CA2510184C - In vivo affinity maturation scheme - Google Patents

Abstract

The present invention relates to the field of evolution of nucleic acids in vivo and provides methods and compositions for introducing diversity into gene products. The present invention allows generation of new sequences that have desirable properties by virtue of high frequency mutation events within a cell. The high frequency mutation of a polynucleotide sequence results in the production of a large population of new sequence variants. Appropriate selection and/or screening permits identification and isolation of mutant forms of the polynucleotide sequence as well as products resulting from expression of the mutant sequences.

Description

In vivo affinity maturation scheme Field of the Invention The present invention relates to the field of evolution of nucleic acids in vivo and provides methods and compositions for introducing diversity into gene products. The present invention allows generation of new sequences that have desirable properties by virtue of high frequency mutation events within a cell. The high frequency mutation of a polynucleotide sequence results in the production of a large population of new sequence variants. Appropriate selection and/or screening permits identification and isolation of mutant forms of the polynucleotide sequence as well as products resulting from expression of the mutant sequences.
Background of the Invention In vitro evolution of proteins involves generating diversity by introducing mutations into known gene sequences to produce a library of mutant sequences, translating the sequences to produce a very large number of mutant gene products, which are then selected for the desired properties. Such schemes can be divided into two distinct groups; i) partial in vitro methods in which the mutated target protein is displayed on the surface of the either phage, bacteria or yeast but the mutation step is performed outside the cell and ii) entirely in vitro methods in which the target is displayed as part of a ribosome quaternary complex or polysome and all other steps including mutation are performed in a cell free environment. These processes have the potential for generating proteins with improved diagnostic and therapeutic utilities.
Unfortunately, however, the potential of such processes has been limited by deficiencies in methods currently available for mutation, library generation and display of correctly folded proteins.
For example, in the case of partial in vitro methods, the DNA must be synthesised in vitro or extracted from the cells for mutagenesis. Although various mutagenesis approaches (including error prone PCR, DNA shuffling, chain shuffling and site directed mutagenesis) have been successfully used to generate mutant libraries, some of this diversity is lost due to limitations in subsequent transformation efficiency.
Consequently, the generation of large libraries (e.g. beyond a library size of l010) of unique individual genes and their encoded proteins has proven difficult particularly with phage display systems. A further disadvantage is that methods which utilise phage display systems require several sequential steps of mutation, amplification, selection and further mutation. Given that extraction and reintroduction of DNA into the cell is required for these systems, their potential to generate large diversity in the target gene library is further restricted.
To circumvent this problem, entirely in vitro methods such as continuous in vitro evolution (CIVE) have been developed and are described, for example, in WO
99/58661. In theory any diversity created through mutagenesis in these systems is not lost. In the case of CIVE, a mutating enzyme is used to introduce nucleic acid base changes into the target sequence. The only factor limiting diversity here is the mutation rate of this enzyme. Reports of mutation rates using other in vitro mutation methods such as error prone PCR, DNA shuffling (sexual PCR), chain shuffling and site directed mutagenesis over selected CDRs which can be used in this scheme vary significantly. Despite using different mechanisms all these approaches operate in an artificial environment in which only defined components required for these processes are present. It is possible there may be additional unknown factors involved, which are not supplied. Furthermore, this cell-free environment lacks the secretory and post-translational machinery required to produce a correctly folded and processed protein.
As a result, this restricts the type of targets which can be "evolved" in these systems and allows incorrectly folded, unmodified mutant proteins which have no functional relevance in a clinical setting to be selected. Bacterial and phage display also have the same associated problems.
In vivo evolution of proteins involves the same steps and principles as previously described for in vitro evolution (i.e. mutation, display, selection and amplification).
However, in vivo systems overcome many of the problems associated with the in vitro approaches. One in vivo cyclical procedure that has been reported involves Escherichia coli mutator cells that were used as a vehicle for mutation of recombinant antibody genes. The E.coli mutator cells, MUTD5-FIT which carried a mutated DNAQ gene were used as the source of the S-30 extracts and therefore allowed mutations to be introduced into DNA during replication as a result of proofreading errors.
However, mutation rates were low compared to the required rate. For example, to mutate residues with the complete permutation of 20 amino acid requires a library size of 1x1026, an extremely difficult task with currently available phage library display methodology. Obviously, the disadvantages with using bacteria and phage in terms of transformation efficiencies, and protein folding etc. make this a less desirable scheme.
In view of the above it is clear that the current affinity maturation schemes are somewhat limited in their ability to generate and select functionally superior binders.
Summary of the invention The present inventors have now developed a novel process for the in vivo evolution of gene products. This process generates mutants of target nucleic acid sequences by somatic hypermutation, yielding mutant products capable of undergoing any post-translational modifications that may be required for biological activity. A selection system particular to the properties of the target product is then utilized to identify desirable mutants.
Accordingly, in a first aspect, the present invention provides a method for producing and selecting a gene product with desired characteristics, the method comprising (i) introducing into a hypermutating cell a target nucleic acid molecule encoding a gene product such that a single copy of the target nucleic acid molecule is integrated into an immunoglobulin locus of the genome of the hypermutating cell, wherein the target nucleic acid molecule is introduced into the cell by way of an integration vector comprising a sequence homologous to a region upstream of a rearranged V allele and a sequence homologous to a region downstream of a rearranged V allele;
(ii) culturing the hypermutating cell such that the target nucleic acid molecule undergoes hypermutation during DNA and/or RNA synthesis, giving rise to a population of cells expressing mutant gene products; and (iii) selecting the mutant gene product with desired characteristics.
The term "immunoglobulin locus" refers to a variable region of an antibody molecule or all or a portion of a regulatory nucleotide sequence that controls expression of an antibody molecule. Immunoglobulin loci for heavy chains may include but are not limited to all or a 3a portion of the V, D, J, and switch regions (including intervening sequences called introns) and flanking sequences associated with or adjacent to the particular heavy chain constant region gene expressed by the antibody-producing cell to be transfected and may include regions located within or downstream of the constant region (including introns).
Immunoglobulin loci for light chains may include but are not limited to the V and J regions, their upstream flanking sequences, and intervening sequences (introns), associated with or adjacent to the light chain constant region gene expressed by the antibody-producing cell to be transfected and may include regions located within or downstream of the constant region (including introns).
Immunoglobulin loci for heavy chain variable regions may include but are not limited to all or a portion of the V, D, and J regions (including introns) and flanking sequences associated with or adjacent to the particular variable region gene expressed by the antibody-producing cell to be transfected. Immunoglobulin loci for light chain variable regions may include but are not limited to the V and J region (including introns) and flanking sequences associated with or adjacent to the light chain variable region gene expressed by the antibody-producing cell to be transfected.
In the human, the immunoglobulin heavy chain (IgH) locus is located on chromosome 14. In the 5'-3' direction of transcription, the locus comprises a large cluster of variable region genes (VH), the diversity (D) region genes, followed by the joining (JH) region genes and the constant (CH) gene cluster. The size of the locus is estimated to be about from 1,500 to about 2,500 kilobases (kb). During B-cell development, discontinuous gene segments from the germ line IgH locus are juxtaposed by means of a physical rearrangement of the DNA. In order for a functional heavy chain Ig polypeptide to be produced, three discontinuous DNA segments, from the VH, D, and JH regions must be joined in a specific sequential fashion; first D to JH then VH to DJH, generating the functional unit VHDJH. Once a VHDJH has been formed, specific heavy chains are produced following transcription of the Ig locus, utilizing as a template the specific VHDJHCH unit comprising exons and introns.
There are two loci for immunoglobulin light chains (IgL), the kappa locus on human chromosome 2 and the lambda locus on human chromosome 22. The organization of the IgL loci is similar to that of the IgH locus, except that the D region is not present.
Following IgH 'rearrangement, rearrangement of a light chain locus is similarly accomplished by VI, to .11, joining of the kappa or lambda chain. The sizes of the lambda and kappa loci are each approximately 1000 kb to 2000 kb. Expression of rearranged IgH and an Ig kappa or Ig lambda light chain in a particular B-cell allows for the generation of antibody molecules.

In a further preferred embodiment of the invention the immunoglobulin locus is a rearranged VH4 gene. In a further preferred embodiment, the immunoglobulin locus is a rearranged VH4-34 allele.

5 By "hypermutation" we mean a mechanism by which mutagenesis occurs at a rate approaching that naturally occurring in the immunoglobulin variable region, which is preferably in the range of 104 to 10-3/base pair/generation/cell but more preferably in the range of 5x10-5 to 5x10-4/base pair/generation/cell.
A "hypermutating cell" is a cell or cell line containing hypermutation elements.
By "hypermutation elements" we mean an intronic enhancer (Ei), matrix attachment regions (MAR), and a 3' enhancer. The intronic enhancer may be, for example, E
mu or E kappa. The 3' enhancer may be, for example, a 3' kappa enhancer or a 3'H
enhancer.
A "matrix attachment region" (MAR) is defined by its ability to bind to the nuclear matrix. Matrix attachment region sequences flank the IgH intronic enhancer.
In one embodiment the hypermutating cell is an immunoglobulin-expressing cell which is capable of expressing at least one immunoglobulin V gene. A V gene may be a variable light chain (VI) or a variable heavy chain (VH) gene, and may be produced as part of an entire immunoglobulin molecule. Preferred hypermutating cells for use in the present invention are derived from B-cell lines. Lymphoma cells may be used for the isolation of constitutively hypermutating cell lines for use in the present invention.
In a preferred embodiment of the present invention, following integration into the immunoglobulin locus of the hypermutating cell, the target nucleic acid molecule is located in proximity to one or more endogenous hypermutation elements.
Preferably, the immunoglobulin locus is a VH gene and the target nucleic acid molecule is located in proximity to an endogenous intronic enhancer, and endogenous matrix attachment regions.
The phrase "located in proximity to" means that hypermutation elements are located close enough to the target nucleic acid molecule to effect hypermutation of the target nucleic acid molecule.

In an alternative embodiment of the invention, following integration into the immunoglobulin locus of the hypermutating cell, the target nucleic acid molecule is located in proximity to at least one exogenous hypermutation element. For example, the target nucleic acid molecule may be located in proximity to an exogenous intronic enhancer, an exogenous matrix attachment region and/or an exogenous 3' kappa enhancer. Any one or more of these exogenous elements may be integrated into the immuno globulin locus simultaneously with the target nucleic acid molecule.
A suitable exogenous "intronic enhancer" may be, for example, the Xbal-EcoRI
fragment described in Grosschedl et al (1985) Cell Vol 41:885-897, the intronic enhancer described in Rabbitts et al (1983) Nature 306 (5945):806-809, or the intronic enhancer described in Ravetch et al (1981) Cell 27 (3 Pt 2): 583-591; or can be one or more sub-fragments thereof determined to have hypermutation activity.
A suitable exogenous "3' kappa enhancer" is the ScaI-XbaI fragment described in Meyer et al (1989) EMBO Journal Vol. 8, no. 7 p. 1959-1964 and can be one or more sub-fragments determined to have hypermutation activity.
Hypermutation-competent fragments of exogenous intronic enhancers or the 3' kappa enhancer can be identified in a number of ways. One way is to perform deletional analysis by constructing hypermutation cassettes containing various enhancer deletion mutants and a reporter gene. The hypermutation efficency of the enhancer deletion mutant can be assessed by determining the rate of mutation of the reporter gene.
Deletion mutants can be prepared in a variety of ways. Oligonucleotides can be designed containing fragment sequences to be tested. Alternatively, a more random approach is to linearize the expression vector by restriction digest within an enhancer, followed by subsequent exonuclease treatment and religation. Yet another method is to simply use restriction digests to remove sections of DNA.
It is preferred that following integration, a 3' enhancer and/or an intronic enhancer are positioned at a location 3' of the target nucleic acid sequence. It is further preferred that the intronic enhancer be located in greater proximity to the target gene than the 3' enhancer. The 5' end of the intronic enhancer is preferably positioned up to 3 kb 3' of the 3' end of the target gene, preferably less than 2 kb, more preferably less than 1 kb, and most preferably immediately adjacent to the target nucleic acid sequence.
The intronic enhancer can be positioned greater than 3 kb 3' of the target gene, but this is less preferred. The 3' enhancer is preferably located up to 20 kb and preferably 5-15 kb 3' of the intronic enhancer. The 3' enhancer can be located as close as 1 kb 3' of the intronic enhancer, but this is less preferred. In another embodiment, the 3' enhancer fragment is located 5' relative to the target gene. The intronic enhancer can also be positioned 5' relative to the target gene, although this embodiment is less preferred.
In a further preferred embodiment, the enhancers are present in a genomic orientation.
The enhancer sequence present in the genomic immunoglobulin gene is present in a "genomic orientation". If it is flipped in the construct so that it now appears in a 3' to 5' orientation (as opposed to the 5' to 3' orientation in the native genomic configuration), it is present in the "reverse orientation". However, the 3' enhancer can be present in reverse orientation. The enhancer can also be present in reverse orientation, but this is less preferred.
In a further preferred embodiment of the first aspect, following integration of the target nucleic acid molecule into the immunoglobulin locus, the target nucleic acid molecule is operatively linked to a promoter. . Preferably, the target nucleic acid molecule is located downstream of the promoter and upstream of an intronic enhancer.
The term "promoter" is well-known in the art and encompasses nucleic acid regions ranging in size and complexity from minimal promoters to promoters including upstream elements and enhancers.
In one preferred embodiment of the invention, the promoter is a naturally occurring promoter that exists within the immunoglobulin locus. In one embodiment, the promoter is an immunoglobulin heavy or light chain promoter. Preferably, the promoter is an immunoglobulin heavy chain promoter of a VH4 allele. There is strong conservation between the promoter sequences of the VH4 alleles (Figure 1). A
wide range of hypermutating cell lines (including RAMOS and BL2 cell lines) carry rearranged VH4 alleles.
Alternatively, the promoter may be a heterologous or exogenous promoter selected from those which are functional in mammalian cells, although prokaryotic promoters and promoters functional in other eukaryotic cells may be used. The promoter may be derived from promoter sequences of viral or eukaryotic genes. For example, it may be a promoter derived from the genome of a cell in which expression is to occur.
With respect to eukaryotic promoters, they may be promoters that function in a ubiquitous manner (such as promoters of a-actin, I3-actin, tubulin) or, alternatively, a tissue-specific manner (such as promoters of the genes for pyruvate kinase). They may also be promoters that respond to specific stimuli, for example promoters that bind steroid hormone receptors. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR) promoter, the rous sarcoma virus (RSV) LTR promoter or the human cytomegalovirus (CMV) IE promoter. In a preferred embodiment, the promoter is an immunoglobulin promoter sequence such as a murine immunoglobulin promoter sequence or a human immunoglobulin heavy chain promoter.
The promoter region of mammalian/human genes can contain several regulatory elements in the DNA sequences, and span several hundred bases or more, it is generally observed that one of these elements, designated 'TATA box" sequence, in eukaryotic promoter regions is usually found approximately 300 bases or more upstream of the translation initiation site (start) sequence, ATG. Rearrangements which bring the V
gene promoter into closer proximity to the ATG translation start signal also brings the promoter closer to the enhancer which is 3' and located in the C region. This activates the promoter which indicates that the close proximity to the enhancer may affect the rate at which the DNA in the VH locus is mutated.
The present inventors have found that in a RAMOS RA-1 cell line in which the rearrangement generates a functional VH4-34 allele, the promoter is significantly closer to the translation initiation start site than in other mammalian/human genes (based upon the number of nucleotides between the 3' end of the "TATA box" and the initiation codon of the immunoglobulin leader sequence). This proximity of the promoter to the start codon and the three dimensional structure of the promoter caused significant difficulties in cloning this region from RAMOS RA-1.
Accordingly, in a further preferred embodiment of the invention, following integration of the target nucleic acid molecule into the immunoglobulin locus, the target nucleic acid molecule is located within close proximity to the promoter. Preferably, the initiation codon of the target nucleic acid molecule is located within 500 bp of the 3' end of the promoter, more preferably within 200 bp of the 3' end of the promoter, more preferably within 100 bp of the 3' end of the promoter and more preferably within 20 bp of the 3' end of the promoter.
It may also be advantageous for the promoters to be inducible so that the levels of expression of the heterologous gene can be regulated during the life-time of the cell.
Inducible means that the levels of expression obtained using the promoter can be regulated.
In addition, any of these promoters may be modified by the addition of further regulatory sequences, for example enhancer sequences. Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above.
In a further preferred embodiment of the present invention, the target nucleic acid molecule is introduced into the cell by way of an integration vector comprising a sequence homologous to a region of at least 500 bp, more preferably at least 2 kb, more preferably approximately 5 kb upstream of a rearranged V gene and a sequence homologous to a region of at least 500 bp, more preferably at least 2 kb, more preferably approximately 5kb downstream of the same rearranged V gene.
Preferably, the sequence homologous to a region downstream of the rearranged V gene comprises an intronic enhancer and matrix attachment regions or portions thereof. It is preferred that the upstream and downstream homologous sequences are at least 500 bp in length, more preferably about 5 kb in length.
A "target nucleic acid molecule" can be any nucleic acid molecule of interest (including DNA and RNA molecules) encoding a gene product where diversification of the gene product is desired.
The "gene product" may be any biologically active molecule of interest. For example, the gene product may be a catalytic molecule such as a ribozyme, a DNAzyme, an LNAzyme or an RNAi/siRNA molecule. Alternatively, the gene product may be an antibody or fragment thereof, an enzyme, a hormone, a receptor, a cell surface molecule, a viral protein, transcription factor or any other biologically active polypeptide.

The selected mutant target sequence may be recycled through the methods of the present invention in order to introduce further diversification. Accordingly, in a further preferred embodiment of the first and second aspects, at least steps (ii) and (iii) of the method are repeated.

The hypermutating cell may be a mammalian, avian, yeast, fungi, insect or bacterial cell. In a preferred embodiment, the hypermutating cell is a mammalian cell.
The mammalian hypermutating cell may be selected from the group consisting of RAMOS, BL2, BL41, BL70 and Nalm.
The method of the present invention may include further steps to increase the rate of mutation of the target nucleic acid sequence. For example, the hypermutating cell may be cultured in the presence of chemical mutagens. Suitable chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid. Other agents which are analogues of nucleotide or nucleoside precursors include nitrosoguanidine, ribavirin, 5-bromouracil, 2-aminopurine, 5-formyl uridine, isoguanosine, acridine and of N4-aminocytidine, N1-methyl-N4-aminocytidine, 3,N4-ethenocytidine, 3 -methylcytidine, 5-hydroxycytidine, N4-dimethylcytidine, 542-hydroxyethyl)cytidine, 5-chlorocytidine, 5-bromocytidine, N4-methyl-N^4- aminocytidine, 5-aminocytidine, 5-nitrosocytidine, 5-(hydroxyalkyl)-cytidine, (thioalkyl)-cytidine and cytidine glycol, 5-hydroxyuridine, 3-hydroxyethyluridine, 3-methyluridine, 02-methyluridine, 02-ethyluridine, 5-aminouridine, 04-methyluridine, 04-ethyluridine, 04-isobutyluridine, 04-alkyluridine, 5-nitrosouridine, 5-(hydroxyalkyl)-uridine, and 5-(thioalkyl)-uridine, 1,N6-ethenoadenosine, 3-methyladeno sine, and N6-methyladenosine, 8-hydroxyguanosine, 06-methylguanosine, 06-ethylguanosine, 06-isopropylguanosine, 3,N2-ethenoguanosine, 06-alkylguanosine, 8 -oxo -guano sine, 2,N3 -etheno guano sine, and 8 -amino guano sineas well as derivatives/analogues thereof. Examples of suitable nucleoside precursors, and synthesis thereof, are described in further detail in USSN 20030119764.
Generally, these agents are added to the replication or transcription reaction thereby mutating the sequence. Intercalating agents such as proflavine, acriflavine, quinacrine and the like can also be used.
Random mutagenesis of the target nucleic acid molecule can also be achieved by irradiation with X-rays or ultraviolet light.

Antigen stimulation of the hypermutating cells, or exposure of the cells to interleukins (such as IL-2, IL-4 or IL-10) or CD40 ligand or B cell activating factor (BAFF) may also be used to increase the mutation frequency.
In one preferred embodiment, the level or activity of activation-induced cytidine deaminase (MD) within the hypermutating cell is increased. This may be achieved, for example, by increasing expression levels of AID within the hypermutating cells. For example, the hypermutating cells may be transfected with plasmid vectors which encode and express AID.
It will be appreciated by those skilled in the art that any process of selecting a mutant product can be used in the methods of the present invention.
In one embodiment, selection can be achieved by binding to a target molecule or by measurement of a biological response affected by the mutant product.
In another example, if the product of interest is an agent that promotes or reduces cell growth or division, the selection process can involve exposing mutant products to a population of cells and monitoring the biological responses of those cells.
In another example, if the mutant product is a receptor ligand, the process can involve exposing mutant proteins to cells expressing the receptor and monitoring a biological response effected by signalling of the receptor.
In one embodiment, the mutant product is selected by way of an assay performed within the hypermutating cell. For example, if the target nucleotide sequence encodes an enzyme, the assay may simply measure enzymatic activity.
Alternatively , the assay performed within the hypermutating cell may be a protein-fragment complementation assay (PCA). PCAs rely on the complementation of enzyme fragments fused to interacting proteins that reconstitute enzymatic activity once dimerised. Examples of PCA protocols are described in Michnick (2001) Current opinion in structural biology 11:472-477; Wehrrnan et al. (2002). PNAS
99(6):3469-3474; and Galarneau et al. (2002). Nature 20:616-622.

Suitable enzyme fragments may be derived, for example, from P-lactamase.
Studies using the P-lactamase system in both bacteria and mammalian cells have successfully validated it as a suitable reporter system to detect protein-protein interactions inside the cell.
The target nucleic acid molecule may therefore encode a fusion protein comprising a binding partner and a p-lactamase fragment. Its binding partner may be introduced into the cell as a fusion protein comprising a complementary p-lactamase fragment.
Selection occurs inside the cell with the binding of the target protein to its cognate partner which is detected by P-lactamase activity on a substrate supplied.
If selection assays such as PCAs are used, it is not necessary to display the target polypeptide on the surface of the hypermutating cell. These selection assays are particularly advantageous for targets which are naturally found intracellularly.
In an alternative embodiment of the present invention, the target nucleic acid molecule is linked to a sequence encoding an anchor domain such that following expression, the mutant gene product is displayed on the surface of the hypermutating cell.
Examples of suitable anchor domains include attachment signals from glycosylphosphatidylinositol (GPI) anchored membrane proteins or transmembrane domains of other cell surface proteins.
If the gene product to be displayed is not normally found on the cell surface, it is preferred that the target nucleic acid molecule is also linked to a sequence encoding an N-terminal signal peptide (or a leader peptide). This signal peptide facilitates targeting of the gene product to the plasma membrane.
Following display on the surface of the hypermutating cell, the mutant gene product may be selected by detecting binding of a binding partner to the mutant gene product.
This may involve, for example, labelling cells with a detectable marker such as a fluorescent dye and allowing binding to occur between the mutant protein on the cell surface and its binding partner. If the binding partner has been immobilized on a plate, a suitable detection system, such as a fluorimeter, can be used to identify wells containing a mutant of interest.

