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United States Patent Application	20120094870
Kind Code	A1
Goldsmith; Neil ;   et al.	April 19, 2012

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Foreign Application Data

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Date	Code	Application Number
Jan 25, 2001	DK	PA 2001 00128
May 1, 2001	DK	PA 2001 00679

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Claims

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1-111. (canceled)

112. A library comprising a collection of individual cells, the cells being denoted cell.sub.1, cell.sub.2, cell;, wherein i.gtoreq.2, each cell comprising an expressible nucleotide sequence, wherein said expressible nucleotide sequence is comprised within a) a concatemer of individual oligonucleotide cassettes, said concatemer comprising a nucleotide sequence of the following formula: [rs.sub.2-SP-PR-X-TR-SP-rs.sub.1].sub.n wherein rs.sub.1 and rs.sub.2 together denote a restriction site, SP denotes a spacer of at least two bases, X denotes an expressible nucleotide sequence, PR denotes a promoter, capable of regulating the expression of X in the cell, TR denotes a terminator, and n.gtoreq.2, or b) a random combination of heterologous oligonucleotides having the general formula: [PR-X] wherein X denotes an expressible nucleotide sequence, and PR denotes an independently controllable promoter being operably associated with X; c) an expression cassette comprising a nucleotide sequence of the following formula: [rs.sub.2-SP-PR-X-TR-SP-rs.sub.1] wherein rs.sub.1 and rs.sub.2 together denote a restriction site, SP denotes a spacer of at least two bases, PR denotes a promoter, capable of functioning in the cell, X denotes an expressible nucleotide sequence, TR denotes a terminator, and wherein said cell comprises at least two expression cassettes, wherein at least one of the expression cassettes comprises an expressible nucleotide sequence heterologous to the cell and wherein at least one concatemer or independently controllable promoter or expression cassette of cell.sub.1 is different from a concatemer or independently controllable promoter or expression cassette of cell.sub.2.

113. The library according to claim 112, said library comprising a collection of sublibraries, wherein a sublibrary is a collection of individual cells having at least one phenotype in common, wherein the at least one phenotype is selected from the group consisting of the ability to grow on unusual substrates, the ability to grow on sublethal concentration of toxins, the ability to grow at a high temperature, the ability to grow at a low temperature, the ability to grow at elevated osmolality, the ability to grow at low osmolality, the ability to grow at high salinity, the ability to grow at low salinity, the ability to grow at elevated metal concentrations, the ability to grow at high CO.sub.2 concentrations, the ability to grow at low CO.sub.2 concentrations, the ability to grow at high O.sub.2 concentrations, the ability to grow at low O.sub.2 concentrations, the ability to provide special spectral properties, the ability to provide a special colour, the ability to have a deviating GST activity and the ability to have a deviating P450 activity.

114. The library according to claim 112, wherein said cells are yeast cells, said yeast being selected from the group consisting of budding yeast, Kluyveromyces marxianus, K. lactis, Candida utilis, Phaffia rhodozyma, Saccharomyces boulardii, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, Candida paraffinica, Schwanniomyces castellii, Pichia stipitis, Candida shehatae, Rhodotorula glutinis, Lipomyces lipofer, Cryptococcos curvatus, Candida spp. (e.g. C. palmioleophila), Yarrowia lipolytica, Candida guilliermondii, Candida, Rhodotorula spp., Saccharomycopsis spp., Aureobasidium pullulans, Candida brumptii, Candida hydrocarbofumarica, Torulopsis, Candida tropicalis, Saccharomyces cerevisiae, Rhodotorula rubra, Candida flaveri, Eremothecium ashbyii, Pichia spp., Kluyveromyces, Hansenula, Kloeckera, Pichia, Pachysolen spp., or Torulopsis bombicola and mutants thereof.

115. The library according to claim 112, wherein substantially all rs.sub.1-rs.sub.2 sequences are recognised by the same restriction enzyme.

116. The library according to claim 112, wherein at least one cassette in one cell comprises an intron between the promoter and the expressible nucleotide sequence.

117. The library according to claim 112, wherein at least two expressible nucleotide sequences come from different expression states, wherein the different expression states represent at least two different tissues.

118. The library according to claim 112, wherein at least one concatemer and/or random combination of heterologous oligonucleotides and/or expression cassette is integrated into the host genome.

119. The library according to claim 112, wherein at least one concatemer and/or random combination of heterologous oligonucleotides and/or expression cassette is integrated into an artificial chromosome in the host cell.

120. The library according to claim 112, wherein the random combinations are made from a two dimensional array of promoters and heterologous expressible nucleotide sequences.

121. The library according to claim 112, comprising an externally controllable promoter.

122. A method of producing a library comprising a collection of individual cells, comprising the steps: i) providing a population of nucleotide cassettes having the general formula [rs.sub.2-SP-PR-X-TR-SP-rs.sub.1], wherein rs.sub.1 and rs.sub.2 together denote a restriction site, SP denotes a spacer of at least two bases, X denotes an expressible nucleotide sequence, PR denotes a promoter, capable of regulating the expression of X in the cell, TR denotes a terminator, and ii) assembling random sub-sets of the cassettes into concatemers comprising at least two cassettes, iii) ligating the concatemers into vectors, iv) introducing vectors into host cells, v) mixing at least two cells so that at least one concatemer of a first cell comprises a random sub-set of cassettes being different from a random sub-set of cassettes of a concatemer of a second cell.

123. The method according to claim 122, whereby the vectors comprise an artificial chromosome.

124. A method of producing a library comprising a collection of individual cells, comprising the steps: inserting at least two expressible nucleotides into the cloning site of at least two primary vectors comprising a cassette, the cassette comprising a nucleotide sequence of the general formula in 5'.fwdarw.3' direction: [RS1-RS2-SP-PR-CS-TR-SP-RS2-RS1'). wherein RS1 and RS1' denote restriction sites, RS2 denote a restriction site different from RS1 and RS1', SP denotes a spacer sequence of at least two nucleotides, PR denotes a promoter, CS denotes a cloning site, and TR denotes a terminator; i) excising the cassettes using at least a restriction enzyme specific for RS1, RS1' RS2 and RS2' obtaining expression cassettes having the general formula [rs.sub.2-SP-PR-X -TR-SP-rs.sub.1], wherein rs.sub.1 and rs.sub.2 together denote a restriction site, and wherein X denotes an expressible nucleotide sequence, ii) inserting the expression cassettes into a vector, iii) transferring the expression cassettes into at least two host cells, and iv) mixing at least two host cells having different cassettes.

125. A method of producing a library comprising a collection of individual cells, comprising the steps: i) providing at least one expressible nucleotide sequence, ii) ligating at least one expressible nucleotide sequence to a controllable promoter capable of functioning in a host cell obtaining a first expression construct, iii) ligating at least one expressible nucleotide sequence to another independently controllable promoter capable of functioning in a host cell, obtaining a second expression construct, iv) inserting constructs of step ii) and iii) into at least two host cells, v) mixing at least two cells having a different combination of independently controllable promoter and expressible nucleotide sequences.

126. An expression library obtainable by the method of claim 122.

127. An expression library obtainable by the method of claim 123.

128. An expression library obtainable by the method of claim 124.

129. An expression library obtainable by the method of claim 125.

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Description

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[0001] This application is a nonprovisional of U.S. provisional application Ser. No. 60/300,863 filed 27, Jun. 2001, which is hereby incorporated by reference in its entirety. The application claims priority from Danish patent application number PA 2001 00128 filed 25, Jan. 2001 and PA 2001 00679 filed on 1, May 2001, which are hereby incorporated by reference in their entirety. All patent and nonpatent references cited in the application, or in the present application, are also hereby incorporated by reference in their entirety.

[0002] The present invention relates to a library of a collection of cells and a method for producing said library. The library is useful as a starting material for evolving cells or compositions having new properties.

TECHNICAL FIELD

[0003] The present invention relates to combinatorial gene expression libraries and methods for making these. Such libraries are useful in discovery of novel and/or enhanced metabolic pathways leading to the production of novel compounds for e.g. drug discovery and/or to the production of known compounds in novel quantities or in novel compartments of the cells.

BACKGROUND OF THE INVENTION

[0004] Methods are known to provide recombined combinatorial gene expression libraries by crossing and recombination between cells comprising expression constructs (WO 00/52180 Terragen Discovery Ltd). Through the recombination, which may be carried out in vitro using the recA recombination enzyme, novel genes are obtained; which may or may not be functional in the host cell.

[0005] One drawback of the libraries of the prior art is that evolution of the libraries may only be obtained through crossing and recombination between cells whereby homologous or homeologous genes are recombined thereby resulting in novel genes yielding gene products with slightly changed properties such as substrate specificity, solubility, cellular location etc.

[0006] Furthermore once the expression constructs have been inserted into the cells the specific gene combinations of a cell is static. Novel combinations may be obtained by crossing and recombination, but this will also lead to formation of novel genes through cross-over. The novel genes may or may not be functional anymore.

[0007] Furthermore, the expression of the cons inserted expression construct is a co-expression of all the genes inserted into any one cell. When a large number of heterologous genes from a wide variety of distantly related species is assembled in one cell, chances are great that some of the heterologous genes are lethal or sub-lethal to the cell, or that several gene products will compete for the same substrates. When only co-expression of the inserts is possible novel metabolic pathways may remain undiscovered due to this fact or due to the fact that the novel metabolite was being further metabolised to a known metabolite by another inserted enzyme.

SUMMARY OF THE INVENTION

[0008] According to a first aspect the invention relates to a library comprising a collection of individual cells, the cells being denoted [0009] cell.sub.1, cell.sub.2, . . . , cell.sub.i, wherein i.gtoreq.2, [0010] each cell comprising at least one concatemer of individual oligonucleotide cassettes, each concatemer comprising a nucleotide sequence of the following formula:

[0010] [rs.sub.2-SP-PR-X-TR-SP-rs.sub.1].sub.n [0011] wherein rs.sub.1 and rs.sub.2 together denote a restriction site, SP denotes a spacer of at least two bases. X denotes an expressible nucleotide sequence, PR denotes a promoter, capable of regulating the expression of X in the cell. TR denotes a terminator, and n.ltoreq.2, and [0012] wherein at least one concatemer of cell.sub.1 is different from a concatemer of cell.sub.2.

[0013] The library according to this embodiment of the invention may in any one cell comprise a unique and preferably random combination of a high number of expression cassettes being heterologous to the host cells. Through this random combination of expression cassettes novel and unique combinations of gene products are obtained in each cell. Such libraries are especially adapted in the discovery of novel metabolic pathways created through the non-native combinations of gene products.

[0014] Due to the common structure of the expression cassettes, these may be assembled 5, easily into concatemers and inserted into the host cells via appropriate vectors. Furthermore, the cassettes may at any point be excised from the host cells again using a restriction enzyme specific for the rs.sub.1-rs.sub.2 restriction site preferably without excising the host cell's native genes. After excision the expression cassettes may be mixed with other expression cassettes of similar structure and be re-concatenated . and re-inserted into another host cell in another combination.

[0015] A further advantage of the common structure of the expression cassettes, is that the common rs.sub.1-rs.sub.2 sequence may be used as a tag for targeted PCR amplification of the expression constructs.

[0016] The expressible nucleotide sequences may conveniently arise from a cDNA library obtained from one or more expression states, wherein the cDNA clones have been inserted into expression constructs. Following excision of the expression construct from the vector comprising the construct in the cDNA library, the multitude of constructs may be concatenated and inserted into a host cell.

[0017] Each unique cell according to the invention may comprise a selection of expressible nucleotide sequences from just one expression state and can thus be assembled from one library representing this expression state or it may comprise cassettes from of a number of different expression states. The variation among and between cassettes in the cells may be such as to minimise the chance of cross over as the host cell undergoes cell division such as through minimising the level of repeat sequences occurring in concatemers, since it is not an object of this embodiment of the invention to obtain inter- or intrachromosomal recombination of the concatemers. Nor to obtain recombination with epitopes of the host cell.

[0018] The contents of the concatemers may be mixed according to any criteria. Thus a library or a sub-library of individual cells may comprise cells having a common phenotype, cells comprising expression cassettes from a common source, cells comprising specific combinations of promoter and expressible nucleotide sequences. A library or sub-library may also or alternatively comprise a collection of individual cells comprising one or more common concatemers in addition to differing concatemers; wherein the common concatemer may represent expression constructs from a common source or coding for genes with a property in common.

[0019] According to another aspect the invention relates to a library comprising a collection of individual cells, the cells being denoted [0020] cell.sub.1, cell.sub.2, . . . , cell.sub.i, wherein i.gtoreq.2, [0021] each cell comprising at least two expression cassettes comprising a nucleotide sequence of the following formula:

[0021] [rs.sub.2-SP-PR-X-TR-SP-rs.sub.1] [0022] wherein rs.sub.1 and rs.sub.2 together denote a restriction site, SP denotes a spacer of at least two bases, X denotes an expressible nucleotide sequence, PR denotes a promoter, capable of regulating the expression of X in cell, TR denotes a terminator, and [0023] wherein at least one of the expression cassettes comprises an expressible nucleotide sequence heterologous to the to cell, and at least one of the cassettes of cell.sub.1 is different from the cassettes of cell.sub.2.

[0024] According to this aspect of the invention, the cells are defined with reference to the expression cassettes. This aspect of the invention shares many advantages with the first aspect of the invention.

[0025] According to a third aspect the invention relates to a library comprising a collection of individual cells, the cells being denoted [0026] cell.sub.1, cell.sub.2, . . . , cell.sub.i, wherein i.gtoreq.2, [0027] each cell comprising a random combination of heterologous oligonucleotides having the general formula:

[0027] [PR-X] [0028] wherein X denotes an expressible nucleotide sequence, and PR denotes an independently controllable promoter being operably associated with X.

[0029] In a library according to this aspect of the invention, the mixing of gene products may not only be done upon insertion of the expressible nucleotide sequences, but also during expression by inducing and/or repressing one or more promoters each regulating the expression of a random group of expressible nucleotide sequences. Thus in each cell, a unique sub-set of genes may be induced and/or repressed at any point

[0030] This feature adds another level of potential variation in the discovery of novel biochemical pathways. By the up and down regulation of independent promoters any combination of sub-sets of genes may be turned on or off in a population of cells having a random combination of promoters and expressible nucleotide sequences.

[0031] In the evolution of novel biochemical pathways based on the insertion and expression of a high number of heterologous genes in a population of cells, it is highly likely that cells will be killed due to the formation of lethal gene products. If each cell comprises just one lethal gene, the co-expression of a number of heterologous genes will not lead to any novel biochemical pathways. By having a random combination of promoters and expressible nucleotide sequences, it may be possible to down regulate lethal or sub-lethal genes without affecting the expression of the other heterologous expression constructs.

[0032] It is also possible to use the co-ordinated expression obtained through the random combination of promoters and expressible nucleotide sequences from the same pool of expressible nucleotide sequences to identify expressible nucleotide sequences involved, in a desired or unwanted property (e.g. lethality or sub-lethality). In a population according to this aspect of the invention, each cell may in principle comprise more or less the same heterologous expressible nucleotide sequences, the difference between the cells being the groups of expressible nucleotide sequences that are induced/repressed by a given promoter. In such a population of cells a desired or unwanted property will be identified in different cells following induction/repression of different promoters. As an illustrative example, in cell A the property may be associated with induction of promoter 1, 2, and 3, and in cell B the property may be associated with induction of promoter 5 and 6. With this information it is possible to target the property (or properties) to the group of expressible nucleotide sequences associated with these promoters in these cells. The expression constructs may be isolated using knowledge about the promoter nucleotide sequence and sequences common for the identified cells may be identified. Thus, by turning on and off only certain sub-sets of genes at a time, it is possible to identify which gene combinations have given a particular phenotype.

[0033] According to a further aspect the invention relates to a library comprising at least one library or at least one sub-library as defined above. In the evolution of novel biochemical pathways, it may be preferable to use a number of libraries- or sub-libraries and to evolve these in parallel or mix the libraries in order to improve the chances of identifying a desired property.

[0034] According to a further aspect the invention relates to a method of producing a library comprising a collection of individual cells, comprising the steps: [0035] i) providing a population of nucleotide cassettes having the general formula [rs.sub.2-SP-PR-X-TR-SP-rs.sub.1], wherein rs.sub.1 and rs.sub.2 together denote a restriction site, SP denotes a spacer of at least two bases, X denotes an expressible nucleotide sequence. PR denotes a promoter, capable of regulating the expression of X in the cell. TR denotes a terminator, and [0036] ii) assembling random sub-sets of the cassettes into concatemers comprising at least two cassettes, [0037] iii) ligating the concatemers into vectors, [0038] iv) introducing vectors into host cells, [0039] v) mixing at least two cells so that at least one concatemer of a first cell comprises a random sub-set of cassettes being different from a random sub-set of cassettes of a concatemer of a second cell.

[0040] The assembly of concatemers is facilitated by the common structure of the expression cassettes. When the rs.sub.1-rs.sub.2 restriction site produces sticky ends with a predetermined nucleotide sequence the assembly of the concatemers becomes especially easy to perform.

[0041] The randomisation of the cassettes may be done at any stage, i.e. during a preceding step in which an entry library (for storing and amplifying cassettes) is produced or during the insertion into vectors and/or during the transformation into host cells. Preferably the randomisation is done during the concatenation step.

[0042] According to another aspect the invention relates to a method of producing a library comprising a collection of individual cells, comprising the steps: [0043] i) inserting at least two expressible nucleotides into the cloning site of at least two primary vectors comprising a cassette, the cassette comprising a nucleotide sequence of the general formula in 5'.fwdarw.3' direction: [RS1-RS2-SP-PR-CS-TR-SP-RS2-RS1'] wherein RS1 and RS1' denote first restriction sites, RS2 denotes another restriction site different from RS1 and RS1', SP denotes a spacer sequence of at least two nucleotides, PR denotes a promoter, CS denotes a cloning site, and TR denotes a terminator. [0044] ii) excising the cassettes using at least a restriction enzyme specific for RS1, RS1' and RS2 obtaining expression cassettes having the general formula [rs.sub.2-SP-PR-X-TR-SP-rs.sub.1], wherein rs.sub.1-rs.sub.2 together denote a restriction site, and wherein X denotes an expressible nucleotide sequence, [0045] iii) inserting the expression cassettes into a vector, [0046] iv) transferring the expression cassettes into at least two host cells, and [0047] v) mixing at least two host cells having different cassettes.

[0048] According to this method for producing a library of individual cells the source expressible nucleotide sequences are first ligated into a primary vector comprising a cloning site and a cloning cassette. This primary vector may be maintained in a cDNA library and reisolated for excision of the expression cassettes and insertion into a host cell. Through this process the expressible nucleotide sequences are given a common structure which makes it possible to clone the cassettes into a predetermined cloning site in a vector and to remove the cassettes selectively from the host cells later.

[0049] According to a final aspect the invention relates to a method of producing a library comprising a collection of individual cells, comprising the steps: [0050] i) providing at least one expressible nucleotide sequence; [0051] ii) ligating at least one expressible nucleotide sequence to a controllable promoter capable of functioning in a host cell obtaining a first expression construct, [0052] iii) ligating at least one expressible nucleotide sequence to another independently controllable promoter capable of functioning in a host cell, obtaining a second expression construct, [0053] iv) inserting constructs of step ii) and iii) into at least two host cells, [0054] v) mixing at least two cells having a different combination of independently controllable promoter and expressible nucleotide sequences.

