagaa tttgatagag atatcgaagt gccgaaagga 960 actgtgagac aggtgctgca tggctgtcgt cagctcgtgt tgtgagatgt tgggttaagt 1020 cccgtaacgg gcgcaaccct tatccttagt tgccaacacg taatggtggg gactctaagg 1080 agactgccgg tgaagaaccg gaggaaggtg gggacgacgt caagtcatca tggcctttat 1140 gggtagggct acacacgtgc tacaatgggg cgtacagagg gttgccaacc tgcgaagggg 1200 agccaatccc ggaaagcgcc tcgtagtcca gattgaagtc tgcaactcga 1250 <210> 93 <211> 1545 <212> DNA <213> Unknown Organ <220> <223> Origin of the sequence: Soil organism <400> 93 ccaggaaaca gctatgacca tgattacgcc aagcttggta ccgagctcgg atccactagt 60 aacggccgcc agtgtgctgg aattcgccct tcaggcctaa cacatgcaag tcgaacggca 120

<Desc / Clms Page number 189>

 gcacagggga gcttgctccc tgggtggcga gtggcggacg ggtgaggaat acatcggaat 180 ctgcccagtc gtgggggata acctcgggaa accgggacta ataccgcata cgaccttagg 240 gtgaaagcgg aggaccgcaa ggcttcgcgc gattggatga gccgatgtcg gattagcttg 300 ttggcggggt aacggcccac caaggcgacg atccgtagct ggtctgagag gatgatcagc 360 cacactggaa ctgagacacg gtccagactc ctacgggagg cagcagtggg gaatattgga 420 caatgggcgc aagcctgatc cagccatgcc gcgtgagtga agaaggcctt cgggttgtaa 480 agctcttttg tccggaaaga aaagctttcg gttaataccc ggaagtcctg acggtaccgg 540 aagaataagc accggctaac ttcgtgccag cagccgcggt aatacgaagg gtgcaagcgt 600 tactcggaat tactgggcgt aaagcgtgcg taggtggttt gttaagtctg atgtgaaagc 660 cctgggctca acctgggaat tgcactggat actggcaggc tagagtgcgg tagaggatgg 720 cggaattccc ggtgtagcag tgaaatgcgt agagatcggg aggaacatct gtggcgaagg 780 cggccatctg gaccagcact gacactgagg cacgaaagcg tggggagcaa acaggattag 840 ataccctggt agtccacgcc ctaaacgatg cgaactggat gttgggagca actaggctct 900 cagtatcgaa gctaacgcgt taagttcgcc gcctggggag tacggtcgca agactgaaac 960 tcaaaggaat tgacggggg c ccgcacaagc ggtggagtat gtggtttaat tcgatgcaac 1020 gcgaagaacc ttacctggcc ttgacatcca cggaacttac cagagatggt ttggtgcctt 1080 cggnaaccgt gagacaggtg ctgcatggct gtcgtcagct cgtgtcgtga gatgttgggt 1140 taagtcccgc aacgagcgca acccttgtcc ttagttgcca gcacgtaatg gtgggaactc 1200 taaggagact gccggtgaca aaccggagga aggtggggat gacgtcaagt catcatggcc 1260 cttacggcca gggctacaca cgtactacaa tggtcggtac agagggttgc aaagccgcga 1320 ggtagagcca atcccagaaa accgatccca gtccggatcg aagtctgcaa ctcgacttcg 1380 tgaagtcgga atcgctagta atcgcggatc agaatgccgc ggtgaatacg ttcccgggcc 1440 ttgtacacac cgcccaaggg cgaattctgc agatatccat cacactggcg gccgctcgag 1500 catgcatcta gagggcccaa ttcgccctat agtgagtcgt attac 1545 <210> 94 <211> 1549 <212> DNA <213> Unknown Organ <220> <223> Origin of the sequence: Soil organism < 400> 94 ttttaaaccg acggccagtg aattgtaata cgactcacta tagggcgaat tgggccctct 60 agatgcatgc tcgagcggcc gccagtgtga tggatatctg cagaattcgc ccttcaggcc 120 taacacatgc aagtcgagcg gcagcgcggg gcaacctggc ggcgagcggc ggacgggtga 180 g gaatgcatc ggaatctacc ctgtcgtggg ggataacgta gggaaactta cgctaatacc 240 gcatacgacc gagaggtgaa agtgggggac cgcaaggcct cacgcgatag gatgagccga 300 tgccggatta gctagttggt gaggtaaagg ctcaccaagg cgacgatccg tagctggtct 360 gagaggatga tcagccacat tgggactgag acacggccca aactcctacg ggaggcagca 420 gtggggaata ttggacaatg ggcgcaagcc tgatccagcc atgccgcgtg tgtgaagaag 480 gccttcgggt tgtaaagcac ttttgttcgg gaagaaatcg tgcgggttaa tacccagtac 540 ggatgacggt accgaaagaa taagcaccgg ctaacttcgt gccagcagcc gcggtaatac 600 gaagggtgca agcgttactc ggaatcactg ggcgtaaagc gtgcgtaggc ggttggttaa 660 gtctgctgtg aaagccctgg gctcaacctg ggaactgcag tggatactgg ccagctagag 720 tgtgatagag gatggtggaa ttcccggtgt agcggtgaaa tgcgtagaga tcgggaggaa 780 caccagtggc gaaggcggcc atctggatca acactgacgc tgaggcacga aagcgtgggg 840 agcaaacagg attagatacc ctggtagtcc acgccctaaa cgatgcgaac tggacgttgg 900 gagcaacttg gctctcagtg tcgaagctaa cgcgctaagt tcgccgcctg gggagtacgg 960 tcgcaagact gaaactcaaa ggaattgacg ggggcccgca caagcggtgg agtatgtggt 1020 ttaattcgat gcaacgcga has gaaccttacc tggccttgac atccacggaa cttaccagag 1080 atggtttggt gccttcggaa ccgtgagaca ggtgctgcat ggctgtcgtc agctcgtgtc 1140 gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gtccttagtt gccagcacgt 1200 aatggtggga actctaagga gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc 1260 aagtcatcat ggcccttacg gccagggcta cacacgtact acaatggtcg gtacaagagg 1320 gttgcaaagc ccgcgaggta gagccaatcc cagaaaaccc gatcccagtc ccggatcgaa 1380 gtctgcaact cgacttcgtg aagtcggaat cgctagtaat cgcggatcag aatgccgcgg 1440 tgaatacgtt cccgggcctt gtacacaccg cccaagggcg aattccagca cactggcggc 1500