Alternatively, the binding partner may be labelled with a fluorescent tag, and cells expressing a mutant gene product of interest may be sorted using flow cytometric techniques. The binding partner may be selected from the group consisting of an antibody, receptor, hormone, enzyme, cell surface molecule, transcription factor, DNA
or RNA molecule.
In another embodiment of the invention, the target gene product is expressed in soluble form.
To prevent repeated mutation after selection, hypermutation may be arrested prior to culturing the selected cells. This can be accomplished in a number of ways, including fusion to a myeloma or repression of an inducible promoter.
In a further preferred embodiment of the invention, the mutant nucleic acid sequence encoding the selected gene product is recovered from the hypermutating cell.
This recovery can be achieved by amplification of the mutant nucleic acid sequence in whole or in part by polymerase chain reaction, using oligonucleotides that will anneal to locations outside the region of hypermutation or within the target sequence itself.
Alternatively, the mutant nucleic acid sequence may be amplified using RT-PCR.
The mutant nucleic acid sequence can then be subcloned for other purposes, such as expression, purification, or characterization.
The conditioned media from the transfected cells can be concentrated if desired and applied to the selection system. Specific binders can be identified directly or indirectly, for example by antibody recognition of either the target gene product itself or an attached tag sequence. The mutant gene products of interest can then be further characterized by a number of protein chemistry techniques such as micro-sequencing.
In a second aspect the present invention provides a gene product produced by a method of the first aspect.
In a third aspect the present invention provides a vector for targeted integration into an immunoglobulin locus of a hypermutating cell, the vector comprising a sequence homologous to a region upstream of a rearranged V gene of the hypermutating cell, a sequence homologous to a region downstream of a rearranged V gene of the hypermutating cell and a site for insertion of a target nucleic acid molecule.

In a preferred embodiment of the third aspect, the region upstream of the rearranged VIA gene of the hypermutating cell is a region within nucleotides 1 to 5190 of SEQ ID
NO: 1.
Preferably, the region is at least 500 bp, more preferably at least 2 kb, more preferably about 5 kb within nucleotides 1 to 5190 of SEQ ID NO: 1. In a further preferred embodiment the region upstream of the rearranged V gene of the hypermutating cell comprises nucleotides 191 to 5190 of SEQ ID NO: 1.
In a further preferred embodiment of the third aspect, the region downstream of the rearranged VH gene of the hypermutating cell is a region within nucleotides 5709 to 8699 of SEQ ID
NO: 1. Preferably, the region is at least 500 bp, more preferably at least 3 kb, 5709 to 8699 of SEQ ID NO: 1. In a further preferred embodiment the region downstream of the rearranged VH gene of the hypermutating cell comprises nucleotides 5709 to 8634 of SEQ ID
NO: 1.
Specifically, the third aspect of the invention relates to a vector for targeted integration into an immunoglobulin locus of a hypermutating cell, the vector comprising a sequence homologous to a region upstream of a rearranged VH gene of the hypermutating cell, a sequence homologous to a region downstream of a rearranged VH gene of the hypermutating cell and a site for integration of a target nucleic acid molecule, wherein the region upstream of the rearranged VH gene of the hypermutating cell comprises nucleotides 191 to 5190 of SEQ ID
NO: 1 and wherein the region downstream of the rearranged VH gene of the hypermutating cell comprises at least 500 contiguous nucleotides of the sequence between nucleotides 5709 and 8699 of SEQ ID NO: 1.
In a further preferred embodiment of the third aspect, the vector further comprises a selectable marker. The term "selection marker" or "selectable marker" includes both positive and negative selection markers. A "positive selection marker" is a nucleic acid sequence that allows the survival of cells containing the positive selection marker under growth conditions that kill or prevent growth of cells lacking the marker. An example of a positive selection marker is a nucleic acid sequence which promotes expression of the neomycin resistance gene, or the kanamycin resistance gene. Cells not containing the neomycin resistance gene 14a are selected against by application of G418, whereas cells expressing the neomycin resistance gene are not harmed by G418 (positive selection). A "negative selection marker" is a nucleic acid sequence that kills, prevents growth of or otherwise selects against cells containing the negative selection marker, usually upon application of an appropriate exogenous agent. An example of a negative selection marker is a nucleic acid sequence which promotes expression of the thymidine kinase gene of herpes simplex virus (HSV-TK). Cells expressing HSV-TK
are selected against by application of ganciclovir or 1-2'-deoxy-2'fluoro-b-D-arabinofuranosy1-5-iodouracil (FIAU); (negative selection), whereas cells not expressing the gene are relatively unharmed by ganciclovir or FIAU.
In a further preferred embodiment the vector encodes an anchor molecule suitable for display of the protein encoded by the target nucleic acid molecule.

In a further preferred embodiment the vector comprises a target nucleic acid molecule with an epitope tag(s) (for example, two flag tags).
In a further preferred embodiment, the vector for targeted integration comprises a 5 sequence as set out in SEQ ID NO:110.
The present invention provides a novel approach for generating diversity in a gene product (see Figure 2). An important feature of the present invention is that the target nucleic acid molecule is integrated into the immunoglobulin locus of a host cell The integration process also ensures that only one copy of the target nucleic acid molecule is present in each cell. This facilitates the recovery of mutant target nucleic acid sequences of interest following the selection process by, for example, PCR or RT-It will be appreciated that the methods of the present invention may be used for a variety of purposes. For example, the methods of the present invention can be used to effect affinity maturation of antibodies. In one aspect, the invention may be applied toward improving the affinity of antibodies from "naive," i.e., non-immune, phage be utilized as improvements over many antibody-based diagnostics and therapeutics currently available.
The methods of the present invention allow a very large library of peptides and single-chain antibodies to be screened and the polynucleotide sequence encoding the desired peptide(s) or single-chain antibodies to be selected. The pool of polynucleotides can then be isolated and shuffled to recombine combinatorially the amino acid sequence of the selected peptide(s) (or predetermined portions thereof) or single-chain antibodies (or just VH, VL, or CDR portions thereof). Using these methods, one can identify a peptide or single-chain antibody as having a desired binding affinity for a molecule and can exploit the process of the invention to converge rapidly to a desired high-affinity peptide or scFv. The peptide or antibody can then be synthesized in bulk by conventional means for any suitable use (e.g., as a therapeutic or diagnostic agent).
The mutagenesis system can also be used to effect receptor or ligand modification. In one aspect, the invention can generate a ligand or receptor with enhanced binding characteristics for its corresponding receptor or ligand. In another aspect, the mutagenesis system can be used to generate an inhibitor of functional receptor-ligand interaction by creating a ligand or receptor that still binds, but does not elicit a functional response. In yet another aspect of the invention, multiple biologically active variants of a target protein can be identified and recovered, thereby providing a means to study structure-function relationships of the protein. Additionally, species diversity can be investigated by comparing results obtained by selections utilizing receptors or other molecules from different species.
A receptor or ligand can be modified such that it can still bind, but does not signal any more. Alternatively, a better signalling ligand can be selected, which would provide a lower effective dosage of a pharmacologically active therapeutic.
The mutagenesis system of the present invention may also be used, for example, on a target such as caspase, an initiation factor target involved in a novel survival mechanism. This involves a cascade of essentially signalling reactions on the route to programmed cell death (apoptosis). Caspase-3 once activated binds to, and cleaves (activates) the 'cell death' proteins (including Id3). In vivo mutation and expression of mutated caspases, especially caspase-3, would have an effect on apoptosis.
Therefore caspase 3 would be a preferred target molecule. This could be relevant to diagnostics in cell signal transduction for monitoring and detection of cancer, and with potential therapeutic outcomes.
Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
A Brief Description of the Figures Figure 1: Alignment of human immunoglobulin heavy chain promoters for VH4 alleles. Sequences provided as SEQ ID NO's 2 to 7.
Figure 2: Schematic representation of a preferred method of the present invention.
Figure 3: Schematic representation of the gene targeting region - the preferred site for direct insertion of the target nucleic acid into a rearranged V gene.
Figure 4: Schematic representation of vector 3kb15a-7-4T.
Figure 5: Schematic representation of vector KW2.
Figure 6: Schematic representation of vector for targeted integration, KW3.
Figure 7: RAMOS cells stained with CFSE showing successive divisions on each day.
Figure 8: Schematic representation of vector pME18SasFP499.
Figure 9: The effect of DNA amount and electroporation parameters on transfection of RAMOS RA-1 cells with pME18SEGFP.
Figure 10: Comparison of RAMOS RA-1 cells transfected with pME18sEGFP, pME18sasFP499 or mock transfected.

Figure 11: (A): Quantum Simply Cellular Beads stained with mouse anti-human IgM-Alexa 488. (B) RAMOS cells stained with mouse anti-human IgM-Alexa 488. (C) Quantitation of IgM molecules on the surface of RAMOS RA-1 cells.
Figure 12: Comparison of IgM expression on RAMOS RA-1 cells of different passage number. (A) RAMOS RA-1 passage 1 (B) RAMOS RA-1 passage 14.
Figure 13: IgM expression on RAMOS RA-1 samples from different sources.
Figure 14: Schematic representation of sequential overlap extension PCR.
Figure 15: Schematic representation of mammalian expression vector pME18sCD26asfp499.
Figure 16: Comparison of expression of asFP499 with and without the CD26 anchor in RAMOS RA-1.
Figure 17: Comparison of expression of asFP499 with and without the CD26 anchor in HEK 293 T.
Key to the Sequence Listing SEQ ID NO:1 - Heavy chain locus of Ramos RA-1 cells.
SEQ ID NO:2 - Sequence of promoter region of a VH4 allele.
SEQ ID NO:3 - Sequence of promoter region of a VH4 allele.
SEQ ID NO:4 - Sequence of promoter region of a VH4 allele.
SEQ ID NO:5 - Sequence of promoter region of a VH4 allele.
SEQ ID NO:6 - Sequence of promoter region of a VH4 allele.
SEQ ID NO:7 - Consensus sequence of SEQ ID NO's 2 to 6.
SEQ ID NO:8 - Plasmid pME18SasFP499.
SEQ ID NO:9 - Sequence of enhancer of human immunoglobulin D segment locus.
SEQ ID NO:10 - Sequence of enhancer of human immunoglobulin heavy locus on chromsome 14.
SEQ ID NO:11 - Sequence of sheep immunoglobulin heavy chain 5' intronic enhancer.
SEQ ID NO:12 - Sequence of mouse 3' IgH regulatory enhancer.
SEQ ID NO:13 - Sequence of murine IgH enhancer.

SEQ ID NO:14 - Sequence of mouse 3' kappa enhancer.
SEQ ID NO:15 - Promoter sequence of mouse imm-unoglobulin VH gene.
SEQ ID NO:16 - Promoter sequence of mouse immunoglobulin V1 gene.
SEQ ID NO:17 - Promoter sequence of mouse immunoglobulin mu heavy chain gene.
SEQ ID NO:18 - Promoter sequence of mouse immunoglobulin VH gene.
SEQ ID NO:19 - Homo sapiens germline IgH chain (ProV4-39) gene fragment.
SEQ ID NO:20 - Homo sapiens germline IgH chain (ProV3-30) gene fragment.
SEQ ID NO:21 - Homo sapiens germline IgH chain (ProV3-9) gene fragment.
SEQ ID NO:22 - Homo sapiens germline IgH chain (ProV1-18) gene fragment.
SEQ ID NO:23 - GPI signal from human decay-accelerating factor.
SEQ ID NO:24 - Polynucleotide encoding SEQ ID NO:23.
SEQ ID NO:25 - GPI signal from porcine membrane dipeptidase.
SEQ ID NO:26 - Polynucleotide encoding SEQ ID NO:25.
SEQ ID NO:27 - GPI signal from rat ceruloplasmin.
SEQ ID NO:28 - Polynucleotide encoding SEQ ID NO:27.
SEQ ID NO:29 - GPI signal from mouse Thy-1.
SEQ ID NO:30 - Polynucleotide encoding SEQ ID NO:29.
SEQ ID NO:31 - Transmembrane domain of murine B7-1.
SEQ ID NO:32 - Polynucleotide encoding SEQ ID NO:31.
SEQ ID NO:33 - Signal sequence of CD59.
SEQ ID NO:34 - Polynucleotide encoding SEQ ID NO:33.
SEQ ID NO's 35 to 86, 88 to 90, 92 to 103, 105, 106, 108 to120 -Oligonucleotide primers.
SEQ ID NO:87 - Plasmid 3kb15a-7-4T.
SEQ ID NO:91 - Plasmid KW2.
SEQ ID NO:104 - Plasmid pME18sCD26asFP499.
SEQ ID NO:107 - Sequence of coding region of AICDA cDNA.
SEQ ID NO:110 - Plasmid KW3.
Detailed Description of the Invention General Techniques The present invention is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase = 79314-39 peptide synthesis, and immunology. Such procedures are described, for example, in the following texts 1. Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of Vols I, 5 LI, and III;
2. DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, whole of text;
3. Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed., 1984) IRL
Press, Oxford, whole of text, and particularly the papers therein by Gait, pp1-22;

10 Atkinson et al., pp35-81; Sproat et al., pp 83-115; and Wu et al., pp 135-151;
4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J.
Higgins, eds., 1985) IRL Press, Oxford, whole of text;
5. Animal Cell Culture: Practical Approach, Third Edition (John R.W.
Masters, ed., 2000), ISBN 0199637970, whole of text;
15 6. Immobilized Cells and Enzymes: A Practical Approach (1986) IRL
Press, Oxford, whole of text;
7. Perbal, B., A Practical Guide to Molecular Cloning (1984);
8. Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series;
20 9. J.F. Ramalho Ortigio, "The Chemistry of Peptide Synthesis" In:
Knowledge database of Access to Virtual Laboratory website (Interactiva, Germany);
10. Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, R.L. (1976).
Biochem.
Biophys. Res. Commun. 73 336-342 11. Merrifield, R.B. (1963). J. Am. Chem. Soc. 2149-2154.

12. Barany, G. and Merrifield, R.B. (1979) in The Peptides (Gross, E. and Meienhofer, J. eds.), vol. 2, pp. 1-284, Academic Press, New York.

13. Wunsch, E., ed. (1974) Synthese von Peptiden in Houben-Weyls Metoden der Organischen Chemie (Mfiler, E., ed.), vol. 15, 4th edn., Parts 1 and 2, Thieme, Stuttgart.

14. Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Verlag, Heidelberg.

15. Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg.

16. Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474.
Hypermutating cells In the context of the present invention, the hypermutating cells may be bacterial, yeast, avian, fungal, insect or mammalian cells. Examples of suitable hypermutating cells are described below.
Bacterial hypermutating strains:
(i) Epicurian coli mutator strain XL1-Red (triple DNA repair deficient ¨
mutD, mutS, mut T) by Stratagene;
(ii) Escherichia coli mutator strain MutD5 (MutD5-FIT, mutated DNAQ
gene) Irving RA, Kortt AA, Hudson PJ (1996). Immunotecbnology 2(2) 127-43;
(iii) Escherichia coli strain FC40. Foster PL (2000) Bioessays 22(12) : 1067-74 and Powell Sc & Wartwell RM (2001) Mutation Research 473 (2) 219-28;
(iv) Serogroup B meningococcal strains BF18, BF21 (defect in methyl ¨
directed mismatch repair ¨ lack DNA adenine methyltransferase (Dam) activity). Bucci et al. (1999) Mol Cell 3 (4) 435-45.
Yeast hypermutating strains:
(i) Saccharomyces cerevisiae strain (mlhl A mutant). Shcherbakova &
Kunkel (1999). Molecular and Cellular Biology 19(4) 3177-3183.
Saccharomyces cerevisiae strain DAG60 (msh2 mutant). (deficient in mismatch repair system). Drotschmann et al. (1999) Proc. Natl Acad Sci USA
96 2970-2975.
Mammalian hypermutating strains:
(i) Human B cell lines from Burldtt's lymphoma strains : RAMOS, BL2, BL41, BL70, Nalm-6. Sale & Neuberger (1998) Immunity 9 (6) 859-869.
(ii) BL2 : Denepouxs et al. (1997) Immunity 6(1) 35-46 and Poltorasky et al.
(2001) Proc Natl Acad Sci USA 98 (14) 7976-81 (iii) Human pre B cell line strain 18.81. Bachl et al. (2001) Journal Immunology 166 (8) 5051-7.

Intronic Enhancers Examples of suitable exogenous intronic enhancers for use in the present invention include the following:
1) 1) DNA sequence of the human immunoglobulin D segment locus (Rabbitts et al (1983) Nature 306 (5945), 806-809):
5' CGGCCCCGATGCGGGACTGCGTTTTGACCATCATAAATCAAGTTTATTTT
TTTAATTAATTGAGCGAAGCTGGAAGCAGATGATGAATTAGAGTCAAGATG
GCTGCATGGGGGTCTCCGGCACCCACAGCAGGTGGCAGGAAGCAGGTCAC
CGCGAGAGTCTATTTTAGGAAGCAAAAAAACACAATTGGTAAATTTATCAC
TTCTGGTTGTGAAGAGGTGGTTTTGCCCAGGCCCAGATCTGAAAGTGCTCT
ACTGAGCAAAACAACACCTGGACAATTTGCGTTTCTAAAATAAGGCGAGGC
TGACCGAAACTGAAAAGGCTTTTTTTAACTATCTGAATTTCATTTCCAATCT
TAGCTTATCAACTGCTAGTTTGTGCAAACAGCATATCAACTTCTAAACTGCA
TTCATTTTTAAAGTAAGATGTTTAAGAAATTAAACAGTCTTAGGGAGAGTTT
ATGACTGTATTCAAAAAGTTTTTTAAATTAGCTTGTTATCCCTTCATGTGTA
ATTAATCTCAAATACTTTTTCGATACCTCAGAGCATTATTTTCATAATGACT
GTGTTCACAATCTTTTT 3' (SEQ ID NO:9) 2) Homo sapiens immunoglobulin heavy locus (IGH.1@) on chromosome 14 (Ravetch et al (1981) Cell 27 (3 Pt 2), 583-591):
5'GGCCCCGATGCGGGACTGCGTTTTGACCATCATAAATCAAGTTTATTTTTT
TAATTAATTGAGCGAAGCTGGAAGCAGATGATGAATTAGAGTCAAGATGG
CTGCATGGGGGTCTCCGGCACCCACAGCAGGTGGCAGGAAGCAGGTCACC
GCGAGAGTCTATTTTAGGAAGCAAAAAAACACAATTGGTAAATTTATCACT
TCTGGTTGTGAAGAGGTGGTTTTGCCCAGGCCCAGATCTGAAAGTGCTCTA
CTGAGCAAAACAACACCTGGACAATTTGCGTTTCTAAAATAAGGCGAGGCT
GACCGAAACTGAAAAGGCTTTTTTTAACTATCTGAATTTCATTTCCAATCTT
AGCTTATCAACTGCTAGTTTGTGCAAACAGCATATCAACTTCTAAACTGCAT
TCATTTTTAAAGTAAGATGTTTAAGAAATTAAACAGTCTTAGGGAGAGTTT
ATGACTGTATTCAAAAAGTTTTTTAAATTAGCTTGTTATCCCTTCATGTGAT
AATTAATCTCAAATACTTTTTCGATACCTCAGAGCATTATTTTCATAATGAC
TGTGTTCACAATCTTTTT 3' (SEQ ID NO:10) 3) Ovis aries immunoglobulin heavy chain 5' intronic enhancer (Dufour et al (1996) J. Immunol 156:2163-2170):
5'CTGCGAATACCGAGACGGGGCCTCTCAAAGCCACCCCTGATAGTCTGGAA
AATTGAAACTTTAAAAAGAGAGATGTTTAAAGTATTTTAAATTTTTATCATT
TAATTAACAACTGCGAATCATGGCTTTGGAGAGTTGAGTAAGAGTTTGGCT
GAAAAGTACTAACTAGGTTCCATCGGCCCTCGGCCCCAATTCAGGGCTGTT
TTGAGAATAATAAATTCAGCTTATTTTTTTAATGTAATTGGTGGTGCCGAGT
TAGTCAAGATGGCCACGGGCCAGACTGACCACCTGCAGCAGGTGGCAGGA
AGCATGTCCACTTGAGAGTCTGTTTTTGGAAGCAAGAAAAAACAGTTGGTA
AATTTATCGCTTCTGGTTTCCAAAAGGTGGTTTGCGGCTGGTTTTGCCCAGC
CCCACAGAACCGAAAGTGTTCCACTGAGCACAACAGCACCTGGCTAATTTG
CATTTCTAAAATAAGGCGCAGATGCTGACCGAAACTGGAAGGTTCCTCTTC
TAACTATTTGAGTTAACTTCAGCTTTAGCTTATCAACTGCTCACTTATCTTCA
TTTTCAAAGTCGATGTTTAAGAAAGCCACCTGTCTCGGGTGCACTGTCTCGG
TGCATTGCTGCACTCTCTGATGAGCCGTCCTTCAAGGTGGTTGAGCTGAG 3' (SEQ ID NO:11) 4) Mouse 3' IgH regulatory enhancers (C alphaTE and hs3) (Saleque et al (1997) J.
Immunol. 158 (10), 4780-4787):
5'CTAGATACTGAGTTCTGGTTCTAATAACTGGCTCCTGTACTGATGGATGG
GTCCTGACTAGTCATTGGGCCCTGATCCTCAACATTGACTTCAAAACCTGAA
CTCTAGCCCCATGCCTCATTCACATTAGGATGATCCCTACAGGGGATTCCTG
CAGAAGATTCCAGAATCCCCACAACACTGTTCACACACTGGGCTGCAACTG
GGACAGTGACCCTTTTGACTCATAGGACTTGCCAGGCACAGAGGCACAGAA
TGGAGACAAAGCAAGCCCAGGACCCTGGAGATGGAGCCTCTGGTGGGGTC
TACAGATGTGGGGTCAGCATCGTAGGGAGGTTTGCAGGGCAGGTGTGGGG
CAGGGCAGAGGTAGTCATGCTTATAGATACTATTTTTCTCTCCTCTGGAGCC
TCCTTTGTCTATCACCTGCTGTCCTGGGATCTCTATCTGGGGTCAACAATGT
TTGCAGTACAGGTGTGGGGGTAGGGCAGGGATGCTCACATTAGCAACTTGT

TTTTCTCTCTTCTGAAGTCTCTGTTGTCTATCACCTGCTGAAACATTCAAAGC
AGCTCTCAGCTGAGGGCAGCTGAGTCATCCTGAGCCTGTCTCAGCACAGGT
GCCCCAAACCAGAGCTACTGTTCTGAGAATCACATCACACTGGACCAGGCC
AGGTGGGCCTGGGACATGGATGAGGGGTGGGAGCCAGGGGAGCCTGCCAG
GGGCTGAGGAGGCCCCAACCCCCACTACCCAAGGCCATCCACACCTGTGCC
TTAGTGAGGCCATGTTCTGTCCCAATGAGAACAAGTCCAATTAAGATTAAG
TATGGTCTTCCCAGGACTATCCAGAGCTAAGGGGTGTCAGCCAGGGACAAC
CCAGACCAGCCTGAGGTCAGCCAGCATCACCCAAGGCCACACAGCTATTCT
GGCTAGAGGACTAGATAGCTAGCTCATCGAGGCCCTGGAGATGCAGAATG
GAAGAGTTTATCCCTGCCAGACAGGGCTCATCAGAAAGGCAGGTATCTCAC
TACACATGACCTCCCTGAATATTTCCCAGAGTCCAGTTGGTTCTAG 3' (SEQ
ID NO:12) 5) Murine IgH enhancer (Kadesch et al (1986) Nucleic Acids Res. 14 (20), 8209-8221):
S'AGTCAAGATGGCCGATCAGAACCAGAACACCTGCAGCAGCTGGCAGGAA
GCAGGTCATGTGGCAAGGCTATTTGGGGAAGGGAAAATAAAACCACTAGG
TAAACTTGTAGCTGTGGTTTGAAGAAGTGGTTTTGAAACACTCTGTCCAGCC
CCACCAAACCGAAAGTCCAGGCTGAGCAAAACACCACCTGGGTAATTTG
CATTTCTAAA ATAAGTTGAGG 3' (SEQ ID NO:13) 3' kappa enhancers An example of a suitable exogenous 3' kappa enhancer for use in the present invention is as follows.
1) Murine 3' kappa enhancer (Meyer and Neuberger (1989) EMBO J. 8 (7), 1964):
S'AGCTCAAACCAGCTTAGGCTACACAGAGAAACTATCTAAAAAATAATTA
CTAACTACTTAATAGGAGATTGGATGTTAAGATCTGGTCACTAAGAGGCAG
AATTGAGATTCGAACCAGTATTTTCTACCTGGTATGTTTTAAATTGCAGTAA