[0055] According to this aspect of the invention there is provided a convenient method for preparation of a library of individual cells comprising expressible nucleotide sequences under the operable control of at least two controllable promoters.

BRIEF DESCRIPTION OF THE DRAWINGS

[0056] FIG. 1 shows a flow chart of the steps leading from an expression state to incorporation of the expressible nucleotide sequences in an entry library (a nucleotide library according to the invention).

[0057] FIG. 2 shows a flow chart of the steps leading from an entry library comprising expressible nucleotide sequences to evolvable artificial chromosomes (EVAC) transformed into an appropriate host cell. FIG. 2a shows one way of producing the EVACs which includes concatenation, size selection and insertion into, an artificial chromosome vector. FIG. 2b shows a one step procedure for concatenation and ligation of vector arms to obtain EVACs.

[0058] FIG. 3 shows a model entry vector. MCS is a multi cloning site for inserting expressible nucleotide sequences. Amp R is the gene for ampicillin resistance. Col E is the origin of replication in E. coli. R1 and R2 are restriction enzyme recognition sites.

[0059] FIG. 4 shows an example of an entry vector according to the invention, EVE4. MET25 is a promoter, ADH1 is a terminator, f1 is an origin of replication for filamentous phages, e.g. M13. Spacer 1 and spacer 2 are constituted by a few nucleotides deriving from the multiple cloning site, MCS, SrfI and AscI are restriction enzyme recognition sites. Other abbreviations, see FIG. 3. The sequence of the vector is set forth in SEQ ID NO 1.

[0060] FIG. 5 shows an example of an entry vector according to the invention, EVE5. CUP1 is a promoter, ADH1 is a terminator, f1 is an origin of replication for filamentous phages, e.g. M13. Spacer 1 and spacer 2 are constituted by a few nucleotides deriving from the multiple cloning site, MCS, SrfI and AscI are restriction enzyme recognition sites. Other abbreviations, see FIG. 3. The sequence of the vector is set forth in SEQ ID NO 2.

[0061] FIG. 6 shows an example of an entry vector according to the invention, EVE8. CUP1 is a promoter, ADH1 is a terminator, f1 is an origin of replication for filamentous phages, e.g. M13. Spacer3 is a 550 bp fragment of lambda phage DNA. Spacer4 is a ARS1 sequence from yeast. SrfI and AscI are restriction, enzyme recognition sites. Other abbreviations, see FIG. 3. The sequence of the vector is set forth in SEQ ID NO 3.

[0062] FIG. 7 shows, a vector (pYAC4-AscI) for providing arms for an evolvable artificial chromosome (EVAC) into which a concatemer according to the invention can be cloned. TRP1, URA3, and HIS3 are yeast auxotrophic marker genes, and AmpR is an E. coli antibiotic marker gene. CEN4 is a centromere and TEL are telomeres. ARS1 and PMB1 allow replication in yeast and E. coli respectively. BamH I and Asc I are restriction enzyme recognition sites. The nucleotide sequence of the vector is set forth in SEQ ID NO 4.

[0063] FIG. 8. shows the general concatenation strategy. On the left is shown a circular entry vector with restriction sites, spacers, promoter, expressible nucleotide sequence and terminator. These are excised and ligated randomly.

TABLE-US-00001 Lane F/Y 1 100/1 2 50/1 3 20/1 4 10/1 5 5/1 6 2/1 7 1/1 8 1/2 9 1/5

Legend: Lane M: molecular weight marker, .lamda.-phage DNA digested w. Pst1. Lanes 1-9, concatenation reactions. Ratio of fragments to yac-arms(F/Y) as in table.

[0064] FIG. 9a and 9b. illustrates the integration of concatenation with synthesis of evolvable artificial chromosomes and how concatemer size can be controlled by controlling the ratio of vector arms to expression cassettes, as described in example 7.

[0065] FIG. 10. Library of EVAC transformed population shown under 4 different growth conditions. Coloured phenotypes can be readily detected upon induction of the Met25 and/or the CapI promoters.

[0066] FIG. 11. EVAC gel Legend: PFGE of EVAC containing clones:

[0067] Lanes. a: Yeast DNA PFGE markers(strain YNN295), b: lambda ladder, c: non-transformed host yeast, 1-9: EVAC containing clones. EVACs in size range 1400-1600 kb. Lane 2 shows a clone containing 2 EVACs sized .about.1500 kb and .about.550 kb respectively. The 550 kb EVAC is comigrating with the 564 kb yeast chromosome and is resulting in an increased intensity of the band at 564 kb relative to the other bands in the lane. Arrows point up to EVAC bands.

DEFINITIONS

[0068] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.

[0069] As used herein, growth under selective conditions, means growth of a cell under conditions that require expression of a selectable marker for survival.

[0070] By a controllable promoter is meant a promoter, which can be controlled through external manipulations such as addition or removal of a compound from the surroundings of the cell, change of physical conditions, etc.

[0071] An independently controllable promoter may be induced/repressed substantially without affecting the induction/repression of other promoters according to the invention. The induction/repression of an independently controllable promoter may affect native promoters in the host cells.

[0072] Co-ordinated expression refers to the expression or a sub-set of genes which are induced or repressed by the same external stimulus.

Oligonucleotides

[0073] Any fragment of nucleic acids having approximately from 2 to 10000 nucleic acids.

Restriction Site

[0074] For the purposes of the present invention the abbreviation RSn (n=1, 2, 3, etc) is used to designate a nucleotide sequence comprising a restriction site. A restriction site is defined by a recognition sequence and a cleavage site. The cleavage site may be located within or outside the recognition sequence. The abbreviation "rs.sub.1" or "rs.sub.2" is used to designate the two ends of a restriction site after cleavage. The sequence "rs.sub.1-rs.sub.2" together designate a complete restriction site.

[0075] The cleavage site of a restriction site may leave a double stranded polynucleotide sequence with either blunt or sticky ends. Thus, "rs.sub.1" or "rs.sub.2" may designate either a blunt or a sticky end.

[0076] In the notation used throughout the present invention, formulae like:

RS1-RS2-SP-PR-X-TR-SP-RS2-RS1

should be interpreted to mean that the individual sequences follow in the order specified. This does not exclude that part of the recognition sequence of e.g. RS2 overlap with the spacer sequence, but it is a strict requirement that all the items except RS1 and RS1' are functional and remain functional after cleavage and re-assemblage. Furthermore the formulae do not exclude the possibility of having additional sequences inserted between the listed items. For example introns can be inserted as described in the invention below and further spacer sequences can be inserted between RS1 and RS2 and between TR and RS2. Important is that the sequences remain functional.

[0077] Furthermore, when reference is made to the size of the restriction site and/or to specific bases within it, only the bases in the recognition sequence are referred to.

Expression State

[0078] An expression state is a state in any specific tissue of any individual organism at any one time. Any change in conditions leading to changes in gene expression leads to another expression state. Different expression states are found in. different individuals, in different species but they may also be found in different organs in the same species or individual, and in different tissue types in the same species or individual. Different expression states may also be obtained in the same organ or tissue in any one species or individual by exposing the tissues or organs to different environmental conditions comprising but not limited to changes in age, disease, infection, drought, humidity, salinity, exposure to xenobiotics, physiological effectors, temperature, pressure, pH, light, gaseous environment, chemicals such as toxins.

Artificial Chromosome

[0079] As used herein, an artificial chromosome (AC) is a piece of DNA that can stably replicate and segregate alongside endogenous chromosomes. For eukaryotes the artificial chromosome may also be described as a nucleotide sequence of substantial length comprising a functional centromer, functional telomeres, and at least one autonomous replicating sequence. It has the capacity to accommodate and express heterologous genes inserted therein. It is referred to as a mammalian artificial chromosome (MAC) when it contains an active mammalian centromere. Plant artificial chromosome and insect artificial chromosome (BUGAC) refer to chromosomes that include plant and insect centromers, respectively. A human artificial chromosome (HAC) refers to a chromosome that includes human centromeres, AVACs refer to avian artificial chromosomes. A yeast artificial chromosome (YAC) refers to chromosomes that are functional in yeast, such as chromosomes that include a yeast centromere.

[0080] As used herein, stable maintenance of chromosomes occurs when at least about 85%, preferably 90%, more preferably 95% of the cells retain the chromosome. Stability is measured in the presence of a selective agent. Preferably these chromosomes are also maintained in the absence of a selective agent. Stable chromosomes also retain their structure during cell culturing, suffering neither intrachromosomal nor interchromosomal rearrangements.

DETAILED DESCRIPTION OF THE INVENTION

[0081] The present invention relates to libraries of individual cells useful for capturing and preserving a diversity of genetic resources from nature, and for expressing the captured genetic resources and allowing them to interact to produce a diversity of chemical structures. The invention also facilitates screening for desirable properties and compounds.

[0082] More particularly, the invention provides methods for constructing and screening libraries of individual cells comprising heterologous expressible nucleotide sequences. These libraries comprise random assortments of expressible nucleotide sequences from multiple expression states and preferably also from multiple species the products of which are allowed to interact with each other in the expression host, and result in some cases in the formation of novel biochemical pathways and/or the production of novel classes of compounds. Moreover, the libraries of the invention provide efficient access to otherwise inaccessible sources of molecular diversity.

[0083] The novel biochemical pathways may carry out processes including but not limited to structural modification of a compound, addition of chemical groups to the compound, or decomposition of the compound.

[0084] The novel classes of compound may include but are not limited to metabolites, secondary metabolites, enzymes, or structural components of an organism. A compound of interest may have one or more potential therapeutic properties, including but not limited to agonist or antagonist to a class of receptor or a particular receptor, antibiotic, antiviral, antitumor, pharmacological or immunomodulating properties or be other commercially-valuable chemicals such as pigments.

[0085] A library of individual cells is a library comprising expression constructs prepared from randomly assembled or even concatenated expressible nucleotide sequences derived from a plurality of species of donor organisms, in which expressible nucleotide sequences are operably associated with regulatory regions that drives expression of the expressible nucleotide sequences in an appropriate host organism. The host organisms used are capable of producing functional gene products of the donor organisms. Upon expression in the host organism, gene products of the donor organism(s) may interact to form novel biochemical pathways.

[0086] Generally, the methods of the invention comprise providing expressible nucleotide sequences derived from one or more donor organism(s), engineering said expressible nucleotide sequences into a context where said expressible nucleotide sequences can be transcribed in a given host organism, and introducing said expressible nucleotide sequences into a host organism via a cloning or expression vector so that one or more expressible nucleotide sequences of the donor organism(s) are transferred to and expressed in the host organism. Such host organisms containing donor expressible nucleotide sequences are pooled to form a library.

[0087] The transferred genetic material, typically comprises a random assortment of expressible nucleotide sequences, the expression of which is driven and controlled by one or preferably by more functional regulatory regions. The expression construct or vector advantageously provide these regulatory regions. The expressible nucleotide sequences of the donor organism(s) are transcribed, translated and processed in the host organism to produce functional proteins that in turn generate the metabolites of interest.

[0088] Once a desirable activity or compound is identified, downstream drug development efforts such as strain improvement and process development, are greatly facilitated. The positive clone can be cultured under standard conditions to produce the desired compound in substantial amounts for further studies or uses. The expressible nucleotide sequences of the biochemical pathway are immediately available for sequencing, mutation, expression, and further rounds of screening. The cloned biochemical pathway is readily amenable to traditional and/or genetic manipulations for overproduction of the desired compound.

[0089] Furthermore, according to the embodiments comprising the expression cassettes with common structure, several positive cells may be identified, their expression cassettes be excised due to the presence of a common restriction site, which is preferably a rare restriction site. The excised expression cassettes may be re-assembled in a random or targeted manner to produce novel combinations of the selected expression cassettes.

[0090] Furthermore, biochemical pathways that are otherwise silent or undetectable in the donor organism may be discovered more easily by virtue of their functional reconstitution in the host organism. Since the biochemical characteristics of the host organism are well known, many deviations as a result of expression of donor genetic material can readily be recognised. Novel compounds may be detected by comparing extracts of a host organism containing donor genetic material against a profile of compounds known to be produced by the control host organism under a given set of environmental conditions. Even very low levels of a desirable activity or compound may be detected when the host biochemical and cellular background of the host organism is well characterised.

[0091] In one embodiment, the methods may be applied to donor organism(s) that cannot be recovered in substantial amounts in nature, or cultured in the laboratory. By transferring genetic material such as cDNA from such organisms into a host organism, the organisms metabolic pathways may be reproduced, and their products tested efficiently for any desirable properties. Thus, the genetic diversity of these organisms is captured and preserved and combined with the genetic diversity of other organisms.

[0092] In another embodiment of the invention, a library can be constructed in which the expressible nucleotide sequences from one or multiple donor organisms are randomly concatenated prior to introduction into the host organism. Thus, each host organism in the library may individually contain a unique, random combination of expressible nucleotide sequences derived from the various donor pathways or organisms. For the most part, such combinations of expressible nucleotide sequences in the library do not occur in nature. Upon expression, the functional gene products of the various donor pathways or organisms interact with each other and with the native host complement of gene products in individual host organisms to generate combinations of biochemical reactions which result in novel metabolic pathways and/or production of novel compounds. Collectively, the genetic resources of the donor organisms in the library are translated into a diversity of chemical compounds that may not be found in individual donor organisms.

[0093] In another aspect of the invention, the methods may be applied to the generation of a multiple kingdom. pathway in the host organism. An example of this would be the introduction of genes from carotenoid pathways (obtained from fungi, algae and/or plants) as well as genes from synthesis of Vitamin A (obtained from animals) or genes coding for the production of visual pigments (obtained from insects). By such targeted selection and combination of elements of biochemical pathways across kingdoms the likelihood of obtaining novel metabolites may be further increased.

[0094] In another aspect of the invention, the species of donor organisms may be selected on the basis of their biological characteristics. Such biological characteristics may include, but are not limited to the capability to utilise certain nutrients, to survive under extreme conditions, to derivatise a chemical structure, and the ability to break down or catalyse formation of certain types of chemical linkages. When expressible nucleotide sequences of the donor organism are expressed in the host organism, the donor gene products can modify and/or substitute the functions of host gene products that constitute host metabolic pathways, thereby generating novel hybrid pathways. Novel activities and/or compounds may be produced by hybrid pathways comprising donor and host-derived components. The target metabolic pathway modified by donor gene products may be native to the host organism. Alternatively, the target metabolic pathway may be provided by products of heterologous genes which are endogenous or have been genetically engineered into every host organism prior to or contemporaneous to construction of the gene expression library. Thus, the present invention also embodies constructing and screening gene expression libraries, wherein DNA fragments encoding metabolic pathway of donor organisms are cloned and coexpressed in host organisms containing a target metabolic pathway.

[0095] In another embodiment of the invention, the host organism may have an enhanced complement of active drug efflux systems which secretes the compounds of interest into the culture medium, thus reducing the toxicity of the compounds to the host organism. Absorptive material, e.g., neutral resins, may be used during culturing of the host organisms, whereby metabolites produced and secreted by the host organism may be sequestered, thus facilitating recovery of the metabolites.

[0096] In many respects, the libraries provides significant convenience and time advantage to the various steps of development of novel small molecules such as the development of drugs up to clinical trials. The libraries of the invention are compatible with e.g. the established multi-well footprint format and robotics for high-throughput screening. The host organisms of the invention are organisms commonly used for genetic manipulation and/or process development. The present invention takes advantage of the fact that such host organisms or production hosts are well-characterised in terms of their biological properties and maintenance requirements. By transferring genetic materials from a donor organism to other more familiar expression systems, the need for difficult culturing conditions for the donor organism is reduced. Thus, the biological activities, the pharmacokinetic and toxic properties of any lead compound discovered in the system of the invention may be studied and optimised more efficiently.

[0097] The novel metabolic pathway generated in a positive clone can be delineated by standard techniques in molecular biology. The lead compound may be synthesised by culturing a clone of the drug-producing host organism under standard or empirically determined culture conditions, so that sufficient quantities of the lead compound may be isolated for further analysis and development. There are already high purity manufacturing protocols, such as Good Manufacturing Practice (GMP) established for some of these standard industrial host organisms. Unlike conventional methods of screening natural product sources, less effort is required to adapt the screening and production technologies to the particular requirements of each potentially drug-producing organism.

[0098] The present invention also provides libraries made according to the methods of the invention from genetic materials of a particular set of donor organisms and/or cell types. Not all organisms or cell types in a set, especially mixed samples, need to be individually identified or characterised to enable preparation of the libraries.

[0099] Any library of the invention may be amplified, replicated, and stored. Amplification is preferably performed by introducing entry vectors containing expressible nucleotide sequences in a initial host organism such as E. coli so that so that multiple clones of the expressible nucleotide sequences are produced. Replication refers to picking and growing of individual clones in the library. A library of the invention may be stored and retrieved by any techniques known in the art that is appropriate for the host organism. Thus, the libraries of the invention are an effective means of capturing and preserving the genetic resources of donor organisms, which may be accessed repeatedly in a drug discovery program or other discovery programs.

Concatemer Assemblage

[0100] Concatemers may be assembled from cDNA libraries on a routine basis. A typical concatemer generation step will pool e.g. 1,000 genes=cDNA expression constructs (from 1 sample) and use this to generate 1,000 concatemers, with an average of 25 genes per concatemer. This means on average each gene will be in 25 different concatemers within a pool. One such concatemer "Source Pool" may be generated per source cDNA library. The Source Pools are suitable for storage of the concatemers.

[0101] However, the invention is not limited to any specific number of genes in a source. pool. Concatemers with approximately 500 genes are easily produced and it is contemplated that this number can be increased even further

[0102] The actual numbers depend on the number of different promoters and/or spacers and/or terminators to be incorporated--i.e. if an expression state gives 1000 different cDNAs. and these are to be combined with 2 promoters and/or spacers and/or terminators the numbers increase proportionally: 1000 cDNAs=2000 expression constructs, so if each construct should still be present in 25 concatemers of 25 constructs then the source pool size would be 2000.

[0103] Certain Source Pools may in fact be generated on a function rather than species basis. Such a source pool may for example be based on sources known for a specific property, such as carotenoid activity, pharmaceutical properties, chemotaxonomic properties, etc.

Host Library Assemblage

[0104] Source Pools may be Mixed and used to generate host libraries or screening libraries with each host containing multiple concatemers In selecting which Source Pools to mix one may use knowledge of the source of given libraries, host pathways, the desired focus of particular programmes and success rates of given libraries in particular screens.

[0105] If each source library is constructed from 1,000 different genes and assembled into EVACs each Containing 25 genes, then for any one given gene, of those EVACS that do contain the gene, 98.8% of them will contain just one copy, 1.2% will contain 2 copies and 0.01% will contain 3 or more copies. Thus for all practical purposes each EVAC can in this situation be regarded as composed of 25 different genes. Should a cell population be created from four such source libraries, then each different gene (assuming no overlap between genes from different sources) will be represented at a frequency of 1 copy per 4,000 genes.