<Desc / Clms Page number 190>

 1549 <210> 95 <211> 1276 <212> DNA <213> Unknown organism <220> <223> Origin of the sequence: Soil organism <400> 95 ctggcggcga ataccgcata cgacctgagg gtgaaagcag gggatcgcaa 120 gaccttgcgc gattggatga gccgatgcc gattagctag ttggtgaggt aaaggctcac 180 caaggcgacg atcggtagct ggtctgagag ggtgatcagc cacactggaa ctgagacacg 240 gtccagactc ctacgggagg cagcagggg gaatattgga caatgggcgc aagcctgatc 300 cagccatgcc gcgtgtgtga agaaggcctt cgggttgtaa agcacttttg ttcgggaaga 360 aatcttccga gttaatacct cgggaggatg acggtaccgg aagaataagc accggctaac 420 ttcgtgccag cagccgcggt aatacgaagg gtgcaagcgt tactcggaat tactgggcgt 480 aaagcgtgcg taggtggttc gttaagctg ccgtgaaagc cccgggctca acctgggaat 540 tgcggtggat actggcggac tagagtgcgg tagagggtgg tggaattccc ggtgtagcag 600 tgaaatgcgt agagatcggg aggaacact gtggcgaagc ggccacctgg accagcactg 660 acactgaggc acgaaagcgt ggggagcaaa caggattaga taccctggta gtcca cgccc 720 taaacgatgc gaactggacg ttgggagcaa ctaggctctc agtgtcgaag ctaacgcgtt 780 aagttcgccg cctggggagt acggtcgcaa gactgaaact caaaggaatt gacgggggcc 840 cgcacaagcg gtggagtgtg tggtttaatt cgatgcaacg cgaagaacct tacctggcct 900 tgacatccac ggaatccttt agagatagag gagtgccttc gggaaccgtg agacaggtgc 960 tgcatggctg tcgtcagctc gtgtcggag atgttgggtt aagtcccgca acgagcgcaa 1020 cccttgtcct tagttgccag cgcgtaatgg cgggaactct aaggagactg ccggtgacaa 1080 accggaggaa ggtggggatg acgtcaagtc atcatggccc ttacggccag ggctacacac 1140 gtactacaat ggtggggaca gagggtcgcg aagccgcgag gtggagccaa tcccagaaac 1200 cccatcctag tccggatcgg agtctgcaac tcgactccgt gaagtcggaa tcgctagtaa 1260 tcgcggtcag catgcc 1276 <210> 96 <211> 1306 <212> DNA <213> Unknown Organ <220> <223> Origin of the sequence: Soil organism < 400> 96 cagggatcag tagagtggca aacggggag taacgcgtgg gcgacctacc ttcgagtggg 60 ggataacctt ccgaaaggag ggctaaacc gcatgacatc ccgtgtttgg atacacggac 120 atcaaagccg gggatcgcaa gacctggcgc ttggagaggg gcccgcgtcc gattagctag 180 ttggtgaggt cacggctcac caaggcccg atcggtatcc ggcctgagag ggcggacgga 240 cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtggg gaattgttcg 300 caatgggcgc aagcctgacg acgcaacgcc gcgtggagga tgaagacctt cgggtcgtaa 360 actcctttcg accgagatga agacccçccg gcctaatacg ccggcggatt gacagtatcg 420 agggaagaag ccccggctaa ctccgtgcca gcagccgcgg taatacgggg ggggcaagcg 480 ttgttcggaa ttactgggcg taaagggttc gtaggtggct cgctaagtca gacgtgaaat 540 ccctcagctc aactggggaa ctgcgtctga gactggcaag cttgagtgca ggagaggaac 600 gcggaattcc aggtgtagcg gtgaaagcg tagatatctg gaggaacacc ggtggcgaag 660 gcggcgttct ggactgcaac tgacactgag gaacgaaagc taggggagca aacgggatta 720 gataccccgg tagtcctagc cctaaacgat gaatgcttgg tgtggcgggt atcgatccct 780 gccgtgccgc agttaacgcg ataagcatc cgcctgggga gtacggtcgc aaggctgaaa 840 ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggttcaa ttcgacgcaa 900