GGATCTAAGTGTAGATATATAATAATAAGATTCTATTGATCTCTGCAACAA
CAGAGAGTGTTAGATTTGTTTGGAAAAAAATATTATCAGCCAACATCTTCT
ACCATTTCAGTATAGCACAGAGTACCCACCCATATCTCCCCACCCATCCCCC
ATACCAGACTGGTTATTGATTTTCATGGTGACTGGCCTGAGAAGATTAAAA

CATAGCTACCGTCACACTGCTTTGATCAAGAAGACCCTTTGAGGAACTGAA
AACAGAACCTTAGGCACATCTGTTGCTTTCGCTCCCATCCTCCTCCAACAGC
CTGGGTGGTGCACTCCACACCCTTTCAAGTTTCCAAAGCCTCATACACCTGC
TCCCTACCCCAGCACCTGGCCAAGGCTGTATCCAGCACTGGGATGAAAATG

TCCCCAGTCCCAATGCTTTTGCACAGTCAAAACTCAACTTGGAATAATCAGT
ATCCTTGAAGAGTTCTGATATGGTCACTGGGCCCATATACCATGTAAGACA
TGTGGAAAAGATGTTTCATGGGGCCCAGACACGTTCTAG 3' (SEQ ID NO:14) 15 Promoter sequences Examples of suitable exogenous promoter sequences for use in the present invention include:
20 1) (Kataoka et al (1982). J Biol. Chem. 257 : 2777-285):
5' AAGCAGCCCTCAGGCAGAGGATAAAAGCTCACACTAACTGAGAAGC
TCCATCCTCTTCTC 3' (SEQ ID NO:15) 25 2) (Clarke et al (1982). Nucleic Acids Res. 10 : 7731):
5' AATTAGGCCACCCTCATCACATGAAAACCAGCCCAGAGTGACTCTAG
CAGTGGGATCCTG 3' (SEQ ID NO:16) 3) Grosschedl and Baltimore D., (1985). Cell 41: 885-897):
5' CATGTGCGACTGTGATGATTAATATAGGGATATCCACACCAAACATC
ATATGAGCCCTAT 3' (SEQ ID NO:17) 4) (Schiff et al (1986). J. Exp. Med. 163 : 573-587):

5' AACATGAGTCTGTGATTATAAATACAGAGATATCCATACCAAACAAC
TTATGAGCACTGT 3' (SEQ ID NO:18) Murine B lymphocyte VH promoters (eg. V1 Ig VH promoter, BCL1 VH promoter) may also be used in the methods of the present invention.
Human Immunoglobulin heavy chain promoters The following human heavy chain promoters are described in in Haino et al (1994) J.
Biol. Chem. 269 (4), 2619-2626:
1) Homo sapiens germline IGH chain (ProV4-39) gene fragment:
S'ATCCCAAAATCTGTCNTTGATCCAGGATCACACTCATCTCTCAGACCAGC
TCCTTCAGCACATCTCTTTACCTGGAAGAAGAGGACTCTGGGCTTGGAGAG
GGGAGCCCCCAAGAAGAGAACTGAGTTCTCAAAGGGCACAGCCAGCATTC
TCCTCCCAGGGTGAGCTCAAAAGACTGGCGCCTCTCTCATCCCTTTTCACTG
CTCCGTACAAACGCACCACCCCCATGCAAATCCTCACTTAGGCGCCCACAG
GAAGCCACCACACATTTCCTTAAATTCAGGTCCAACTCATAAGGGAAATGC
TTTCTGAGAGTCATGGATCTCATGTGCAAGAAA 3' (SEQ ID NO:19) 2) Homo sapiens germline IGH chain (ProV3-30) gene fragment:
S'AGATATAACTATATTTTCCTGAATGATGGAATTACTACCAGTCTCCCCCA
GGACACTTCATCTGCCCTGAGCCCAGCCTCTCCTCAGATGTCCCACCCAGA
GCTTGCTATATAGTGGGGGACATGCAAATAGGGCCCTCCCTCTACTGATGA
AAACCAGCCCAGCCCTGACCCTGCAGCTCTGGGAGAGGAGCCCAGCACTA
GAAGTCGGCGGTGTTTCCATTCGGTGATCAGCACTGAACACAGAGGAC
TCACC 3' (SEQ ID NO:20) 3) Homo sapiens germline IGH chain (ProV3-9) gene fragment:
5' CAGTAGAAATGCTAATAAGAATTAATTGTTTATGAAGTGTAATCACTCTG
GGACACAGCCCACTCAGAGGCATCCCTTCCAGAACCCGCTATATAGTAGGA
GACATGCAAATAGGGCCCTCCCTCTGCTGATGAAAACCAGCCCAGCCCTGA

CCCTGCAGCTCTGGGAGAGGAGCCCCAGCCCTGAGATTCCCAGGTGTT
TCCATTCAGTGATCAGCACTGAACACAGAGGACTCACC 3' (SEQ ID NO:21) 4) Homo sapiens germline IGH chain (ProV1-18) gene fragment:
5' GATGGGTAGGGGATGCGTGTCCTCTAACAGGATTACGTCTTGAACCCTCA
GCTTCTACAATTGTGTCGTCCATGTGTCATGTATTTGCTCTTTCTCATCCTGG
GTCAGGAATTGGGCTATTAAATAGCATCCTTCATGAATATGCAAATAACTG
AGGTGAATATAGATATCTGTGTGCCCTGAGAGCATCACCCAAAAACCACAC
CCCTCCTTGGGAGAATCCCCTAGATCACAGCTCCTCACC 3' (SEQ ID NO:22) Methods for selection of nucleic acids or proteins/peptides with an altered phenotype The terms "altered phenotype", "desired activity" and "altered activity" are generally used interchangeably herein.
, In particular embodiments of the present invention, the mutated nucleic acid, or gene product encoded thereby, is subjected to an assay for identifying an altered phenotype.
Suitable procedures for identifying altered phenotypes include, but are not limited to, those described below.
Protein/peptide display and selection In one embodiment of the invention, proteins encoded by nucleic acids obtained using the methods of the invention are displayed on the surface of the hypermutating cells.
One well-known peptide display method involves the presentation of a peptide sequence on the surface of a filamentous bacteriophage, typically as a fusion with a bacteriophage coat protein. The bacteriophage library can be incubated with an immobilized, predetermined macromolecule or small molecule (e.g., a receptor) so that bacteriophage particles which present a peptide sequence that binds to the immobilized macromolecule can be differentially partitioned from those that do not present peptide sequences that bind to the predetermined macromolecule. The bacteriophage particles (i.e., library members) which are bound to the immobilized macromolecule are then recovered and replicated to amplify the selected bacteriophage subpopulation for a subsequent round of affinity enrichment and phage replication. After several rounds of affinity enrichment and phage replication, the bacteriophage library members that are thus selected are isolated and the nucleotide sequence encoding the displayed peptide sequence is determined, thereby identifying the sequence(s) of peptides that bind to the predetermined macromolecule (e.g., receptor). Such methods are further described in WO 91/17271, WO 91/18980, WO 91/19818 and WO 93/08278.
WO 93/08278 describes a recombinant DNA method for the display of peptide ligands that involves the production of a library of fusion proteins with each fusion protein composed of a first polypeptide portion, typically comprising a variable sequence, that is available for potential binding to a predetermined macromolecule, and a second polypeptide portion that binds to DNA, such as the DNA vector encoding the individual fusion protein. When transformed host cells are cultured under conditions that allow for expression of the fusion protein, the fusion protein binds to the DNA
vector encoding it. Upon lysis of the host cell, the fusion protein/vector DNA
complexes can be screened against a predetermined macromolecule in much the same way as bacteriophage particles are screened in the phage-based display system, with the replication and sequencing of the DNA vectors in the selected fusion protein/vector DNA complexes serving as the basis for identification of the selected library peptide sequence(s).
The displayed protein/peptide sequences can be of varying lengths, typically from 3-5000 amino acids long or longer, frequently from 5-100 amino acids long, and often from about 8-15 amino acids long. A library can comprise library members having varying lengths of displayed peptide sequence, or may comprise library members having a fixed length of displayed peptide sequence. Portions or all of the displayed peptide sequence(s) can be random, pseudorandom, defined set kernal, fixed, or the like. The display methods include methods for display of single-chain antibodies, such as nascent scFv on polysomes or scFv displayed on phage, which enable large-scale screening of scFv libraries having broad diversity of variable region sequences and binding specificities.
Another method of display is bacterial surface display. The protein of interest is expressed on the bacterial cell surface as a fusion with one of the following proteins, OmpA, LamB, PhoE, FliC, PALD, and EaeA intimin. Alternatively it may be expressed in the peiiplasm (periplasmic expression with cytometric screening, (PECS)) (Chen et al., 2001). Whilst experiments demonstrate expression on the bacterial cell surface, this display system is not operating as well or used as frequently as the yeast surface display. The nature of a prokaryotic system itself may explain why the system is not always preferred. Firstly, bacteria lack the protein folding and post-translational modification machinery required for the presentation of an eukaryotic protein.
Secondly, the protein of interest needs to be expressed in a soluble form.
Thirdly, steric interference from the bacterial lipopolysaccharide layer can potentially impede binding to larger macromolecular antigens. Despite being a single cell system (one plasmid to each bacterium), capable of displaying thousands of copies of the protein of interest on the cell surface and being amenable to screening using flow cytometry, this system offers no additional advantages over the yeast surface display system.
A further method of display is yeast surface display. The protein of interest is fused to an alpha agglutinin subunit called Aga2p and expressed on the surface of the yeast cell.
This form of display appears to be very successful and offers several advantages over other display systems. These include; correct protein folding and secretion (homologous to that in mammalian cells), display of large numbers of protein on yeast cell surface, single cell system (i.e. each yeast cell contains only one plasmid copy), system is amenable to screening using flow cytometry which offers finer quantitative discrimination between mutants and the dissociation constant (KD) can be estimated in situ in the display format without having to subclone. The major disadvantage of this system is that the library size is restricted due to low transfection efficiency. To date no one has managed to overcome this limitation of the system.
Proteins can be anchored to the surface of mammalian cells via a number of different anchors including, but not limited to, Type I transmembrane domains (TM I), type II
transmembrane (TM II) and glycosylphosphatydlinisitol (GPI).
Examples of suitable GPI anchor attachment signals include:
(i) GPI signal from human decay-accelerating factor (DAF). Caras IW, Weddell GN, Davits MA, Nussenzweig W, Martin DW. (1987) Science 238(4831):1280-3 Accession No.: AY055758 Peptide sequence: PNKGSTTSG'TTRLLSGHTCFTLTGLLGTLVTMGLLT (SEQ ID
NO:23) Nucleotide sequence: 1158-1268 CCAAATAAAGGAAGTGGAACCACTTCAGGTACTACCCGTCTTCTATCTGGG
CACACGTGTTTCACGTTGACAGGTTTGCTTGGGACGCTAGTAACCATGGGC
TTGCTGACT (SEQ ID NO:24) 5 (ii) GPI signal from porcine membrane dipeptidase. Hooper NM, Low MG, Turner AJ. (1987) Biochem. J. 244(2):465-9 Accession No.: E04233 Peptide sequence: CRTNYGYSAAPSLHLPPGSLLASLVPLLLLSLP (SEQ ID
NO:25) 10 Nucleotide sequence: 1248-1346 TGCCGGACGAATTACGGCTACTCAGCCGCCCCCAGCCTCCACCTCCCGCCG
GGCTCGCTGCTGGCCTCCCTCGTGCCCCTCCTCCTCCTCAGTCTTCCG (SEQ
ID NO:26) 15 (iii) GPI signal from rat ceruloplasmin. Patel BN, Dunn RJ & David S.
(2000) J.
Biol. Chem. 275(6):4305-10 Accession No.: AF202115 Peptide sequence: ASSQSYRMTWNILYTLLISMTTLFQISTKE (SEQ ID NO:27) Nucleotide sequence:

ATCAGCATGACTACTTTATTCCAAATATCTACCAAGGAG (SEQ ID NO:28) (iv) GPI signal from mouse Thy-1. Bernasconi E, Fasel N & Wittek R. (1996) J.
Cell Sci. 109(6):1195-201 Peptide sequence:
SSNKSISVYRDKLVKCGGISLLVQNTSWMLLLLLSLSLLQALDFISL (SEQ ID
NO:29) Nucleotide sequence:
AGCTCCAATAAAAGTATCAGTGTGTATAGAGACAAGCTGGTCAAGTGTGGC

CCCTCTCCCTCCTCCAAGCCCTGGACTTCATT (SEQ ID NO:30) (v) Dictyostelium discoideum protein 11. Stevens BA, White IJ, flames BD
Hooper NM. (2001) Biochimica et Biophysica Acta 1511: 317-329.

(vi) Plasmodium falciparum merozoite surface protein-1. Burghas PA, Gerold P, Pan W, Schwartz RT, Lingelbach K, Burjard H. (1999) Molecular and Biochemical Parisitology 104:171-183.
An example of a suitable transmembrane domain for use as an anchor domain in the present invention is:
(i) The transmembrane domain of murine B7-1. Chou W., Liao K., Jiang S.Y., Yeh M.Y. and Roffler S.R.(1999) Biotechnol Bioeng. 1999 Oct 20;65(2):160-9.
Accession No.:AH00465S3 Peptide sequence: PEDPPDSKNTLVLFGAGFGAVITVVVIVVIIKCFCKH (SEQ ID
NO:31) Nucleotide sequence: 171-281:
CCCAGAAGACCCTCCTGATAGCAAGAACACACTTGTGCTCTTTGGGGCAGG
ATTCGGCGCAGTAATAACAGTCGTCGTCATCGTTGTCATCATCAAATGCTTC
TGTAAGCAC (SEQ ID NO:32) Other suitable examples include those provided in Table 1.
Table 1 : Examples of anchors suitable for surface display:
Anchor type Molecule name Accession No.
Transmembrane type I (TM I) CD1a X04450 (gi 32495) CD68 BC05557(gi 33869409) Transmembrane type II (TM II) CD10 Y00811 (gi 29625) CD13 X13276 (gi 28677) CD26 M74777 (gi 180082) Glycosylphosphatydlinisitol (GPI) CD14 X113334 (gi 29740) CD24 M58664 (gi 180167) CD48 M59904 (gi 180138) CDw52 X62466 (gi 29645) CD55 M31516 (gi 181467) CD59 X16447(gi 29805) CD67 X52378 (gi 29918) Signal peptide sequences For TM1 and GPI anchors, an N-terminal signal sequence as well as a C-terminal signal sequence is preferably be added to the polypeptide if it is not normally found on the cell surface. For TM2 the signal and anchor sequence are one and the same and are added to the N-terminus of the polypeptide. To achieve adequate levels of surface expression, it may be necessary to mutate the initiation codon of the target gene so that only chimeric proteins consisting of the signal and/or anchor fused to the target protein are produced.
An example of an appropriate signal sequence is the signal sequence of CD59:
Accession No.:X16447 (gi 180082) Peptide sequence: MGIQGGSVLFGLLLVLAVFCHSGHS (SEQ ID NO:33) Nucleotide sequence: 64-138 S'ATGGGAATCCAAGGAGGGTCTGTCCTGTTCGGGCTGCTGCTCGTCCTGGC
TGTCTTCTGCCATTCAGGTCATAGC 3' (SEQ ID NO:34) Proteins/peptides encoded by mutant nucleic acids obtained using the methods of the invention can be used in a number of yeast based methods to detect protein-protein interactions. One well known system is the yeast two-hybrid system (Fields and Song 1989) which has been used to identify interacting proteins and to isolate the corresponding encoding genes. In this system, prototrophic selectable markers which allow positive growth selection are used as reporter genes to facilitate identification of protein-protein interactions. Related systems which may be employed include the yeast three-hybrid system (Licitra and Liu 1996) and the yeast reverse two-hybrid system (Vidal et al. 1996). Such procedures are known to those skilled in the art.
Examples Example 1: Defining a region in RAMOS cells for integration of target genes Methods & Materials Cell line and cell culture conditions õ

The RAMOS strain RA 1 was obtained from the American Tissue Culture Collection (ATCC-CLR-1596). This strain is IgM positive and expresses the interleukin 4 (IL-4) and CD23 receptors. Cells were maintained in RPM! 1640 medium (Gibco BRL) supplemented with 10% heat inactivated fetal calf serum (FCS) and penicillin (100 U /
ml) and streptomycin (100 g / ml), and incubated at 37 C with 5% CO2.
=
Extraction of DNA from cells Cells were harvested and centrifuged at 1500 rpm and resuspended in PBS. DNA
was extracted from cells using a Genoprep DNA isolation kit (Scientifix, Australia) according to manufacturer's instructions. Briefly, after removing the supernatant 375 I
of lysis and binding solution was added to cells (5 x 105), together with 20 I GenoprepTh DNA magnetic beads. This mixture was vortexed for five seconds then incubated at room temperature for a minimum of ten minutes on a rocker, or rotating wheel.
Beads with attached DNA were collected using a magnet, the supernatant was removed and 450 p.1 of washing solution was added. The mixture was subsequently vortexed for five seconds and the beads were collected as described previously. This washing procedure was repeated twice, with the final wash solution being 70 % ethanol. Beads were resuspended in 450 pl of 70 % ethanol and transferred to a new tube. After removing the supernatant, 450 1 of sterile water was added to the beads and removed immediately. The beads were then resuspended in 200 1 of sterile water and incubated at 70 C for two minutes to elute the DNA. The beads were collected again using a magnet and the supematent containing the eluted DNA was transferred to a new tube.
The quantity and quality of isolated DNA was determined by spectrometry and electrophoresis. A culture containing 5 x 105 cells consistently yielded 10 ng / p1 of DNA. This DNA migrated- as a single band at approximately 23 kbp on a 0.9 %
gel indicating that the genomic DNA was intact.
Sequencing of the 5' region upstream of the rearranged Vii allele The homologous sequence upstream of the site of integration chosen for the vector corresponds to a ¨ 5kb fragment between the VH7_35 and VH4_34 alleles of the immunog,lobulin heavy chain locus of the RAMOS cell line (corresponding to the sequence gi 4512287 nucleotides 54521 ¨59517).

RAMOS RA-1 genomic DNA was prepared using the Genoprep DNA isolation kit (Scientifix, Australia). Platimun PfXN DNA polymerase (GibcoBRL, Life Technologies) was used to amplify fragments varying from 300bp to 1000 bp from genomic DNA. The reaction included 1 X PfX amplification buffer, 50 mM
Magnesium sulfate, 1 X PCR enhancer solution, forward primer (10 pM), reverse primer (10 pM), dNTPs (10 mM each) template DNA (100 ng), platinum PfX DNA
polymerase (0.6 U) and sterile water in a final volume of 20 1. Cycling conditions were as follows; one cycle of 95 C for 5 minutes, 30 cycles of 95 C for seconds, 60 C
for 30 seconds, 68 C for one and a half seconds, and one cycle of 72 C for 7 minutes.
Annealing temperatures and extension times for some primer sets varied, ranging from 55 C to 65 C and one minute to two and a half minutes respectively. Primers were designed based on the human germline DNA for immunoglobulin heavy-chain variable region, complete sequence gi 4512287 nucleotides 54786 to 59721 (Table 2). A
second PCR reaction was performed using 0.5 / 20 I of the first PCR as a template to gain sufficient DNA for cloning.
PCR products were run on 1.0 % agarose gels and DNA was extracted using Nucleospin Extraction Kit (Nagel-Macherey, Germany) according to manufacturer's instructions. The purified DNA was digested with restriction enzymes EcoR I
and Hind III. Digested products were cleaned up and concentrated using phenol extraction followed by ethanol precipitation. These products were ligated into into pBluescript SKN
+ (Stratagene, Texas, USA) and transformed into Escherichia coil XL1 Blue electro-competent bacteria. Minipreps were prepared from 5 ml overnight cultures using QIAprelimminiprep spin kit (Qiagen, CA, USA) and sequenced using an ABI 373 DNA
sequencer with primers T3 and T7.
Sequences were analysed using BLAST program (NCBI, National Library of Medicine, Building 38A, Bethesda MD USA 20894) and assembled in Clone Manager rm Suite 7 (Scientific and Educational Software). The assembled sequence is set out as nucleotides 1 to 5190 of SEQ ID NO:1. This sequence shares 99 % similarity with the published sequence gi 4512287 nucleotides 54521-59517.
Cloning of the 5kb fragment upstream of the rearranged VH allele from genomic DNA
Genomic DNA was prepared using Genoprep DNA isolation kit (Scientifix, Australia).
Platinum PfX DNA polymerase (Invitrogen, CA. USA) was used to amplify the 5 kb õ

fragment from genomic DNA. The reaction included 1 X PfX amplification buffer, mM magnesium sulfate, lx PCR enhancer solution, forward primer 8771 (5' CCATCGATAATTTAGTTTTCACGGGGCATCTGCAGGGT 3') 10 pM, reverse primer 8872 (5' GGGGTACCGTTCTTGTGCAGGAGGTCCATGACTCTCAG 3') 10 5 pM, dNTPs (10 mM each) template DNA (100 ng), platinum PfX DNA polymerase (0.6 U) and sterile water in a final volume of 20 pl. Cycling conditions were as follows ; one cycle of 94 C for 15 seconds, fifteen cycles of 94 C for 10 seconds, 68 C for 3.5 minutes, 15 cycles of 94 C for 10 seconds, 68 C for 3.5 minutes with and an extra 15 seconds added each cycle, and one cycle of 72 C for 7 minutes. A second PCR

reaction was performed using 0.5 / 20.0 pi of the first PCR reaction as a template to gain sufficient DNA for detection using ethidium bromide.
PCR products were run on a 0.9 % agarose gel and DNA was extracted using Gel Extraction Kit (Qiagen, CA, USA). Purified DNA was cloned into pPCRScript using 15 the PCR-Script Amp cloning kit (Stratagene, Texas, USA) at the Srf I site. The resulting construct was referred to as 5kbPCRScript 15a-7 (7971 bp). This construct was sequenced using primers in Table 3.
Table 2: Primers used for genomic PCR to sequence the 5' region upstream of the 20 rearranged VII allele (V114..34) in RAIVIOS RA-1 Primer Sequence Homologous sequence Name in gi AB019439 8444 5' CCGGAATTCAAITI'GAGATTGTGTGTGAGATCTCAGGAG 3' NT 58890 - 58920 (SEQ ID NO:35) 8436 5' CCGGAATTC ATAGACAGCGCAGGTGAGGGACAG GGTCTC 3' NT 59702- 59731 (SEQ ID NO:36) 8438 5' CCGGAATTCCTG AGA ACTCAG TTCTCTTCCTGTGGCCTC 3' NT 59281 - 59310 (SEQ ID NO:37) 8440 5' CCGGAATTCAA rri GAGATTGTGTGTGAGATCTCAGGAG 3' NT 58890 - 58920 (SEQ ID NO:38) 8441 5' CCCAAGCTTTCCTGTTACAACATCCATGGAGATATITI G 3' NT 58420 - 58450 (SEQ ID NO:39) 8442 5' CCGGAATTC TGAATTGCAAGAACATACCCTAGGGTGTGC 3' NT 58500 - 58530 (SEQ ID NO:40) 8464 5' CCGGAATTCTAGGGCAAACAGAGGCCAGATGTTIGAGGAG NT 57720 - 57750 3' (SEQ ID NO:41) 8466 5' CCGGATfCAA1TIAACAGCATAkkACGATCAGTCCAjS NT 57330 - 57360 3' (SEQ ED NO:42) 8470 5' CCGGAATTCCGTG Fri CTGGAGCAGGGCATGGCTTTGGG 3' NT 56550 - 56580 (SEQ ID NO:43) 8472 5' CCGGAATTCGTTGGGTTCCCAGTGTAGGTGATGATCCAT 3' NT 56160 - 56190 (SEQ BD NO:44) 8550 5' CCGGAA'TTCTCCCAGGAAGTGGGTTA cirri AAATAGTA 3' NT 58951- 58981 (SEQ ID NO:45) 8553 5' CCGGAATTCACTATAGTCACCTCAGTTAATTGCATATTC 3' NT 55770 - 55800 (SEQ BD NO:46) 8554 5' CCCAAGCTTGACTTCCTTTAAAAATATCTAAAATAAGTA 3' NT 55300 - 55330 (SEQ ID NO:47) 8555 5' CCGGAATTCGGTTCTCATTACAACATCCAG1-11 GATAAA 3' NT 55380 - 55410 (SEQ 11D NO:48) 8557 5' CCGGAATTCCTCCAAG.AAAAGATCTCATGCATCACCAGG NT 54990 - 55020 3' (SEQ ID NO:49) 8558 5' CCCAAGCTTAAMAGITrtCACGGGGCATCTGCAGGGT 3' NT 54520 - 54550 (SEQ ID NO:50) 8606 5' CCCAAGC ITIGCACACCCTAGGGTATGTTCTTGCAATTC 3' NT 58500 - 58530 (SEQ ID NO:51) 8607 5' CCCAAGCTTCCCAAAGCCATGCCCTGCTCCAGAAACACG NT 56550 - 56580 3' (SEQ ID NO:52) 8608 5' CCGGAATTC CCATAATATGTGAATGCGTTATTTAGG GAA NT 55500 - 55529 3' (SEQ ID NO:53) 8687 5' CCCAAGCTTCATGTTCCACGCATTACGTC 3' (SEQ ID NO:54) NT 54336 - 54355 8689 5' CCCAAGCTTAAGAGTGTTTGGGTTCACCG 3' (SEQ ID NT 54927 - 54946 NO:55) SUBSTITUTE SHEET (RULE 26) RO/AU