[0106] In a cell population where each cell contains four EVACS, generated from a pool of 4 source libraries, then in respect of any one of the source libraries, statistically: [0107] 0.4% of cells will have all four of their EVACS from this source [0108] 4.3% of cells will have three out of four EVACS from this source [0109] 25.5% of cells will have two EVACS from this source [0110] 38.3% of cells will have just one EVAC from this source [0111] 31.6% of cells will not contain any EVACs from this source

[0112] From these figures the probability of any two-gene combination can be calculated using standard statistical tools

[0113] For more focused evolutionary approaches, such as the evolution of novel carotenoids or other known structural classes or metabolite pathways, EVACs can be enriched for enzymes, and homologs or functional analogs of these enzymes, that conduct different stages of the metabolic pathway. Such an approach can lead to significant probabilities that essentially all steps of a given pathway are represented, at least at the transcription level, in a cell. Thus if a 10-step pathway is required, and 50-gene EVACS are constructed randomly from genes encoding for homologous or analogous enzymes to those responsible for each step then any given step will be encoded in >85% of EVACs between 3-9 times (inclusive) and will be entirely missing in just 0.52% of EVACs. Thus it can be seen that a 10.sup.8 member cell population where each cell contains 4 EVACs of 50 genes each, constructed from 4 enzyme encoding gene pools, will contain a large number of cells in which all steps of the Potential pathway are represented, in most cases multiple times.

Sub-Libraries

[0114] Initial screens are designed to sort host lines into "collections", sub-libraries, based on whether novel activity has been induced by the concatemers, and the type of activity that has been induced. As such initial screens should be reasonably high throughput and should be arbitrary in their selection criteria.

[0115] A large number of such screens can be considered. An illustrative example of such screens may include but are not limited to: [0116] Novel spectral properties . [0117] Induced cytochrome oxidase activity [0118] Changed size, morphology, stickiness or adhesive properties or lack thereof [0119] Ability to grow on substrates they cannot normally grow on [0120] Ability to grow on sublethal substrates [0121] Ability to grow in the absence of normal essential requirements [0122] Ability to grow on media comprising one or more inhibitors [0123] Ability to grow under changed physical conditions, such as temperature, osmolarity, electromagnetic radiation including light of certain wavelengths. [0124] Ability to grow under magnetic field of certain force. [0125] Secretion or the lack of it from the cell [0126] The inhibition or prevention of inhibition of an enzyme [0127] The activation of a receptor. [0128] The prevention of an activating molecule binding to a receptor. [0129] The inhibition or promotion of binding of small molecules or proteins to nucleic acid or peptide sequences. [0130] The inhibition or promotion of transcription or translation of post translational processing. [0131] Changes in the transport or localisation of molecules within the cell or within organelles. [0132] Changes in the DNA content or morphology of the cell. [0133] The production of small molecules with certain properties that allow their selective isolation (e.g. all the chromoatography principles available to the skilled practitioner). [0134] The production of small molecules with certain spectroscopic properties (defined broadly to include visible light, microwaves, IR, UV, X-ray, etc.). [0135] Changes in the morphology of the cell, including the prevention or promotion of cell differentiation: [0136] The induction of apoptotic pathways.

[0137] For each Host Library (of 10,000 host lines) the 1-2% of host lines that are most extreme on each of such criteria may be grouped into a sub-library. These initial sorting screens will in general be conducted under conditions that maximise the number of genes expressed per concatemer.

[0138] The output of a sorting screen may be host lines that are characterised on one or more broad criteria. These may be categorised as sub-libraries.

[0139] A sub-library may be defined with reference to a common phenotype of the cells in the sub-library. But sublibrary may also be defined as a collection of individual cells, said cells having--for at least one identical expressible DNA sequence--different promotors, i.e. with reference to the presence of specific expressible nucleotide sequences. Furthermore, a sub-library may be described with reference to a cassette and/or in a concatemer of cassettes comprised in the host cells. A sub-library may thus be defined as a collection of individual cells, each cell having--in at least one cassette of the concatemer--identical expressible DNA sequences. A sublibrary may also be looked upon as a collection of individual cells, said cells having--for at least one identical expressible DNA sequence, more preferably for substantially all identical expressible nucleotide sequences--different promotors:

[0140] The common phenotype of a given sub-library may be at least one phenotype selected from the group comprising the ability to grow on unusual substrates, the ability to grow on sublethal concentration of toxins, the ability to grow at a high temperature, the ability to grow at a low temperature, the ability to grow at elevated osmolality, the ability to grow at low osmolality, the ability to grow at high salinity, the ability to grow at low salinity, the ability to grow at elevated metal concentrations, the ability to grow at high CO.sub.2 concentrations, the ability to grow at low CO.sub.2 concentrations, the ability to grow at high O.sub.2 concentrations, the ability to grow at low O.sub.2 concentrations, the ability to provide special spectral properties, the ability to provide a special colour, the ability to have a deviating GST activity, the ability to have a deviating P450 activity.

Size of Library

[0141] A library of cells may in principle comprise just two cells differing with respect to one of the features discussed below. However, normally a library comprises at least 20 individual cells, such as at least 50 individual cells. More preferably, a library comprises at least 100 individual cells, such as at least 1,000 cells, for example at least 10,000 cells such as at least 100,000 cells, for example at least 1,000,000 cells, such as at least 1,000,000,000 cells.

[0142] The number of cells in a sub-library depends on the selection criterion or criteria used. At the beginning a sub-library typically comprises less cells than a library, but the cells of the sub-library may be combined or allowed to sexually propagate to produce increased variation and in this way the number of different cells in a sub-library may increase.

Variation Among Cells

[0143] The difference between cells in a library may be defined with reference to differences between expression cassettes, between concatemers or differences between promoters controlling the expression of an expressible nucleotide sequence.

[0144] Thus in a library according to the invention a concatemer of each cell may comprise at least a first cassette and a second cassette, said first cassette being different from said second cassette. More preferably substantially, all cassettes of a concatemer in a given cell are different.

[0145] The difference between the expression cassettes, which may be reflected in the difference between concatemers in different cells may be a difference in the spacer sequences and/or the promoter, and/or the expressible nucleotide sequence and/or the intron and/or terminator sequence.

[0146] When the differences lie in the expressible nucleotide sequences. these different expressible nucleotide sequences may come from the same or from different expression states. The different expression states may represent at least two different tissues, such as at least two organs, such as at least two species, such as at least two genera. The different species are from at least two different phylae, such as from at least two different classes, such as from at least two different divisions, more preferably from at least two different sub-kingdoms, such as from at least two different kingdoms. In this way cells and libraries representing an extremely wide array of gene combinations is obtained.

[0147] Preferably substantially all cells in a library are different. This increases the number of available combinations of expressible nucleotide sequences. Further variation may be obtained by having one library in cells of one mating type and another library in cells of another mating type. For yeast this may be obtained by having one library in Meta cells and another library in Mat.alpha. cells. These may then be sexually crossed to obtain further variation.

[0148] According to an especially preferred embodiment of the invention the library comprises a random combination of promoter and expressible nucleotide sequences made from a two dimensional array of promoters and heterologous expressible nucleotide sequences. Thereby, it is possible to get--in principle--all expressible nucleotide sequences from a given pool represented in a library under the control of different promoters.

[0149] When each cell furthermore comprises an individual selection of combinations of promoters and heterologous expressible nucleotide sequences drawn individually from the same pool of promoters and heterologous expressible nucleotide sequences completely random combinations of promoter and expressible nucleotide sequences are inserted into all cells. Each expressible nucleotide sequence may then be found in the library under the control of different promoters and in a number of combinations with a number of other expressible nucleotide sequences.

[0150] Each library may comprise at least 2 different independently controllable promoters, such as at least 3, for example at least 4, such as at least 5, for example at least 6, such as at least 7, for example at least 8, such as at least 9, for example at least 10, such as at least 15, for example at least 25, such as at least 50, for example at least 75, such as at least 100. The higher the number of promoters in the library, the number of sub-sets of genes may be constructed within any one cell and within any one library. Preferably the regulation of the promoters should not interact on each other. The absence of interaction sets an upper limit to the number of promoters that can be used under practical circumstances. However, new promoters are discovered and synthetic promoters are being developed continuously so it is likely that in the future combinations of different non-interacting promoters can be made.

[0151] At least one heterologous expressible nucleotide sequence may be found in at least 2 cells, such as at least 3 cells, for example at least 5 cells, such as at least 10 cells, for example at least 25 cell, such as at least 50 cells, for example at least 100 cells, such as at least 500 cells, for example at least 1000 cells. By having the same expressible nucleotide represented in several preferably in many cells, any one expressible nucleotide sequence may be found in many combinations with different expressible nucleotide sequences.

[0152] The combination of promoter and expressible nucleotide sequences in any one cell may be laid out so that at least one cell comprises a group of heterologous expressible nucleotide sequences under the control of a first promoter, the group comprising at least 5 heterologous expressible nucleotide sequences, such as at least 1.0 heterologous expressible nucleotide sequences, for example at least 15 heterologous expressible nucleotide sequences, such as at least 25 heterologous expressible nucleotide sequences, for example at least 50 heterologous expressible nucleotide sequences, such as at least 75 heterologous expressible nucleotide sequences, for example at least 100 heterologous expressible nucleotide sequences, such as at least 250 heterologous expressible nucleotide sequences, for example at least 500 heterologous expressible nucleotide sequences. Thereby a sub-set of expressible nucleotide sequences of different size can be turned on and off in the cells.

[0153] By furthermore having in a cell at least a second group of heterologous expressible nucleotide sequences under the independent control of second promoter, such as at least a third group of heterologous expressible nucleotide sequences under the independent control of a third promoter, for example at least a fourth group of heterologous expressible nucleotide sequences under the independent control of a fourth promoter, such as at least a fifth group of heterologous expressible nucleotide sequences under the independent control of a fifth promoter, for example at least a sixth group of heterologous expressible nucleotide sequences under the independent control of a sixth promoter, such as at least a seventh group of heterologous expressible nucleotide sequences under the independent control of a seventh promoter, such as at least a eighth group of heterologous expressible nucleotide sequences under the independent control of a eighth promoter, for example at least a ninth group of heterologous expressible nucleotide sequences under the independent control of a ninth promoter, such as at least a tenth group of heterologous expressible nucleotide sequences under the independent control of a tenth promoter, groups of expressible nucleotide sequences, sub-sets, may be turned on and off in the cells.

Origin of Expressible Nucleotide Sequences

[0154] The expressible nucleotide sequences that can be inserted into the vectors, concatemers, and cells according to this invention encompass any type of nucleotide such as RNA, DNA. Such a nucleotide sequence could be obtained e.g. from cDNA, which by its nature is expressible. But it is also possible to use sequences of genomic DNA, coding for specific genes. Preferably, the expressible nucleotide sequences correspond to full length genes such as substantially full length cDNA, but nucleotide sequences coding for shorter peptides than the original full length mRNAs may also be used. Shorter peptides may still retain the catalytic activity similar to that of the native proteins.

[0155] Another way to obtain expressible nucleotide sequences is through chemical synthesis of nucleotide sequences coding for known peptide or protein sequences. Thus the expressible DNA sequences does not have to be a naturally occurring sequence, although it may be preferable for practical purposes to primarily use naturally occurring nucleotide sequences. Whether the DNA is single or double stranded will depend on the vector system used.

[0156] In most cases the orientation with respect to the promoter of an expressible nucleotide sequence will be such that the coding strand is transcribed a proper mRNA. It is however conceivable that the sequence may be reversed generating an antisense transcript in order to block expression of a specific gene.

Cassettes

[0157] An important aspect of the invention concerns a cassette of nucleotides in a highly ordered sequence, the cassette having the general formula in 5'.fwdarw.3' direction:

[RS1-RS2-SP-PR-CS-TR-SP-RS2'-RS1]

wherein RS1 and RS1' denote restriction sites, RS2 and RS2 denote restriction sites different from RS1 and RS1', SP individually denotes a spacer sequence of at least two nucleotides, PR denotes a promoter, CS denotes a cloning site, and TR denotes a terminator.

[0158] It is an advantage to have two different restriction sites flanking both sides of the expression construct. By treating the primary vectors with restriction enzymes cleaving both restriction sites, the expression construct and the primary vector will be left with two non-compatible ends. This facilitates a concatenation process, since the empty vectors do not participate in the concatenation of expression constructs.

Restriction Sites

[0159] In principle, any restriction site, for which a restriction enzyme is known can be used. These include the restriction enzymes generally known and used in the field of molecular biology such as those described in Sambrook, Fritsch, Maniatis, "A laboratory Manual", 2.sup.nd edition. Cold Spring Harbor Laboratory Press, 1989.

[0160] The restriction site recognition sequences preferably are of a substantial length, so that the likelihood of occurrence of an identical restriction site within the cloned oligonucleotide is minimised. Thus the first restriction site may comprise at least 6 bases, but more preferably the recognition sequence comprises at least 7 or 8 bases. Restriction sites having 7 or more non N bases in the recognition sequence are generally known as "fare restriction sites" (see example 6). However, the recognition sequence may also be at least 10 bases, such as at least 15 bases, for example at least 16 bases, such as at least 17 bases, for example at least 18 bases, such as at least 18 bases, for example at least 19 bases, for example at least 20 bases, such as at least 21 bases, for example at least 22 bases, such as at least 23 bases, for example at least 25 bases, such as at least 30 bases, for example at least 35 bases, such as at least 40 bases, for example at least 45 bases, such as at least 50 bases.

[0161] Preferably the first restriction site RS1 and RS1' is recognised by a restriction enzyme generating blunt ends of the double stranded nucleotide sequences. By generating blunt ends at this site, the risk that the vector participates in a subsequent concatenation is greatly reduced. The first restriction site may also give rise to sticky ends, but these are then preferably non-compatible with the sticky ends resulting from the second restriction site, RS2 and RS2' and with the sticky ends in the AC.

[0162] According to a preferred embodiment of the invention, the second restriction site, RS2 and RS2' comprises a rare restriction site. Thus, the longer the recognition sequence of the rare restriction site the more rare it is and the less likely is it that the restriction enzyme recognising it will cleave the nucleotide sequence at other--undesired--positions.

[0163] The rare restriction site may furthermore serve as a PCR priming site. Thereby it is possible to copy the cassettes via PCR techniques and thus indirectly "excise" the cassettes from a vector.

Spacer Sequence

[0164] The spacer sequence located between the RS2 and the PR sequence is preferably a non-transcribed spacer sequence. The purpose of the spacer sequence(s) is to minimise recombination between different concatemers present in the same cell or between cassettes present in the same concatemer, but it may also serve the purpose of making the nucleotide sequences in the cassettes more "host" like. A further purpose of the spacer sequence is to reduce the occurrence of hairpin formation between adjacent palindromic sequences, which may occur when cassettes are assembled head to head or tail to tail. Spacer sequences may also be convenient for introducing short conserved nucleotide sequences that may serve e.g. as PCR primer sites or as target for hybridization to e.g. nucleic acid or PNA or LNA probes allowing affinity purification of cassettes.

[0165] The cassette may also optionally comprise another spacer sequence of at least two nucleotides between TR and RS2. When cassettes are cut out from a vector and concatenated into concatemers of cassettes, the spacer sequences together ensure that there is a certain distance between two successive identical promoter and/or terminator sequences. This distance may comprise at least 50 bases, such as at least 60 bases, for example at least 75 bases, such as at least 100 bases, for example at least 150 bases, such as at least 200 bases, for example at least 250 bases, such as at least 300 bases, for example at least 400 bases, for example at least 500 bases, such as at least 750 bases, for example at least 1000 bases, such as at least 1100 bases, for example at least 1200 bases, such as at least 1300 bases, for example at least 1400 bases, such as at least 1500 bases, for example at least 1600 bases, such as at least 1700 bases, for example at least 1800 bases, such as at least 1900 bases, for example at least 2000 bases, such as at least 2100 bases, for example at least 2200 bases, such as at least 2300 bases, for example at least 2400 bases, such as at least 2500 bases, for example at least 2600 bases, such as at least 2700 bases, for example at least 2800 bases, such as at least 2900 bases, for example at least 3000 bases, such as at least 3200 bases, for example at least 3500 bases, such as at least 3800 bases, for example at least 4000 bases, such as at least 4500 bases, for example at least 5000 bases, such as at least 6000 bases.

[0166] The number of the nucleotides between the spacer located 5' to the PR sequence and the one located 3' to the TR sequence may be any. However, it may be advantageous to ensure that at least one of the spacer sequences comprises between 100 and 2500 bases, preferably between 200 and 2300 bases, more preferably between 300 and 2100 bases, such as between 400 and 1900 bases, more preferably between 500 and 1700 bases, such as between 600 and 1500 bases, more preferably between 700 and 1400 bases.

[0167] If the intended host cell is yeast, the spacers present in a concatemer should preferably comprise a combination of a few ARSes with varying lambda phage DNA fragments.

[0168] Preferred examples of spacer sequences include but are not limited. to: Lamda phage DNA, prokaryotic genomic DNA such as E. coli genomic DNA, ARSes.

[0169] Promoter

[0170] A promoter is a DNA sequence to which RNA polymerase binds and initiates transcription. The promoter determines the polarity of the transcript by specifying which strand will be transcribed. [0171] Bacterial promoters normally consist of -35 and -10 (relative to the transcriptional start) consensus sequences which are bound by a specific sigma factor and RNA polymerase. [0172] Eukaryotic promoters are more complex. Most promoters utilized in expression vectors are transcribed by RNA polymerase II. General transcription factors (GTFs) first bind specific sequences . near the transcriptional start and then recruit the binding of RNA polymerase II. In addition to these minimal promoter elements, small sequence elements are recognized specifically by modular DNA-binding/trans-activating proteins (e.g. AP-1, SP-1) which regulate the activity of a given promoter. [0173] Viral promoters may serve the same function as bacterial and eukaryotic promoters. Upon viral . infection of their host, viral promoters direct transcription either by using host transcriptional machinery or by supplying virally encoded enzymes to substitute part of the host machinery. Viral promoters are recognised by the transcriptional machinery of a large number of host organisms and are therefore often used in cloning and expression vectors.

[0174] Promoters may furthermore comprise regulatory elements, which are DNA sequence elements which act in conjunction with promoters and bind either repressors (e.g., IacO/LAC Iq repressor system in E. coli) or inducers (e.g., gal1/GAL4 inducer system in yeast). In either case, transcription is virtually "shut off" until the promoter is derepressed or induced, at which point transcription is "turned-on". The choice of promoter in the cassette is primarily dependent on the host organism into which the cassette is intended to be inserted. An important requirement to this end is that the promoter should preferably be capable of functioning in the host cell, in which the expressible nucleotide sequence is to be expressed.

[0175] Preferably the promoter is an externally controllable promoter, such as an inducible promoter and/or a repressible promoter. The promoter may be either controllable (repressible/inducible) by chemicals such as the absence/presence of chemical inducers, e.g. metabolites, substrates, metals, hormones, sugars. The promoter may likewise be controllable by certain physical parameters such as temperature, pH, redox status, growth stage, developmental stage, or the promoter may be inducible/repressible by a synthetic inducer/repressor such as the gal inducer.