<Desc / Clms Page number 191>

 cgcgaagaac cttacctagg ctcgaagtgc agatgaccat cggtgaaagc cgactttcgc 960 aagaacatct gtagaggtgc tgcatggctg tcgtcagctc gtgtcgtgag atgttgggtt 1020 aagtcccgca acgagcgcaa cccttgtttc ctgttgccat caggttaagc tgggcactct 1080 ggagagactg ccggtgacaa accggaggaa ggtggggatg acgtcaagtc agcatggcct 1140 ttatgtctag ggctacacac gtgctacaat ggccggtaca aagcgctgca aacccgcgag 1200 ggtgagccaa tcgcagaaag ccggtctcag ttcggatagc aggctgcaac tcgcctgctt 1260 gaagttggaa tcgctagtaa tcgcggatca gcatgccgcg gtgaat 1306 <210 > 97 <211> 1300 <212> DNA <213> Unknown Organ <220> <223> Origin of the sequence: Soil organism <400> 97 cccgcagggt gagtagatgg caaacgggtg agtaacacgt gggtgacctg cctcagagtg 60 ggggataacg acccgaaagg gtcgctaata ccgcataaca tcctgtcttt ggatagacgg 120 agatcaaagc cggggatcgc aagacctggc gcttagagag gggcccgcgg ccgattagct 180 agttggtgag gtaacggctc accaaggcaa cgatcggtat ccggcctgag agggcggacg 240 gacacactgg gactgagaca cggcccagac tcctacggga ggcagcagtg gggaattgtt 300 cgcaatgggc gcaagcctga cgacgcaacg ccgcgtggag gatgaagatc ttcgggtcgt 360 aaactccttt cgatcgggaa gaacgccrct ggtgtgaaca ccatcagagg gtgacggtac 420 cgagagaaga agccccggct aactctgtgc cagcagccgc ggtaatacag ggggggcaag 480 cgttgttcgg aattactggg cgtaaagggc tcgtaggcgg ccggctaagt ccgacgtgaa 540 atccccaggc ttaacctggg aactgcgtcg gatactggcg ggcttgaatc cgggagaggg 600 atgcggaatt ccaggtgtag cggtgaaatg cgtagatatc tggaggaaca ccggtggcga 660 aggcggcatc ctggaccggt attgacgctg aatagcgaaa gccaggggag caaacgggat 720 tagatacccc ggtagtcctg gccctaaacg atgaatgttt ggtgtggcgg gtatcgatcc 780 ctgccgtgcc gaagctaacg cattaaacat tccgcctggg gagtacggtc gcaaggctga 840 aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttc aattcgacgc 900 aacgcgaaga accttaccca ggctcgaacg gcattggaca tccggcgaaa gccggctccc 960 gcaagggccg atgtcgaggt gctgcatggc tgtcgtcagc tcgtgtcgtg agatgttggg 1020 ttaagtcccg caacgagcgc aacccttgtc cgctgttgcc atcacgttat ggtgggcact 1080 ctgcggagac tgccggtgat aaaccggagg aaggtgggga tgacgtcaag tcagcatggc 1140 ctttatgtct ggggctacac acgtgctaca atggccggta caaaccgttg cgatctcgca 1200agagtgagct aatcggagaa agccggtctc agttcggatt gcaggctgca actcgcctgc 1260 atgaagttgg aatcgctagt aatcgcggat cagcacgccg 1300 <210> 98 <211> 1233 <212> DNA <213> Unknown Organ <220> <223> Origin of the sequence: Soil organism <400> 98 acggagcggc agacgggaga gtaacacgtg ggaacgtgcc ctttggttcg gaacaacaca 60 gggaaacttg tgctaatacc ggataagccc ttacggggaa agatttatcg ccaaaggatc 120 ggcccgcgtc tgattagcta gttggtgagg taacggctca ccaaggcgac gatcagtagc 180 tggtctgaga ggatgatcag cctcactggg actgagacac ggcccagact cctacgggag 240 gcagcagtgg ggaatattgg acaatgggcg caagcctgat ccagccatgc cgcgtggatg 300 atgaaggccc tagggttgta aagtcctttc ggcggggaag ataatgacgg tacccgcaga 360 agaagccccg gctaacttcg tgccagcagc cgcggtaata cgaagggggc tagcgttgct 420 cggaatcact gggcngtaaa gcgcacgtag gcggcttttt aagtcagggg tgaaatcctg 480 gagctcaact ccagaactgc ctttgatact gagaagcttg agtccgggag aggtgagtgg 540