Table 3 : Primers used for sequencing clones containing the 5 kb region upstream of the rearranged VII allele (VH4_34) in RA1VIOS RA-1 Primer Sequence Homologous Name sequence in gi 8445 5' CCCAAGCriTIAACTCAGGAGGACTCAATACACCCTGGA NT 57640-57670 3' (SEQ ID NO:56) 8443 5' CCCAAGCTTAAACAATACCTACAAATTCAGAAGCTC1T1 NT 58030-58060 3' (SEQ ID NO:57) 8471 5' CCCAAGCTT AAGTCTTCTGGTTACACCTTCACCAT TAT 3' NT 56080-56110 (SEQ ID NO:58) 8465 5'CCCAAGCTTACTCTCTTCCCTCTGTGACTAGAGCTCTGT 3' NT 57250-57280 (SEQ ID NO:59) 8473 5' CCCAAGCTTTCAGCTTCTACAGTTGTGTCACCCATGTGT 3' NT 55690-55720 (SEQ ID NO:60) 8609 5' CCCAAGCITIOCAGAGTTCACTGGGTTTCCTAAAGG CAA NT 55027-55056 3' (SEQ ID NO:61) 8467 5' CCCAAGC1T1CACCACAAAGGAACTTTCATCTCTCCTGG 3' NT 56860-56890 (SEQ ID NO:62) 8439 5' CCCAAGC1'1-1-1"1 CACACAGAAATGTTTAGAGGT CAGGCC NT 58810-58840 3' (SEQ ID NO:63) 8606 5' CCCAAGC rri GCACACCCTAGGGTATGTTCTTGCAATTC 3' NT 58500- 58530 (SEQ ID NO:64) 8551 5' CCCAAGCTTCCCAAAGCCATGCCCTGCTCCAGAAACACG NT 56550- 56580 3' (SEQ ID NO:65) 0003 5' CCCAAGCTTATGATGTAACCCTCATTGGCCTCA 3' (SEQ ID NT 54497-54520 NO:66) 0006 5' CCGGAAT'TCGAGACTCCAAGAAAAGATCTCATG 3' (SEQ NT 55001-55024 ID NO:67) 0007 5' CCCAAGCTTATGTTCT'TGCAATTCAGCGGAGGA 3' (SEQ ID NT 58514-58537 NO:68) 0008 5' CCGGAATTCTGTGTGAGATCTCAGGAGAAGGTA 3' (SEQ NT 58885-58908 ID NO:69) PCR amplification of rearranged VII, D and JH segments from genomic DNA
SUBSTITUTE SHEET (RULE 26) RO/AU

Genomic DNA was prepared using the Genoprep DNA isolation kit (Scientifix, Australia). Platinum PfX DNA polymerase (Invitrogen, CA, USA) was used to amplify the rearranged VH, D and JH genes from genomic DNA. The PCR reaction included lx PfX amplification buffer, 50 mM magnesium sulfate, 1X PCR enhancer solution, forward primer (10 pM), reverse primer (10 pM), dNTPs (10 mM each), template (100 ng), platinum PfX DNA polymerase (0.6 U) and sterile water in a final volume of 20 IA. Cycling conditions were as follows; one cycle of 95 C for 5 minutes, 30 cycles of 95 C for 30 seconds, 65 C for 30 seconds, 68 C for 2.5 minutes, and one cycle of 72 C
for 7 minutes. Primers used were specific for each of the seven VH family leader sequences together with a previously described consensus JH primer, J0L48, that anneals to all six human JH segments (Table 4). A second PCR reaction was performed using 0.5 / 20 pl of the first PCR as template to gain sufficient DNA for detection using ethidium bromide.
PCR products were run on 1.5 % agarose gels and DNA was extracted from bands using Nucleo spin, Extraction Kit (Nagel-Macherey, Germany) according to manufacturer's instructions. A third PCR was performed on the purified DNA as described above. Products were cloned into pBluescript SK + (Stratagene, Texas, USA) using EcoR I and Hind III sites and sequenced as previously described.
Table 4 : Primers for PCR amplification of rearranged VH, D and JH segments from genomic DNA.
Primer Sequence Specificity Name 8111 5 'CCCAAGCTTATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCA 3' VH1 (SEQ ID NO:113) leader 8116 5 ' CCCAAGCTTATGGACACAC ITt GCTCCACGCTCCTGCTGCTGACC ATCCCT VH2 3' (SEQ ID NO:114) leader 8112 5' CCCAAGCTTATGGAGTTTGGGCTGAGCTGGG1TITCCTTGTTGCTATT 3' VH3 (SEQ ID NO:115) leader 8113 5 ' CCCAAGCTTATGAAACACCTGTGGTTCTT'CCTCCTGCTGGTGGCAGCT 3' VH4 (SEQ ID NO:116) leader 8118 5 ' CCCAAGCTTATGGGGTCAACCGCCATCCTCGCCCTCCTCCTGGCTGTTCTC VH5 3' (SEQ ID NO:117) leader 8114 5' CCCAAGCTTATGTCTGTCTCCTTCCTCATCTTCCTGCCCGTGCTGGGCCTC 3' VH6 (SEQ ID NO:118) leader 8115 5 ' CCCAAGCTTATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCA H7 leader 3' (SEQ ID NO:119) N8336 5 ' CCCAAGCTTCCCCAAGCTTCCCAGGTGCAGCTACAGCAG 3' (SEQ ID Consensus (J0L48) NO:120) SUBSTITUTE SHEET (RULE 26) RO/AU

Table 5 : Primer sequences used for genomic PCR to sequence the 3 kb region downstream of the rearranged VII allele (VH4_34) in RAMOS RA-1 Primer Sequence Homologous Name sequence in NCBI
database 8604 5' CCCAAGCTTCGGCCCCGATGCGGGACTGCGTTTTGACCA 3' Gi 34819 NT 1-30 (SEQ ID NO:70) 8605 5' CCGGAATTCATAACAAGCTAAMAAAAAACTITTTGAA 3' Gi 34819 NT 450 -(SEQ ID NO:71) 500 8559 5' CCCAAGCTTGCACAGACGGGAGGTACGGTATGGACGTCT 3' Gi 33100 NT 460-(SEQ ID NO:72) 490 8562 5' CCGGAATTCAAAAAAATAAACTTGAITJATGAT GGTCAA 3' Gi 33100 NT 705-(SEQ ID NO:73) 734 8603 5' CCGGAA'TTCCGCGGTGACCTGCTTCCTGCCACCTGCTGT 3' Gi 34819 NT 126-(SEQ ID NO:74) 155 8692 5' CCGGAATTC AGT'TAGTGCAGCCAAGCCCT 3' (SEQ ID NO:75) Gi 33101 NT 301-8695 5' CCGGAATTCAAAAGGCAAGTGGAC'TTCGGTGCTTACCTG 3' Gi 188910 NT 945-(SEQ ID NO:76) 974 8697 5' CCCAAGCTT CAGCTCAGCTCAGTTCAGTTCAGCCCT 3' (SEQ Gi 188910 NT 4-30 ID NO:77) 8508 5' CCCAAGCTTATGCGAGGGTCTGGACGGCTGAGGACCCCC 3' Gi 188910 NT 370-(SEQ ID NO:78) 400 8696 5' CCGGAATTCATGCGGCAAGGGTTGCGGACCGCTGGCTGG 3' Gi 188910 NT 715-(SEQ ID NO:79 744 8694 5' CCGGAATTCGCCCAGCCCAGCCTAGCTCA 3' (SEQ ID NO:80) Gi 33101 NT 770-8691 5' CCCAAGCIT1TATCAACTGCTAGTTTGTG 3' (SEQ ID NO:81) Gi 34819 NT 361-9090 5' CCGGAATTCAGGGCTGAACTGAACTGAGCTGAGCTG 3' (SEQ Gi 188910 NT 1-30 ID NO:82) 9879 5' CGGCTGATATCTGGGAGCCTCTGTGGAI1-1-1CCGA 3' (SEQ ID Gi 33100 NT 1-24 NO:83) 9805 5' AGCCGGATATCGCCCAGCCCAGCCTAGCTCA 3' (SEQ ID Gi 33101 NT 770-N
O:84) 790 SUBSTITUTE SHEET (RULE 26) RO/AU

0002 5' GAAAGTTAAATGGGAGTGACCCAG 3' (SEQ ID NO:85) GI 29502084 NT

0021 5' GAGTGACCATCGCACCCTTGACAG 3' (SEQ 113 NO:86) 01 29502084 Identification of rearranged VW segment in RAMOS RA-1 5 The sequenced VH allele for RAMOS RA-1 was identified as VH4-34 (DP 63) using V- Base available from the MRC Centre for Protein Engineering (Cambridge, UK). A
schematic diagram of the gene targeting region is shown in Figure 3 and the genomic sequence is set out in SEQ ID NO:!. The immunoglobulin heavy chain promoter shown in Figure 3 corresponds to nucleotides 4852-5190 of SEQ ID NO:!. The leader sequence (including inton 1) shown 10 in Figure 3 corresponds to nucleotides 5191-5329 of SEQ ID NO: 1. The VDJ segment shown in Figure 3 corresponds to nucleotides 5330-5708 of SEQ ID NO:l.
The consensus nucleotide sequence differs from the published VH4.34 sequence (V ¨
Base) by six nucleotides only (C68->G, C72->T, C228->G, T2,32->C, C244->T, C248->A) of 15 which four were coding changes (A23->G; N76->K, F78->L, T83->N). The sequence was further classified as VH4-34 subgroup 2 (Journal of Molecular Biology, 1987, Vol 195, 761-768) using the Kabat database (Kabat et al., 1991, Sequences of proteins of immunological interest, vol 1, 5th edition). The C68-> G mutation in framework 1 and N76- > K mutation in framework 4 were unique to RAMOS RA-1 and occurred in 20 otherwise conserved residues in the other four members of the group (Lee et al. 1987, Journal of Immunology, 142, 4054-4061, Sanz et al. 1988, Clinical Experimental Immunology 71, 508-516).
The nucleotide sequence generated from RAMOS DNA corresponding to the D allele 25 differed significantly from the published alleles in V Base. Although no significant homology was identified, the closest related sequence similarity (and sequence length) was to D3-16 (Corbett, S et al, Journal of Molecular Biology, 270, 587-597).
The sequenced JH allele for RAMOS RA-1 was identified as JH6b (Mathis et al.
1995) 30 using V-Base. No nucleotide base changes were detected between the consensus sequence and the published JH6b sequence.

Sequencing of the 3 ' region downstream of the rearranged VII allele The homologous sequence downstream of the site of integration chosen for the vector corresponds to a ¨ 3kb region between the rearranged VII) genes through to the mu enhancer of the human immunoglobulin heavy chain locus of the RAMOS cell line (corresponding to sequence gi 29502084 nucleotides 960091 ¨ 962947).
The region 3' downstream of the rearranged VDJ segment was sequenced using methods previously described. Primers were designed based on published sequences, human immunoglobulin heavy chain enhancer on chromosome 14 (gi 34819), human J6 to enhancer DNA of the immunoglobulin heavy-chain gene (gi 33100), human (AW-Ramos) translocated t(8;14) c-myc oncogene, exon 1 (gi 188910) and human mu switch DNA of the immunoglobulin heavy-chain gene locus (gi 33101)(Table 5).
Sequences were analysed using BLAST program (NB CI, http://www.ncbi.nlm.nih.gov/BLAST/) and assembled in Clone Manager Suite 7 (Scientific and Educational Software). The assembled sequence is set out as nucleotides 5709 to 8634 of SEQ ID NO:l. This sequence shares 98 % similarity with the published sequence gi 29502084 nucleotides 960091-962947.
Cloning of the 3kb fragment downstream of the rearranged VII allele from genomic DNA
The 3 kb fragment was amplified from genomic DNA extracted from RAMOS RA-1 cells using Platinum Pa DNA polymerase (Invitrogen, CA, USA). The PCR reaction was the same as previously described except the forward primer 9779 (5' CCGCTCGAGTGGGAGCCTCTGTGGATTTTCCGA 3') (SEQ ID NO:111) and reverse primer 9801 (5' TGACCGGACGTCGCCCAGCCCAGCCTAGCTCA 3') (SEQ ID NO:112) were used and the cycling conditions were as follows ; one cycle of 94 C for 15 seconds, fifteen cycles of 94 C for 10 seconds, 68 C for 2 minutes, 15 cycles of 94 C for 10 seconds, 68 C for 2 minutes with and an extra 15 seconds added each cycle, and one cycle of 72 C for 7 minutes. A second PCR reaction was performed using 0.5 / 20.0 Ill of the first PCR reaction as a template to gain sufficient DNA for detection using ethidium bromide.

The 3 kb fragment was cloned into pPCRScript using the PCR-Script Amircloning kit (Stratagene, Texas, USA) at the Srf I site. The resulting construct was referred to as 3kbPCRScript 10-1-3 (5822 bp). This construct was sequenced using primers in Table 5.
The size and GC rich stretches present in the 5 kb and 3 kb homology regions gave rise to structural regions which caused difficulties in cloning and thereafter problems with stability of the cloned regions.
Example 2: Design and construction of a vector for integration into the rearranged VH
allele, VH4-34 in RAMOS RA-1 1) Construction of Vector For Integration A 3 kb fragment, containing sequence homologous to the region downstream of the rearranged allele VH4_34 was amplified from the construct 3kbPCRScript 10-1-3 with the forward primer 9879 (5' CGGCTGATATCTGGGAGCCTCTGTGGATTiTCCGA
3') (SEQ ID NO:83) and the reverse primer 9805 (5' AGCCGGATATCGCCCAGCCCAGCCTAGCTCA 3') (SEQ ID NO:84) using Platinum PfX DNA polymerase (Invitrogen, CA. USA). PCR products were purified using QlAquickni PCR Purification Kit (Qiagenlm) according to manufacturer's instructions then digested with EcoRV and ethanol precipitated. Digested fragments were ligated into construct 5kbPCRScript 15a-7 containing the 5 kb sequence upstream of the rearranged VII allele. The DNA was transformed into Esherichia coli and grown at 37 C overnight.
Bacterial colonies were screened by Southern blotting and probed using 32P
labeled oligonucleotides 8604 (5' CCCAAGCTTCGGCCCCGATGCGGGACTGCGIT1TGACCA 3') (SEQ ID NO:70) to detect the 3 kb fragment and a pool of labeled oligonucleotides 8687 (5' CCCAAGCTTCATGTTCCACGCATTACGTC 3') (SEQ ID NO:54), 8440 (5' CCGGAATTCAATTTGAGATTGTGTGTGAGATCTCAGGAG 3') (SEQ ID NO:38) and 8472 (5' CCGGAATTCGTTGGGTTCCCAGTGTAGGTGATGATCCAT 3') (SEQ ID NO:44) to detect the 5 kb fragment. Colonies that were positive for both fragments were subcultured and DNA was extracted using QIAprep Miniprep kit (QiagenTm). Clones were analysed by diagnostic restriction enzymes and PCR for each =
õ

fragment and sequenced using an ABI 373 DNA sequencer with T7 and T3 primers.
This new construct is referred to as 3kb15a-7- 4T is 10 826 bp long (Figure 4). The sequence of is shown in SEQ ID NO:87.
2) Transfection of 3kb15a-7-4T into RAMOS and screening for integration Clone 3kb15a-7-4T (5 pz) is transfected into 3 x 106 RAMOS RA-1 cells using the following protocol ; cells are centrifuged to remove spent media then resuspended at 1 x 106 cells / ml in RPMI containing DEAE Dextran (25 p,g / m1). Resuspended cells (1 Three days after transfection, cells are stained for surface IgM and sorted by flow cytometry into an IgM positive population and IgM negative population. Prior to this Genomic DNA is extracted from IgM negative cells using the Genoprep DNA
isolation kit (Scientifx, Australia) according the manufacturers' instructions. The DNA
is then digested using Xba I enzyme and run on a 0.6 % agarose gel. DNA is transferred onto 32P labeled oligonucleotides 0041 (5' GACGGTATCGATAAGCTTGATATCGAATTCCTGCAGCCCGGG 3') (SEQ ID
NO:88) and 0042 (5' GACCTCCTGCACAAGAACGGTACCGGGCTAGAGCGGCCGCCA 3') (SEQ ID
The following patterns of radioactive probe binding to DNA extracted from cells when the 5kb and 3kb recombination occurs with the chromosomal DNA integrating the middle sequence; the binding of the radioactive 9403 probe shows this DNA
from untransfected cells show no binding with the 9403 radioactive probe, iv) the lanes on the agarose gel with the plasmid 3kb15a-7-4T show radioactive signal with all probes. Obtaining this pattern i-iv) is indicative of 3kb15a-7-4T integration into the host cell genome.
Example 3: Design and construction of a vector for integration into the rearranged VH
allele, VH4-34 in RAMOS RA-1 and mutation of the asFP499 gene The components of the vector for integration are as follows:
i) Construct 5 kb in PCRScript 15a-7 is modified by removing the Nae I ¨ Xho I

fragment which effectively deletes an additional Kpn I site. This new construct is herein referred to as 5kbPCRScript minus Nae I ¨ Xho I and is 7608 bp in length.
ii) The cloned gene for asFP499, which is a fluorescent protein isolated from the sea anemone Anemonia suleata, was obtained from J. Wiedenmann (University of Ulm, Germany) in the plasmid pQE32 (Qiagen, CA. USA). Four sequential PCR reactions were performed to introduce restriction sites Kpn I and Not I at the 5' and 3' ends of the asFP499 gene respectively and two C-terminal flag tags with a Sal I site at the 3' end of the second flag tag. This final product (-770 bp) is subcloned into pPCRscript (Stratagene, Texas, USA) and herein is referred to as targetPCRScriptasFP499-1.
iii) The gene encoding thymidine kinase is amplified by PCR and restriction sites Hind III and Cla I are introduced at the 5' and 3' end of the gene respectively.
This product (1260 bp) is subcloned into pPCRscript (Stratagene, Texas, USA) and herein is referred to as construct TKPCRscript -64.
iv) pMClneo Poly A (3800 bp) was obtained from Stratagene (Texas, USA).
These components are assembled in the following order:
Constructs targetPCRScriptasFP499-1 and 5kbPCRScript minus Nae I ¨ Xho I are digested with Kpn I and Sal I and run on 1.0 and 0.9 % agarose gels respectively. The desired products (-7608 bp and ¨770 bp) are cut out and DNA extracted using , QIAquick gel extraction kit (QiagenTM). The asFP499 gene (-770 bp) is ligated into 5kbPCRScript minus Nae I ¨ Xho I (-7608 bp) to create 5kb-asFP499PCRscript minus Nae I ¨ XhoI (8289 bp).
5 Constructs TKPCRscript -64 and 5kb-asFP499PCRscript minus Nae I ¨ XhoI
are subsequently digested with Cla I and Sal I and run on agarose gels and DNA
is extracted as described above. The asFP499-5kb fragment (-5770 bp) is ligated to the TKPCRScript-64 backbone (¨ 4737 bp) to generate TK-5kb-asFP499 PCRscript (10464 bp).
The TK-5kb-asFP499 cassette (-7558 bp) is digested out of TK-5kb-asFP499 PCRscript with Sal I and Hind III and ligated into pMClneo Poly A, cleaved with Sal I
and Hind III (3837 bp). This new construct is designated KW 1.
Finally, 3kbPCRscript 10-1-3 and KW1 are digested with restriction enzymes Xho I
and Aat II and gel purified as described above. The 3kb fragment is ligated into the KW1 backbone (-10868 bp) yielding the final integration vector designated KW2 (13722 bp) (Figure 5). The sequence of integration vector KW2 is set out in SEQ ID
NO:91.
The asFP499 gene was deleted from vector KW2 to generate vector KW3 (Figure 6).
The sequence of integration vector KW3 is set out in SEQ ID NO:110. It will be appreciated that any target nucleic acid molecule of interest can be inserted into this vector for use in the affinity maturation process of the present invention.
Example 4: Optimal culturing conditions for RAMOS RA-1 cells Optimal growth conditions for RAMOS cells were determined by performing growth =
curve experiments using different supplements to the medium and different seeding concentrations. RAMOS cells were seeded at 1 x 104, 5 x 104, lx 105, and 2 x (cells / ml) and cultured as 25 ml cultures in either RPMI medium with 10 %
heat inactivated FBS and 1mM sodium pyruvate or RPMI medium with 15 % heat inactivated FBS, 1 mM sodium pyruvate and 50 % conditioned medium. Every 24 hours, 1 ml samples of cells were taken and the number of viable cells was determined using the Vi-Cell r Viability Analyser (Beckman Coulter, CA, USA,). The results õ