[0176] In order to avoid unintentional interference with the gene regulation systems of the host cell, and in order to improve controllability of the co-ordinated gene expression the promoter is preferably a synthetic promoter. Suitable promoters are described in U.S. Pat. No. 5,798,227, U.S. Pat. No. 5,667,986. Principles for designing suitable synthetic eukaryotic promoters are disclosed in U.S. Pat. No. 5,559,027, U.S. Pat. No. 5,877,018 or U.S. Pat. No. 6,072,050.

[0177] Synthetic inducible eukaryotic promoters for the regulation of transcription of a gene may achieve improved levels of protein expression and lower basal levels of gene expression. Such promoters preferably contain at least two different classes of regulatory elements, usually by modification of a native promoter containing one of the inducible elements by inserting the other of the inducible elements. For example, additional metal responsive elements IR:Es) and/or glucocorticoid responsive elements (GREs) may be provided to native promoters. Additionally, one or more constitutive elements may be functionally disabled to provide the lower basal levels of gene expression.

[0178] Preferred examples of promoters include but is not limited to those promoters being induced and/or repressed by any factor selected from the group comprising carbohydrates, e.g. galactose; low inorganic phosphase levels; temperature, e.g. low or high temperature shift; metals or metal ions, e.g. copper ions; hormones, e.g. dihydrotestosterone; deoxycorticosterone; heat shock (e.g. 39.degree. C.); methanol; redox-status; growth stage, e.g. developmental stage; synthetic inducers, e.g. gal inducer. Examples of such promoters include ADH 1, PGK 1, GAP 491, TPI, PYK, ENO, PMA 1, PHO5, GAL 1, GAL 2, GAL 10, MET25, ADH2, MEL 1, CUP 1, HSE, AOX, MOX, SV40, CaMV, Opaque-2, GRE, ARE, PGK/ARE hybrid, CYC/GRE hybrid, TPI/.alpha.2 operator, AOX 1, MOX A.

[0179] More preferably, however the promoter is selected from hybrid promoters such as PGK/ARE hybrid, CYC/GRE hybrid or from synthetic promoters. Such promoters can be controlled without interfering too much with the regulation of native genes in the expression host.

Yeast Promoters

[0180] In the following, examples of known yeast promoters that may be used in conjunction with the present invention are shown. The examples are by no way limiting and only serve to indicate to the skilled practitioner how to select or design promoters that are useful according to the present invention.

[0181] Although numerous transcriptional promoters which are functional in yeasts have been described in the literature, only some of them have proved effective for the production of polypeptides by the recombinant route. There may be mentioned in particular the promoters of the PGK genes (3-phosphoglycerate kinase, TDH genes encoding GAPDH (Glyceraldehyde phosphate dehydrogenase), TEF1 genes (Elongation factor 1), MF.alpha.1 (.alpha. sex pheromone precursor) which are considered as strong constitutive promoters or alternatively the regulatable promoter CYCI which is repressed in the presence of glucose or PHO5 which can be regulated by thiamine. However, for reasons which are often unexplained, they do not always allow the effective expression of the genes which they control. In this context, it is always advantageous to be able to have new promoters in order to generate new effective host/vector systems. Furthermore, having a choice of effective promoters in a given cell also makes it possible to envisage the production of multiple proteins in this same cell (for example several enzymes of the same metabolic chain) while avoiding the problems of recombination between homologous sequences.

[0182] In general, a promoter region is situated in the 5' region of the genes and comprises all the elements allowing the transcription of a DNA fragment placed under their control, in particular: [0183] (1) a so-called minimal promoter region comprising the TATA box and the site of initiation of transcription, which determines the position of the Site of initiation as well as the basal level of transcription. In Saccharomyces cerevisiae, the length of the minimal promoter region is relatively variable. Indeed, the exact location of the TATA box varies from one gene to another and may be situated from -40 to -120 nucleotides upstream of the site of the initiation (Chen and Struhl, 1985, EMBO J., 4, 3273-3280) [0184] (2) sequences situated upstream of the TATA box (immediately upstream up to several hundreds, of nucleotides) which make it possible to ensure an effective level of transcription either constitutively (relatively constant level of transcription all along the cell cycle, regardless of the conditions of culture) or in a regulatable manner (activation of transcription in the presence of an activator. and/or repression in the presence of a repressor). These sequences, may be of several types: activator, inhibitor, enhancer, inducer, repressor and may respond to cellular factors or varied culture conditions.

[0185] Examples of such promoters are the ZZA1 and ZZA2 promoters disclosed in U.S. Pat. No. 5,641,661, the EF1-.alpha. protein promoter and the ribosomal protein S7 gene promoter disclosed in WO 97/44470, the COX 4 promoter and two unknown promoters (SEQ ID No: 1 and 2 in the document) disclosed in U.S. Pat. No. 5,952,195. Other useful promoters include the HSP150 promoter disclosed in WO 98/54339 and the SV40 and RSV promoters disclosed in U.S. Pat. No. 4,870,013 as well as the PyK and GAPDH promoters disclosed in EP 0 329 203 A1.

Synthetic Yeast Promoters

[0186] More preferably the invention employs the use of synthetic promoters. Synthetic promoters are often constructed by combining the minimal promoter region of one gene with the upstream regulating sequences of another gene. Enhanced promoter control may be obtained by modifying specific sequences in the upstream regulating sequences, e.g. through substitution or deletion or through inserting multiple copies of specific regulating sequences. One advantage of using synthetic promoters is that they may be controlled without interfering too much with the native promoters of the host cell.

[0187] One such synthetic yeast promoter comprises promoters or promoter elements of two different yeast-derived genes, yeast killer toxin leader peptide, and amino terminus of IL-1.beta. (WO 98/54339).

[0188] Another example of a yeast synthetic promoter is disclosed in U.S. Pat. No. 5,436,136 (Hinnen et al), which concerns a yeast hybrid promoter including a 5' upstream promoter element comprising upstream activation site(s) of the yeast PHO5 gene and a 3' downstream promoter element of the yeast GAPDH gene starting at nucleotide -300 to -180 and ending at nucleotide -1 of the GAPDH gene.

[0189] Another example of a yeast synthetic promoter is disclosed in U.S. Pat. No. 5,089,398 (Rosenberg et al). This disclosure describes a promoter with the general formula--

(P.R.(2)-P.R.(1))-

wherein:

[0190] P.R.(1) is the promoter region proximal to the coding sequence and having the transcription initiation site, the RNA polymerase binding site, and including the TATA box, the CAAT sequence, as well as translational regulatory signals, e.g., capping sequence, as appropriate;

[0191] P.R.(2) is the promoter region joined to the 5'-end of P.R.(1) associated with enhancing the efficiency of transcription of the RNA polymerase binding region;

[0192] In U.S. Pat. No. 4,945,046 (Horii et al) discloses a further example of how to design a synthetic yeast promoter. This specific promoter comprises promoter elements derived both from yeast and from a mammal. The hybrid promoter consists essentially of Saccharomyces cerevisiae PHO5 or GAP-DH promoter from which the upstream activation site (UAS) has been deleted and replaced by the early enhancer region derived from SV40 virus.

Cloning Site

[0193] The cloning site in the cassette in the primary vector should be designed so that any nucleotide sequence can be cloned into it.

[0194] The cloning site in the cassette preferably allows directional cloning. Hereby is ensured that transcription in a host cell is performed from the coding strand in the intended direction and that the translated peptide is identical to the peptide for which the original nucleotide sequence codes.

[0195] However according to some embodiments it may be advantageous to insert the sequence in opposite direction. According to these embodiments, so-called antisense constructs may be inserted which prevent functional expression of specific genes involved in specific pathways. Thereby it may become possible to divert metabolic intermediates from a prevalent pathway to another less dominant pathway.

[0196] The cloning site in the cassette may comprise multiple cloning sites, generally known as MCS or polylinker sites, which is a synthetic DNA sequence encoding a series of restriction endonuclease recognition sites. These sites are engineered for convenient cloning of DNA into a vector at a specific position and for directional cloning of the insert.

[0197] Cloning of cDNA does not have to involve the use of restriction enzymes. Other alternative systems include but are not limited to: [0198] Creator.TM. Cre-loxP system from Clontech, which uses recombination and loxP sites [0199] use of Lambda attachment sites (att-.lamda.), such as the Gateway.TM. system from Life Technologies.

[0200] Both of these systems are directional.

Terminator

[0201] The role of the terminator sequence is to limit transcription to the length of the coding sequence. An optimal terminator sequence is thus one, which is capable of performing this act in the host cell.

[0202] In prokaryotes, sequences known as transcriptional terminators signal the RNA polymerase to release the DNA template and stop transcription of the nascent RNA.

[0203] In eukaryotes, RNA molecules are transcribed well beyond the end of the mature mRNA molecule. New transcripts are enzymatically cleaved and modified by the addition of a long sequence of adenylic acid residues known as the poly-A tail. A polyadenylation consensus sequence is located about 10 to 30 bases upstream from the actual cleavage site.

[0204] Preferred examples of yeast derived terminator sequences include, but are not limited to: ADN1, CYC1, GPD, ADH1 alcohol dehydrogenase.

Intron

[0205] Optionally, the cassette in the vector comprises an intron sequence, which may be located 5' or 3' to the expressible nucleotide sequence. The design and layout of introns is well known in the art. The choice of intron design largely depends on the intended host cell, in which the expressible nucleotide sequence is eventually to be expressed. The effects of having intron sequence in the expression cassettes are those generally associated with intron sequences.

[0206] Examples of yeast introns can be found in the literature and in specific databases such as Ares Lab Yeast Intron Database (Version 2.1) as updated on 15 Apr. 2000. Earlier versions of the database as well, as extracts of the database have been published in: "Genome-wide bioinformatic and molecular analysis of introns in Saccharomyces cerevisiae." by Spingola M, Grate L, Haussler D, Ares M Jr. (RNA 1999 February; 5(2):221-34) and "Test of intron predictions reveals novel splice sites, alternatively spliced mRNAs and new introns in meiotically regulated genes of yeast" by Davis C A, Grate L, Spingola M, Ares M Jr, (Nucleic Acids Res 2000 Apr. 15; 28(8):1700-6).

Primary Vectors (Entry Vectors)

[0207] By the term entry vector is meant a vector for storing and amplifying cDNA or other expressible nucleotide sequences using the cassettes according to the present invention. The primary vectors are preferably able to propagate in E. coli or any other suitable standard host cell. It should preferably be amplifiable and amenable to standard normalisation and enrichment procedures.

[0208] The primary vector may be of any type of DNA that has the basic requirements of a) being able to replicate itself in at least one suitable host organism and b) allows insertion of foreign DNA which is then replicated together with the vector and c) preferably allows selection of vector molecules that contain insertions of said foreign DNA. In a preferred embodiment the vector is able to replicate in standard hosts like yeasts, and bacteria and it should preferably have a high copy number per host cell. It is also preferred that the vector in addition to a host specific origin of replication, contains an origin of replication for a single stranded virus, such as e.g. the f1 origin for filamentous phages. This will allow the production of single stranded nucleic acid which may be useful for normalisation and enrichment procedures of cloned sequences. A vast number of cloning vectors have been described which are commonly used and references may be given to e.g. Sambrook, J; Fritsch, E. F; and Maniatis T. (1989) Molecular Cloning: A laboratory manual. Cold Spring Harbour Laboratory Press, USA, Netherlands Culture Collection of Bacteria (www.cbs.knaw.nl/NCCB/collection.htm) or Department of Microbial Genetics, National Institute of Genetics, Yata 1111 Mishima Shizuoka 411-8540, Japan (www.shigen.nig.ac.jp/cvector/cvector.html). A few type-examples that are the parents of many popular derivatives are M13mp10, pUC18. Lambda gt 10, and pYAC4. Examples of primary vectors include but are not limited to M13K07, pBR322, pUC18, pUC19, pUC118, pUC119, pSP64, pSP65, pGEM-3, pGEM-3Z, pGEM-3Zf(-), pGEM-4, pGEM-4Z, .pi.AN13, pBluescript II, CHARON 4A, .lamda.*, CHARON 21A, CHARON 32, CHARON 33, CHARON 34, CHARON 35, CHARON 40, EMBL3A, .lamda.2001, .lamda.DASH, .lamda.FIX, .lamda.gt10, .lamda.gt11, .lamda.gt18, .lamda.gt20, .lamda.gt22, .lamda.ORF8, .lamda.ZAP/R, pJB8, c2RB, pcos1EMBL

[0209] Methods for cloning of cDNA or genomic DNA into a vector are well known in the art. Reference may be given to J. Sambrook, E. F. Fritsch, T. Maniatis: Molecular Cloning, A Laboratory Manual (2.sup.nd edition, Cold Spring Harbor Laboratory Press, 1989).

[0210] One example of a circular model entry vector is described in FIG. 3. The vector, EVE contains the expression cassette, R1-R2-Spacer-Promoter-Multi Cloning Site-Terminator-Spacer-R2-R1. The vector furthermore contains a gene for ampicillin resistance, AmpR, and an origin of replication for E. coli, ColE1.

[0211] The entry vectors EVE4, EVE5, and EVE8 shown in FIGS. 4, 5, and 6. These all contain SrfI as R1 and AscI as R2. Both of these sites are palindromic and are regarded as rare restriction sites having 8 bases in the recognition sequence. The vectors furthermore -contain the AmpR ampicillin resistance gene, and the ColE1 origin or replication for E. coli as well as f1, which is an origin of replication for filamentous phages, such as M13. EVE4 (FIG. 4) contains the MET25 promoter and the ADH1 terminator. Spacer 1 and spacer 2 are short sequences deriving from the multiple cloning site, MCS. EVE5 (FIG. 5) contains the CUP1 promoter and the ADH1 terminator. EVE8 (FIG. 6) contains the CUP1 promoter and the ADH1 terminator. The spacers of EVE8 are a 550 bp lambda phage DNA (spacer 3) and an ARS sequence from yeast (spacer 4).

Nucleotide Library (Entry Library)

[0212] Methods as well as suitable vectors and host cells for constructing and maintaining a library of nucleotide sequences in a cell are well known in the art. The primary requirement for the library is that is should be possible to store and amplify in it a number of primary vectors (constructs) according to this invention, the vectors (constructs) comprising expressible nucleotide sequences from at least one expression state and wherein at least two vectors (constructs) are different.

[0213] One specific example of such a library is the well known and widely employed cDNA libraries. The advantage of the cDNA library is mainly that it contains only DNA sequences corresponding to transcribed messenger RNA in a cell. Suitable methods are also present to purify the isolated mRNA or the synthesised cDNA so that only substantially full-length cDNA is cloned into the library.

[0214] Methods for optimisation of the process to yield substantially full length cDNA may comprise size selection, e.g. electrophoresis, chromatography, precipitation or may comprise ways of increasing the likelihood of getting full length cDNAs, e.g. the SMART.TM. method (Clonetech) or the CapTrap.TM. method (Stratagene).

[0215] Preferably the method for making the nucleotide library comprises obtaining a substantially full length cDNA population comprising a normalised representation of cDNA species. More preferably a substantially full length cDNA population comprises a normalised representation of cDNA species characteristic of a given expression state.

[0216] Normalisation reduces the redundancy of clones representing abundant mRNA species and increases the relative representation of clones from rare mRNA species.

[0217] Methods for normalisation of cDNA libraries are well known in the art. Reference may be given to suitable protocols for normalisation such as those described in U.S. Pat. No. 5,763,239 (DIVERSA) and WO 95/08647 and WO 95/11986. and Bonaldo, Lennon, Soares, Genome Research 1996, 6:791-806; Ali, Holloway, Taylor, Plant Mol Biol Reporter, 2000, 18:123-132.

[0218] Enrichment methods are used to isolate clones representing mRNA which are characteristic of a particular expression state. A number of variations. of the method broadly termed as subtractive hybrisation are known in the art. Reference may be given to Sive, John, Nucleic Acid Res, 1988, 16:10937; DiatChenko, Lau, Campbell et al, PNAS, 1996, 93:6025-6030; Caminci, Shibata, Hayatsu, Genome Res, 2000, 10:1617-30, Bonaldo, Lennon, Soares, Genome Research 1996, 6:791-806; Ali, Holloway, Taylor, Plant Mol Blot Reporter, 2000, 18:123-132. For example, enrichment may be achieved by doing additional rounds of hybridization similar to normalization procedures, using e.g. cDNA from a library of abundant clones or simply a library representing the uninduced state as a driver against a tester library from the induced state. Alternatively mRNA or PCR amplified cDNA derived from the expression state of choice can be used to subtract common sequences from a tester library. The choice of driver and tester population will depend on the nature of target expressible nucleotide sequences in each particular experiment

[0219] In the library an expressible nucleotide sequence coding for one peptide is preferably found in different but similar vectors under the control of different promoters. Preferably the library comprises at least three primary vectors with an expressible nucleotide sequence coding for the same peptide under the control of three different promoters. More preferably the library comprises at least four primary. vectors with an expressible nucleotide sequence coding for the same peptide under the control of four different promoters. More preferably the library comprises at least five primary vectors with an expressible nucleotide sequence coding for the same peptide under the control of five different promoters, such as comprises at lest six primary vectors with an expressible nucleotide sequence coding for the same peptide under the control of six different promoters, for example comprises at least seven primary vectors with an expressible nucleotide sequence coding for the same peptide under the control of seven different promoters, for example comprises at least eight primary vectors with an expressible nucleotide sequence coding for the same peptide under the control of eight different promoters, such as comprises at least nine primary vectors with an expressible nucleotide sequence coding for the same peptide under the control of nine different promoters, for example comprises at least ten primary vectors with an expressible nucleotide sequence coding for the same peptide under the control of ten different promoters.

[0220] The expressible nucleotide sequence coding for the same peptide preferably comprises essentially the same nucleotide sequence, more preferably the same nucleotide sequence.

[0221] By having a library with what may be termed one gene under the control of a number of different promoters different vectors, it is possible to construct from the nucleotide library an array of combinations of genes and promoters. Preferably, one library comprises a complete or substantially complete combination such as a two dimensional array of genes and promoters, wherein substantially all genes are found under the control of substantially all of a selected number of promoters.

[0222] According to another embodiment of the invention the nucleotide library comprises combinations of expressible nucleotide sequences combined in different vectors with different spacer sequences and/or different intron sequences. Thus any one expressible nucleotide sequence may be combined in a two, three, four or five dimensional array with different promoters and/or different spacers and/or different introns and/or different terminators. The two, three, four or five dimensional array may be complete or incomplete, since not all combinations will have to be present.

[0223] The library may suitably be maintained in a host cell comprising prokaryotic cells or eukaryotic cells. Preferred prokaryotic host organisms may include but are not limited to Escherichia coli, Bacillus subtilis, Streptomyces lividans, Streptomyces coelicolor Pseudomonas aeruginosa, Myxococcus xanthus.

[0224] Yeast species such as Saccharomyces cerevisiae (budding yeast), Schizosaccharomyces pombe (fission yeast), Pichia pastoris, and Hansenula polymorpha (methylotropic yeasts) may also be used. Filamentous ascomycetes, such as Neurospora crassa and Aspergillus nidulans may also be used. Plant cells such as those derived from Nicotiana and Arabidopsis are preferred: Preferred mammalian host cells include but are not limited to those derived from humans, monkeys and rodents, such is chinese hamster ovary (CHO) cells, NIH/3T3, COS, 293, VERO, HeLa etc (see Kriegler M. in "Gene Transfer and Expression: A Laboratory Manual", New York, Freeman & Co. 1990).