<Desc / Clms Page number 192>

 aactgcgagt gtagaggtga aattcgtaga tattcgcaag aacaccagtg gcgaaggcgg 600 ctcactggcc cggtactgac gctgaggtgc gaaagcgtgg ggagcaaaca ggattagata 660 ccctggtagt ccacgctgta aacgatggat gctagccgtt gtcgggttta ctcgtcagtg 720 gcgcagctaa cgcattaagc atcccgcctg gggagtacgg tcgcaagatt aaaactcaaa 780 ggaattgacg ggggcccgca caagcggtgg agcatgtggt tcaattcgaa gcaacgcgca 840 gaaccttacc agcccttgac atgtcccgta tgagtaccag agatggaact cttcagttcg 900 gctggcggga acacaggtgc tgcatggctg tcgtcagctc gtgtcgtgag atgttgggtt 960 aagtcccgca acgagcgcaa ccctcgccct tagttgccat catttagttg ggcactctaa 1020 ggggactgcc ggtgataagc cgcgaggaag gtggggatga cgtcaagtcc tcatggccct 1080 tacgggctgg gctacacacg tgctacaatg gcggtgacag tgggatgcag aggggtaacc 1140 ccgagcaaat ctcaaaaagc cgtctcagtt cggattgtgc tctgcaactc gagcacatga 1200 agttggaatc gctagtaatc gcagatcagc acg 1233 <210> 99 <211> 1304 <212> DNA <213> Organime Unknown < 220> <223> Origin of the sequence: Soil organism <400> 99 cgaaatccg cagggatcag tagagtggca aacgggtgag taacacgtgg gtgacctgcc 60 ttcgagtggg ggataacgtc ccgaaaggga cgctaatacc gcatgacatc ctgctcttga 120 acgagtggag atcaaagctg gggatcgcaa gacctagcgc tcaaagaggg gcccgcgcct 180 gattagctag ttggtggggt aacggctcac caaggcgacg atcagtatcc ggcctgagag 240 ggcggacgga cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtggg 300 gaattgttcg caatgggcgc aagcctgacg acgcaacgcc gcgtggagga tgaagatctt 360 cgggtcgtaa actcctttcg atcgagacga acggcctccg ggtgaacaat ccggaggagt 420 gacggtaccg agagaagaag ccccggctaa ctccgtgcca gcagccgcgg taatacgggg 480 ggggcaagcg ttgttcggaa ttactgggcg taaagggctc gtaggcggcc aactaagtca 540 gacgtgaaat ccctcggctt aaccggggaa ctgcgtctga tactggatgg ctagaggttg 600 ggagagggat gcggaattcc aggtgtagcg gtgaaatgcg tagatatctg gaggaacacc 660 ggtggcgaag gcggcatcct ggaccaattc tgacgctgag gagcgaaagc caggggagca 720 aacgggatta gataccccgg tagtcctggc cctaaacgat gaatgcttgg tgtggcgggt 780 atcgatccct gccgtgccga agctaacgca ttaagcattc cgcctgggga gtacggtcgc 840 aaggctgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggttcaa 900 ttcgacgcaa cgcgaaga ac cttacccagg cttgaacagc gagtgaccac tcctgaaaag 960 gagcttccgc aaggacactc gtagaggtgc tgcatggctg tcgtcagctc gtgtcgtgag 1020 atgttgggtt aagtcccgca acgagcgcaa cccttgtttg ctgttgccat cacgttatgg 1080 tgggcactct gcaaagactg ccggtgataa accggaggaa ggtggggatg acgtcaagtc 1140 agcatggcct ttatgtctgg ggctacacac gtgctacaat ggccggtaca aaccgtcgca 1200 aaaccgtaag gtcgagctaa tcggagaaag ccggtctcag ttcggatcgt cggctgcaac 1260 tcgccggcgt gaagttggaa tcgctagtaa tcgcggatca CCGA 1304 <210> 100 <211> 1197 <212> DNA <213> Organime Unknown <220> <223> Origin of sequence: soil Organism <400> 100 tctagtggcg cacgggtgcg taacgcgtgg gaatctgccc ttgggttcgg gataacagtt 60 ggaaacgact gctaataccg gatgatgtct tcggaccaaa gatttatcgc ccagggatga 120 gcccgcgtcg gattagctag ttggtgaggt aaaggctcac caaggcgacg atccgtagct 180 ggtctgagag gatgatcagc cacactggga ctgagacacg gcccagactc ctacgggagg 240

<Desc / Clms Page number 193>

 cagcagtggg gaatattgga caatgggcga aagcctgatc cagcaatgcc gcgtgagtga 300 tgaaggcctt agggttgtaa agctcttttg cccgggatga taatgacagt accgggagaa 360 taagccccgg ctaactccgt gccagcagcc gcggtaatac ggagggggct agcgttgttc 420 ggaattactg ggcgtaaagc gcacgtaggc ggcttgtaa gttagaggtg aaagcccgga 480 gctcaactcc ggaactgcct ttaagactgc atcgcttgaa cgtcggagag gtaagtggaa 540 ttccgagtgt agaggtgaaa ttcgtagata ttcggaagaa caccagtggc gaaggcgact 600 tactggacga ctgttgacgc tgaggtgcga aagcgtgggg agcaaacagg attagatacc 660 ctggtagtcc acgccgtaaa cgatgatgac tagctgtcgg ggctcatgga gtttcggtgg 720 cgcagctaac gcgttaagtc atccgcctgg ggagtacggc cgcaaggtta aaactcaaag 780 aaattgacgg gggcctgcac aagcggtgga gcagtggtt taattcgaag caacgcgcag 840 aaccttacca gcgtttgaca tggtaggacg gtttccagag atggattcct tcccttacgg 900 gacctacaca caggtgctgc atggctgtcg tcagctcgtg tcgtgagatg ttgggttaag 960 tcccgcaacg agcgcaaccc tcgtcttag ttgctaccat ttagttgggc actctaaaga 1020 aactgccggt gataagccgg aggaaggtgg ggatgacgtc aagtcctcat ggcccttacg 1080 cgctgggcta cacacgtgct acaatggcgg tgacagtggg cagcaaactc gcgagagtga 1140 gcaaatcccc aaaaaccgtc tcagttcgga ttgttctctg caactcgaga gcatgaa 1197 <210> 101 <211> 1352 <212> DNA <213> Unknown Organ <220> <223> Origin of the Sequence-Soil Organism <400> 101 cgacggccag tgaattgtaa tacgactcac taagggcga attgggccct ctagatgcat 60 gctcgagcgg ccgccagtgt gatggatatc tgcagaattc gcccttcagg cctaacacat 120 gcaagtcgca cgagaaaggg cttcggcccc ggacagtgg cgcacgggtg agtaacacgt 180 aggcaatctc ccccgagtg gtggataacc ttccgaaagg agggctaata cagcatgaga 240 ccacgagctc gcagagcttg tggccaaagc ggacctcttc ttgaaagttc gcgcttgagg 300 atgagcctgc ggcccatcag ctagttggta gggtaatggc ctaccaaggc taagacgggt 360 agctggtctg agaggacgga cagccacact ggaactgaga cacggtccag actcctacgg 420 gaggcagcag tggggaatct tgcgcaatgg acgaaagtct gacgcagcga cgccgcgtga 480 gcgatgaagg ccttcgggtt gtaaagctct gtggggagag acgaataagg tgcagctaat 540 acctgcatcg atgacggtat ctccttagca agcaccggct aactctgtgc cagcagccgc 600 ggtaagacag agggtgcaaa cgttgttcgg aattactggg cgtaaagcgc gtgtaggcgg 6 60 ctgtgtaagt cgggcgtgaa atcccatggc tcaaccatgg aagtgcaccc gaaactgcgt 720 agctagagtc ctggagagga aggtggaatg cttggtgtag aggtgaaatt cgtagatatc 780 aagcggaaca ccggtggcga agcggccttc tggacagtga ctgacgctga gacgcgaaag 840 cgtggggagc aaacaggatt agataccctg gtagtccacg ccgtaaacga tgaatgctag 900 acgctggggt gcatgcactt cggtgtcgcc gctaacgcat taagcattcc gcctggggag 960 tacggccgca aggttaaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat 1020 gtggtttaat tcgaagcaac gcgcaaacct taccaaccct tgacatgtcc attgccggtc 1080 cgagagattg gaccttcagt tcggctggat ggaacacagg tgctgcatgg ctgtcgtcag 1140 ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccctac cgccagttgc 1200 catcattcag ttgggcactc tggtggaact gccggtgaca agccggagga agcggggatg 1260 acgtcaagtc ctcatggccc ttatgggttg ggctacacac gtgctacaat ggcggtgaca 1320 gtgggacgcg aagccaaga tggacaaatc cc 1352 <210> 102 <211> 1361 <212> DNA <213> Organime Unknown <220> <223> Origin of the sequence: Soil organism