showed that RAMOS cells had a lag phase of 48 hours regardless of the seeding concentrations. Cultures reach an exponential growth phase at a density of 0.25 ¨ 0.5 x 106 cells / ml RPMI medium with 10 % heat inactivated FBS and 1 mM sodium pyruvate whereas cells growing in RPMI medium with 15 % heat inactivated FBS, mM sodium pyruvate and 50 % conditioned medium did not enter an exponential phase. The optimal seeding rate for RAMOS was 1 x 105 cells / ml.
Mycoplasma infection can affect transfection rates, therefore we tested RAMOS

monthly. Cells were tested using 4', 6-diamidino-2-phenylindole (DAPI) staining in which all DNA is stained specifically with this very bright dye. If mycoplasma is present small bright specks of dye are seen in the cytoplasm. Cells were fixed onto glass slides and viewed under a 100 X objective with a UV filter system. Cells were negative for mycoplasma over a period of 12 months.
Example 5: RAMOS RA-1 cell division rate Transfection rates in RAMOS are affected by the viability of cells in culture.
To identify the optimal time during the growth cycle to transfect RAMOS cells, we first determined the cell division rate. RAMOS cells were stained with cell-permeant fluorescein-based dye carboxyfluorescein diacetate succinimidyl ester (CFSE) and analysed by flow cytometry based on established techniques in Current Protocols in Cytometry 9.11.2.
Briefly, RAMOS cells in exponential log phase growth were washed and re-suspended in phosphate buffered saline at 5x106 cells / ml. A volume of 2 pi of 5 mM
CFSE was added per ml of cells. Cells were incubated for 10 minutes at 37 C after which volumes of ice cold RPMI + 10 % FBS was added. Cells were then incubated on ice for a further 5 minutes and washed three times in culture medium before transferring to flasks and culturing under normal conditions (see example 1). Initially cells were not analysed until 12 hours post staining to allow for the initial fluorescence decay.
Thereafter cells were anlaysed by flow cytometry every 24 hours. The CFSE
fluorescence was plotted against the number of cells anlaysed (Figure 7). Upon cell division, the dye is equally distributed between daughter cells, allowing the resolution of up to eight cycles of cell division by flow cytometry. In the case of using cultured cells that divide in concert, the resulting fluorescent distribution is halved per cell after , = CA 02510184 2011-05-09 each division. Therefore, these results indicate that the cell population was dividing at least once every 24 hours.
Example 6: Optimisation of conditions for transfection of RAMOS RA-1 cells 1) Construction of vector for expression of asFP499 in RAMOS RA-1 Primers were designed to add a Xho I site to the 5' end of the asFP499 gene and two flag tags, two stop codons and a Xba Ito the 3' end of the gene to allow cloning into pME18s. The primers used are 8934 (5' CCGCTCGAGATGTATCCTTCCATCAAGGAAACC 3') (SEQ ID NO:92), 8935 (5' TCTAGATTATTATITATCATCATCATCTTTATAATCTTIATCATCATCATCTT
TATAATCAGCGGCCGC 3' (SEQ ID NO:93); 8936 (5' CTAGTCTAGATTATTATTTATCATCATCATC 3') (SEQ ID NO:94), and 8398 (5' GTTATGTCCTAATTTCGAAGGCACTTGGGAGTA 3') (SEQ ID NO:95). The cloned sequence was confirmed by DNA sequence analysis. The resulting construct, referred to as pME18sasFP499 (3693 bp) (Figure 8) (SEQ ID NO:8) was used for subsequent transfection of RAMOS cells.
ii) Transfection of pME18sasFP499 into RAMOS RA-1 cells A number of different transfection reagents or methods can be used to transfect mammalian cells including Calcium Phosphate coprecipitation, LipofectaminTM
(Invitrogen), FuGENE6T"(Roche, Germany), SuperFecim(Qiagen, CA, USA), Effectene (Qiagen, CA, USA), GenePORTERni (Gene Therapy Systems, CA, USA) and MetafectenTem(Biontex, Germany). Electroporation is another method commonly used to transfect mammalian cells. Electroporation can be carried out using different electroporators such as the Gene Pulse?' (BioRad, CA, USA) or the Electro Square PoratorTm ECM 830 (BTX, Fisher Biotech, Australia) and various voltage and capacitance settings. can also be used. Since RAMOS RA-1 cells are of B-lymphoid origin and it is known that lymphoid cells can be difficult to transfect, we optimised transfection of this cell line.
The efficiency of transfection of RAMOS RA-1 cells was monitored by flow cytometric analysis using an EPICS Elitem(Beckman Coulter, CA, USA). Samples of transfected cells were stained with 1 tig / mL Propidium Iodode (PI). The live cell õ

population (based on forward and side scatter characteristics and PI staining) was gated and the percentage of Fluorescent Protein (FP) positive cells was assessed. FP

expression was also assessed by fluorescence microscopy using an Olympus IX70A

microscope and Olympus U-RFL-T burner.
In order to optimise the transfection process, a number of different transfection parameters were tested including: set voltage, set capacitance, amount of DNA, concentration of DEAE Dextran and analysis time post transfection.
Figures 9 show the results of a representative set of optirnisation experiments. The combination of 550 V and 25 Fd resulted in the highest transfection efficiency. No significant effect on the transfection efficiency was observed using 1 pg or 30 pg of pME18sEGFP. The optimum time to analyse FP expression was between 24 and 36 hours post electroporation. DEAE Dextran varying from 25 pg / ml to 1 mg / nil was tested and concentrations between 25 pg / ml and 100 pg / ml were optimal for transfection but concentrations above 500 pg / ml were found to be toxic to the cells (data not shown).
Using the optimum conditions, transfection rates of between 0.5 % and 3.5 %
were achieved with the average being approximately 1.0 %.
Figure 10 shows the comparison of RAMOS RA-1 cells transfected with pME18sEGFP, pME18sasFP499 or mock transfected. The level of expression of asFP499 in RAMOS RA-1 was assessed 24 hours post transfection and was found to be approximately 10 fold lower than that of EGFP.
Example 7: Quantitation of the number of IgM molecules on the surface of RAMOS

RA-1 cells It is known in the art that the loss of surface IgM may be used to monitor integration into the rearranged VII allele.
We therefore determined the number of natural IgM molecules on the surface of RAMOS cells. RAMOS cells (50 .1) and Quantum Simply Cellular Tm beads (Bangs Laboratories, EN, USA) (50 pl) were separately incubated on ice for 1 hour with a saturating amount (2.5 ug) of a mouse anti-human IgM monoclonal antibody (Southern Biotech, AL, USA) conjugated to Alexa Fluor 488TM (Molecular Probes, OR, USA).

Cells and beads were then analysed by flow cytometry using an EPICS Elite (Beckman-Coutler). Fluorescence intensity (at 488 nm) was plotted against the number of cells or beads analysed (Figures 11 a and 1 lb). Five distinct populations of beads (labeled B, C, D, E, G) which have defined numbers of anti-mouse IgG binding sites (0, 2000, 20 000, 46 000 and 68 000) were observed (Figure 11a). Figure 1 lb (H) shows a normal distribution of fluorescence intensity, ranging from 2 to 100, as expected for a population of cells. The mean fluorescence for each of these populations was plotted against the corresponding number of predetermined binding sites to generate a linear line graph (Figure 11c) from which we extrapolated the average number of IgM molecules on RAMOS cells. The mean fluorescence intensity was calculated as 23.5 which corresponded to 1.25 X 106 molecules for RAMOS RA-1.
Example 8 : Monitoring cell surface IgM loss on RAMOS RA-1 RAMOS RA-1 cell surface IgM decreases with time in culture. We therefore established the rate of natural loss of surface IgM in order to use this characteristic as a marker for integration. To evaluate the percentage of the population that were IgM
positive, RAMOS RA-1 cells were stained for IgM and analysed by flow cytometry.
Briefly, cells were washed and resuspended at lx106 cells / 100 ul. A sheep polyclonal antibody against human IgM OA chain specific) FITC conjugated (Chemicon, CA, USA) (10 ul) was added to cells which were incubated on ice for lhour. Cells were then washed with 2 ml of cold wash buffer (PBS + 2 % FBS, 0.01 % sodium azide) and resuspended in 500 ul this buffer with 5 p1 propidium iodide (1mg / ml) (Sigma-Aldrich, Australia). Cells were anlaysed by flow cytometry using an EPICS
Elite (Beckman Coulter, CA, USA). Cells were gated on the basis of forward and side scatter and negative propidium iodide. In passage one of RAMOS RA-1 98.39 % of the cell population was positive for surface IgM (Figure 12a) by passage 14, 26.55 % of the cell population was positive (Figure 12b). This loss is attributed to either a high mutation rate or a growth advantage of IgM negative mutants. Therefore, RAMOS
RA-1 cells were presorted to remove IgM negative cells and the IgM+ cells quantitated prior to transfection as the IgM loss is used as a measure of integration.

To further investigate IgM loss on RAMOS, we quantitated the number of cell surface IgM molecules using the methodology described above. We observed that the average number of cell surface IgM molecules significantly decreased over time. In passage 4, RAMOS RA 1 cells possessed an average 9.0 x 105 molecules of IgM on their surface, 5 however by passage 16 the number of IgM molecules had decreased to 4.1 x molecules (Figure 12). Together these data indicate that the decrease in cell surface IgM is dependent on passage number.
We have also observed and quantitated differences in the rate of IgM loss between 10 RAMOS strains, 'RAMOS RA-1' supplied by American Tissue Culture Collection and 'RAMOS' supplied by European Collection of Cell Culture (ECAC) (Figure 13).
Together these data suggest that although IgM loss is a marker for integration reported widely in the literature, we have opted to use an antibiotic marker, such as neomycin as a positive selection marker and thymidine kinase as a negative selection marker for 15 integration in our system.
Example 9: Surface display of asFP499 using the anchor domain of CD26 For cell surface display of the asFP499 gene product on RAMOS cells we used the 20 transmembrane domain of CD26 (Tanaka,T et al., 1992, J. Immunol. 149 (2), 481-486) as an anchor.
Transmembrane domain of CD26 Accession No: M74777 (gi 180082) 25 Peptide sequence: MKTPWKVLLGLLGAAALVTIITVPVVLLNK (SEQ ID NO:96) Nucleotide sequence: 10 -100 5'ATGAAGACACCGTGGAAGGTTCTCCTGGGACTGCTGGGTGCTGCTGCGC
TTGTCACCATCATCACCGTGCCCGTGGTTCTGCTGAACAAA 3' (SEQ ID
NO:97) This anchor sequence was added to the 3' end of the asFP499 gene, to provide an N-terminal anchor on the protein (CD26asFP499). This was achieved by sequential overlap extension PCR using primers that partially overlapped the existing DNA
end and also added new sequence. A representation of this sequential overlap PCR
is shown in Figure 14. This method can be used to add sequence to either the 3' or 5' end of a sequence. Primers are listed in Table 6.

Table 6: Primers used for addition of CD26 anchor sequence to asFP499 Primer Sequence 9712 5' CGTGCCCGTGGTTCTGCTGAACAAAATGTATCCTIVCATCAAGGAAACCA3 ' (SEQ ID NO:98) 9713 5' CCGTGCCCGTGGTTCTGCTGAACAAAGTGTATCCTTCCATCAAGGAAACCA3' (SEQ ID NO:99) 9726 5 ' TGCTGCTGCGCTTGTCACCATCATCACCGTGCCCGTGGTTCTGCTGAACAA_A3 ' (SEQ ID NO: 100) 9727 5' GGAAGGTTCTCCTGGGACTGCTGGGTGCTGCTGCGCTTGTCACCATCATCA3 ' (SEQ ID NO:101) 9728 5' CCGGAATTCATGAAGACACCGTGGAAGGTTCTCCTGGGACTGCTGGG3 ' (SEQ ID NO:102) 9963 5 ' GACTAG1T1ATTAl" lATCATCATCATC iTIATAATC1TTATCATCATC3 ' (SEQ ID NO:103) The final PCR product was cloned into pME18s as described previously except that the restriction sites SpeI and EcoRI were used. The cloned sequence was confirmed by DNA sequence analysis. The resulting vector was designated pME18sCD26asFP and is shown in Figure 15. The sequence of pME18sCD26asFP is set out in SEQ ID
NO:104.
Example 10: Transfection and analysis of CD26asFP499 To demonstrate cell surface display of CD26asFP499, the plasmid pME18sasFP499CD26 was transfected into RAMOS RA-1 and control HEK 293T
cells.
RAMOS RA-1 cells were transfected as previously described (Example 6).
Transfections in HEK 293T cells, which were 60 % ¨ 90 % confluent, were carried out using FuGENE6 (Roche, Germany) according to the manufacturer's instructions. 5 pg of DNA was used in each transfection and pME18sasFP499 was used as a positive control.
Efficiency of transfection was assessed by flow cytometry as previously described.
Figure 16 shows the flow cytometry data for transfection of RAMOS RA-1 cells with = CA 02510184 2011-05-09 pME18sCD26asFP499. It can be seen that the efficiency of transfection with this vector in this cell line is very low (0.01 % corresponds to 4 positive cells in this case).
The data for transfection of HEK 293T cells is shown in Figure 17. In this cell line, the transfection efficiency of pME18sCD26asFP499 is equal to that of pME18sasFP499 and the expression of both vectors is improved in this cell line. The mean fluorescence intensity of pME18sCD26asFP499 was lower than that of pME18sasFP499.
HEK 293 T cells transfected with pME18sCD26asFP499 were analysed by confocal microscopy using a NikonTmC1 (Coherent Life Sciences, Australia). The majority .of cells showed a diffuse fluorescence at their periphery indicating expression of asFP499CD26 at the cell membrane, whereas bright fluorescence was observed uniformly throughout control cells transfected with pME18sasFP499 (data not shown).
Example 11: Cloning of the coding region of AICDA (AID) gene into plvfEl 8s Activation Induced Cytidine Deaminase (AID), the protein product of the differentiation specific AICDA gene has been shown to be a B-cell-specific factor required and essential for the processes, of Class Switch Recombination (CSR) and Somatic Hypermutation (SHM) in B-cells. Its ectopic expression in a number of mammalian cell systems including non B-cell systems has shown the ability of AID
protein to induce and/or enhance hypermutation (Martin et. al 2002, Okazaki et. al 2002, Yoshikawa et. al 2002).
Extraction of total mRNA from RAMOS RA-1 Cells were harvested and centrifuged at 1500 rpm and resuspended in PBS. Total cellular mRNA was extracted using a GenoPrepTg mRNA isolation kit (Scientifix, Australia) according to the manufacturer's instructions. Briefly, after removing the supernatant, 700 I of Lysis and Binding solution was added to cells (1x106).
This mixture was combined with 50 1 (250 lig) of GenoPrepTM mRNA magnetic beads and incubated at room temperature for 5 minutes. Beads were magnetically collected and washed with 500111 of washing solution I. Beads were then washed twice with 500 gl of washing solution II. mRNA-bead complexes were resuspended in 20 I
sterile water and then incubated at 65 C for 2 minutes. Beads were magnetically collected and the mRNA-containing supernatant was transferred to a new tube.
=
=

The human AICDA (AID) gene was amplified from RAMOS RA-1 total RNA using the Superscript One-step RT-PCR with platinum Tali kit (Invitrogen, CA. USA).
The reaction included 1 X reaction mix, forward primer 9645 (5' ATG
GACAGCCTCTTGATGAACCGGAGGA 3') (SEQ ID NO:105) 1 OpM, reverse primer 9646 (5' CAAAGTCCCAAAGTACGAAATGCGT 3') (SEQ ID NO:106) 1 OpM, template RNA (-150 ng), RT / Taq mix (1.0 [II) and sterile water in a final volume of 50 p1. Cycling conditions were as follows; one cycle of 55 C for 30 minutes, 94 C for 2 minutes, 35 cycles of 94 C for 30 seconds, 55 C for 30 seconds, 68 C for one minute, and one cycle of 72 C for 7 minutes.
The RT-PCR product (596 bp) was amplified by PCR using Platinum Pfx polymerase (Invitrogen CA., USA) as previously described. This product was then cloned into pPCRScript using the PCR-Script Amp cloning kit (Stratagene, Texas, USA) at the Sif / site. The coding region of AID was then subcloned into the pME18s using Xho I and Xba I restriction sites with primers 9792 (5' CCCTCGAGATGGACAGCCTCTTGATGAACCGGA 3') (SEQ ID NO:108) and 9793 (5' GCTCTAGACAAAGTCCCAAAGTACGAAATGCGT 3') (SEQ ID
NO:109). The PCR reaction and cycling conditions were as described above.
These constructs were verified by sequencing as previously described. The DNA
sequence of the coding region of the AICDA gene is set out in SEQ ID NO:107.
Example 12: Affinity maturation of an antibody fragment The gene targeting vector KW2 is modified such that following integration and target nucleic acid expression, a chimeric protein consisting of an antibody fragment fused to an anchor is produced. This is achieved by using standard molecular biology techniques to clone the CD26 anchor sequence into KW2 and then inserting the sequence for the antibody fragment downstream of the CD26 anchor sequence. The resulting vector is called KW3.
RAMOS RA-1 cells are transfected with the gene targeting vector KW3, using the optimised protocol previously described. The cells are allowed to recover for 48 to 72 hours before G418 at 5 mg / ml and gancyclovir or FIAU are added to the media for 7 to 9 days. This results in the selection of stable transfectants and also allows time for mutation and surface display to occur. The live cell population, which consists of the stable transfectants, is sorted using flow cytometry and allowed to react with the fluorescently labelled binding partner of the displayed antibody fragment. The cells with the highest fluorescence intensity (ie highest binding) are then single cell sorted into 96 well U bottomed plates containing 100 p.1 of 50 % conditioned media and 50 %
fresh growth media. Single cell sorting is achieved using the Auto cloneTmfunction of the EPICS Elite (Beckman Coulter, CA, USA).
It is also possible to select the cells with the highest affinity gene products on the cell surface, by capturing cells with immoblised binding partner, and selecting for the highest affinity gene product by competitive elution.
If necessary, the cells can be cycled through the in vivo strategy (Figure 2).
The single cells can be expanded to between 1x105 and 1x106 to allow further mutation to occur and then reacted with the labelled binding partner and single cell sorted again. This cycle of single cell sorting/expansion and mutation/re-selection can be carried out until cells displaying the molecule with the desired characteristic, in this case increased binding affinity, have been isolated. The DNA from these cells can be extracted and the mutated gene can be amplified by PCR. Alternatively, the RNA can be extracted and the gene amplified by RT-PCR. The amplified gene can then be inserted into an appropriate expression vector for high-level production of the affinity matured antibody fragment.
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