Concatemers

[0225] A concatemer is a series of linked units. In the present context a concatemer is used to denote a number of serially linked nucleotide cassettes, wherein at least two of the serially linked nucleotide units comprises a cassette having the basic structure

[rs.sub.2-SP-PR-X-TR-SP-rs.sub.1] [0226] wherein [0227] rs.sub.1 and rs.sub.2 together denote a restriction site, [0228] SP individually denotes a spacer of at least two nucleotide bases, [0229] PR denotes a promoter, capable of functioning in a cell, [0230] X denotes an expressible nucleotide sequence, [0231] TR denotes a terminator, and [0232] SP individually denotes a spacer of at least two nucleotide bases.

[0233] Optionally the cassettes comprise an intron sequence between the promoter and the expressible nucleotide sequence and/or between the terminator and the expressible sequence.

[0234] The expressible nucleotide sequence in the cassettes of the concatemer may comprise a DNA sequence selected from the group comprising cDNA and genomic DNA.

[0235] According to one aspect of the invention, a concatemer comprises cassettes with expressible nucleotide from different expression states, so that non-naturally occurring combinations or non-native combinations of expressible nucleotide sequences are obtained. These different expression states may represent at least two different tissues, such as at least two organs, such as at least two species, such as at least two genera. The different spies may be from at least two different phylae, such as from at least two different classes, such as from at least two different divisions, more preferably from at least two different sub-kingdoms, such as from at least two different kingdoms.

[0236] For example, the expressible nucleotide sequences may originate from eukaryots such as mammals such as humans, mice or whale, from reptiles such as snakes crocodiles or turtles, from tunicates such as sea squirts, from lepidoptera such as butterflies and moths, from coelenterates such as jellyfish, anenomes, or corals, from fish such as bony and cartilaginous fish, from plants such as dicots, e.g. coffee, oak or monocots such as grasses, lilies, and orchids; from lower plants such as algae and gingko, from higher fungi such as terrestrial fruiting fungi, from marine actinomycetes. The expressible nucleotide sequences may also originate from protozoans such as malaria or trypanosomes, or from prokaryotes such as E. coli or archaebacteria. Furthermore, the expressible nucleotide sequences may originate from one or more preferably from more expression states from the species and genera listed in the table below.

TABLE-US-00002 Bacteria Streptomyces, Micromonospora, Norcadia, Actinomadura, Actinoplanes, Streptosporangium, Microbispora, Kitasatosporiam, Azobacterium, Rhizobium, Achromobacterium, Enterobacterium, Brucella, Micrococcus, Lactobacillus, Bacillus (B.t. toxins), Clostridium (toxins), Brevibacterium, Pseudomonas, Aerobacter, Vibrio, Halobacterium, Mycoplasma, Cytophaga, Myxococcus Fungi Amanita muscaria (fly agaric, ibotenic acid, muscimol), Psilocybe (psilocybin) Physarium, Fuligo, Mucor, Phytophtora, Rhizopus, Aspergillus, Penicillium (penicillin), Coprinus, Phanerochaete, Acremonium (Cephalosporin), Trochoderma, Helminthosporium, Fusarium, Alternaria, Myrothecium, Saccharomyces Algae Digenea simplex (kainic acid, antihelminthic), Laminaria anqustata (laminine, hypotensive) Lichens Usnea fasciata (vulpinicacid, antimicrobial; usnic acid, antitumor) Higher Artemisia (artemisinin), Coleus (forskolin), Desmodium (K channel agonist), Plants Catharanthus (Vinca alkaloids), Digitalis (cardiac glycosides), Podophyllum (podophyllotoxin), Taxus (taxol), Cephalotaxus (homoharringtonine), Camptotheca (Camptothecin), Camellia sinensis (Tea), Cannabis indica, Cannabis sativa (Hemp), Erythroxylum coca (Coca), Lophophora williamsii (Peyote Myristica fragrans (Nutmeg), Nicotiana, Papaver somniferum (Opium Poppy), Phalaris arundinacea (Reed canary grass) Protozoa Ptychodiscus brevis; Dinoflagellates (brevitoxin, cardiovascular) Sponges Microciona prolifera (ectyonin, antimicrobial) Cryptotethya cryta (D-arabino furanosides) Coelenterata Portuguese Man o War & other jellyfish and medusoid toxins. Corals Pseudoterogonia species (Pseudoteracins, anti-inflammatory), Erythropodium (erythrolides, anti-inflammatory) Aschelminths Nematode secretory compounds Molluscs Conus toxins, sea slug toxins, cephalapod neurotransmitters, squid inks Annelida Lumbriconereis heteropa (nereistoxin, insecticidal) Arachnids Dolomedes ("fishing spider" venoms) Crustacea Xenobalanus (skin adhesives) Insects Epilachna (mexican bean beetle alkaloids) Spinunculida Bonellia viridis (bonellin, neuroactive) Bryozoans Bugula neritina (bryostatins, anti cancer) Echinoderms Crinoid chemistry Tunicates Trididemnum solidum (didemnin, anti-tumor and anti-viral; Ecteinascidia turbinata ecteinascidins, anti-tumor) Vertebrates Eptatretus stoutii (eptatretin, cardioactive), Trachinus draco (proteinaceous toxins, reduce blood pressure, respiration and reduce heart rate). Dendrobatid frogs (batrachotoxins, pumiliotoxins, histrionicotoxins, and other polyamines); Snake venom toxins; Orinthorhynohus anatinus (duck-billed platypus venom), modified carotenoids, retinoids and steroids; Avians: histrionicotoxins, modified carotenoids, retinoids and steroids

[0237] According to a preferred embodiment of the invention the concatemer comprises at least a first cassette and a second cassette, said first cassette being different from said second cassette. More preferably, the concatemer comprises cassettes, wherein substantially all cassettes are different. The difference between the cassettes may arise from differences between promoters, and/or expressible nucleotide sequences, and/or spacers, and/or terminators, and/or introns.

[0238] The number of cassettes in a single concatemer is largely determined by the host species into which the concatemer is eventually to be inserted and the vector through which the insertion is carried out. The concatemer thus may comprise at least 10 cassettes, such as at least 15, for example at least 20, such as at least 25, for example at least 30, such as from 30 to 60 or more than 60, such as at least 75, for example at least 100, such as at least 200, for example at least 500, such as at least 750, for example at least 1000, such as at least 1500, for example at least 2000 cassettes.

[0239] Each of the cassettes may be laid out as described above.

[0240] Once the concatemer has been assembled or concatenated it may be ligated into a suitable vector. Such a vector may advantageously comprise an artificial chromosome. The basic requirements for a functional artificial chromosome have been described in U.S. Pat. No. 4,464,472, the contents of which is hereby incorporated by reference. An artificial chromosome or a functional minichromosome, as it may also be termed must comprise a DNA sequence capable of replication and stable mitotic maintenance in a host cell comprising a DNA segment coding for centromere-like activity during mitosis of said host and a DNA sequence coding for a replication site recognized by said host.

[0241] Suitable artificial chromosomes include a Yeast Artificial Chromosome (YAC) (see e.g. Murray et al. Nature 305:189-193; or U.S. Pat. No. 4,464,472), a mega Yeast Artificial Chromosome (mega YAC), a Bacterial Artificial Chromosome (BAC), a mouse artificial chromosome, a Mammalian Artificial Chromosome (MAC) (see e.g. U.S. Pat. No. 6,133,503 or U.S. Pat. No. 6,077,697), an Insect Artificial Chromosome (BUGAC), an Avian Artificial Chromosome (AVAC), a Bacteriophage Artificial Chromosome, a Baculovirus Artificial Chromosome, a plant artificial chromosome (U.S. Pat. No. 5,270,201), a BIBAC vector (U.S. Pat. No. 5,977,439) or a Human Artificial Chromosome (HAC).

[0242] The artificial chromosome is preferably so large that the host cell perceives it as a "real" chromosome and maintains it and transmits it as a chromosome. For yeast and other suitable host species, this will often correspond approximately to the size of the smallest native chromosome in the species. For Saccharomyces, the smallest chromosome has a size of 225 Kb.

[0243] MACs may be used to construct artificial chromosomes from other species, such as insect and fish species. The artificial chromosomes preferably are fully functional stable chromosomes. Two types of artificial chromosomes may be used. One type, referred to as SATACs [satellite artificial chromosomes] are stable heterochromatic chromosomes, and the other type are minichromosomes based on amplification of euchromatin.

[0244] Mammalian artificial chromosomes provide extra-genomic specific integration sites for introduction of genes encoding proteins of interest and permit megabase size DNA integration, such as integration of concatemers according to the invention.

[0245] According to another embodiment of the invention, the concatemer may be integrated into the host chromosomes or cloned into other types of vectors, such as a plasmid vector, a phage vector, a viral vector or a cosmid vector.

[0246] A preferable artificial chromosome vector is one that is capable of being conditionally amplified in the host cell, e.g. in yeast. The amplification preferably is at least a 10 fold amplification. Furthermore, it is advantageous that the cloning site of the artificial chromosome vector can be modified to comprise the same restriction site as the one bordering the cassettes described above, i.e. RS2 and/or RS2'.

Concatenation

[0247] Cassettes to be concatenated are normally excised from a vector either by digestion with restriction enzymes or by PCR. After excision the cassettes may be separated from the vector through size fractionation such as gel filtration or through tagging of known sequences in the cassettes. The isolated cassettes may then be joined together either through interaction between sticky ends or through ligation of blunt ends.

[0248] Single-stranded compatible ends may be created by digestion with restriction enzymes. For concatenation a preferred enzyme for excising the cassettes would be a rare cutter, i.e. an enzyme that recognises a sequence of 7 or more nucleotides. Examples of enzymes that cut very rarely are the meganucleases, many of which are intron encoded, like e.g. I-Ceu I, I-Sce I, I-Ppo I, and PI-Psp I (see example 6d for more). Other preferred enzymes recognize a sequence of 8 nucleotides like e.g. Asc I, AsiS I, CciN I, CspB I, Fse I, MchA I, Not I, Pac I, Sbf I, Sda I, Sgf I, SgrA I, Sse232 I, and Sse8387 I, all of which create single stranded, palindromic compatible ends.

[0249] Other preferred rare cutters, which may also be used to control orientation of individual cassettes in the concatemer are enzymes that recognize non-palindromic sequences like e.g. Aar I, Sap I, Sfi I, Sdi I, and Vpa (see example 6c for more).

[0250] Alternatively, cassettes can be prepared by the addition of restriction sites to the ends, e.g. by PCR or ligation to linkers (short synthetic dsDNA molecules). Restriction enzymes are continuously being isolated and characterised and it is anticipated that many of such novel enzymes can be used to generate single-stranded compatible ends according to the present invention.

[0251] It is conceivable that single stranded compatible ends can be made by cleaving the vector with synthetic cutters. Thus, a reactive chemical group that will normally be able to cleave DNA unspecifically can cut at specific positions when coupled to another molecule that recognises and binds to specific sequences. Examples of molecules that recognise specific dsDNA sequences are DNA, PNA, LNA, phosphothioates, peptides, and amides. See e.g. Armitage, B.(1998) Chem. Rev. 98: 1171-1200, who describes photocleavage using e.g. anthraquinone and UV light; Dervan P. B. & Bildt R. W. (1999) Curr. Opin. Chem. Biol. 3: 688-93 describes the specific binding of polyamides to DNA; Nielsen, P. E. (2001) Curr. Opin. Biotechnol. 12: 16-20 describes the specific binding of PNA to DNA, and Chemical Reviews special thematic issue: RNA/DNA Cleavage (1998) vol. 98 (3) Bashkin J. K. (ed.) ACS publications, describes several examples of chemical DNA cleavers.

[0252] Single-stranded compatible ends may also be created by using e.g. PCR primers including dUTP and then treating the PCR product with Uracil-DNA glycosylase (Ref: U.S. Pat. No. 5,035,996) to degrade part of the primer. Alternatively, compatible ends can be created by tailing both the vector and insert with complimentary nucleotides using Terminal Transferase (Chang, L M S, Bottum T J (1971) J Biol Chem 246:909).

[0253] It is also conceivable that recombination can be used to generate concatemers, e.g. through the modification of techniques like the Creator.TM. system (Clontech) which uses the Cre-loxP mechanism (Sauer B 1993 Methods Enzymol 225:890-900) to directionally join DNA molecules by recombination or like the Gateway.TM. system (Life Technologies, U.S. Pat. No. 5,888,732) using lambda att attachment sites for directional recombination (Landy A 1989, Ann Rev Biochem 58:913). It is envisaged that also lambda cos site dependent systems can be developed to allow concatenation.

[0254] More preferably the cassettes may be concatenated without an intervening purification step through excision from a vector with two restriction enzymes, one leaving sticky ends on the cassettes and the other one leaving blunt ends in the vectors. This is the preferred method for concatenation of cassettes from vectors having the basic structure of [RS1-RS2-SP-PR-X-TR-SP-RS2'-RS1'].

[0255] An alternative way of producing concatemers free of vector sequences would be to PCR amplify the cassettes from a single stranded primary vector. The PCR product must include the restriction sites RS2 and RS2' which are subsequently cleaved by its cognate enzyme(s). Concatenation can then be performed using the digested , PCR product, essentially without interference from the single , stranded primary vector template or the small double stranded fragments, which have been cut from the ends.

[0256] The concatemer may be assembled or concatenated by concatenation of at least two cassettes of nucleotide sequences each cassette comprising a first sticky end, a spacer sequence, a promoter, an expressible nucleotide sequence, a terminator, a spacer sequence, and a second sticky end. A flow chart of the procedure is shown in FIG. 2a.

[0257] Preferably concatenation further comprises [0258] starting from a primary vector [RS1-RS2-SP-PR-X-TR-SP-RS2'-RS1'], [0259] wherein X denotes an expressible nucleotide sequence, [0260] RS1 and RS1' denote restriction sites, [0261] RS2 and RS2' denote restriction sites different from RS1 and RS1', [0262] SP individually denotes a Spacer sequence of at least two nucleotides, [0263] PR denotes a promoter, [0264] TR denotes a terminator, [0265] i) cutting the primary vector with the aid of at least one restriction enzyme specific for RS2 and RS2' obtaining cassettes having the general formula [rs.sub.2-SP-PR-X-TR-SP-rs.sub.1] wherein rs.sub.1 and rs.sub.2 together denote a functional restriction site RS2 or RS2', [0266] ii) assembling the cut out cassettes through interaction, between rs.sub.1 and rs.sub.2.

[0267] In this way at least 10 cassettes. can be concatenated, such as at least 15, for example at least 20, such as at least 25, for example at least 30, such as from 30 to 60 or more than 60, such as at least 75, for example at least 100, such as at least 200, for example at least 500, such as at least 750, for example at least 1000, such as at least 1500, for example at least 2000.

[0268] According to an especially preferred embodiment, vector arms each having a RS2 or RS2' in one end and a non-complementary overhang or a blunt end in the other end are added to the concatenation mixture together with the cassettes described above to further simplify the procedure (see FIG. 2b). One example of a suitable vector for providing vector arms is disclosed in FIG. 7 TRP1, URA3, and HIS3' are auxotrophic marker genes, and AmpR is an E. coli antibiotic marker gene. CEN4 is a centromer and TEL are telomeres. ARS1 and PMB1 allow replication in yeast and E. coli respectively. BamH I and Asc I are restriction enzyme recognition sites. The nucleotide sequence of the vector is set forth in SEQ ID NO 4. The vector is digested with BamHI and AscI to liberate the vector arms, which are used for ligation to the concatemer.

[0269] The ratio of vector arms to cassettes determines the maximum number of cassettes in the concatemer as illustrated in FIG. 8. The vector arms preferably are artificial chromosome vector arms such as those described in FIG. 7.

[0270] It is of course also possible to add stopper fragments to the concatenation solution, the stopper fragments each having a RS2 or RS2' in one end and a non-complementary overhang or a blunt end in the other end. The ratio of stopper fragments to cassettes can likewise control the maximum size of the concatemer.

[0271] The complete sequence of steps to be taken when starting with the isolation of mRNA until inserting into an entry vector may include the following steps [0272] i) isolating mRNA from an expression state, [0273] ii) obtaining substantially full length cDNA corresponding to the mRNA sequences, [0274] iii) inserting the substantially full length cDNA into a cloning site in a cassette in a primary vector, said cassette being of the general formula in 5'.fwdarw.3' direction:

[0274] [RS1-RS2-SP-PR-CS-TR-SP-RS2'-RS1'] [0275] wherein CS denotes a cloning site.

[0276] In preparation of the concatemer, genes may be isolated from different entry libraries to provide the desired selection of genes. Accordingly, concatenation may further comprise selection of vectors having expressible nucleotide sequences from at least two different expression states, such as from two different species. The two different species may be from two different classes, such as from two different divisions, more preferably from two different sub-kingdoms, such as from two different kingdoms.

[0277] As an alternative to including vector arms in the concatenation reaction it is possible to ligate the concatemer into an artificial chromosome selected from the group comprising yeast artificial chromosome, mega yeast artificial chromosome, bacterial artificial chromosome, mouse artificial chromosome, human artificial chromosome.

[0278] Preferably at least one inserted concatemer further comprises a selectable marker. The marker(s) are conveniently not included in the concatemer as such but rather in an artificial chromosome vector, into which the concatemer is inserted. Selectable markers generally provide a means to select, for growth, only those cells which contain a vector. Such markers are of two types: drug resistance and auxotrophy. A drug resistance marker enables cells to grow in the presence of an otherwise toxic compound. Auxotrophic markers allow cells to grow in media lacking an essential component by enabling cells to synthesise the essential component (usually an amino acid).

[0279] Illustrative and non-limiting examples of common compounds for which selectable markers are available with a brief description of their mode of action follow:

Prokaryotic

[0280] Ampicillin: interferes with a terminal reaction in bacterial cell wall synthesis. The resistance gene (bla) encodes beta-lactamase which cleaves the beta-lactam ring of the antibiotic thus detoxifying it. [0281] Tetracycline: prevents bacterial protein synthesis by binding to the 30S ribosomal subunit. The resistance gene (tet) specifies a protein that modifies the bacterial membrane and prevents accumulation of the antibiotic in the cell. [0282] Kanamycin: binds to the 70S ribosomes and causes misreading of messenger RNA. The resistant gene (nptH) modifies the antibiotic and prevents interaction with the ribosome. [0283] Streptomycin: binds to the 30S ribosomal subunit, causing misreading of messenger RNA. The resistance gene (Sm) modifies the antibiotic and prevents interaction with the ribosome. [0284] Zeocin: this new bleomycin-family antibiotic intercalates into the DNA and cleaves it. The Zeocin resistance gene encodes a 13,665 dalton protein. This protein confers resistance to Zeocin by binding to the antibiotic and preventing it from binding DNA. Zeocin is effective on most aerobic cells and can be used for selection in mammalian cell lines, yeast, and bacteria.