<Desc / Clms Page number 194>

 <400> 102 aacagctatg accatgatta cgccaagctt ggtaccgagc tcggatccac tagtaacggc 60 cgccagtgtg ctggaattcg cccttcaggc ctaacacatg caagtcgaac ggatccttcg 120 ggattagtgg cggacgggtg agtaacacgt gggaacgtgc cctttggttc ggaacaactc 180 agggaaactt gagctaatac cggataagcc tttcgaggga aagatttatc gccattggag 240 cggcccgcgt aggattagct agttggtgag gtaaaagctc accaaggcga cgatccttag 300 ctggtctgag aggatgatca gccacattgg gactgagaca cggcccaaac tcctacggga 360 ggcagcagtg gggaatcttg cgcaatgggc gcaagcctga tccagccatg ccgcgtgagt 420 gatgaaggcc ttagggttgt aaagctcttt caccggagaa gataatgacg gtatccggag 480 aagaagcccc ggctaacttc gtgccagcag ccgcggtaat acgaaggggg ctagcgttgt 540 tcggaattac tgggcgtaaa gcgcacgtag gcggatattt aagtcagggg tgaaatccca 600 gagctcaact ctggaactgc ctttgatact gggtatcttg agtatggaag aggtaagtgg 660 aattccgagt gtagaggtga aattcgtaga tattcggagg aacaccagtg gcgaaggcgg 720 cttactggtc cattactgac gctgaggtgc gaaagcgtgg ggagcaaaca ggattagata 780 ccctggtagt ccacgccgta aacgatgaat gttagccgtc gggcagtata ctgttcggtg 840 gcgcagctaa cgcattaaac attccgcctg gggagtacgg tcgcaagatt aaaactcaaa 900 ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa gcaacgcgca 960 gaaccttacc agctcttgac attcggggtt tgggcagtgg agacattgtc cttcagttag 1020 gctggcccca gaacaggtgc tgcatggctg tcgtcagctc gtgtcgtgag atgttgggtt 1080 aagtcccgca acgagcgcaa ccctcgccct tagttgccag catttagttg ggcactctaa 1140 ggggactgcc ggtgataagc cgagaggaag gtggggatga cgtcaagtcc tcatggccct 1200 tacgggctgg gctacacacg tgctacaatg gtggtgacag tgggcagcga gacagcgatg 1260 tcgagctaat ctccaaaagc 1325 <210> 103 <211> 1300 <212> DNA <213> Unknown organism <220> <223> Origin of the sequence:: Soil organism <400> 103 catgttagt agcaatacta aatgatgacg catctcagtt cggattgcat ctgcaactcg agtgcatgaa 1320 gttggaatcg ctagtaatcg cagatcagca tgctgcggtg a agcggcggac gggtgaggaa cacgtaggaa 60 cctgcccaag agagggggac aaccaaggga aactttggct aataccgcat aatctctacg 120 gagaaaagtt gcccgtaagg gtggcgcttt tggaggggcc tgcgtccgat tagttagttg 180 gtgaggtaat agctcaccaa gactgtgatc ggtaactggt ctgagaggac gaccagtcac 240 actgggactg agacacggcc cagactccta cgggaggcag cagtggggaa tcttggacaa 300 tgggggcaac cctgatccag cgatgccgcg tgggtgaaga aggccttcgg gttgtaaagc 360 cctttaggcg gggaagaagg atatgggatg aataagcctg tattttgacg gtacccgcag 420 aataagcacc ggcaaactct gtgccagcag ccgcggtaat acagagggtg cgagcgttaa 480 tcggatttac tgggcgtaaa gggcgcgtag gcggttgtgt gagtgtgatg tgaaagcccc 540 gggctcaacc tgggaagtgc atcgcaaacg acacaactgg agtatatgag agggtggcgg 600 aatttccggt gtagcggtga aatgcgtaga gatcggaagg aacgtcgatg gcgaaggcag 660 ccacctggca taatactgac gctgaggcgc gaaagcgtgg ggagcgaaca ggattagata 720 ccctggtagt ccacgccgtaaacgatgaga actagatgtt ggagggggaa cccttcagta 780 tcgaagctaa cgcgataagt tctccgcctg ggaagtacag tcgcaagact gaaactcaaa 840 agaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgat gcaacgcgaa 900 gaaccttacc Lacccttgac atcctgcgaa tcttgccgag aggtgagagt gccgcaagga 960 gcgcagagac aggtgctgca tggctgtcgt cagctcgtgt tgtgagatgt tgggttaagt 1020 cccgtaacga gcgcaaccct tgtccttagt tgccatcatt tagttgggga ctctaaggag 1080 accgccggtg atg aaccgga ggaaggcggg gacgacgtca agtcatcatg gcctttatgg 1140 gtagggctac acacgtgcta caatggggcg tacagagggt cgccaacccg cgagggggag 1200 ccaatctctt aaagcgtctc gtagtccgga ttggagtctg caactcgact ccatgaagtc 1260 ggaatcgcta gtaatcgcgg atcagcagtg ccgcggtgaa 1300 <210> 104