SEQUENCE LISTING
<110> Diatech Pty Ltd <120> In vivo affinity maturation scheme <130> 501974 <150> AU 2002953381 <151> 2002-12-18 <160> 120 <170> PatentIn version 3.1 <210> 1 <211> 8699 <212> DNA
<213> Homo sapiens <220>
<221> misc feature <222> (2697)-(2697) <223> n = unknown <220>
<221> misc feature <222> (4014)-(4014) <223> n = unknown <400> 1 gcttcattgt tccacgcatt acgtctcacc agtttagtca atatggatta aatatgagag 60 tgtggcaatt cgcaaactct atctgaggag gaaaatcgga taaaaaatgt tatgaaaaat 120 aaagcaattt gaagcctctg acttcagcaa cttcaccact aatgaaatga tgtaaccctc 180 attggcctca aatttagttt tcacggggca tctgcagggt tccaaagtga gaccaggtga 240 attcaatgtg catgcacttc ccaagtgtcc acttgtattc tgtttcttta cttctgttta 300 cagaaagtag acacatattc agtcttagta ccagtgtagg gagcgctttc catgagatgg 360 ataccagaaa aaaatggcaa acatgggatc cgttaatata aaaattagcc acgatgtata 420 tatatatatg tgtgtgtgtg tgtgtgtgtg tgtgtacaca cgcgcgcgca tgtgtgagtt 480 gaatagcaga gttggagtgg gtttctatcc acatgtacct gcacctgcag gtattctcag 540 gtgccataat caactgtagg accctaaagg aaataagagt ctcccctcaa cccctgaaga 600 gtgtttgggt tcaccgtgtg tccaatgatt ctgtgcctct tgagctccag gaaagggctc 660 cctggtgatg catgagatct tttcttggag tctctctgca gagttcactg ggtttcctaa 720 aggcaattca ctatttcaaa agatggtgtg aggagcatgt ggtgtcccta aaggagaatt 780 ctgagccagg gcacaaccac tttatactga gctggataca ctggtaggaa tatactctgt 840 cagctcagat agaaacctcc ctgcatggtt ggggctgggc tgcagggggc gctccggata 900 cacccagcac aggctcccgc cccagagcag gtgcacagga ggctggggag aggttcctcc 960 cagggcctgg gacttccttt aaaaatatct aaaataagta tttcacaaag actgctgaag 1020 tttgtataaa tatctattca attgtgagca tttatcaaac tggatgttgt aatgagaacc 1080 acttttacaa tggggatttc aaactctgct ggaggtcagg aagagatcct ttcttataaa 1140 taaatgcaat ttttggataa acacagtcat tccctaaata acgcattcac atattatggt 1200 ctagaaatga tgcaagttga ccctgagaca gtcaaatgtg gtttcaaagt gaggtgctgt 1260 ccttgaggag cttgttctcc agtgggggaa gctctgtcaa cacagagttc agggatgtgt 1320 aggggacaca tggcctctaa caggattacg gcttgaaccc tcagcttcta cagttgtgtc 1380 acccatgtgt ctgtttctca tactgggtca ggaattgggc tattaaatag catccttcat 1440 gaatatgcaa ttaactgagg tgactatagt atctctgtac cctgaaagca tcacccaaca 1500 acaacatccc tccttgggag aatcccctag agcacagctc ctcaccatgg actggacctg 1560 gagcatcctc ttcttggtgg cagcagcaac aggtaagggg ctccccagtc tcggggttga 1620 ggcagaaacc aggccactca agtgaggctt tacccacccc tgtgtcctct ccacaggtac 1680 ctactcccag gtgcagctgg tgcagtctgg ccatgaggtg aagcagcctg gggcctcagt 1740 gaaggtctcc tgcaagtctt ctggttacac cttcaccatc tatggtatga attgggtatg 1800 atagacccct ggacagggct ttgagtggat gtgatggatc atcacctaca ctgggaaccc 1860 aacgtatacc cacggcttca caggatggtt tgtcttctcc atggacacgt ctgtcagcac 1920 ggcgtgtctt cagatcagca gcctaaaggc tgaggacacg gccgagtatt actgtgcgaa 1980 gtacacagtg tggaaaccca catcccgaga gtttcagaaa gcctgaggaa ggaggcagct 2040 gtgctgagct gaggcagtgg tacagcagtt ttctgaactt ccatagtatc tcattttgca 2100 ttgagttccg ctttaatatt agccaagaat atgggataga cgggtgctcc taagagatcc 2160 ttaacttgcc cattttgatg ggttttccca aagacgtgag aagccacttt tttcgcaaag 2220 catcccaaag ccatgccctg ctccagaaac acgtgtatcc atttcctggt ctttgattaa 2280 ctgacaaact ctcatcagcg cacctgggct aatttcacat caggtagaaa tatgtgcttt 2340 aaagcaaggc taacgttgta atagcaattc ctgcttaata accttcagca ttgttgttgt 2400 gtgctccatc aactaattac gttagttcaa ggttctcaat gggagtttct aataaatata 2460 agggatgtat agaagttccc ctaattaaaa caattgtgaa gacaacctca gtgttcaacc 2520 atatttcaac ccttcaccac aaaggaactt tcatctctcc tggaagttgg gttcattttc 2580 aaattagttt ttttatttta atatctcaag attattgtat gtgactattt tagcagaaag 2640 1(59 tgaattatgg gaacttgaac taaccaactg aaaatacatt cagaactaat taaacangat 2700 gccagaatgt gattggctcc aggcatttta aattcaacag gttatgtaac caggctttaa 2760 atttgcacat cttcgtgtta ccttcatgac acagtcaact cccattatgt aagaaatggt 2820 gagtgcattc ccaagggtct tgcacagtta taaaaataga cttgatgagg tgaggagttg 2880 tttaaattcc cctctgaaga agcagcatca acccaacaaa ccactctctt ccctctgtga 2940 ctagagctct gtcacaggcc acatggacct aaatccttga tggagattac aggactacgt 3000 aaattggact gatcgttttt atgctgttaa attaataggt gagtctgcac tccagcctgg 3060 gcaacagaat aatcttgtct gtaaaataca aaagaaagat aaattaatag atactgactt 3120 tgacatttcg gataataata ttttcataaa ccgaatttaa ttatacccac attgttacct 3180 acaccttcac tgaaaagttc ctagttatgt tgagttccat caacactcca catgttcaaa 3240 tctggacatc caagagagtc tagagaataa aaCgcaatga gggcagtgaa acttgcgtat 3300 attcagcacc tcttaactca ggaggactca atacaccctg gaacactctg cttttctgaa 3360 tggctcacaa tgactccagc tcactctcca acctccgcaa acatctggcc tctgtttgcc 3420 ctaagttcac gctctgctct tagtctatgt tctgaagtct ttgtagaggt gaaaatgagc 3480 tgtcagatgg atcttccttc tcactgcaac atggaatttg ctatttcact taatgaccac 3540 tctttccaca atggttgatt tcttttggcc tgttcattac tggtgatttt caagggaatc 3600 tcagttgaat ctttactgtt ttgcattttg tctccatgac aatgttggga agtttttctt 3660 ctagcagcat aacatgatct agtgacctga cacatttgca gcaaacaata cctacaaatt 3720 cagaagctct ttggttttct ttccacgaaa tataattctt gctcttctgt gtatgagcac 3780 atcctagcat ccctgtacac acccaggtag atgtctacac gccgatgaaa tattccctgt 3840 aaataaaaaa agtatctcag tttctctcaa tgttcataat tctcctgagg gtgaggaagg 3900 tacttctggg tctgctcaaa caaatggccc agagaccacc tggtaggtag gtaaggagct 3960 cacctcgctc tggatattga gtctgtctct ttccctctgt cgtctcatag aagnccagcc 4020 cacttgttca gctcctaaga agagagccca ggtttatcca gattatacaa cacaaccagc 4080 ttctgatgac tctcctgtta caacatccat ggagatattt tgtgtattat ataattcacc 4140 aaactaatgt gaaatgccca agttgcaata ctgcacaccc tagggtatgt tcttgcaatt 4200 cagcggagga gaaattcttt cagagacaga tggatctgaa ttggtaaata tgtgggtacg 4260 aattctgggc ttgagtgtca ttgtccagcc atgtttcaca ggtgtgacct gtcagggaag 4320 aaccagagtt ccttgttctc tcagagggta gagctcacag aggtcctctc tggttcccag 4380 gaaaggtaat ttcactaatc ttggtgatga gactatcctc cagtgctgat gtactataga 4440 gttttcatct gaagctgtca ctgctatccc caatgtacat cttttcacac agaaatgttt 4500 agaggtcagg ccatattctc agggttacac attgagaagg atggagatat attctactac 4560 cttctcctga gatctcacac acaatctcaa atttcaaaag gtctcagaag ggcagctctc 4620 aggtactatt taaaaataac ccacttcctg ggacaggtag catccttcta accatgatgg 4680 atgttctgaa ctacagtaca cattgcatgg atccaggttt gtctcaattc actgtgatta 4740 ttacactcag cagctgtttc aatatgtctg aaggggtaaa tgacaattta ggtgacctgg 4800 gtgtatggtt ggtgttatat gaatctttaa atgtagaaca gtattaactg tattccaaaa 4860 tctgtctttg atccatgatc acacttgtct cccagaccag ctccttcagc acatttccta 4920 cctggaagaa gaggactctg ggtttggtga ggggaggcca caggaagaga actgagttct 4980 cagagggcac agccagcata cacctcccag ggtgagccca aaagactggg gcctccctca 5040 tcccttttta cctatccata caaaggcacc acccacatgc aaatcctcac ttaggcaccc 5100 acaggaaatg actacacatt tccttaaatt cagggtccag ctcacatggg aagtgctttc 5160 tgagagtcat ggacctcctg cacaagaaca tgaaacacct gtggttcttc ctcctcctgg 5220 tggcagctcc cagatgtgag tgtctcagga atgcggatat gaagatatga gatgctgcct 5280 ctgatcccag ggctcactgt gggtttttct gttcacaggg gtcctgtccc aggtgcagct 5340 acagcagtgg ggcgcaggac tgttgaagcc ttcggagacc ctgtccctca cctgcggtgt 5400 ttatggtggg tccttcagtg gttactactg gagctggatc cgccagcccc cagggaaggg 5460 gctggagtgg attggggaaa tcaatcatag tggaagcacc aactacaacc cgtccctcaa 5520 , gagtcgagtc accatatcag tagacacgtc caagaagcag ctctccctga agttgagctc 5580 tgtgaacgcc gcggacacgg ctgtgtatta ctgtgcgaga gttattacta gggcgagtcc 5640 tggcacagac gggaggtacg gtatggacgt ctggggccaa gggaccacgg tcaccgtctc 5700 ctcaggtgag aatggccact ctagggcctc tgttctctgc tactgcctgt ggggtttcct 5760 gagcattgca ggttggtcct cggggcatgt tccgagggga cctgggcgga ctggccagga 5820 ggggacgggc actggggtgc cttgaggatc tgggagcctc tgtggatttt ccgatgcctt 5880 tggaaaatgg gactcaggtt gggtgcgtct gatggagtaa ctgagcctgg gggcttgggg 5940 agccacattt ggacgagatg cctgaacaaa ccaggggtct tagtgatggc tgaggaatgt 6000 gtctcaggag cggtgtctgt aggactgcaa gatcgctgca cagcagcgaa tcgtgaaata 6060 ttttctttag aattatgagg tgcgctgtgt gtCaacctgc atcttaaatt ctttattggc 6120 tggaaagaga actgtcggag tgggtgaatc cagccaggag ggacgcgtag ccccggtctt 6180 gatgagagca gggttggggg caggggtagc ccagaaacgg tggctgccgt cctgacaggg 6240 gcttagggag gctccaggac ctcagtgcct tgaagctggt ttccatgaga aaaggattgt 6300 ttatcttagg aggcatgctt actgttaaaa gacaggatat gtttgaagtg gcttctgaga 6360 aaaatggtta agaaaattat gacttaaaaa tgtgagagat tttcaagtat attaattttt 6420 ttaactgtcc aagtatttga aattcttatc atttgattaa cacccatgag tgatatgtgt 6480 ctggaattga ggccaaagca agctcagcta agaaatacta gcacagtgct gtcggccccg 6540 atgcgggact gcgttttgac catcataaat caagtttatt tttttaatta attgagcgaa 6600 gctggaagca gatgatgaat tagagtcaag atggctgcat gggggtctcc ggcacccaca 6660 gcaggtggca ggaagcaggt caccgcgaga gtctatttta ggaagcaaaa aaacacaatt 6720 ggtaaattta tcacttctgg ttgtgaagag gtggttttgc ccaggcccag atctgaaagt 6780 gctctactga gcaaaacaac acctggacaa tttgcgtttc taaaataagg cgaggctgac 6840 cgaaactgaa aaggcttttt ttaactatct gaatttcatt tccaatctta gcttatcaac 6900 tgctagtttg tgcaaacagc atatcaactt ctaaactgca ttcattttta aagtaagatg 6960 tttaagaaat taaacagtct tagggagagt ttatgactgt attcaaaaag ttttttaaat 7020 tagcttgtta tcccttcatg tgataattaa tctcaaatac tttttcgata cctcagagca 7080 ttattttcat aatgactgtg ttcacaatct ttttaggtta actcgttttc tctttgtgat 7140 taaggagaaa cactttgata ttctgataga gtggccttca ttttagtatt tttcaagacc 7200 acttttcaac tactcacttt aggataagtt ttaggaaaaa tgtgcatcat tatcctgaat 7260 tatttcagtt aagcatgtta gttggtggca taagagaaaa ctcaatcaga tagtgctgaa 7320 gacaggactg tggagacacc ttagaaggac agattctgtt ccgaatcacc gatgcggcgt 7380 cagcaggact ggcctagcgg aggctctggg agggtggctg ccaggcccgg cctgggctct 7440 gggtctcccc ggactaccca gagctgggat gcgtggcttc tgctgccggg ccgactggct 7500 gctcaggccc cagcccttgt taatggactt ggaggaatga ttccatgcca aagctttgca 7560 aggctcgcag tgaccaggcg cccgacatgg taagagacag gcagccgccg ctgctgcatt 7620 tgcttctctt aaaactttgt atttgacgtc ttatttccac tagaagggga actggtctta 7680 attgcttgat gaagagcagg agactcattt atgtgagtct tttgagtgac cattgtctgg 7740 gtcactccca tttaactttc cctaaagccc atttgaagga gaggtcgcac gagctgctcc 7800 acaacctctg aatggggatg gcatgggtaa tgatgcttga gaacatacca agtcccactg 7860 gcatcgccct tgtctaagtc attgactgta ggtcatcatc gcacccttga aagtagccca 7920 tgccttccaa agcgatttat ggtaaatggc agaattttaa gtggcaaatt cagataaaat 7980 gcatttcttg gttgtttcca atgatgactg ttatctagag ggaatttaaa ggcaggggtt 8040 tactgcagac tcagaaggga ggggatgctc cgggaaggtg gaggctctga gcatctcaat 8100 accctcctct tggtgcagaa gatatgctgc cacttctaga gcaaggggac ctgctcattt 8160 ttatcacagc acaggctcct aaattcttgg tctcattctc aagatgtttt aatgacttta 8220 aagcagcaaa gaaatattcc acccaggtag tggagggtgg taatgattgg taatgctttg 8280 gaaccaaaac ccaggtggcg ctggggcagg actgcaggga actggggtat caagtagagg 8340 gagacaaaag atggaagcca gcctggctgt gcaggaaccc ggcaatgaga tggctttagc 8400 tgagacaagc aggtctggtg ggctgaccat ttctggccat gacaactcca tccagttttc 8460 agaaatggac tcagatgggc aaaactgacc taagttgacc tagactaaac aaggccgaac 8520 tgggctgagc tgagctgaac tgggctgagt tgaactgggc tgagctgagc tgagctgagc 8580 tgggctaagt tgcaccaggt gagctgagtt gagctgggct tggctgcatt aaggtgggct 8640 gagctgggca gggctgggct gaattgagct gggctgggct gagctaggct gggctgggc 8699 <210> 2 <211> 338 <212> DNA
<213> Home sapiens <220>
<221> misc_feature <222> (54)..(54) <223> n = unknown <220>
<221> misc_feature <222> (86)..(86) <223> n = unknown <400> 2 taagtgaatc ctggtgtgtc tgaactcaag tgattgttac attaagctgc tgtnccaatc 60 tgtttcctca cctgggaaaa gagganccag gacatagtga gttgaggccc caggaagata 120 actgaattct cagagggcac agccagcatc ctcctcccag ggagagtcta aaagactggg 180 gcctccctca tcccttttca cctgtccata cagaggcacc acccacatgc aaatctcact 240 taggcaccca cagaaaacca ccacacattt ccttaaattc agggtcctgc tcacatggga 300 aatactttct gagagtcctg gacctcctgt gcaagaac 338 <210> 3 <211> 339 <212> DNA
<213> Homo sapiens <400> 3 taagtgaatc ctggtgtgtc tgaactcaca tgattgttac attaagctgc tgttgcaatc 60 tgtttcctca cctgggaaaa gaggagccag gacatagtga gttgaggccc caggaagata 120 actgaattct cagagggcac aaccagcatc ctccttgcca gggagagcct aaaagactgg 180 ggcctccctc atcccttttc acctctccat acagaggcac cacccacatg caaatctcac 240 ttaggcaccc aagggaaacc atcacacatt tccttaaatt cagggtcctg ctcacatggg 300 aaatactttc tgagagctct ggacctcctg tgcaagaac 339 <210> 4 <211> 338 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <222> (106)..(106) <223> n = unknown <400> 4 attccaaaat ctgtctttga tccatgatca cacttgtctc ccagaccagc tccttcagca 60 catttcctac ctggaagaag aggactctgg gtttggtgag gggagnccac aggaagagaa 120 ctgagttctc agagggcaca gccagcatac acctcccagg gtgagcccaa aagactgggg 180 cctccctcat ccctttttac ctatccatac aaaggcacca cccacatgca aatcctcact 240 taggcaccca caggaaatga ctacacattt ccttaaattc agggtccagc tcacatggga 300 agtgctttct gagagtcatg gacctcctgc acaagaac 338 <210> 5 <211> 339 <212> DNA
<213> Homo sapiens =
<400> 5 attccaaaat ctgtccctga tccaagatca cactgatctc ccagagcagc atcttcagca 60 catttcccta cctggaagaa gaggactatg ggcttggtaa ggggaggcca caggaagaga 120 actgagttct cagagggcac agccagcttc ctactcccag ggcaagccca aaagactggg 180 gcctccctcc tcccttttca cctgtccata caaagtcacc gcccacatgc aaatcctcac 240 ttaggcacct acaggaaacc agcacacatt tccttaaatt tgggatccag ctcacatggg 300 aaatactttc tgagactcat gggcctcctg cacaagaac 339 <210> 6 <211> 338 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <222> (16)¨(16) <223> n = unknown <400> 6 atcccaaaat ctgtcnttga tccaggatca cactcatctc tcagaccagc tccttcagca 60 catctcttta cctggaagaa gaggactctg ggcttggaga ggggagcccc caagaagaga 120 actgagttct caaagggcac agccagcatt ctcctcccag ggtgagctca aaagactggc 180 gcctctctca tcccttttca ctgctccgta caaacgcacc acccccatgc aaatcctcac 240 ttaggcgccc acaggaagcc accacacatt tccttaaatt caggtccaac tcataaggga 300 aatgctttct gagagtcatg gatctcatgt gcaagaaa 338 <210> 7 <211> 334 <212> DNA
<213> Artificial Sequence <220>
<223> Consensus sequence for human immunoglobulin heavy chain promoter for VH4 alleles <400> 7 atccaaaatc tgtctctgat ccaagatcac acttatctcc agagcagctc ttcagcacat 60 ttccttacct ggaagaagag gactctgggc ttggtgaggg gaggccccag gaagagaact 120 gagttctcag agggcacagc cagcatcctc ctcccagggg agcccaaaag actggggcct 180 ccctcatccc ttttcacctt ccatacaaag gcaccaccca catgcaaatc ctcacttagg 240 cacccacagg aaaccaccac acatttcctt aaattcaggg tccagctcac atgggaaata 300 ctttctgaga gtcatggacc tcctgtgcaa gaac 334 <210> 8 <211> 3693 <212> DNA
<213> Artificial Sequence <220>
<223> Plasmid pME18Sasfp499 <400> 8 aagcttggct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag 60 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc 120 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa 180 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt 240 gacttcttta agcagtctct acctggtggt ttttcttggg aaagagtaag cacctatgaa 1020 gatggaggag tgctttcagc tacccaagaa acaagtttgc agggtgattg catcatctgc 1080 tacaagtcga agaaagaggt aaagcttcca gaacttcact ttcatcattt gcgtatggaa 1320 aagctgaaca taagtgacga ttggaagacc gttgagcagc acgagtctgt ggtggctagc 1380 tactcccaag tgccttcgaa attaggacat aacgcggccg ctgattataa agatgatgat 1440 gataaagatt ataaagatga tgatgataaa gattataaag atgatgatga taaagattat 1500 aaagatgatg atgataaata ataaactagt ctagagaaaa aacctcccac acctccccct 1560 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa 1620 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 1980 tcgacgctca agtcagaggt ggcgaaaccc ga:caggacta taaagatacc aggcgtttcc 2040 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 2100 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag 2160 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 2220 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 2280 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 2340 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg 2400 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 2460 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 2520 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 2580 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 2640 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag 2700 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 2760 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 2820 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa 2880 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 2940 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 3000 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 3060 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 3120 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 3180 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 3240 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 3300 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct 3360 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 3420 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 3480 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 3540 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 3600 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 3660 tccgcgcaca tttccccgaa aagtgccacc tgc 3693 <210> 9 <211> 581 <212> DNA
<213> Homo sapiens , <400> 9 cggccccgat gcgggactgc gttttgacca tcataaatca agtttatttt tttaattaat 60 tgagcgaagc tggaagcaga tgatgaatta gagtcaagat ggctgcatgg gggtctccgg 120 cacccacagc aggtggcagg aagcaggtca ccgcgagagt ctattttagg aagcaaaaaa 180 acacaattgg taaatttatc acttctggtt gtgaagaggt ggttttgccc aggcccagat 240 ctgaaagtgc tctactgagc aaaacaacac ctggacaatt tgcgtttcta aaataaggcg 300 aggctgaccg aaactgaaaa ggcttttttt aactatctga atttcatttc caatcttagc 360 ttatcaactg ctagtttgtg caaacagcat atcaacttct aaactgcatt catttttaaa 420 gtaagatgtt taagaaatta aacagtctta gggagagttt atgactgtat tcaaaaagtt 480 .
ttttaaatta gcttgttatc ccttcatgtg taattaatct caaatacttt ttcgatacct 540 cagagcatta ttttcataat gactgtgttc acaatctttt t 581 <210> 10 <211> 582 <212> DNA
<213> Homo sapiens <400> 10 cggccccgat gcgggactgc gttttgacca tcataaatca agtttatttt tttaattaat 60 tgagcgaagc tggaagcaga tgatgaatta gagtcaagat ggctgcatgg gggtctccgg 120 cacccacagc aggtggcagg aagcaggtca ccgcgagagt ctattttagg aagcaaaaaa 180 acacaattgg taaatttatc acttctggtt gtgaagaggt ggttttgccc aggcccagat 240 ctgaaagtgc tctactgagc aaaacaacac ctggacaatt tgcgtttcta aaataaggcg 300 aggctgaccg aaactgaaaa ggcttttttt aactatctga atttcatttc caatcttagc 360 ttatcaactg ctagtttgtg caaacagcat atcaacttct aaactgcatt catttttaaa 420 gtaagatgtt taagaaatta aacagtctta gggagagttt atgactgtat tcaaaaagtt 480 ttttaaatta gcttgttatc ccttcatgtg ataattaatc tcaaatactt tttcgatacc 540 tcagagcatt attttcataa tgactgtgtt cacaatcttt tt 582 <210> 11 <211> 666 <212> DNA
<213> Ovis aries <400> 11 ctgcgaatac cgagacgggg cctctcaaag ccacccctga tagtctggaa aattgaaact 60 ttaaaaagag agatgtttaa agtattttaa atttttatca tttaattaac aactgcgaat 120 catggctttg gagagttgag taagagtttg gctgaaaagt actaactagg ttccatcggc 180 1/(59 cctcggcccc aattcagggc tgttttgaga ataataaatt cagcttattt ttttaatgta 240 attggtggtg ccgagttagt caagatggcc acgggccaga ctgaccacct gcagcaggtg 300 gcaggaagca tgtccacttg agagtctgtt tttggaagca agaaaaaaca gttggtaaat 360 ttatcgcttc tggtttccaa aaggtggttt gcggctggtt ttgcccagcc ccacagaacc 420 gaaagtgttc cactgagcac aacagcacct ggctaatttg catttctaaa ataaggcgca 480 gatgctgacc gaaactggaa ggttcctctt ctaactattt gagttaactt cagctttagc 540 ttatcaactg ctcacttatc ttcattttca aagtcgatgt ttaagaaagc cacctgtctc 600 gggtgcactg tctcggtgca ttgctgcact ctctgatgag ccgtccttca aggtggttga 660 gctgag 666 <210> 12 <211> 1067 <212> DNA
<213> Mus musculus <400> 12 ctagatactg agttctggtt ctaataactg gctcctgtac tgatggatgg gtcctgacta 60 gtcattgggc cctgatcctc aacattgact tcaaaacctg aactctagcc ccatgcctca 120 ttcacattag gatgatccct acaggggatt cctgcagaag attccagaat ccccacaaca 180 ctgttcacac actgggctgc aactgggaca gtgacccttt tgactcatag gacttgccag 240 gcacagaggc acagaatgga gacaaagcaa gcccaggacc ctggagatgg agcctctggt 300 ggggtctaca gatgtggggt cagcatcgta gggaggtttg cagggcaggt gtggggcagg 360 gcagaggtag tcatgattat agatactatt tttctctcct ctggagcctc ctttgtctat 420 cacctgctgt cctgggatct ctatctgggg tcaacaatgt ttgcagtaca ggtgtggggg 480 tagggcaggg atgctcacat tagcaacttg tttttctctc ttctgaagtc tctgttgtct 540 atcacctgct gaaacattca aagcagctct cagctgaggg cagctgagtc atcctgagcc 600 tgtctcagca caggtgcccc aaaccagagc tactgttctg agaatcacat cacactggac 660 caggccaggt gggcctggga catggatgag gggtgggagc caggggagcc tgccaggggc 720 tgaggaggcc ccaaccccca ctacccaagg ccatccacac ctgtgcctta gtgaggccat 780 gttctgtccc aatgagaaca agtccaatta agattaagta tggtcttccc aggactatcc 840 agagctaagg ggtgtcagcc agggacaacc cagaccagcc tgaggtcagc cagcatcacc 900 caaggccaca cagctattct ggctagagga ctagatagct agctcatcga ggccctggag 960 atgcagaatg gaagagttta tccctgccag acagggctca tcagaaaggc aggtatctca 1020 ctacacatga cctccctgaa tatttcccag agtccagttg gttctag 1067 <210> 13 <211> 221 <212> DNA