Eukaryotic

[0284] [0285] Hygromycin: a aminocyclitol that inhibits protein synthesis by disrupting ribosome. translocation and promoting mistranslation. The resistance gene (hph) detoxifies hygromycin -B- phosphorylation. [0286] Histidinol: cytotoxic to mammalian cells by inhibiting histidyl-tRNA synthesis in histidine free media. The resistance gene (hisD) product inactivates histidinol toxicity by converting it to the essential amino acid, histidine. [0287] Neomycin (G418): blocks protein synthesis by interfering with ribosomal functions. The resistance gene ADH encodes amino glycoside phosphotransferase which detoxifies G418. [0288] Uracil: Laboratory yeast strains carrying a mutated gene which encodes orotidine -5'-phosphate decarboxylase, an enzyme essential for uracil biosynthesis, are unable to grow in the absence of exogenous uracil. A copy of the wild-type gene (ura4+, S. pombe or URA3 S. cerevisiae) carried on the vector will complement this defect in transformed cells. [0289] Adenosine: Laboratory strains carrying a deficiency in adenosine synthesis may be complemented by a vector carrying the wild type gene. ADE 2. [0290] Amino acids: Vectors carrying the wild-type genes for LEU2, TRP 1, HIS 3 or LYS 2 may be used to complement strains of yeast deficient in these genes. [0291] Zeocin: this new bleomycin-family antibiotic intercalates into the DNA and cleaves it. The Zeocin resistance gene encodes a 13,665 dalton protein. This protein confers resistance to Zeocin by binding to the antibiotic and preventing it from binding DNA. Zeocin is effective on most aerobic cells and can be used for selection in mammalian cell lines, yeast, and bacteria.

Transgenic Cells

[0292] In one aspect of the invention, the concatemers comprising the multitude of cassettes are introduced into a host cell, in which the concatemers can be maintained and the expressible nucleotide sequences can be expressed in a co-ordinated way. The cassettes comprised in the concatemers may be isolated from the host cell and re-assembled due to their uniform structure with--preferably--concatemer restriction sites between the cassettes.

[0293] The host cells selected for this purpose are preferably cultivable under standard laboratory conditions using standard culture conditions, such as standard media and protocols. Preferably the host cells comprise a substantially stable cell line, in which the concatemers can be maintained for generations of cell division. Standard techniques for transformation of the host cells and in particular methods for insertion of artificial chromosomes into the host cells are known.

[0294] It is also of advantage if the host cells are capable of undergoing meiosis to perform sexual recombination. It is also advantageous that meiosis is controllable through external manipulations of the cell culture. One especially advantageous host cell type is one where the cells can be manipulated through external manipulations into different mating types.

[0295] The genome of a number of species have already, been sequenced more or less completely and the sequences can be found in databases. The list of species for which the whole genome has been sequenced increases constantly. Preferably the host cell is selected from the group of species, for which the whole genome or essentially the whole genome has been sequenced. The host cell should preferably be selected from a species that is well described in the literature with respect to genetics, metabolism, physiology such as model organism used for genomics research.

[0296] The host organism should preferably be conditionally deficient in the abilities to undergo homologous recombination. The host organism should preferably have a codon usage similar to that of the donor organisms. Furthermore, in the case of genomic DNA, if eukaryotic donor organisms are used, it is preferable that the host organism has the ability to process the donor messenger RNA properly, e.g., splice out introns.

[0297] The host cells can be bacterial, archaebacteria, or eukaryotic and can constitute a homogeneous cell line or mixed culture. Suitable cells include the bacterial and eukaryotic cell lines commonly used in genetic engineering and protein expression.

[0298] Preferred prokaryotic host organisms may include but are not limited to Escherichia coli, Bacillus subtilis, B. licehniformis, B. cereus, Streptomyces lividans, Streptomyces coelicolor, Pseudomonas aeruginosa, Myxococcus xanthus, RhOdococcus, Streptomycetes, Actinomycetes, Corynebacteria, Bacillus, Pseudomonas, Salmonella, and Erwinia. The complete genome sequences of E. coli and Bacillus subtilis are described by Blattner et al., Science 277, 1454-1462 (1997); Kunst et al., Nature 390, 249-256 (1997)).

[0299] Preferred eukaryotic host organisms are mammals, fish, insects, plants, algae and fungi.

[0300] Examples of mammalian cells include those from, e.g., monkey, mouse, rat, hamster, primate, and human, both cell lines and primary cultures. Preferred mammalian host cells include but are not limited to those derived from humans, monkeys and rodents, such as chinese hamster ovary (CHO) cells, NIH/3T3. COS, 293, VERO, HeLa etc (see Kriegler M. in "Gene Transfer and Expression: A Laboratory Manual", New York, Freeman & Co. 1990), and stem cells, including embryonic stem cells and hemopoietic stem cells, zygotes, fibroblasts, lymphocytes, kidney, liver, muscle, and skin cells.

[0301] Examples of insect cells include baculo lepidoptera.

[0302] Examples of plant cells include maize, rice, wheat, cotton, soybean, and sugarcane. Plant cells such as those derived from Nicotiana and Arabidopsis are preferred

[0303] Examples of fungi include penicillium, aspergillus, such as Aspergillus nidulans, podospora, neurospora, such as Neurospora crassa, saccharomyces, such as Saccharomyces cerevisiae (budding yeast), Schizosaccharomyces, such as Schizosaccharomyces pombe (fission yeast), Pichia spp, such as Pichia pastoris, and Hansenula polymorpha (methylotropic yeasts).

[0304] In a preferred embodiment the host cell is a yeast cell, and an illustrative and not limiting list of suitable yeast host cells comprise: bakers yeast, Kluyveromyces marxianus, K. lactis, Candida utilis, Phaffia rhodozyma, Saccharomyces boulardii, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, Candida paraffinica, Schwanniomyces castellii, Pichia stipitis, Candida shehatae, Rhodotorula glutinis, Lipomyces lipofer, Cryptococcos curvatus, Candida spp. (e.g. C. palmioleophila), Yarrowia lipolytica, Candida guilliermondii, Candida, Rhodotorula spp., Saccharomycopsis spp., Aureobasidium pullulans, Candida brumptii, Candida hydrocarbofumarica, Torulopsis, Candida tropicalis, Saccharomyces cerevisiae, Rhodotorula rubra, Candida flayeri, Eremothecium ashbyii, Pichia spp., Pichia pastoris, Kluyveromyces, Hansenula, Kloeckera, Pichia, Pachysolen spp., or Torulopsis bombicola.

[0305] The choice of host will depend on a number of factors, depending on the intended use of the engineered host, including pathogenicity, substrate range, environmental hardiness, presence of key intermediates, ease of genetic manipulation, and likelihood of promiscuous transfer of genetic information to other organisms. Particularly advantageous hosts are E. coli, lactobacilli, Streptomycetes, Actinomycetes, Saccharomyces and filamentous fungi.

[0306] In any one host cell it is possible to make all sorts of combinations of expressible nucleotide sequences from all possible sources. Furthermore, it is possible to make combinations of promoters and/or spacers and/or introns and/or terminators in combination with one and the same expressible nucleotide sequence.

[0307] Thus in any one cell there may be expressible nucleotide sequences from two different expression states. Furthermore, these two different expression states may be from one species or advantageously from two different species. Any one host cell may also comprise expressible nucleotide sequences from at least three species, such as from at least four, five, six, seven, eight, nine or ten species, or from more than 15 species such as from more than 20 species, for example from more than 30, 40 or 50 species, such as from more than 100 different species, for example from more than 300 different species, such as form more than 500 different species, for example from more than 1000 different species, thereby obtaining combinations of large numbers of expressible nucleotide sequences from a large number of species. In this way potentially unlimited numbers of combinations of expressible nucleotide sequences can be combined across different expression states. These different expression states may represent at least two different tissues, such as at least two organs, such as at least two species, such as at least two genera. The different species may be from at least two different phylae, such as from at least two different classes, such as from at least two different divisions, more preferably from at least two different sub-kingdoms, such as from at least two different kingdoms.

[0308] Any two of these species may be from two different classes, such as from two different divisions, more preferably from two different sub-kingdoms, such as from two different kingdoms. Thus expressible nucleotide sequences may be combined from a eukaryot and a prokaryot into one and the same cell.

[0309] According to another embodiment of the invention, the expressible nucleotide sequences may be from one and the same expression state. The products of these sequences may interact with the products of the genes in the host cell and form new enzyme combinations leading to novel biochemical pathways. Furthermore, by putting the expressible nucleotide sequences under the control of a number of promoters it becomes possible to switch on and off groups of genes in a co-ordinated manner. By doing this with expressible nucleotide sequences from only one expression states, novel combinations of genes are also expressed.

[0310] The number of concatemers in one single cell may be at least one concatemer per cell, preferably at least 2 concatemers per cell, more preferably 3 per cell, such as 4 per cell, more preferably 5 per cell, such as at least 5 per cell, for example at least 6 per cell, such as 7, 8, 9 or 10 per cell, for example more than 10 per cell. As described above, each concatemer may preferably comprise up to 1000 cassettes, and it is envisages that one concatemer may comprise up to 2000 cassettes. By inserting up to 10 concatemers into one single cell, this cell may thus be enriched with up to 20,000 heterologous expressible genes, which under suitable conditions may be turned on and off by regulation of the regulatable promoters.

[0311] Often it is more preferable to provide cells having anywhere between 10 and 1000 heterologous genes, such as 20-900 heterologous genes, for example 30 to 800 heterologous genes, such as 40 to 700 heterologous genes, for example 50 to 600 heterologous genes, such as from 60 to 300 heterologous genes or from 100 to 400 heterologous genes which are inserted as 2 to 4 artificial chromosomes each containing one concatemer of genes. The genes may advantageously be located on 1 to 10 such as from 2 to 5 different concatemers in the cells. Each concatemer may advantageously comprise from 10 to 1000 genes, such as from 10 to 750 genes, such as from 10 to 500 genes, such as from 10 to 200 genes, such as from 20 to 100 genes, for example from 30 to 60 genes, or from 50 to 100 genes.

[0312] The concatemers may be inserted into the host cells according to any known transformation technique, preferably according to such transformation techniques that ensure stable and not transient transformation of the host cell. The concatemers may thus be inserted as en artificial chromosome which is replicated by the cells as they divide or they may be inserted into the chromosomes of the host cell. The concatemer may also be inserted in the form of a plasmid such as a plasmid vector, a phage vector, a viral vector, a cosmid vector, that is replicated by the cells as they divide. Any combination of the three insertion methods is also possible. One or more concatemers may thus be integrated into the chromosome(s) of the host cell and one or more concatemers may be inserted as plasmids or artificial chromosomes. One or more concatemers may be inserted as artificial chromosomes and one or more may be inserted into the same cell via a plasmid.

EXAMPLES

Example 1

[0313] In the examples 1-3 an AscI site was introduced into the EcoR1 site in pYAC4 (Sigma, Burke D T et al. 1987, Science vol 236, p 806), so that sticky ends match the Asc1 site(=RS2 in general formula of this patent) of the cassettes in pEVE vectors

Preparation of EVACS (Evolvable Artificial Chromosomes) Including Size Fractioning

[0314] Preparation of pYAG4-Asc Arms 1. inoculate 150 ml of LB (sigma) with a single colony of E. coli DH5.alpha. containing pYAC4-Asc 2. grow to OD600.about.1, harvest cells and make plasmid preparation 3. digest 100 .mu.g pYAC4-Asc w, BamH1 and Asc1 4. dephosphorylate fragments and heat inactivate phosphatase (20 min, 80 C) 5. purify fragments(e.g. Qiaquick Gel Extraction Kit) 6. run 1% agarose gel to estimate amount of fragment

Preparation of Expression Cassettes

[0315] 1. take 100 .mu.g of plasmid preparation from each of the following libraries a) pMA-CAR b) pCA-CAR c) Phaffia cDNA library d) Carrot-cDNA library 2. digest w. Srf1(10 units/prep, 37 C overnight) 3. dephosphorylate (10 units/prep, 37 C, 2 h) 4. heat inactivate 80 C, 20 min 5. concentrate and change buffer (precipitation or ultra filtration), 6. digest w. Asc1 (10 units/prep, 37 C, overnight) 7. adjust volume of preps to 100 .mu.L

Preparation of EVACs

[0316] Different types of EVACs have been made by varying the ratio of the different libraries that goes into the ligation reaction.

TABLE-US-00003 EVAC pMA-CAR pCA-CAR Phaffia cDNA Carrot cDNA A 40% 40% 10% 10% B 25% 25% 25% 25%

[0317] 1. add .about.100 ng arms of pYAC4-Asc /100 .mu.g of cassette mixture [0318] 2. concentrate to <33.5 .mu.L [0319] 3. add 2.5 units of T4 DNA-ligase+4 pt 10.times. ligase buffer. Adjust to 40 .mu.L [0320] 4. ligate 3 h, 16 C [0321] 5. stop reaction by adding 2 .mu.L of 500 mM EDTA [0322] 6. bring reaction volume to 125 .mu.L, add 25 .mu.L loading mix, heat at 60 C for 5 min [0323] 7. distribute evenly in 10 wells of a 1% LMP agarose gel [0324] 8. run pulsed field gel (CHEF III, 1% LMP agarose, 1/2 strength TBE (BioRad), angle 120, temperature 12 C, voltage 5.6V/cm, switch time ramping 5-25 s, run time 30 h) [0325] 9. Stain part of the gel that contains molecular weight markers+1 sample lane for quality check [0326] 10. cut remaining 9 sample lanes corresponding to mw. 97-194 kb(fraction 1); [0327] 194-291 kb(fraction 2); 291-365 kb(fraction 3) from the gel [0328] 11. agarase gel in high NaCl agarase buffer . 1 U agarase/100 .mu.g gel. 40 C 3 h [0329] 12. concentrate preparation to <20 .mu.L [0330] 13. transform suitable yeast strain w. preparation using alkali/cation transformation [0331] 14. plate on selective minimal media plates [0332] 15. incubate 30 C for 4-5 days [0333] 16. pick colonies [0334] 17. analyse colonies

Example 2

Preparation of EVACs (Evolvable Artificial Chromosomes) with Direct Transformation

[0335] Preparation of pYAC4-Asc Arms 1. inoculate 150 ml of LB with a single colony of DH5.alpha. containing pYAC4-Asc 2. grow to OD600.about.1, harvest cells and make plasmid preparation 3. digest 100 .mu.g pYAC4-Asc w. BamH1 and Asc1 4. dephosphorylate fragments and heat inactivate phosphatase(20 Min, 80 C) 5. purify fragments(e.g. Qiaquick Gel Extraction Kit). 1. run 1% agarose gel to estimate amount of fragment

Preparation of Expression Cassettes

[0336] 1. take 100 .mu.g of plasmid preparation from each of the following libraries e) pMA-CAR f) pCA-CAR g) Phaffia cDNA library h) Carrot cDNA library 2: digest w. Srf1 (10 units/prep, 37 C overnight) 3. dephosphorylate (10 units/prep, 37 C, 2 h) 4. heat inactivate 80 C, 20 min 5. concentrate and change buffer (precipitation or ultra filtration), 6. digest w. Asc1. (10 units/prep, 37 C, overnight) 7. adjust volume of preps to 100 .mu.L

Preparation of EVACs

[0337] Different types of EVACs have been made by varying the ratio of the different libraries that goes into the ligation reaction.

TABLE-US-00004 EVAC pMA-CAR pCA-CAR Phaffia cDNA Carrot cDNA A 40% 40% 10% 10% B 25% 25% 25% 25%

[0338] 1. concentrate to <32 .mu.l [0339] 2. add 1 unit of T4 DNA-ligase+4 .mu.L 10.times. ligase buffer. Adjust to 40 .mu.L [0340] 3. ligate 2 h, 16 C [0341] 4. stop reaction by adding 2 .mu.L of 500 mM EDTA, heat inactivate 60 C, 20 min [0342] 5. bring reaction volume to 500 with dH.sub.2O, concentrate to 30 .mu.l [0343] 6. add 10 U Asc1, 4 .mu.L 10.times. Asc1 buffer, bring to 40 .mu.L [0344] 7. incubate at 37 C for 1 h (alternatively 15 min 30 min) [0345] 8. heat inactivate 60 C, 20 min [0346] 9. add 2 .mu.g YAC4-Asc arms, 1 U T4 DNA ligase, 10 .mu.L 10.times. ligase buffer, bring to 100 .mu.L [0347] 10. incubate ON, 16 C [0348] 11. add water to 500 .mu.L [0349] 12. concentrate to 25 .mu.L [0350] 13. transform suitable yeast strain w. preparation using alkali/cation transforination or other suitable transformation method [0351] 14. plate on selective minimal media plates [0352] 5. incubate 30 C for 4-5 days [0353] 6. pick colonies [0354] 17. analyse colonies

Example 3

Preparation of EVACs (EVolvable Artificial Chromosomes) (Small Scale Preparation)

Preparation of Expression Cassettes

[0354] [0355] 1. inoculate 5 ml of LB-medium (Sigma) with library inoculum corresponding to a 10+fold representation of library. Grow overnight [0356] 2. make plasmid miniprep from 1.5 ml of culture (E.g. Qiaprep spin miniprep kit) [0357] 3. digest plasmid w. Sri 1 [0358] 4. dephosphorylate fragments and heat inactivate phosphatase (20 min, 80 C) [0359] 5. digest w. Asc1 [0360] 6. run 1/10 of reaction in 1% agarose to estimate amount of fragment Preparation of pYAC4-Asc Arms [0361] 1. inoculate 150 ml of LB with a single colony of E. coli DH5.alpha. containing pYAC4-Asc [0362] 2. grow to OD600.about.1, harvest cells and make plasmid preparation [0363] 3. digest 100 .mu.g pYAC4-Asc w. BamH1 and Asc1 [0364] 4. dephosphorylate fragments and heat inactivate phosphatase (20 min, 80 C) [0365] 5. purify fragments(E.g. Qiaquick Gel Extraction Kit) [0366] 6. run 1% agarose gel to estimate amount of fragment

Preparation of EVACs

[0366] [0367] 1. mix expression cassette fragments with YAC-arms so that cassette/arm ration is .about.1000/1 [0368] 2. if needed concentrate mixture(use e.g. Microcon YM30) so fragment concentration>75 ng/.mu.L reaction [0369] 3. add 1 U T4 DNA ligase, incubate 16 C, 1-3 h . Stop reaction by adding 1 .mu.L of 500 mM EDTA [0370] 4. run pulsed field gel (CHEF III, 1% LMP agarose, 1/2 strength TBE, angle 120, temperature 12 C. voltage 5.6V/cm, switch time ramping 5-25 s, run time 30 h) Load sample in 2 lanes. [0371] 5. stain part of the gel that contains molecular weight markers [0372] 6. cut sample lanes corresponding to mw. 100-200 kb [0373] 7. agarase gel in high NaCl agarase buffer. 1 U agarase/100 mg gel [0374] 8. concentrate preparation to <20 .mu.L [0375] 9. transform suitable yeast strain w. preparation using electroporation [0376] 10. plate on selective minimal media plates [0377] 11. incubate 30 C for 4-5 days [0378] 12. pick colonies