<Desc / Clms Page number 195>

 <211> 1250 <212> DNA <213> Unknown Organ <220> <223> Origin of the sequence: Soil organism <400> 104 tgtagcaata catcagtggc agacgggtga gtaacacgtg ggaaccttcc tcgttgtacg 60 ggacaactca gggaaacttg agctaatacc gtatacgtcc gagaggagaa agatttatcg 120 caatgagacg ggcccgcgtc ggattagcta gttggtaagg taacggctta ccaaggcgac 180 gatccgtagc tgatctgaga ggatgatcag ccacactggg actgagacac ggcccagact 240 cctacgggag gcagcagtgg ggaatcttgg acaatgggcg caagcctgat ccagccatgc 300 cgcgtgagtg aagaaggcct tagggttgta aagctctttt gccagggacg ataatgacgg 360 tacctgagaa taagccccgg caaacttcgt gccagcagcc gcggtaatac gaagggggct 420 agcgttgttc ggatttactg ggcgtaaagc gcacgtaggc gggtcgttaa gtcaggggtg 480 aaatcccgga gctcaactcc ggaactgcct ttgatactgg cgaccttgag gctggaagag 540 gttagtggaa ttcccagtgt agaggtgaaa ttcgtagata ttgggaagaa caccagtggc 600 gaaggcggct aactggtcca gatctgacgc tgaggtgcga aagcgtgggg agcaaacagg 660 attagatacc ctggtagtcc acgccgtaaa ctatgggtgc tagctgtcag cgggcttgct 720 cgttggtggc gcagctaacg cattaagcac cccgcctggg gagtacggt c gcaagattaa 780 aacttaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc 840 aacgcgcaga accttaccaa cccttgacat cccgatcgcg gacaccagag atggagtcct 900 tcagttcggc tggatcggag acaggtgctg catggctgtc gtcagctcgt gtcgtgagat 960 gttgggttaa gtcccgcaac gagcgcaacc ctcgccttta gttgccatca tttagttggg 1020 cactctaaag ggactgccgg tgataagccg gaggaaggtg gggatgacgt caagtcctca 1080 tggcccttac gggttgggct acacacgtgc tacaatggcg gtgacaatgg gcagctactt 1140 cgcaaggaga agctaatccc aaaaagccgt ctcagttcag attgcactct gcaactcggg 1200 tgcatgaagt tggaatcgct agtaatcgct aatcagcagg tagcggtgaa 1250 <210> 105 <211> 1302 <212> DNA <213> Unknown Organ <220> <223> Origin of the sequence: Soil organism <400> 105 ggcttcggct ccccggtaga gggcggacg ggtgagtaac acgtgggtaa tctgcctttg 60 The invention relates to the following: The invention relates to the following aspects of the application of the present invention: - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -. ccagactcct acgggaggca gcagtgggga 300 attttgggca atgggcgcaa gcctgaccca gcaacgccgc gtgaaggacg aaatccctct 360 gggatgtaaa cttcgaaagt tggggaagaa atccgtgtga ggataatgca cacgggatga 420 cggtacccaa cgtaagcccc ggctaactac gtgccagcag ccgcggtaat acgtaggggg 480 caagcgttgt tcggaattac tgggcgtaaa gggcgcgtag gcggtacgac aagtctggag 540 tgaaagcccg gggctcaacc ccggaatgtc tttggaaact gtcgaacttg agtgcggaag 600 aggcatctgg aattcccagt gtagcggtga aatgcgtaga tattgggaag aacacctgag 660 gcgaaggcgg gatgctgggc cgacactgac gctgaggcgc gaaagccagg ggagcgaacg 720 ggattagata ccccggtagt cctggcccta aacgatggat acttggtgtg tggggttctc 780 gaagtccccg cgtgccggag caacgcggt aagtatcccg cctggggagt acggtcgcaa 840 ggctgaaact caaaggaatt gacggggacc cgcacaagcg gtggagcatg tggttcaatt 900 cgacgcaacg cgaagaacct tacctgggtt aaatcctacc tcgtcgcctc agagatgagg 960 tttcccttcg ggggaggtag gacggtgctg catggctgtc gtcagctcgt gccgtgaggt 1020 gttgggttaa gtcccgcaac gagcgcaacc cttaccacta gttgccagcg gttcggccgg 1080 gcactctatt gggactgccg gtgacaaacc ggaggaaggt ggggatga cg tcaagtcatc 1140 atggccttta tgtccagggc tacacacgtg ctacaatggc cggaacaaag cgcagcaaac 1200