<213> Mus musculus <400> 13 agtcaagatg gccgatcaga accagaacac ctgcagcagc tggcaggaag caggtcatgt 60 ggcaaggcta tttggggaag ggaaaataaa accactaggt aaacttgtag ctgtggtttg 120 aagaagtggt tttgaaacac tctgtccagc cccaccaaac cgaaagtcca ggctgagcaa 180 aacaccacct gggtaatttg catttctaaa ataagttgag g 221 <210> 14 <211> 808 <212> DNA
<213> Mus musculus <400> 14 agctcaaacc agcttaggct acacagagaa actatctaaa aaataattac taactactta 60 ataggagatt ggatgttaag atctggtcac taagaggcag aattgagatt cgaaccagta 120 ttttctacct ggtatgtttt aaattgcagt aaggatctaa gtgtagatat ataataataa 180 gattctattg atctctgcaa caacagagag tgttagattt gtttggaaaa aaatattatc 240 agccaacatc ttctaccatt tcagtatagc acagagtacc cacccatatc tccccaccca 300 tcccccatac cagactggtt attgattttc atggtgactg gcctgagaag attaaaaaaa 360 gtaatgctac cttattggga gtgtcccatg gaccaagata gcaactgtca tagctaccgt 420 cacactgctt tgatcaagaa gaccctttga ggaactgaaa acagaacctt aggcacatct 480 gttgctttcg ctcccatcct cctccaacag cctgggtggt gcactccaca ccctttcaag 540 tttccaaagc ctcatacacc tgctccctac cccagcacct ggccaaggct gtatccagca 600 ctgggatgaa aatgataccc cacctccatc ttgtttgata ttactctatc tcaagcccca 660 ggttagtccc cagtcccaat gcttttgcac agtcaaaact caacttggaa taatcagtat 720 ccttgaagag ttctgatatg gtcactgggc ccatatacca tgtaagacat gtggaaaaga 780 tgtttcatgg ggcccagaca cgttctag 808 <210> 15 <211> 60 *
<212> DNA
<213> Mus musculus <400> 15 aagcagccct caggcagagg ataaaagctc acactaactg agaagctcca tcctcttctc 60 <210> 16 <211> 60 <212> DNA
<213> Mus musculus <400> 16 aattaggcca ccctcatcac atgaaaacca gcccagagtg actctagcag tgggatcctg 60 <210> 17 <211> 60 <212> DNA
<213> Mus musculus <400> 17 catgtgcgac tgtgatgatt aatataggga tatccacacc aaacatcata tgagccctat 60 <210> 18 <211> 60 <212> DNA
<213> Mus musculus <400> 18 aacatgagtc tgtgattata aatacagaga tatccatacc aaacaactta tgagcactgt 60 <210> 19 <211> 338 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <222> (16)..(16) <223> n = unknown <400> 19 atcccaaaat ctgtcnttga tccaggatca cactcatctc tcagaccagc tccttcagca 60 catctcttta cctggaagaa gaggactctg ggcttggaga ggggagcccc caagaagaga 120 actgagttct caaagggcac agccagcatt ctcctcccag ggtgagctca aaagactggc 180 gcctctctca tcccttttca ctgctccgta caaacgcacc acccccatgc aaatcctcac 240 ttaggcgccc acaggaagcc accacacatt tccttaaatt caggtccaac tcataaggga 300 aatgctttct gagagtcatg gatctcatgt gcaagaaa 338 <210> 20 <211> 255 <212> DNA
<213> Homo sapiens <400> 20 agatataact atattttcct gaatgatgga attactacca gtctccccca ggacacttca 60 tctgccctga gcccagcctc tcctcagatg tcccacccag agcttgctat atagtggggg 120 acatgcaaat agggccctcc ctctactgat gaaaaccagc ccagccctga ccctgcagct 180 ctgggagagg agcccagcac tagaagtcgg cgqtgtttcc attcggtgat cagcactgaa 240 cacagaggac tcacc 255 <210> 21 <211> 238 <212> DNA
<213> Homo sapiens <400> 21 cagtagaaat gctaataaga attaattgtt tatgaagtgt aatcactctg ggacacagcc 60 cactcagagg catcccttcc agaacccgct atatagtagg agacatgcaa atagggccct 120 ccctctgctg atgaaaacca gcccagccct gaccctgcag ctctgggaga ggagccccag 180 ccctgagatt cccaggtgtt tccattcagt gatcagcact gaacacagag gactcacc 238 <210> 22 <211> 244 <212> DNA
<213> Homo sapiens .
<400> 22 gatgggtagg ggatgcgtgt cctctaacag gattacgtct tgaaccctca gcttctacaa 60 ttgtgtcgtc catgtgtcat gtatttgctc tttctcatcc tgggtcagga attgggctat 120 taaatagcat ccttcatgaa tatgcaaata actgaggtga atatagatat ctgtgtgccc 180 tgagagcatc acccaaaaac cacacccctc cttgggagaa tcccctagat cacagctcct 240 cacc 244 <210> 23 <211> 36 <212> PRT
<213> Homo sapiens <400> 23 Pro Asn Lys Gly Ser Thr Thr Ser Gly Thr Thr Arg Leu Leu Ser Gly His Thr Cys Phe Thr Leu Thr Gly Leu Leu Gly Thr Leu Val Thr Met Gly Leu Leu Thr <210> 24 <211> 111 <212> DNA
<213> Homo sapiens 16)(59 <400> 24 ccaaataaag gaagtggaac cacttcaggt actacccgtc ttctatctgg gcacacgtgt 60 ttcacgttga caggtttgct tgggacgcta gtaaccatgg gcttgctgac t 111 <210> 25 <211> 33 <212> PRT
<213> Sus scrofa <400> 25 Cys Arg Thr Asn Tyr Gly Tyr Ser Ala-Ala Pro Ser Leu His Leu Pro Pro Gly Ser Leu Leu Ala Ser Leu Val Pro Leu Leu Leu Leu Ser Leu Pro <210> 26 <211> 99 <212> DNA
<213> Sus scrofa <400> 26 tgccggacga attacggcta ctcagccgcc cccagcctcc acctcccgcc gggctcgctg 60 ctggcctccc tcgtgcccct cctcctcctc agtcttccg 99 <210> 27 <211> 30 <212> PRT
<213> Rattus rattus <400> 27 Ala Ser Ser Gln Ser Tyr Arg Met Thr Trp Asn Ile Leu Tyr Thr Leu Leu Ile Ser Met Thr Thr Leu Phe Gln Ile Ser Thr Lys Glu <210> 28 <211> 90 <212> DNA
<213> Rattus rattus <400> 28 gcatcgtctc agagctacag gatgacctgg aacatactct atacactgtt aatcagcatg 60 actactttat tccaaatatc taccaaggag 90 <210> 29 <211> 47 <212> PRT
<213> Mus musculus <400> 29 Ser Ser Asn Lys Ser Ile Ser Val Tyr Arg Asp Lys Leu Val Lys Cys Gly Gly Ile Ser Leu Leu Val Gin Asn Thr Ser Trp Met Leu Leu Leu Leu Leu Ser Leu Ser Leu Leu Gin Ala Leu Asp Phe Ile Ser Leu <210> 30 <211> 135 <212> DNA
<213> Mus musculus <400> 30 agctccaata aaagtatcag tgtgtataga gacaagctgg tcaagtgtgg cggcataagc 60 ctgctggttc agaacacatc ctggatgctg ctgctgctgc tttccctctc cctcctccaa 120 gccctggact tcatt 135 <210> 31 <211> 37 <212> PRT
<213> Mus musculus <400> 31 Pro Glu Asp Pro Pro Asp Ser Lys Asn Thr Leu Val Leu Phe Gly Ala Gly Phe Gly Ala Val Ile Thr Val Val Val Ile Val Val Ile Ile Lys Cys Phe Cys Lys His <210> 32 <211> 111 <212> DNA
<213> Mus musculus <400> 32 ccagaagacc ctcctgatag caagaacaca cttgtgctct ttggggcagg attcggcgca 60 gtaataacag tcgtcgtcat cgttgtcatc atcaaatgct tctgtaagca c 111 <210> 33 <211> 25 <212> PRT
<213> Homo sapiens <400> 33 Met Gly Ile Gin Gly Gly Ser Val Leu Phe Gly Leu Leu Leu Val Leu Ala Val Phe Cys His Ser Gly His Ser <210> 34 <211> 75 <212> DNA
<213> Homo sapiens <400> 34 atgggaatcc aaggagggtc tgtcctgttc gggctgctgc tcgtcctggc tgtcttctgc 60 cattcaggtc atagc 75 <210> 35 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 35 ccggaattca atttgagatt gtgtgtgaga tctcaggag 39 <210> 36 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 36 ccggaattca tagacagcgc aggtgaggga cagggtctc 39 <210> 37 <211> 39 <212> DNA
<213> Artificial Sequence =
<220>
<223> Oligonucleotide primer <400> 37 ccggaattcc tgagaactca gttctcttcc tgtggcctc 39 <210> 38 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 38 ccggaattca atttgagatt gtgtgtgaga tctcaggag 39 <210> 39 <211> 39 <212> DNA .
<213> Artificial Sequence , <220>
<223> Oligonucleotide primer <400> 39 cccaagcttt cctgttacaa catccatgga gatattttg 39 <210> 40 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 40 ccggaattct gaattgcaag aacataccct agggtgtgc , 39 <210> 41 <211> 40 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 41 ccggaattct agggcaaaca gaggccagat gtttgaggag 40 <210> 42 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 42 ccggaattca atttaacagc ataaaaacga tcagtccaa 39 <210> 43 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 43 ccggaattcc gtgtttctgg agcagggcat ggctttggg 39 <210> 44 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 44 ccggaattcg ttgggttccc agtgtaggtg atgatccat 39 <210> 45 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 45 ccggaattct cccaggaagt gggttatttt taaatagta 39 <210> 46 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 46 ccggaattca ctatagtcac ctcagttaat tgcatattc 39 <210> 47 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 47 cccaagcttg acttccttta aaaatatcta aaataagta 39 <210> 48 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 48 ccggaattcg gttctcatta caacatccag tttgataaa 39 <210> 49 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 49 ccggaattcc tccaagaaaa gatctcatgc atcaccagg 39 <210> 50 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 50 cccaagctta atttagtttt cacggggcat ctgcagggt 39 <210> 51 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 51 cccaagcttt gcacacccta gggtatgttc ttgcaattc 39 <210> 52 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 52 cccaagcttc ccaaagccat gccctgctcc agaaacacg 39 <210> 53 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 53 .
ccggaattcc cataatatgt gaatgcgtta tttagggaa 39 <210> 54 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 54 cccaagcttc atgttccacg cattacgtc 29 <210> 55 <211> 29 <212> DNA
<213> Artificial Sequence <220>
=
<223> Oligonucleotide primer <400> 55 cccaagctta agagtgtttg ggttcaccg 29 <210> 56 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 56 cccaagcttt taactcagga ggactcaata caccctgga 39 <210> 57 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 57 cccaagctta aacaatacct acaaattcag aagctcttt 39 <210> 58 <211> 38 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 58 cccaagctta agtcttctgg ttacaccttc accattat 38 <210> 59 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 59 cccaagctta ctctcttccc tctgtgacta gagctctgt 39 <210> 60 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 60 cccaagcttt cagcttctac agttgtgtca cccatgtgt 39 <210> 61 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 61 cccaagcttt gcagagttca ctgggtttcc taaaggcaa 39 <210> 62 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 62 cccaagcttt caccacaaag gaactttcat ctptcctgg 39 <210> 63 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 63 cccaagcttt ttcacacaga aatgtttaga ggtcaggcc 39 <210> 64 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 64 cccaagcttt gcacacccta gggtatgttc ttgcaattc 39 <210> 65 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 65 cccaagcttc ccaaagccat gccctgctcc agaaacacg 39 <210> 66 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 66 cccaagctta tgatgtaacc ctcattggcc tca 33 <210> 67 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 67 ccggaattcg agactccaag aaaagatctc atg 33 <210> 68 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 68 cccaagctta tgttcttgca attcagcgga gga 33 <210> 69 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 69 ccggaattct gtgtgagatc tcaggagaag gta 33 <210> 70 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 70 cccaagcttc ggccccgatg cgggactgcg ttttgacca 39 <210> 71 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400>' 71 ccggaattca taacaagcta atttaaaaaa ctttttgaa 39 <210> 72 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 72 cccaagcttg cacagacggg aggtacggta tggacgtct 39 <210> 73 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 73 ccggaattca aaaaaataaa cttgatttat gatggtcaa 39 <210> 74 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 74 ccggaattcc gcggtgacct gcttcctgcc acctgctgt 39 <210> 75 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer .
<400> 75 ccggaattca gttagtgcag ccaagccct 29 <210> 76 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 76 ccggaattca aaaggcaagt ggacttcggt gcttacctg 39 <210> 77 <211> 36 <212> DNA
<213> Artificial Sequence -<220>
<223> Oligonucleotide primer <400> 77 cccaagcttc agctcagctc agttcagttc agccct 36 <210> 78 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 78 cccaagctta tgcgagggtc tggacggctg aggaccccc 39 <210> 79 <211> 39 <212> DNA .
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 79 ccggaattca tgcggcaagg gttgcggacc gctggctgg 39 <210> 80 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 80 ccggaattcg cccagcccag cctagctca 29 <210> 81 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 81 cccaagcttt tatcaactgc tagtttgtg 29 <210> 82 <211> 36 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 82 ccggaattca gggctgaact gaactgagct gagctg 36 <210> 83 <211> 35 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 83 cggctgatat ctgggagcct ctgtggattt tccga 35 <210> 84 <211> 31 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 84 agccggatat cgcccagccc agcctagctc a 31 <210> 85 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 85 gaaagttaaa tgggagtgac ccag 24 <210> 86 <211> 24 <212> DNA
<213> Artificial Sequence <220>
=
<223> Oligonucleotide primer <400> 86 gagtgaccat cgcacccttg acag 24 <210> 87 <211> 10826 <212> DNA
<213> Artificial Sequence <220>
<223> Plasmid 3kb15a-7-4T
<400> 87 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 180 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 240 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 300 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 420 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 480 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 600 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg 660 gccccccctc gaggtcgacg gtatcgataa gcttgatatc tgggagcctc tgtggatttt 720 ccgatgcctt tggaaaatgg gactcaggtt gggtgcgtct gatggagtaa ctgagcctgg 780 gggcttgggg agccacattt ggacgagatg cctgaacaaa ccaggggtct tagtgatggc 840 tgaggaatgt gtctcaggag cggtgtctgt aggactgcaa gatcgctgca cagcagcgaa 900 tcgtgaaata ttttctttag aattatgagg tgpgctgtgt gtcaacctgc atcttaaatt 960 ctttattggc tggaaagaga actgtcggag tgggtgaatc cagccaggag ggacgcgtag 1020 ccccggtctt gatgagagca gggttggggg caggggtagc ccagaaacgg tggctgccgt 1080 cctgacaggg gcttagggag gctccaggac ctcagtgcct tgaagctggt ttccatgaga 1140 aaaggattgt ttatcttagg aggcatgctt actgttaaaa gacaggatat gtttgaagtg 1200 gcttctgaga aaaatggtta agaaaattat gacttaaaaa tgtgagagat tttcaagtat 1260 attaattttt ttaactgtcc aagtatttga aattcttatc atttgattaa cacccatgag 1320 tgatatgtgt ctggaattga ggccaaagca agctcagcta agaaatacta gcacagtgct 1380 gtcggccccg atgcgggact gcgttttgac catcataaat caagtttatt tttttaatta 1440 attgagcgaa gctggaagca gatgatgaat tagagtcaag atggctgcat gggggtctcc 1500 ggcacccaca gcaggtggca ggaagcaggt caccgcgaga gtctatttta ggaagcaaaa 1560 aaacacaatt ggtaaattta tcacttctgg ttgtgaagag gtggttttgc ccaggcccag 1620 =
atctgaaagt gctctactga gcaaaacaac acctggacaa tttgcgtttc taaaataagg 1680 cgaggctgac cgaaactgaa aaggcttttt ttaactatct gaatttcatt tccaatctta 1740 gcttatcaac tgctagtttg tgcaaacagc atatcaactt ctaaactgca ttcattttta 1800 aagtaagatg tttaagaaat taaacagtct tagggagagt ttatgactgt attcaaaaag 1860 ttttttaaat tagcttgtta tcccttcatg tgataattaa tctcaaatac tttttcgata 1920 cctcagagca ttattttcat aatgactgtg ttcacaatct ttttaggtta actcgttttc 1980 tctttgtgat taaggagaaa cactttgata ttctgataga gtggccttca ttttagtatt 2040 tttcaagacc acttttcaac tactcacttt aggataagtt ttaggaaaaa tgtgcatcat 2100 tatcctgaat tatttcagtt aagcatgtta gttggtggca taagagaaaa ctcaatcaga 2160 tagtgctgaa gacaggactg tggagacacc ttagaaggac agattctgtt ccgaatcacc 2220 gatgcggcgt cagcaggact ggcctagcgg aggctctggg agggtggctg ccaggcccgg 2280 cctgggctct gggtctcccc ggactaccca gagctgggat gcgtggcttc tgctgccggg 2340 ccgactggct gctcaggccc cagcccttgt taatggactt ggaggaatga ttccatgcca 2400 aagctttgca aggctcgcag tgaccaggcg cccgacatgg taagagacag gcagccgccg 2460 ctgctgcatt tgcttctctt aaaactttgt atttgacgtc ttatttccac tagaagggga 2520 actggtctta attgcttgat gaagagcagg agactcattt atgtgagtct tttgagtgac 2580 cattgtctgg gtcactccca tttaactttc cctaaagccc atttgaagga gaggtcgcac 2640 gagctgctcc acaacctctg aatggggatg gcatgggtaa tgatgcttga gaacatacca 2700 agtcccactg gcatcgccct tgtctaagtc attgactgta ggtcatcatc gcacccttga 2760 aagtagccca tgccttccaa agcgatttat ggtaaatggc agaattttaa gtggcaaatt 2820 cagataaaat gcatttcttg gttgtttcca atgatgactg ttatctagag ggaatttaaa 2880 ggcaggggtt tactgcagac tcagaaggga ggggatgctc cgggaaggtg gaggctctga 2940 gcatctcaat accctcctct tggtgcagaa gatatgctgc cacttctaga gcaaggggac 3000 ctgctcattt ttatcacagc acaggctcct aaattcttgg tctcattctc aagatgtttt 3060 aatgacttta aagcagcaaa gaaatattcc acccaggtag tggagggtgg taatgattgg 3120 taatgctttg gaaccaaaac ccaggtggcg ctggggcagg actgcaggga actggggtat 3180 caagtagagg gagacaaaag atggaagcca gcctggctgt gcaggaaccc ggcaatgaga 3240 tggctttagc tgagacaagc aggtctggtg ggctgaccat ttctggccat gacaactcca 3300 tccagttttc agaaatggac tcagatgggc aaaactgacc taagttgacc tagactaaac 3360 aaggccgaac tgggctgagc tgagctgaac tgggctgagt tgaactgggc tgagctgagc 3420 tgagctgagc tgggctaagt tgcaccaggt gagctgagtt gagctgggct tggctgcatt 3480 aaggtgggct gagctgggca gggctgggct gaattgagct gggctgggct gagctaggct 3540 gggctgggcg atatcgaatt cctgcagccc gggggatccg cccccatcga taatttagtt 3600 ttcacggggc atctgcaggg ttccaaagtg agaccaggtg aattcaatgt gcatgcactt 3660 cccaagtgtc cacttgtatt ctgtttcttt acttctgttt acagaaagta gacacatatt 3720 cagtcttagt accagtgtag ggagcgcttt ccatgagatg gataccagaa aaaaatggca 3780 aacatgggat ccgttaatat aaaaattagc cacgatgtat atatatatat gtgtgtgtgt 3840 gtgtgtgtgt gtgtgtacac acgcgcgcgc atgtgtgagt tgaatagcag agttggagtg 3900 ggtttctatc cacatgtacc tgcacctgca ggtattctca ggtgccataa tcaactgtag 3960 gaccctaaag gaaataagag tctcccctca acccctgaag agtgtttggg ttcaccgtgt 4020 gtccaatgat tctgtgcctc ttgagctcca ggaaagggct ccctggtgat gcatgagatc 4080 ttttcttgga gtctctctgc agagttcact gggtttccta aaggcaattc actatttcaa 4140 aagatggtgt gaggagcatg tggtgtccct aaaggagaat tctgagccag ggcacaacca 4200 ctttatactg agctggatac actggtagga atatactctg tcagctcaga tagaaacctc 4260 cctgcatggt tggggctggg ctgcaggggg cgctccggat acacccagca caggctcccg 4320 ccccagagca ggtgcacagg aggctgggga gaggttcctc ccagggcctg ggacttcctt 4380 taaaaatatc taaaataagt atttcacaaa gactgctgaa gtttgtataa atatctattc 4440 aattgtgagc atttatcaaa ctggatgttg taatgagaac cacttttaca atggggattt 4500 caaactctgc tggaggtcag gaagagatcc tttcttataa ataaatgcaa tttttggata 4560 aacacagtca ttccctaaat aacgcattca catattatgg tctagaaatg atgcaagttg 4620 accctgagac agtcaaatgt ggtttcaaag tgaggtgctg tccttgagga gcttgttctc 4680 cagtggggga agctctgtca acacagagtt cagggatgtg taggggacac atggcctcta 4740 acaggattac ggcttgaacc ctcagcttct acagttgtgt cacccatgtg tctgtttctc 4800 atactgggtc aggaattggg ctattaaata gcatccttca tgaatatgca attaactgag 4860 gtgactatag tatctccgtt ccctgagagc ctcacccaac aaccacaccc ctcctctgga 4920 gaagccccta gatcacagct cctcaccatg gactggacct gaaggatcct cttcttgatg 4980 gcagcagcaa caggtaaggg gctccccagt ctcagggctg aggaagaaac caggccagtc 5040 atgtgagact tcacccactc ttgtgtccac tccacaggtg cccactccct gcagctggtg 5100 cagtctgggc ctgaggtgaa gaagcctggg gcctcagtga aggtctccta taagtcttct 5160 ggttacacct tcaccatcta tggtatgaat tgggtatgat agacccctgg acagggcttt 5220 gagtggatgt gatggatcat cacctacact gggaacccaa cgtataccca cggcttcaca 5280 ggatggtttg tcttctccat ggacacgtct gtcagcacgg cgtgtcttca gatcagcagc 5340 ctaaaggctg aggacacggc cgagtattac tgtgcgaagt acacagtgtg gaaacccaca 5400 tcccgagagt ttcagaaagc ctgaggaagg aggcagctgt gctgagctga ggcagtggta 5460 cagcagtttt ctgaacttcc atagtatctc attttgcatt gagttccgct ttaatattag 5520 ccaagaatat gggatagacg ggtgctccta agagatcctt aacttgccca ttttgatggg 5580 ttttcccaaa gacgtgagaa gccacttttt tcgcaaagca tcccaaagcc atgccctgct 5640 ccagaaacac gtgtatccat ttcctggtct ttgattaact gacaaactct catcagcgca 5700 cctgggctaa tttcacatca ggtagaaata tgtgctttaa agcaaggcta acgttgtaat 5760 agcaattcct gcttaataac cttcagcatt gttgttgtgt gctccatcaa ctaattacgt 5820 tagttcaagg ttctcaatgg gagtttctaa tap.atataag ggatgtatag aagttcccct 5880 aattaaaaca attgtgaaga caacctcagt gttcaaccat atttcaaccc ttcaccacaa 5940 aggaactttc atctctcctg gaagttgggt tcattttcaa attagttttt ttattttaat 6000 atctcaagat tattgtatgt gactatttta gcagaaagtg aattatggga acttgaacta 6060 accaactgaa aatacattca gaactaatta aacaagatgc cagaatgtga ttggctccag 6120 gcattttaaa ttcaacaggt tatgtaacca ggctttaaat ttgcacatct tcgtgttacc 6180 ttcatgacac agtcaactcc cattatgtaa gaaatggtga gtgcattccc aagggtcttg 6240 cacagttata aaaatagact tgatgaggtg aggagttgtt taaattcccc tctgaagaag 6300 cagcatcaac ccaacaaacc actctcttcc ctctgtgact agagctctgt cacaggccac 6360 atggacctaa atccttgatg gagattacag gactacgtaa attggactga tcgtttttat 6420 gctgttaaat taataggtga gtctgcactc cagcctgggc aacagaataa tcttgtctgt 6480 aaaatacaaa agaaagataa attaatagat actgactttg acatttcgga taataatatt 6540 ttcataaacc gaatttaatt atacccacat tg.ttacctac accttcactg ..aaagttcct 6600 agttatgttg agttccatca acactccaca tgttcaaatc tggacatcca agagagtcta 6660 gagaataaaa cgcaatgagg gcagtgaaac ttgcgtatat tcagcacctc ttaactcagg 6720 aggactcaat acaccctgga acactctgct tttctgaatg gctcacaatg actccagctc 6780 actctccaac ctcctcaaac atctggcctc tgtttgccct aagttcacgc tctgctctta 6840 gtctatgttc tgaagtcttt gtagaggtga aaatgagctg tcagatggat cttccttctc 6900 actgcaacat ggaatttgct atttcactta atgaccactc tttccacaat ggttgatttc 6960 ttttggcctg ttcattactg gtgattttca agggaatctc agttgaatct ttactgtttt 7020 gcattttgtc