Example 4

cDNA Libraries Used in the Production of EVACs

[0379] 1. Daucus carota, carrot root library: [0380] Full length [0381] Oligo dT primed, directional cDNA library [0382] cDNA library made using a pool of 3 Evolva EVE 4, 5 & 8 vectors (FIG. 4, 5, 6) [0383] Number of independent clones; 41.6.times.10.sup.6 [0384] Average size: 0.9-2.9 kb [0385] Number of different genes present: 5000-10000 2. Xanthophyllomyces dendrorhous, (yeast), hole organism library [0386] Full length [0387] Oligo dT primed, directional cDNA library [0388] cDNA library made using a pool of 3 Evolva EVE 4, 5 & 8 vectors (FIG. 4, 5, 6) [0389] Number of independent clones: 48.0.times.10.sup.6 [0390] Average size: 1.0-3.8 kb [0391] Number of different genes present: 5000-10000 3. Target carotenoid gene cDNA library [0392] Full length and normalised [0393] Directional cDNA cloning [0394] Library made by cloning each gene individually in 2 Evolva EVE 4, 5 & 8 vectors (FIG. 4, 5, 6) [0395] Number of different genes: 48. [0396] Species and genes used: [0397] Gentiana sp., ggps, psy, pds, zds, lcy-b, lcy-e, bhy, zep [0398] Rhodobacter capsulatus, idi, crtC, crtF [0399] Erwinia uredovdra, crtE, crtB, crtI, crtY, crtZ [0400] Nostoc anabaena, zds [0401] Synechococcus PCC7942, pds [0402] Erwinia herbicola, crtE, crtB, crtl, crtY, crtZ [0403] Staphylococcus aureus, crtM, crtN [0404] Xanthophyllomyces dendrorhous, crtl, crtYb [0405] Capsicum annuum, ccs, crtL [0406] Nicotiana tabacum, crtL, bchy [0407] Prochlorococcus sp., lcy-b, lcy-e [0408] Saccharomyces cerevisiae, idi [0409] Corynebacterium sp., crtl, crtYe, crtYf, crtEb [0410] Lycopersicon esculentum, psy-1 [0411] Neurospora crassa, al1

Example 5

Transformation of EVACs

Example 5a

Transformation

[0411] [0412] 1. Inoculate a single colony into 100 ml YPD broth and grow with aeration at 30.degree. C. to mid log, 2.times.10.sup.8 to 2.times.10.sup.7 cells/ml. [0413] 2. Spin to pellet cells at 400.times.g for 5 minutes; discard supernatant. [0414] 3. Resuspend cells in a total of 9 ml TE, pH 7:5. Spin to pellet cells and discard supernatant. [0415] 4. Gently resuspend cells in 5 ml 0.1 M Lithium/Cesium Acetate solution, pH 7.5. [0416] 5. Incubate at 30.degree. C. for 1 hour with gentle shaking. [0417] 6. Spin at 400.times.g for 5 minutes to pellet cells and discard supernatant. [0418] 7. Gently resuspend in 1 ml TE, pH 7.5. Cells are now ready for transformation. [0419] 8. In a 1.5 ml tube combine: [0420] 100 .mu.l yeast cells [0421] 5 .mu.l Carrier DNA (10 mg/ml) [0422] 5 .mu.l Histamine Solution [0423] 1/5 of an EVAC preparation in a 10 .mu.l volume (max). (One EVAC preparation is made of 100 .mu.g of concatenation reaction mixture) [0424] 9. Gently mix and incubate at room temperature for 30 minutes. [0425] 10. In a separate tube, combine 0.8 ml 50% (w/v) PEG 4000 and 0.1 ml TE and 0.1 ml of 1M LiAc for each transformation reaction. Add 1 ml of this PEG/TE/LiAc mix to each transformation reaction. Mix cells into solution with gentle pipetting. [0426] 11. Incubate at 30.degree. C. for 1 hour. [0427] 12. Heat shock at 42.degree. C. for 15 minutes; cool to 30.degree. C. [0428] 13. Pellet cells in a microcentrifuge at high speed for 5 seconds and remove supernatant. [0429] 14. Resuspend in 200 .mu.l of rich media and plate in appropriate selective media [0430] 15. Incubate at 30.degree. C. for 48-72 hours until transformant colonies appear.

Example 5b

Transformation of EVACs Using Electroporation

[0431] 100 ml of YPD is inoculated with one yeast colony and grown to OD.sub.600=1.3 to 1.5. The culture is harvested by centrifuging at 4000.times.g and 4.degree. C. The cells are resuspended in 16 ml sterile H.sub.2O. Add 2 ml 10.times.TE buffer, pH 7.5 and swirl to mix. Add 2 ml 10.times. lithium acetate solution (1 M, pH 7.5) and swirl to mix. Shake gently 45 min at 30.degree. C. Add 1.0 ml 0.5 M DTE while swirling. Shake gently 15 min at 30.degree. C. The yeast suspension is diluted to 100 ml with sterile water. The cells are washed and concentrated by centrifuging at 4000.times.g, resuspending the pellet in 50 ml ice-cold sterile water, centrifuging at 4000.times.g, resuspending the pellet in 5 ml sterile water, centrifuging at 4000.times.g and resuspending the pellet in 0.1 ml ice-cold sterile 1 M sorbitol. The electroporation was done using a Bio-Rad Gene Pulser. In a sterile 1.5-ml microcentrifuge tube 40 .mu.l concentrated yeast cells were mixed with 5 .mu.l 1:10 diluted EVAC preparation. The yeast-DNA mix is transferred to an ice-cold 0.2-cm-gap disposable electroporation cuvette and pulsed at 1.5 kV, 25 .mu.F, 200.OMEGA.. 1 ml ice-cold 1 M sorbitol is added to the cuvette to recover the yeast. Aliquots are spread on selective plates containing 1 M sorbitol. Incubate at 30.degree. C. until colonies appear.

Example 6

Rare Restriction Enzymes with Recognition Sequence and Cleavage Points

[0432] In this example, rare restriction enzymes are listed together with their recognition sequence and cleavage points. ( ) indicates cleavage points 5'-3' sequence and (_) indicates cleavage points in the complementary sequence.

W=A or T; N=A, C, G, or T

TABLE-US-00005 [0433] 6a) Unique, palindromic overhang AscI GG{circumflex over ( )}CGCG_C AsiSI GCG_T{circumflex over ( )}CGC CciNI GC{circumflex over ( )}GGCC_GC CspBI GC{circumflex over ( )}GGCC_GC FseI GG_CCGG{circumflex over ( )}CC MchAI GC{circumflex over ( )}GGCC_GC NotI GC{circumflex over ( )}GGCC_GC PacI TTA_AT{circumflex over ( )}TAA SbfI CC_TGCA{circumflex over ( )}GG SdaI CC_TGCA{circumflex over ( )}GG SgfI GCG_AT{circumflex over ( )}CGC SgrAI CR{circumflex over ( )}CCGG_YG Sse232I CG{circumflex over ( )}CCGG_CG Sse8387I CC_TGCA{circumflex over ( )}GG

TABLE-US-00006 6b) No overhang BstRZ246I ATTT{circumflex over ( )}AAAT BstSWI ATTT{circumflex over ( )}AAAT MspSWI ATTT{circumflex over ( )}AAAT MssI GTTT{circumflex over ( )}AAAC PmeI GTTT{circumflex over ( )}AAAC SmiI ATTT{circumflex over ( )}AAAT SrfI GCCC{circumflex over ( )}GGGC SwaI ATTT{circumflex over ( )}AAAT

TABLE-US-00007 6c) Non-palindromic and/or variable overhang AarI CACCTGCNNNN{circumflex over ( )}NNNN_ AbeI CC{circumflex over ( )}TCA_GC AloI {circumflex over ( )}NNNNN_NNNNNNNGAACNNNNNNTCCNNNNNNN_NNNNN N BaeI {circumflex over ( )}NNNNN_NNNNNNNNNNACNNNNGTAYCNNNNNNN_NNNN N{circumflex over ( )} BbvCI CC{circumflex over ( )}TCA_GC CpoI CG{circumflex over ( )}GWC_CG CspI CG{circumflex over ( )}GWC_CG Pfl27I RG{circumflex over ( )}GWC_CY PpiI {circumflex over ( )}NNNNN_NNNNNNNGAACNNNNNCTCNNNNNNNN_NNNN N PpuMI RG{circumflex over ( )}GWC_CY PpuXI RG{circumflex over ( )}GWC_CY Psp5II RG{circumflex over ( )}GWC_CY PspPPI RG{circumflex over ( )}GWC_CY RsrII CG{circumflex over ( )}GWC_CG Rsr2I CG{circumflex over ( )}GWC_CG SanDI GG{circumflex over ( )}GWC_CC SapI GCTCTTCN{circumflex over ( )}NNN_ SdiI GGCCN_NNN{circumflex over ( )}NGGCC SexAI A{circumflex over ( )}CCWGG_T SfiI GGCCN_NNN{circumflex over ( )}NGGCC Sse1825I GG{circumflex over ( )}GWC_CC Sse8647I AG{circumflex over ( )}GWC_CT VpaK32I GCTCTTCN{circumflex over ( )}NNN_

TABLE-US-00008 6d) Meganucleases I-Sce I TAGGGATAA_CAGG{circumflex over ( )}GTAAT I-Ceu I ACGGTC_CTAA{circumflex over ( )}GGTAG I-Cre I AAACGTC_GTGA{circumflex over ( )}GACAGTTT I-Sce II GGTC_ACCC{circumflex over ( )}TGAAGTA I-Sce III GTTTTGG_TAAC{circumflex over ( )}TATTTAT Endo. Sce I GATGCTGC_AGGC{circumflex over ( )}ATAGGCTTGTTTA PI-Sce I GG_GTGC{circumflex over ( )}GGAGAA PI-Psp I TGGCAAACAGCTA_TTAT{circumflex over ( )}GGGTATTATGGGT I-Ppo I CTCTC_TTAA{circumflex over ( )}GGTAG HO TTTCCGC_AACA{circumflex over ( )}GT I-Tev I NN_NN{circumflex over ( )}NNTCAGTAGATGTTTTTCTTGGTCTACCGTTT

[0434] More meganucleases have been identified, but their precise sequence of recognition has not been determined, see e.g. www.meganuclease.com

Example 7

Concatemer Size Limitation Experiments (Use of Stoppers)

[0435] Materials used:

[0436] pYAC4 (Sigma. Burke et al. 1987, science, vol 236, p 806) was digested w. EcoR1 and BamH1 and dephosphorylated

[0437] pSE420 (invitrogen) was linearised using EcoR1 and used as the model fragment for concatenation.

[0438] T4 DNA ligase (Amersham-pharmacia biotech) was used for ligation according to manufacturers instructions.

[0439] Method: Fragments and arms were mixed in the ratios(concentrations are arbitrary units) indicated on FIGS. 9a and 9b. Ligation was allowed to proceed for 1 h at 16 C. Reaction was stopped by the addition of 1 .mu.L 500 mM EDTA. Products were analysed by standard agarose GE (1% agarose, 1/2 strength TBE) or by PFGE(CHEF III, 1% LMP agarose, 1/2 strength TBE, angle 120, temperature 12 C, voltage 5.6V/cm, switch time ramping 5-25 s, run time 30 h)

[0440] The results are shown in FIG. 9, wherein it is shown that the size of concatemers is proportional to the ratio of cassettes per YAC arms.

Example 8

Integration of Expression Cassettes into Artificial Chromosomes

[0441] Integration of expression cassettes into YAC12 was done essentially as done by Sears D. D., Hieter P., Simchen G., Genetics, 1994, 138, 1055-1065.

[0442] An AscI site was introduced into the BgI II site of the integration vectors pGS534 and pGS525.

[0443] A .beta.-galactosidase gene, as well as crtE, crtB, crtl and crtY from Erwinia Uredovora were cloned into pEVE4. These expression cassettes were ligated into AscI of the modified integration vectors pGS534 and pGS525.

[0444] Linearised pGS534 and pGS525 containing the expression cassettes were transformed into haploid yeast strains containing the appropriate target YAC which carries the Ade'' gene. Red Ade- transformants were selected (the parent host strain is red due to the ade2-101 mutation).

[0445] Additional confirmation of correct integration of the .beta.-galactosidase expression cassette was done using a .beta.-galactosidase assay.

Example 9

Re-Transformation of Cells that Already Contain Artificial Chromosomes to Obtain at Least 2 Artificial Chromosomes Per Cell

[0446] Yeast strains containing YAC12, Sears D. D., Hieter P., Simchen G., Genetics, 1994, 138, 1055-1065 were transformed with EVACs following the protocol described in example 4a. The transformed cells were plated on plates that select for cells that contained both YAC12 and EVACs.

Example 10

Example of Different Expression Patterns "Phenotypes" Obtained Using the Same Yeast Clones Under Different Expression Conditions

[0447] Colonies were picked with a sterile toothpick and streaked sequentially onto plates corresponding to the four repressed and/or induced conditions (-Ura/-Trp, -Ura/-Trp/-Met, -Ura/-Trp/+200 .mu.M Cu.sub.2SO.sub.4, -Ura/-Trp/-Met/+200 .mu.M Cu.sub.2SO.sub.4). 20 mg adenin was added to the media to suppress the ochre phenotype.