<Desc / Clms Page number 196>

 ccgcgagggg gagccaatcg caaaaatccg gtctcagttc ggattggagt ctgcaactcg 1260 actccatgaa gttggaatcg ctagtaatcg cggatcagca tg 1302 <210> 106 <211> 1281 <212> DNA <213> Unknown Organ <220> <223> Origin of the sequence: Soil organism <400> 106 tgcttctctt gagagcggcg gacgggtgag taatgcctag gaatctgcct ggtagtgggg 60 gataacgttc ggaaacggac gctaataccg catacgtcct acgggagaaa gcaggggacc 120 ttcgggcctt gcgctatcag atgagcctag gtcggattag ctagttggtg aggtaatggc 180 tcaccaaggc gacgatccgt aactggtctg agaggatgat cagtcacact ggaactgaga 240 cacggtccag actcctacgg gaggcagcag tggggaatat tggacaatgg gcgaaagcct 300 gatccagcca tgccgcgtgt gtgaagaagg tcttcggatt gtaaagcact ttaagttgga 360 aggaagggca gtaaattaat actttgctgt tttgacgtta ccgacagaat aagcaccggc 420 taactctgtg ccagcagccg cggtaataca The invention relates to the following: The invention relates to a method for controlling the operation of a vehicle. actaa 660 tactgacact gaggtgcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca 720 cgccgtaaac gatgtcaact agccgttgga agccttgagc ttttagtggc gcagctaacg 780 cattaagttg accgcctggg gagtacggcc gcaaggttaa aactcaaatg aattgagggg 840 ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag 900 gccttgacat ccaatgaact ttctagagat agattggtgc cttcgggaac attgagacag 960 gtgctgcatg gctgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgtaacgagc 1020 gcaacccttg tccttagtta ccagcacgac atggtgggca ctctaaggag actgccggtg 1080 acfaaccgga ggaaggtggg gatgacgtca agtcatcatg gcccttacgg cctgggctac 1140 acacgtgcta caatggtcgg tacagagggt tgccaagccg cgaggtggag ctaatcccac 1200 aaaaccgatc gtagtccgga tcgcagtctg caactcgact gcgtgaagtc ggaatcgcta 1260 gtaatcgcga atcagaaatg t 1281

Claims (55)