Sequence CWU 1

2313417DNAArtificial SequenceSynthetic 1ctgatttgcc cgggcagttc aggctcatca ggcgcgccat gcagggattc ttcggatgca 60agggttcgaa tcccttagct ctcattattt tttgcttttt ctcttgaggt cacatgatcg 120caaaatggca aatggcacgt gaagctgtcg atattgggga actgtggtgg ttggcaaatg 180actaattaag ttagtcaagg cgccatcctc atgaaaactg tgtaacataa taaccgaagt 240gtcgaaaagg tggcaccttg tccaattgaa cacgctcgat gaaaaaaata agatatatat 300aaggttaagt aaagcgtctg ttagaaagga agtttttcct ttttcttgct ctcttgtctt 360ttcatctact atttccttcg tgtaatacag ggtcgtcaga tacatagata caattctatt 420acccccatcc atacaagctt ggcgccgaat tcgtcgaccc ggggatccgc ggccgcaggc 480ctaaattgat ctagagcttt ggacttcttc gccagaggtt tggtcaagtc tccaatcaag 540gttgtcggct tgtctacctt gccagaaatt tacgaaaaga tggaaaaggg tcaaatcgtt 600ggtagatacg ttgttgacac ttctaaataa gcgaatttct tatgatttat gatttttatt 660attaaataag ttataaaaaa aataagtgta tacaaatttt aaagtgactc ttaggtttta 720aaacgaaaat tcttgttctt gagtaactct ttcctgtagg tcaggttgct ttctcaggta 780tagcatgagg tcgctcttat tgaccacacc tctaccggca tgcccatggg ttaactgatc 840aatgcatcct gcatggcgcg cctgatgagc ctgaactgcc cgggcaaatc agctggacgt 900ctgcctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc 960ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 1020agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa 1080catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 1140tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 1200gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 1260ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 1320cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 1380caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 1440ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 1500taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 1560taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 1620cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 1680tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 1740gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 1800catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 1860atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 1920ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 1980gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 2040agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 2100gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 2160agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 2220catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 2280aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 2340gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 2400taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac 2460caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg 2520ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc 2580ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg 2640tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac 2700aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat 2760actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 2820catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 2880agtgccacct gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 2940cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 3000ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 3060gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 3120acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 3180ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 3240ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 3300acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttcca ttcgccattc 3360aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccag 341723501DNAArtificial SequenceSynthetic 2ctgatttgcc cgggcagttc aggctcatca ggcgcgccat gcagggataa gccgatccca 60ttaccgacat ttgggcgcta tacgtgcata tgttcatgta tgtatctgta tttaaaacac 120ttttgtatta tttttcctca tatatgtgta taggtttata cggatgattt aattattact 180tcaccaccct ttatttcagg ctgatatctt agccttgtta ctagttagaa aaagacattt 240ttgctgtcag tcactgtcaa gagattcttt tgctggcatt tcttctagaa gcaaaaagag 300cgatgcgtct tttccgctga accgttccag caaaaaagac taccaacgca atatggattg 360tcagaatcat ataaaagaga agcaaataac tccttgtctt gtatcaattg cattataata 420tcttcttgtt agtgcaatat catatagaag tcatcgaaat agatattaag aaaaacaaac 480tgtacaatca atcaatcaat catcacataa aatgttcaaa gcttggcgcc gaattcgtcg 540acccggggat ccgcggccgc aggcctaaat tgatctagag ctttggactt cttcgccaga 600ggtttggtca agtctccaat caaggttgtc ggcttgtcta ccttgccaga aatttacgaa 660aagatggaaa agggtcaaat cgttggtaga tacgttgttg acacttctaa ataagcgaat 720ttcttatgat ttatgatttt tattattaaa taagttataa aaaaaataag tgtatacaaa 780ttttaaagtg actcttaggt tttaaaacga aaattcttgt tcttgagtaa ctctttcctg 840taggtcaggt tgctttctca ggtatagcat gaggtcgctc ttattgacca cacctctacc 900ggcatgccca tgggttaact gatcaatgca tcctgcatgg cgcgcctgat gagcctgaac 960tgcccgggca aatcagctgg acgtctgcct gcattaatga atcggccaac gcgcggggag 1020aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 1080cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 1140atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 1200taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 1260aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 1320tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 1380gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 1440cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 1500cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 1560atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 1620tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat 1680ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 1740acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 1800aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 1860aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 1920tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 1980cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 2040catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 2100ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 2160aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 2220ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 2280caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 2340attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 2400agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 2460actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 2520ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 2580ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 2640gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 2700atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 2760cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 2820gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 2880gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 2940ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg ccctgtagcg gcgcattaag 3000cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc 3060cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 3120tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 3180aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 3240ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 3300actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta 3360ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac 3420gcttacaatt tccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg 3480gcctcttcgc tattacgcca g 350134188DNAArtificial SequenceSynthetic 3ctgatttgcc cgggcagttc aggctcatca ggcgcgccat gcagggattc tggaaattgc 60aacgaaggaa gaaacctcgt tgctggaagc ctggaagaag tatcgggtgt tgctgaaccg 120tgttgataca tcaactgcac ctgatattga gtggcctgct gtccctgtta tggagtaatc 180gttttgtgat atgccgcaga aacgttgtat gaaataacgt tctgcggtta gttagtatat 240tgtaaagctg agtattggtt tatttggcga ttattatctt caggagaata atggaagttc 300tatgactcaa ttgttcatag tgtttacatc accgccaatt gcttttaaga ctgaacgcat 360gaaatatggt ttttcgtcat gttttgagtc tgctgttgat atttctaaag tcggtttttt 420ttcttcgttt tctctaacta ttttccatga aatacatttt tgattattat ttgaatcaat 480tccaattacc tgaagtcttt catctataat tggcattgta tgtattggtt tattggagta 540gatgcttgct tttctgagcc atagctctga tatcagatct tcttcggatg caagggttcg 600aatcccttag ctctcattat tttttgcttt ttctcttgag gtcacatgat cgcaaaatgg 660caaatggcac gtgaagctgt cgatattggg gaactgtggt ggttggcaaa tgactaatta 720agttagtcaa ggcgccatcc tcatgaaaac tgtgtaacat aataaccgaa gtgtcgaaaa 780ggtggcacct tgtccaattg aacacgctcg atgaaaaaaa taagatatat ataaggttaa 840gtaaagcgtc tgttagaaag gaagtttttc ctttttcttg ctctcttgtc ttttcatcta 900ctatttcctt cgtgtaatac agggtcgtca gatacataga tacaattcta ttacccccat 960ccatacaagc ttggcgccga attcgtcgac ccggggatcc gcggccgcag gcctaaattg 1020atctagagct ttggacttct tcgccagagg tttggtcaag tctccaatca aggttgtcgg 1080cttgtctacc ttgccagaaa tttacgaaaa gatggaaaag ggtcaaatcg ttggtagata 1140cgttgttgac acttctaaat aagcgaattt cttatgattt atgattttta ttattaaata 1200agttataaaa aaaataagtg tatacaaatt ttaaagtgac tcttaggttt taaaacgaaa 1260attcttgttc ttgagtaact ctttcctgta ggtcaggttg ctttctcagg tatagcatga 1320ggtcgctctt attgaccaca cctctaccgg catgcccatg ggttcttttg aaaagcaagc 1380ataaaagatc taaacataaa atctgtaaaa taacaagatg taaagataat gctaaatcat 1440ttggcttttt gattgattgt acaggaaaat atacatcgca gggggttgac ttttaccatt 1500tcaccgcaat ggaatcaaac ttgttgaaga gaatgttcac aggcgcatac gctacaatga 1560cccgattctt gctagccttt tctcggtctt gcaaacaacc gccaactgat caatgcatcc 1620tgcatggcgc gcctgatgag cctgaactgc ccgggcaaat cagctggacg tctgcctgca 1680ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 1740ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 1800aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 1860aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 1920gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 1980gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 2040tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 2100ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 2160ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 2220tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 2280tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 2340ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 2400aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 2460ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 2520tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 2580atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 2640aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 2700ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 2760tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 2820ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 2880tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 2940aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 3000gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 3060tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 3120cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 3180tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 3240ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 3300cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 3360actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 3420ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 3480aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 3540ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 3600atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 3660tgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 3720cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 3780cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 3840tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 3900gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 3960tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 4020ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 4080taacgcgaat tttaacaaaa tattaacgct tacaatttcc attcgccatt caggctgcgc 4140aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccag 4188411466DNAArtificial SequenceSynthetic 4ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa 60ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg 120caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt 180gcgggatatc gtccattccg acagcatcgc cagtcactat ggcgtgctgc tagcgctata 240tgcgttgatg caatttctat gcgcacccgt tctcggagca ctgtccgacc gctttggccg 300ccgcccagtc ctgctcgctt cgctacttgg agccactatc gactacgcga tcatggcgac 360cacacccgtc ctgtggatca attcccttta gtataaattt cactctgaac catcttggaa 420ggaccggtaa ttatttcaaa tctctttttc aattgtatat gtgttatgtt atgtagtata 480ctctttcttc aacaattaaa tactctcggt agccaagttg gtttaaggcg caagacttta 540atttatcact acggaattgg cgcgccaatt ccgtaatctt gagatcgggc gttcgatcgc 600cccgggagat ttttttgttt tttatgtctt ccattcactt cccagacttg caagttgaaa 660tatttctttc aagggaattg atcctctacg ccggacgcat cgtggccggc atcaccggcg 720ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc 780gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg 840ggggactgtt gggcgccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg 900gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgac 960cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta 1020tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag 1080cgctctgggt cattttcggc gaggaccgct ttcgctggag cgcgacgatg atcggcctgt 1140cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca 1200ccaaacgttt cggcgagaag caggccatta tcgccggcat ggcggccgac gcgctgggct 1260acgtcttgct ggcgttcgcg acgcgaggct ggatggcctt ccccattatg attcttctcg 1320cttccggcgg catcgggatg cccgcgttgc aggccatgct gtccaggcag gtagatgacg 1380accatcaggg acagcttcaa ggatcgctcg cggctcttac cagcctaact tcgatcactg 1440gaccgctgat cgtcacggcg atttatgccg cctcggcgag cacatggaac gggttggcat 1500ggattgtagg cgccgcccta taccttgtct gcctccccgc gttgcgtcgc ggtgcatgga 1560gccgggccac ctcgacctga atggaagccg gcggcacctc gctaacggat tcaccactcc 1620aagaattgga gccaatcaat tcttgcggag aactgtgaat gcgcaaacca acccttggca 1680gaacatatcc atcgcgtccg ccatctccag cagccgcacg cggcgcatcc ccccccccct 1740ttcaattcaa ttcatcattt tttttttatt cttttttttg atttcggttt ctttgaaatt 1800tttttgattc ggtaatctcc gaacagaagg aagaacgaag gaaggagcac agacttagat 1860tggtatatat acgcatatgt agtgttgaag aaacatgaaa ttgcccagta ttcttaaccc 1920aactgcacag aacaaaaacc tgcaggaaac gaagataaat catgtcgaaa gctacatata 1980aggaacgtgc tgctactcat cctagtcctg ttgctgccaa gctatttaat atcatgcacg 2040aaaagcaaac aaacttgtgt gcttcattgg atgttcgtac caccaaggaa ttactggagt 2100tagttgaagc attaggtccc aaaatttgtt tactaaaaac acatgtggat atcttgactg 2160atttttccat ggagggcaca gttaagccgc taaaggcatt atccgccaag tacaattttt 2220tactcttcga agacagaaaa tttgctgaca ttggtaatac agtcaaattg cagtactctg 2280cgggtgtata cagaatagca gaatgggcag acattacgaa tgcacacggt gtggtgggcc 2340caggtattgt tagcggtttg aagcaggcgg cagaagaagt aacaaaggaa cctagaggcc 2400ttttgatgtt agcagaattg tcatgcaagg gctccctatc tactggagaa tatactaagg 2460gtactgttga cattgcgaag agcgacaaag attttgttat cggctttatt gctcaaagag 2520acatgggtgg aagagatgaa ggttacgatt ggttgattat gacacccggt gtgggtttag 2580atgacaaggg agacgcattg ggtcaacagt atagaaccgt ggatgatgtg gtctctacag 2640gatctgacat tattattgtt ggaagaggac tatttgcaaa gggaagggat gctaaggtag 2700agggtgaacg ttacagaaaa gcaggctggg aagcatattt gagaagatgc ggccagcaaa 2760actaaaaaac tgtattataa gtaaatgcat gtatactaaa ctcacaaatt agagcttcaa 2820tttaattata tcagttatta ctcgggcgta atgattttta taatgacgaa aaaaaaaaaa 2880ttggaaagaa aagggggggg gggcagcgtt gggtcctggc cacgggtgcg catgatcgtg 2940ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa 3000tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca 3060acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg 3120ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca 3180cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc 3240cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca 3300tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac 3360agaaattccc ccttacacgg aggcatcaag tgaccaaaca ggaaaaaacc gcccttaaca 3420tggcccgctt tatcagaagc cagacattaa cgcttctgga gaaactcaac gagctggacg 3480cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca 3540gccctcgagg gataagcttc atttttagat aaaatttatt aatcatcatt aatttcttga 3600aaaacatttt atttattgat cttttataac aaaaaaccct tctaaaagtt tatttttgaa 3660tgaaaaactt ataaaaattt atgaaaacta caaaaaataa aatttttaat taaaataatt 3720ttgataagaa cttcaatctt

tgactagcta gcttagtcat ttttgagatt taattaatat 3780tttatgttta ttcatatata aactattcaa aatattatag aatttaaaca ttttaacatc 3840ttaatcattc ataaataact aaaaatcaaa gtattacatc aataaataac ttttactcaa 3900tgtcaaagaa ttattggggt tggggttggg gttggggttg gggttggggt tggggttggg 3960gttggggttg gggttggggt tggggttggg gttggggttg gggttggggt tggggttggg 4020gttggggttg gggttggggt tggggttggg gttggggttg gggttggggt tggggttggg 4080gttggggttg gggttggggt tggggttggg gttggggttg gggttggggt tggggttggg 4140gttggggttg gggttggggt tggggttggg gttggggttg gggtgggaaa acagcattca 4200ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgcggg atcctcgggg 4260acaccaaata tggcgatctc ggccttttcg tttcttggag ctgggacatg tttgccatcg 4320atccatctac caccagaacg gccgttagat ctgctgccac cgttgtttcc accgaagaaa 4380ccaccgttgc cgtaaccacc acgacggttg ttgctaaaga agctgccacc gccacggcca 4440ccgttgtagc cgccgttgtt gttattgtag ttgctcatgt tatttctggc acttcttggt 4500tttcctctta agtgaggagg aacataacca ttctcgttgt tgtcgttgat gcttaaattt 4560tgcacttgtt cgctcagttc agccataata tgaaatgctt ttcttgttgt tcttacggaa 4620taccacttgc cacctatcac cacaactaac tttttcccgt tcctccatct cttttatatt 4680ttttttctcg atcgagttca agagaaaaaa aaagaaaaag caaaaagaaa aaaggaaagc 4740gcgcctcgtt cagaatgaca cgtatagaat gatgcattac cttgtcatct tcagtatcat 4800actgttcgta tacatactta ctgacattca taggtataca tatatacaca tgtatatata 4860tcgtatgctg cagctttaaa taatcggtgt cactacataa gaacaccttt ggtggaggga 4920acatcgttgg taccattggg cgaggtggct tctcttatgg caaccgcaag agccttgaac 4980gcactctcac tacggtgatg atcattcttg cctcgcagac aatcaacgtg gagggtaatt 5040ctgctagcct ctgcaaagct ttcaagaaaa tgcgggatca tctcgcaaga gagatctcct 5100actttctccc tttgcaaacc aagttcgaca actgcgtacg gcctgttcga aagatctacc 5160accgctctgg aaagtgcctc atccaaaggc gcaaatcctg atccaaacct ttttactcca 5220cgcgccagta gggcctcttt aaaagcttga ccgagagcaa tcccgcagtc ttcagtggtg 5280tgatggtcgt ctatgtgtaa gtcaccaatg cactcaacga ttagcgacca gccggaatgc 5340ttggccagag catgtatcat atggtccaga aaccctatac ctgtgtggac gttaatcact 5400tgcgattgtg tggcctgttc tgctactgct tctgcctctt tttctgggaa gatcgagtgc 5460tctatcgcta ggggaccacc ctttaaagag atcgcaatct gaatcttggt ttcatttgta 5520atacgcttta ctagggcttt ctgctctgtc atctttgcct tcgtttatct tgcctgctca 5580ttttttagta tattcttcga agaaatcaca ttactttata taatgtataa ttcattatgt 5640gataatgcca atcgctaaga aaaaaaaaga gtcatccgct aggtggaaaa aaaaaaatga 5700aaatcattac cgaggcataa aaaaatatag agtgtactag aggaggccaa gagtaataga 5760aaaagaaaat tgcgggaaag gactgtgtta tgacttccct gactaatgcc gtgttcaaac 5820gatacctggc agtgactcct agcgctcacc aagctcttaa aacgagaatt aagaaaaagt 5880cgtcatcttt cgataagttt ttcccacagc aaagcaatag tagaaaaaaa caatgggaaa 5940cgttgaatga agacaaagcg tcgtggttta aaaggaaata cgctcacgta catgctaggg 6000aacaggaccg tgcagcggat cccgcgcatc aacaatattt tcacctgaat caggatattc 6060ttctaatacc tgaatgctgt tttcccaccc caaccccaac cccaacccca accccaaccc 6120caaccccaac cccaacccca accccaaccc caaccccaac cccaacccca accccaaccc 6180caaccccaac cccaacccca accccaaccc caaccccaac cccaacccca accccaaccc 6240caaccccaac cccaacccca accccaaccc caaccccaac cccaacccca accccaaccc 6300caaccccaac cccaacccca accccaaccc caaccccaac cccaacccca accccaataa 6360ttctttgaca ttgagtaaaa gttatttatt gatgtaatac tttgattttt agttatttat 6420gaatgattaa gatgttaaaa tgtttaaatt ctataatatt ttgaatagtt tatatatgaa 6480taaacataaa atattaatta aatctcaaaa atgactaagc tagctagtca aagattgaag 6540ttcttatcaa aattatttta attaaaaatt ttattttttg tagttttcat aaatttttat 6600aagtttttca ttcaaaaata aacttttaga agggtttttt gttataaaag atcaataaat 6660aaaatgtttt tcaagaaatt aatgatgatt aataaatttt atctaaaaat gaagcttatc 6720cctcgagggc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct 6780cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg 6840cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag 6900cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat 6960atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc 7020gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 7080cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 7140tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 7200cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 7260aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 7320cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 7380gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 7440ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 7500cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 7560aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 7620tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 7680ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 7740tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 7800ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 7860agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 7920atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 7980cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 8040ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 8100ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 8160agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 8220agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tgcaggcatc 8280gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 8340cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 8400gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 8460tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 8520tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aacacgggat 8580aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 8640cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 8700cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 8760aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 8820ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 8880tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 8940ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc 9000acgaggccct ttcgtcttca agaattaatt cggtcgaaaa aagaaaagga gagggccaag 9060agggagggca ttggtgacta ttgagcacgt gagtatacgt gattaagcac acaaaggcag 9120cttggagtat gtctgttatt aatttcacag gtagttctgg tccattggtg aaagtttgcg 9180gcttgcagag cacagaggcc gcagaatgtg ctctagattc cgatgctgac ttgctgggta 9240ttatatgtgt gcccaataga aagagaacaa ttgacccggt tattgcaagg aaaatttcaa 9300gtcttgtaaa agcatataaa aatagttcag gcactccgaa atacttggtt ggcgtgtttc 9360gtaatcaacc taaggaggat gttttggctc tggtcaatga ttacggcatt gatatcgtcc 9420aactgcatgg agatgagtcg tggcaagaat accaagagtt cctcggtttg ccagttatta 9480aaagactcgt atttccaaaa gactgcaaca tactactcag tgcagcttca cagaaacctc 9540attcgtttat tcccttgttt gattcagaag caggtgggac aggtgaactt ttggattgga 9600actcgatttc tgactgggtt ggaaggcaag agagccccga aagcttacat tttatgttag 9660ctggtggact gacgccagaa aatgttggtg atgcgcttag attaaatggc gttattggtg 9720ttgatgtaag cggaggtgtg gagacaaatg gtgtaaaaga ctctaacaaa atagcaaatt 9780tcgtcaaaaa tgctaagaaa taggttatta ctgagtagta tttatttaag tattgtttgt 9840gcacttgcct gcaggccttt tgaaaagcaa gcataaaaga tctaaacata aaatctgtaa 9900aataacaaga tgtaaagata atgctaaatc atttggcttt ttgattgatt gtacaggaaa 9960atatacatcg cagggggttg acttttacca tttcaccgca atggaatcaa acttgttgaa 10020gagaatgttc acaggcgcat acgctacaat gacccgattc ttgctagcct tttctcggtc 10080ttgcaaacaa ccgccggcag cttagtatat aaatacacat gtacatacct ctctccgtat 10140cctcgtaatc attttcttgt atttatcgtc ttttcgctgt aaaaacttta tcacacttat 10200ctcaaataca cttattaacc gcttttacta ttatcttcta cgctgacagt aatatcaaac 10260agtgacacat attaaacaca gtggtttctt tgcataaaca ccatcagcct caagtcgtca 10320agtaaagatt tcgtgttcat gcagatagat aacaatctat atgttgataa ttagcgttgc 10380ctcatcaatg cgagatccgt ttaaccggac cctagtgcac ttaccccacg ttcggtccac 10440tgtgtgccga acatgctcct tcactatttt aacatgtgga attaattcta aatcctcttt 10500atatgatctg ccgatagata gttctaagtc attgaggttc atcaacaatt ggattttctg 10560tttactcgac ttcaggtaaa tgaaatgaga tgatacttgc ttatctcata gttaactcta 10620agaggtgata cttatttact gtaaaactgt gacgataaaa ccggaaggaa gaataagaaa 10680actcgaactg atctataatg cctattttct gtaaagagtt taagctatga aagcctcggc 10740attttggccg ctcctaggta gtgctttttt tccaaggaca aaacagtttc tttttcttga 10800gcaggtttta tgtttcggta atcataaaca ataaataaat tatttcattt atgtttaaaa 10860ataaaaaata aaaaagtatt ttaaattttt aaaaaagttg attataagca tgtgaccttt 10920tgcaagcaat taaattttgc aatttgtgat tttaggcaaa agttacaatt tctggctcgt 10980gtaatatatg tatgctaaag tgaactttta caaagtcgat atggacttag tcaaaagaaa 11040ttttcttaaa aatatatagc actagccaat ttagcacttc tttatgagat atattataga 11100ctttattaag ccagatttgt gtattatatg tatttacccg gcgaatcatg gacatacatt 11160ctgaaatagg taatattctc tatggtgaga cagcatagat aacctaggat acaagttaaa 11220agctagtact gttttgcagt aatttttttc ttttttataa gaatgttacc acctaaataa 11280gttataaagt caatagttaa gtttgatatt tgattgtaaa ataccgtaat atatttgcat 11340gatcaaaagg ctcaatgttg actagccagc atgtcaacca ctatattgat caccgatata 11400tggacttcca caccaactag taatatgaca ataaattcaa gatattcttc atgagaatgg 11460cccaga 11466515DNAArtificial SequenceAar1 recognition site 5cacctgcnnn nnnnn 15637DNAArtificial SequenceAloI recognition site 6nnnnnnnnnn nngaacnnnn nntccnnnnn nnnnnnn 37738DNAArtificial SequenceBaeI recognition site 7nnnnnnnnnn nnnnnacnnn ngtaycnnnn nnnnnnnn 38837DNAArtificial SequencePpiI recognition site 8nnnnnnnnnn nngaacnnnn nctcnnnnnn nnnnnnn 37911DNAArtificial SequenceSapI recognition site 9gctcttcnnn n 111013DNAArtificial SequenceSdiI recognition site 10ggccnnnnng gcc 131113DNAArtificial SequenceSfiI recognition site 11ggccnnnnng gcc 131211DNAArtificial SequenceVpaK32I recognition site 12gctcttcnnn n 111318DNAArtificial SequenceI-Sce I recognition site 13tagggataac agggtaat 181415DNAArtificial SequenceI-Ceu I recognition site 14acggtcctaa ggtag 151519DNAArtificial SequenceI-Cre I recognition site 15aaacgtcgtg agacagttt 191615DNAArtificial SequenceI-Sce II recognition site 16ggtcaccctg aagta 151718DNAArtificial SequenceI-Sce III recognition site 17gttttggtaa ctatttat 181825DNAArtificial SequenceEndo. Sce I recognition site 18gatgctgcag gcataggctt gttta 251912DNAArtificial SequencePI-Sce I recognition site 19gggtgcggag aa 122030DNAArtificial SequencePI-Psp I recognition site 20tggcaaacag ctattatggg tattatgggt 302114DNAArtificial SequenceI-Ppo I recognition site 21ctctcttaag gtag 142213DNAArtificial SequenceHO recognition site 22tttccgcaac agt 132336DNAArtificial SequenceI-Tev I recognition site 23nnnnnntcag tagatgtttt tcttggtcta ccgttt 36

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