A method of preparing a nucleic acid collection from a soil sample containing organisms, said method comprising the following sequence of steps: (a) Obtaining micro-particles by grinding a sample of previously dried or desiccated soil, and then suspending the microparticles in a liquid buffer medium; and (b) extracting the nucleic acids present in the microparticles; and (c) - passing the nucleic acid-containing solution over a molecular sieve, then recovering the nucleic acid-enriched elution fractions and passing the nucleic acid-enriched elution fractions on an exchange media chromatography medium. anions and then recovering the elution fractions containing the purified nucleic acids.  2. Method according to claim 1, characterized in that step I- (a) is followed by the complementary steps of: - treatment of microparticles in suspension in a liquid buffer medium by sonication and - extraction and recovery of nucleic acids .  3. Method according to claim 1, characterized in that step I- (a) is followed by the following additional steps: - treatment of microparticles in suspension in a liquid buffer medium by sonication; incubation of the suspension at 37 ° C. after sonication in the presence of lysozyme and of achromopeptidase; - addition of SDS - recovery of nucleic acids.  4. Method according to claim 1, characterized in that step - (a) is followed by the following complementary steps: homogenization of the microparticles by means of a violent mixing stage (vortex) followed by a stage of simple agitation; freezing of the homogeneous suspension followed by thawing; treatment by sonication of the suspension after thawing; <Desc / Clms Page number 198>  incubation of the suspension at 37 ° C. after sonication in the presence of lysozyme and of achromopeptidase; - addition of SDS; - centrifugation and precipitation of nucleic acids; and - recovery of nucleic acids.  5. Method according to one of claims 1 to 4, characterized in that the nucleic acids are DNA molecules.  6. Process for preparing a recombinant vector collection, characterized in that the nucleic acids obtained by the method according to one of claims 1 to 5 are inserted into a cloning and / or expression vector.  7. Method according to claim 6, characterized in that the nucleic acids are separated according to their size prior to their insertion into the cloning and / or expression vector.  8. Method according to claim 6, characterized in that the average size of the nucleic acids is made substantially uniform by physical rupture, prior to their insertion into the cloning and / or expression vector.  9. Process according to claim 6, characterized in that the cloning and / or expression vector is of the plasmid type.  10. Process according to claim 6, characterized in that the cloning and / or expression vector is of the cosmid type.  11. The method of claim 10, characterized in that it is a replicative cosmid in E. coli and integrative Streptomyces.  12. The method of claim 11, characterized in that it is cosmos pOS7001.  13. The method of claim 10, characterized in that it is a cosmid conjugative and integrative Streptomyces.  14. The method of claim 13, characterized in that it is the cosmid pOSV303. <Desc / Clms Page number 199>  15. The method of claim 10, characterized in that it is a cosmid replicative both in E. coli and Streptomyces.  16. The method of claim 15, characterized in that it is cosmos pOS 700R.  17. The method of claim 10, characterized in that it is a replicative cosmid in E. coli and Streptomyces and conjugative Streptomyces.  18. Process according to claim 6, characterized in that the cloning and / or expression vector is of the BAC type.  19. Method according to claim 18, characterized in that it is an integrative and conjugative BAC vector in Streptomyces.  20. The method of claim 19, characterized in that it is the BAC vector pOSV403.  21. Nucleic acid collection consisting of the nucleic acids obtained by the method according to one of claims 1 to 5.  22. A nucleic acid characterized in that it is contained in the nucleic acid collection according to claim 21.  23. Nucleic acid according to claim 22, characterized in that it comprises a nucleotide sequence encoding at least one operon, or part of an operon.  24. Nucleic acid according to claim 23, characterized in that the operon encodes all or part of a metabolic pathway.  25. Nucleic acid according to claim 24, characterized in that the metabolic pathway is the synthetic route of the polyketides.  Nucleic acid according to Claim 25, characterized in that it is chosen from polynucleotides comprising the sequences SEQ ID N 30 to 44. <Desc / Clms Page number 200>  27. Nucleic acid according to claim 22, characterized in that it comprises all of a nucleotide sequence coding for a polypeptide. 28. Nucleic acid according to one of claims 22 to 27, characterized in that it is of prokaryotic origin.  29. Nucleic acid according to claim 28, characterized in that it comes from a bacterium or a virus.  30. Nucleic acid according to one of claims 22 to 24 and 27, characterized in that it is of eukaryotic origin.  31. Nucleic acid according to claim 30, characterized in that it comes from a fungus, a yeast, a plant or an animal.  32. Recombinant vector characterized in that it is chosen from vectors comprising a nucleic acid according to one of claims 26 to 31; 33. Vector according to claim 32 characterized in that it is cosmos pOS 7001.  34. The vector of claim 32 characterized in that it is the cosmid pOSV303.  35. Vector according to claim 32 characterized in that it is the cosmid pOS 700R.  36. Vector according to claim 32 characterized in that it is the BAC vector pOSV403.  37. Recombinant vector collection as obtained according to the method of one of claims 6 to 20.  38. Recombinant cloning and / or expression vector characterized in that it is contained in the recombinant vector collection according to claim 37.  Recombinant host cell comprising a nucleic acid according to one of claims 22 to 31 or a recombinant vector according to claim 38. <Desc / Clms Page number 201>  40. Recombinant host cell according to claim 39, characterized in that it is a prokaryotic or eukaryotic cell.  41. Recombinant host cell according to claim 40, characterized in that it is a bacterium.  42. Recombinant host cell according to claim 41, characterized in that it is a bacterium selected from E. coli and Streptomyces.  43. Recombinant host cell according to claim 40, characterized in that it is a yeast or a filamentous fungus.  44. Collection of recombinant host cells, each of the host cells constituting the collection comprising a nucleic acid of the nucleic acid collection according to claim 21.  45. Collection of recombinant host cells, each of the host cells constituting the collection comprising a recombinant vector according to one of claims 32 or 38.  46. A method for detecting a nucleic acid of a given nucleotide sequence, or of a nucleotide sequence structurally related to a determined nucleotide sequence, in a recombinant host cell collection according to one of claims 44 or 45, characterized in that comprises the following steps: - bringing the collection of recombinant host cells into contact with a pair of primers hybridizing with the determined nucleotide sequence or hybridizing with the nucleotide sequence structurally related to a determined nucleotide sequence; - perform at least three amplification cycles; detect the optionally amplified nucleic acid.  47. A method for detecting a nucleic acid of a given nucleotide sequence, or of a nucleotide sequence structurally related to a determined nucleotide sequence, in a recombinant host cell collection according to one of claims 44 or 45, characterized in that includes the following steps: <Desc / Clms Page number 202>  bringing the collection of recombinant host cells into contact with a probe hybridizing with the determined nucleotide sequence or hybridizing with a nucleotide sequence structurally related to the determined nucleotide sequence; detect the hybrid possibly formed between the probe and the nucleic acids included in the vectors of the collection.  48. A method for identifying the production of a compound of interest by one or more recombinant host cells in a collection of recombinant host cells according to one of claims 44 or 45, characterized in that it comprises the following steps: culturing the recombinant host cells of the collection in a suitable culture medium; detection of the compound of interest in the culture supernatant or in the cell lysate of one or more of the recombinant cultured host cells.  A method for selecting a recombinant host cell producing a compound of interest in a recombinant host cell collection according to one of claims 44 or 45, characterized in that it comprises the following steps: - culture of the recombinant host cells of the collection in a suitable culture medium; detection of the compound of interest in the culture supernatant or in the cell lysate of one or more of the recombinant cultured host cells.  selection of the recombinant host cells producing the compound of interest.  50. Process for the production of a compound of interest characterized in that it comprises the following steps: cultivating a recombinant host cell selected according to the method of claim 49; recovering and, if necessary, purifying, the compound produced by said recombinant host cell.  51. A compound of interest characterized in that it is obtained according to the process of claim 50.  52. A compound according to claim 51, characterized in that it is a polyketide.  53. Polyketide characterized in that it is produced by the expression of at least one nucleotide sequence comprising a sequence chosen from the sequences SEQ ID N 30 to 44. <Desc / Clms Page number 203>  54. A composition comprising a polyketide according to claim 52 or 53.  A pharmaceutical composition comprising a pharmacologically active amount of a polyketide according to claim 52 or 53, in association with a pharmaceutically compatible carrier.