tccatgacaa tgttgggaag tttttcttct agcagcataa catgatctag 7080 tgacctgaca catttgcagc aaacaatacc tacaaattca gaagctcttt ggttttcttt 7140 ccacgaaata taattcttgc tcttctgtgt atgagcacat cctagcatcc ctgtacacac 7200 ccaggtagat gtaaacacgc cgatgaaata ttccctgtaa ataaaaaaag tatctcagtt 7260 tctctcaatg ttcataattc tcctgagggt gaggaaggta cttctgggtc tgctcaaaca 7320 aatggcccag agaccacctg gtaggtaggt aaggagctca cctcgctctg gatattgagt 7380 ctgtctcttt ccctctgtcg tctcatagaa ggccagccca cttgttcagc tcctaagaag 7440 agagcccagg tttatccaga ttatacaaca caaccagctt ctgatgactc tcctgttaca 7500 acatccatgg agatattttg tgtattatat aattcaccaa actaatgtga aatgcccaag 7560 ttgcaatact gcacacccta gggtatgttc ttgcaattca gcggaggaga aattctttca 7620 gagacagatg gatctgaatt ggtaaatatg tgggtacgaa ttctgggttt gagtgtcatt 7680 gtccagccat gtttcacagg tgtgacctgt cagggaagaa ccagagttcc ttgttctctc 7740 agagggtaga gctcacagag gtcctctctg gttcccagga aaggtaattt cactaatctt 7800 ggtgatgaga ctatcctcca gtgctgatgt actatagagt tttcatctga agctgtcact 7860 gctatcccca atgtacatct tttcacacag aaatgtttag aggtcaggcc atattctcag 7920 ggttacacat tgagaaggat ggagatatat tctactacct tctcctgaga tctcacacac 7980 aatctcaaat ttcaaaaggt ctcagaaggg cagctctcag gtactattta aaaataaccc 8040 acttcctggg acaggtagca tccttctaac catgatggat gttctgaact acagtacaca 8100 ttgcatggat ccaggtttgt ctcaattcac tgtgattatt acactcagca gctgtttcaa 8160 tatgtctgaa ggggtaaatg acaatttagg tgacctgggt gtatggttgg tgttatatga 8220 atctttaaat gtagaacagt attaactgta ttccaaaatc tgtctttgat ccatgatcac 8280 acttgtctcc cagaccagct ccttcagcac atttcctacc tggaagaaga ggactctggg 8340 tttggtgagg ggaggccaca ggaagagaac tgagttctca gagggcacag ccagcataca 8400 cctcccaggg tgagcccaaa agactggggc ctccctcatc cctttttacc tatccataca 8460 aaggcaccac ccacatgcaa atcctcactt aggcacccac aggaaatgac tacacatttc 8520 cttaaattca gggtccagct cacatgggaa gtgctttctg agagtcatgg acctcctgca 8580 caagaacggt accgggctag agcggccgcc accgcggtgg agctccagct tttgttccct 8640 ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa 8700 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg 8760 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca 8820 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg 8880 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 8940 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 9000 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 9060 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 9120 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 9180 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 9240 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 9300 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 9360 ctgcgcctta tccggtaact atcgtcttga gt"ccaacccg gtaagacacg acttatcgcc 9420 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 9480 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 9540 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 9600 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 9660 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 9720 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 9780 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 9840 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 9900 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 9960 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 10020 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 10080 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 10140 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 10200 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 10260 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 10320 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 10380 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 10440 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 10500 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 10560 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 10620 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 10680 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta 10740 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 10800 gcgcacattt ccccgaaaag tgccac 10826 <210> 88 <211> 42 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide Primer <400> 88 gacggtatcg ataagcttga tatcgaattc ctgcagcccg gg 42 <210> 89 <211> 42 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 89 gacctcctgc acaagaacgg taccgggcta gagcggccgc ca 42 <210> 90 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 90 cagtgctgca atgataccgc gagac 25 <210> 91 <211> 13722 <212> DNA
<213> Artificial Sequence <220>
<223> Plasmid KW2 <400> 91 cagtgtggtt ttgcaagagg aagcaaaaag cctctccacc caggcctgga atgtttccac 60 ccaatgtcga gcagtgtggt tttgcaagag gaagcaaaaa gcctctccac ccaggcctgg 120 aatgtttcca cccaatgtcg agcaaacccc gcccagcgtc ttgtcattgg cgaattcgaa 180 cacgcagatg cagtcggggc ggcgcggtcc caggtccact tcgcatatta aggtgacgcg 240 tgtggcctcg aacaccgagc gaccctgcag ccaatatggg atcggccatt gaacaagatg 300 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac 360 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg 420 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc 480 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg 540 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc 600 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc 660 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta 720 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg 780 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg 840 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat 900 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc 960 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta 1020 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag 1080 gggatcggca ataaaaagac agaataaaac gcacgggtgt tgggtcgttt gttcggatcc 1140 gtcgacttta tcatcatcat ctttataatc tttatcatca tcatctttat aatcgttatg 1200 tcctaatttc gaaggcactt gggagtagct agCcaccaca gactcgtgct gctcaacggt 1260 cttccaatcg tcacttatgt tcagcttttc catacgcaaa tgatgaaagt gaagttctgg 1320 aagctttacc tctttcttcg acttgtaagt tgtttccatg aagcaagaaa gatgacctcc 1380 gtcagccagc ataagtgcgg gggtatcgcg aagcagaagt ccaccatctc gtgggatgac 1440 tgtttcagtt gatggctccc atccacaggt cttcttttgc atcactggac cgtttgcggg 1500 aaaattggtg ccaaggactt taactttgca gatgatgcaa tcaccctgca aacttgtttc 1560 ttgggtagct gaaagcactc ctccatcttc ataggtgctt actctttccc aagaaaaacc 1620 accaggtaga gactgcttaa agaagtcagg aatttctttg gggtacttgg cgaagacctt 1680 gatgccatac tgaaaggcgt gtgacagaat gtcaaaagca aatggcagag gacctccttc 1740 agttattgta atattcaggc tttgggtgcc ttcgtatggt tttccctctc cttttccagt 1800 gcacttgaag gcgtggtagt taacactacc ctccatagaa agctgaacgc gcatggtttc 1860 cttgatggaa ggatacatgg taccatgact ctcagaaagc acttcccatg tgagctggac 1920 cctgaattta aggaaatgtg tagtcatttc ctgtgggtgc ctaagtgagg atttgcatgt 1980 gggtggtgcc tttgtatgga taggtaaaaa gggatgaggg aggccccagt cttttgggct 2040 caccctggga ggtgtatgct ggctgtgccc tctgagaact cagttctctt cctgtggcct 2100 cccctcacca aacccagagt cctcttcttc caggtaggaa atgtgctgaa ggagctggtc 2160 tgggagacaa gtgtgatcat ggatcaaaga cagattttgg aatacagtta atactgttct 2220 acatttaaag attcatataa caccaaccat acacccaggt cacctaaatt gtcatttacc 2280 ccttcagaca tattgaaaca gctgctgagt gtaataatca cagtgaattg agacaaacct 2340 ggatccatgc aatgtgtact gtagttcaga acatccatca tggttagaag gatgctacct 2400 gtcccaggaa gtgggttatt tttaaatagt acctgagagc tgcccttctg agaccttttg 2460 aaatttgaga ttgtgtgtga gatctcagga gaaggtagta gaatatatct ccatccttct 2520 caatgtgtaa ccctgagaat atggcctgac ctctaaacat ttctgtgtga aaagatgtac 2580 attggggata gcagtgacag cttcagatga aaactctata gtacatcagc actggaggat 2640 agtctcatca ccaagattag tgaaattacc tttcctggga accagagagg acctctgtga 2700 gctctaccct ctgagagaac aaggaactct ggttcttccc tgacaggtca cacctgtgaa 2760 acatggctgg acaatgacac tcaaacccag aattcgtacc cacatattta ccaattcaga 2820 tccatctgtc tctgaaagaa tttctcctcc gctgaattgc aagaacatac cctagggtgt 2880 gcagtattgc aacttgggca tttcacatta gtttggtgaa ttatataata cacaaaatat 2940 ctccatggat gttgtaacag gagagtcatc agaagctggt tgtgttgtat aatctggata 3000 aacctgggct ctcttcttag gagctgaaca agtgggctgg ccttctatga gacgacagag 3060 ggaaagagac agactcaata tccagagcga ggtgagctcc ttacctacct accaggtggt 3120 ctctgggcca tttgtttgag cagacccaga agtaccttcc tcaccctcag gagaattatg 3180 aacattgaga gaaactgaga tacttttttt atttacaggg aatatttcat cggcgtgttt 3240 acatctacct gggtgtgtac agggatgcta ggatgtgctc atacacagaa gagcaagaat 3300 tatatttcgt ggaaagaaaa ccaaagagct tctgaatttg taggtattgt ttgctgcaaa 3360 tgtgtcaggt cactagatca tgttatgctg ctagaagaaa aacttcccaa cattgtcatg 3420 gagacaaaat gcaaaacagt aaagattcaa ctgagattcc cttgaaaatc accagtaatg 3480 aacaggccaa aagaaatcaa ccattgtgga aagagtggtc attaagtgaa atagcaaatt 3540 ccatgttgca gtgagaagga agatccatct gacagctcat tttcacctct acaaagactt 3600 cagaacatag actaagagca gagcgtgaac ttagggcaaa cagaggccag atgtttgagg 3660 aggttggaga gtgagctgga gtcattgtga gccattcaga aaagcagagt gttccagggt 3720 gtattgagtc ctcctgagtt aagaggtgct gaatatacgc aagtttcact gccctcattg 3780 cgttttattc tctagactct cttggatgtc cagatttgaa catgtggagt gttgatggaa 3840 ctcaacataa ctaggaactt ttcagtgaag gtgtaggtaa caatgtgggt ataattaaat 3900 tcggtttatg aaaatattat tatccgaaat gtcaaagtca gtatctatta atttatcttt 3960 cttttgtatt ttacagacaa gattattctg ttgcccaggc tggagtgcag actcacctat 4020 taatttaaca gcataaaaac gatcagtcca at.ttacgtag tcctgtaatc tccatcaagg 4080 atttaggtcc atgtggcctg tgacagagct ctagtcacag agggaagaga gtggtttgtt 4140 gggttgatgc tgcttcttca gaggggaatt taaacaactc ctcacctcat caagtctatt 4200 tttataactg tgcaagaccc ttgggaatgc actcaccatt tcttacataa tgggagttga 4260 ctgtgtcatg aaggtaacac gaagatgtgc aaatttaaag cctggttaca taacctgttg 4320 aatttaaaat gcctggagcc aatcacattc tggcatcttg tttaattagt tctgaatgta 4380 ttttcagttg gttagttcaa gttcccataa ttcactttct gctaaaatag tcacatacaa 4440 taatcttgag atattaaaat aaaaaaacta atttgaaaat gaacccaact tccaggagag 4500 atgaaagttc ctttgtggtg aagggttgaa atatggttga acactgaggt tgtcttcaca 4560 attgttttaa ttaggggaac ttctatacat cccttatatt tattagaaac tcccattgag 4620 aaccttgaac taacgtaatt agttgatgga gcacacaaca acaatgctga aggttattaa 4680 gcaggaattg ctattacaac gttagccttg ctttaaagca catatttcta cctgatgtga 4740 aattagccca ggtgcgctga tgagagtttg tcagttaatc aaagaccagg aaatggatac 4800 acgtgtttct ggagcagggc atggctttgg gatgctttgc gaaaaaagtg gcttctcacg 4860 tctttgggaa aacccatcaa aatgggcaag ttaaggatct cttaggagca cccgtctatc 4920 ccatattctt ggctaatatt aaagcggaac tcaatgcaaa atgagatact atggaagttc 4980 agaaaactgc tgtaccactg cctcagctca gcacagctgc ctccttcctc aggctttctg 5040 aaactctcgg gatgtgggtt tccacactgt gtacttcgca cagtaatact cggccgtgtc 5100 ctcagccttt aggctgctga tctgaagaca cgccgtgctg acagacgtgt ccatggagaa 5160 gacaaaccat cctgtgaagc cgtgggtata cgttgggttc ccagtgtagg tgatgatcca 5220 tcacatccac tcaaagccct gtccaggggt ctatcatacc caattcatac catagatggt 5280 gaaggtgtaa ccagaagact tataggagac cttcactgag gccccaggct tcttcacctc 5340 aggcccagac tgcaccagct gcagggagtg ggcacctgtg gagtggacac aagagtgggt 5400 gaagtctcac atgactggcc tggtttcttc ctcagccctg agactgggga gccccttacc 5460 tgttgctgct gccatcaaga agaggatcct tcaggtccag tccatggtga ggagctgtga 5520 tctaggggct tctccagagg aggggtgtgg ttgttgggtg aggctctcag ggaacggaga 5580 tactatagtc acctcagtta attgcatatt catgaaggat gctatttaat agcccaattc 5640 ctgacccagt atgagaaaca gacacatggg tgacacaact gtagaagctg agggttcaag 5700 ccgtaatcct gttagaggcc atgtgtcccc tacacatccc tgaactctgt gttgacagag 5760 cttcccccac tggagaacaa gctcctcaag gacagcacct cactttgaaa ccacatttga 5820 ctgtctcagg gtcaacttgc atcatttcta gaccataata tgtgaatgcg ttatttaggg 5880 aatgactgtg tttatccaaa aattgcattt atttataaga aaggatctct tcctgacctc 5940 cagcagagtt tgaaatcccc attgtaaaag tggttctcat tacaacatcc agtttgataa 6000 atgctcacaa ttgaatagat atttatacaa acttcagcag tctttgtgaa atacttattt 6060 tagatatttt taaaggaagt cccaggccct gggaggaacc tctccccagc ctcctgtgca 6120 cctgctctgg ggcgggagcc tgtgctgggt gtatccggag cgccccctgc agcccagccc 6180 caaccatgca gggaggtttc tatctgagct gacagagtat attcctacca gtgtatccag 6240 ctcagtataa agtggttgtg ccctggctca gaattctcct ttagggacac cacatgctcc 6300 tcacaccatc ttttgaaata gtgaattgcc tttaggaaac ccagtgaact ctgcagagag 6360 actccaagaa aagatctcat gcatcaccag ggagcccttt cctggagctc aagaggcaca 6420 gaatcattgg acacacggtg aacccaaaca ctcttcaggg gttgagggga gactcttatt 6480 tcctttaggg tcctacagtt gattatggca cctgagaata cctgcaggtg caggtacatg 6540 tggatagaaa cccactccaa ctctgctatt caactcacac atgcgcgcgc gtgtgtacac 6600 acacacacac acacacacac acatatatat atatacatcg tggctaattt ttatattaac 6660 ggatcccatg tttgccattt ttttctggta tccatctcat ggaaagcgct ccctacactg 6720 gtactaagac tgaatatgtg tctactttct gtaaacagaa gtaaagaaac agaatacaag 6780 tggacacttg ggaagtgcat gcacattgaa ttcacctggt ctcactttgg aaccctgcag 6840 atgccccgtg aaaactaaat tatcgattgc cagccctggg accgaacccc gcgtttatga 6900 acaaacgacc caacacccgt gcgttttatt ctgtcttttt attgccgtca tagcgcgggt 6960 tccttccggt attgtctcct tccgtgtttc agttagcctc ccccatctcc cgggcaaacg 7020 tgcgcgccag gtcgcagatc gtcggtatgg agcctggggt ggtgacgtgg gtctggacca 7080 tcccggaggt aagttgcagc agggcgtccc ggtagccggc gggcgattgg tcgtaatcca 7140 ggataaagac gtgcatggga cggaggcgtt tggccaagac gtccaaggcc caggcaaaca 7200 cgttatacag gtcgccgttg ggggccagca actcgggggc ccgaaacagg gtaaataacg 7260 tgtccccgat atggggtcgt gggcccgcgt tgctctgggg ctcggcaccc tggggcggca 7320 cggccgtccc cgaaagctgt ccccaatcct cccgccacga cccgccgccc tgcagatacc 7380 gcaccgtatt ggcaagcagc ccgtaaacgc ggcgaatcgc ggccagcata gccaggtcaa 7440 gccgctcgcc ggggcgctgg cgtttggcca ggcggtcgat gtgtctgtcc tccggaaggg 7500 cccccaacac gatgtttgtg ccgggcaagg tcggcgggat gagggccacg aacgccagca 7560 cggcctgggg ggtcatgctg cccataaggt atcgcgcggc cgggtagcac aggagggcgg 7620 cgatgggatg gcggtcgaag atgagggtga gggccggggg cggggcatgt gagctcccag 7680 cctccccccc gatatgagga gccagaacgg cgtcggtcac ggcataaggc atgcccattg 7740 ttatctgggc gcttgtcatt accaccgccg cgtccccggc cgatatctca ccctggtcga 7800 ggcggtgttg tgtggtgtag atgttcgcga ttgtctcgga agcccccagc acctgccagt 7860 aagtcatcgg ctcgggtacg tagacgatat cgtcgcgcga acccagggcc accagcagtt 7920 gcgtggtggt ggttttcccc atcccgtgag gaccgtctat ataaacccgc agtagcgtgg 7980 gcattttctg ctccaggcgg acttccgtgg cttcttgctg ccggcgaggg cgcaacgccg 8040 tacgtcggtt gctatggccg cgagaacgcg cagcctggtc gaacgcagac gcgtgttgat 8100 ggcaggggta cgaagccata cgcgcttcta caaggcgctt gccgaagagg tgcgggagtt 8160 tcacgccacc aagatctgcg gcacgctgtt gacgctgtta agcgggtcgc tgcaggtcga 8220 aaggcccgga gatgaggaag aggagaacag cgCggcagac gtgcgctttt gaagcgtgca 8280 gaatgccggg cctccggagg accttcgggc gcccgccccg cccctgagcc cgcccctgag 8340 cccgcccccg gacccacccc ttcccagcct ctgagcccag aaagcgaagg agcaaagctg 8400 ctattggccg ctgccccaaa ggcctacccg cttccattgc tcagcggtgc tgtccatctg 8460 cacgagacta gtgagacgtg ctacttccat ttgtcacgtc ctgcacgacg cgagctgcgg 8520 ggcggggggg aacttcctga ctaggggagg agtagaaggt ggcgcgaagg ggccaccaaa 8580 gaacggagcc ggttggcgcc taccggtgga tgtggaatgt gtgcgaggcc agaggccact 8640 tgtgtagcgc caagtgccca gcggggctgc taaagcgcat gctccagact gcaagcttgg 8700 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca 8760 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg aggtaactca 8820 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 8880 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattggcgct cttccgcttc 8940 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 9000 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 9060 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 9120 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 9180 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 9240 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 9300 ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 9360 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 9420 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 9480 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 9540 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 9600 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 9660 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 9720 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 9780 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 9840 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 9900 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 9960 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 10020 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 10080 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 10140 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 10200 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 10260 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 10320 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 10380 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 10440 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 10500 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 10560 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 10620 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 10680 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 10740 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 10800 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 10860 tgacgtcgcc cagcccagcc tagctcagcc cagcccagct caattcagcc cagccctgcc 10920 cagctcagcc caccttaatg cagccaagcc cagctcaact cagctcacct ggtgcaactt 10980 agcccagctc agctcagctc agctcagccc agttcaactc agcccagttc agctcagctc 11040 agcccagttc ggccttgttt agtctaggtc aaCttaggtc agttttgccc atctgagtcc 11100 atttctgaaa actggatgga gttgtcatgg ccagaaatgg tcagcccacc agacctgctt 11160 gtctcagcta aagccatctc attgccgggt tcctgcacag ccaggctggc ttccatcttt 11220 tgtctccctc tacttgatac cccagttccc tgcagtcctg ccccagcgcc acctgggttt 11280 tggttccaaa gcattaccaa tcattaccac cctccactac ctgggtggaa tatttctttg 11340 ctgctttaaa gtcattaaaa catcttgaga atgagaccaa gaatttagga gcctgtgctg 11400 tgataaaaat gagcaggtcc ccttgctcta gaagtggcag catatcttct gcaccaagag 11460 gagggtattg agatgctcag agcctccacc ttcccggagc atcccctccc ttctgagtct 11520 gcagtaaacc cctgccttta aattccctct agataacagt catcattgga aacaaccaag 11580 aaatgcattt tatctgaatt tgccacttaa aattctgcca tttaccataa atcgctttgg 11640 aaggcatggg ctactttcaa gggtgcgatg atgacctaca gtcaatgact tagacaaggg 11700 cgatgccagt gggacttggt atgttctcaa gcatcattac ccatgccatc cccattcaga 11760 ggttgtggag cagctcgtgc gacctctcct tcaaatgggc tttagggaaa gttaaatggg 11820 agtgacccag acaatggtca ctcaaaagac tcacataaat gagtctcctg ctcttcatca 11880 agcaattaag accagttccc cttctagtgg aaataagacg tcaaatacaa agttttaaga 11940 gaagcaaatg cagcagcggc ggctgcctgt ctcttaccat gtcgggcgcc tggtcactgc 12000 gagccttgca aagctttggc atggaatcat tcctccaagt ccattaacaa gggctggggc 12060 ctgagcagcc agtcggcccg gcagcagaag ccacgcatcc cagctctggg tagtccgggg 12120 agacccagag cccaggccgg gcctggcagc caccctccca gagcctccgc taggccagtc 12180 ctgctgacgc cgcatcggtg attcggaaca gaatctgtcc ttctaaggtg tctccacagt 12240 cctgtcttca gcactatctg attgagtttt ctcttatgcc accaactaac atgcttaact 12300 gaaataattc aggataatga tgcacatttt tcctaaaact tatcctaaag tgagtagttg 12360 aaaagtggtc ttgaaaaata ctaaaatgaa ggccactcta tcagaatatc aaagtgtttc 12420 tccttaatca caaagagaaa acgagttaac ctaaaaagat tgtgaacaca gtcattatga 12480 aaataatgct ctgaggtatc gaaaaagtat ttgagattaa ttatcacatg aagggataac 12540 aagctaattt aaaaaacttt ttgaatacag tcataaactc tccctaagac tgtttaattt 12600 cttaaacatc ttactttaaa aatgaatgca gtttagaagt tgatatgctg tttgcacaaa 12660 ctagcagttg ataagctaag attggaaatg aaattcagat agttaaaaaa agccttttca 12720 gtttcggtca gcctcgcctt attttagaaa cgcaaattgt ccaggtgttg ttttgctcag 12780 tagagcactt tcagatctgg gcctgggcaa aaccacctct tcacaaccag aagtgataaa 12840 tttaccaatt gtgttttttt gcttcctaaa atagactctc gcggtgacct gcttcctgcc 12900 acctgctgtg ggtgccggag acccccatgc agccatcttg actctaattc atcatctgct 12960 tccagcttcg ctcaattaat taaaaaaata aacttgattt atgatggtca aaacgcagtc 13020 ccgcatcggg gccgacagca ctgtgctagt atttcttagc tgagcttgct ttggcctcaa 13080 ttccagacac atatcactca tgggtgttaa tcaaatgata agaatttcaa atacttggac 13140 agttaaaaaa attaatatac ttgaaaatct ctcacatttt taagtcataa ttttcttaac 13200 catttttctc agaagccact tcaaacatat cctgtctttt aacagtaagc atgcctccta 13260 agataaacaa tccttttctc atggaaacca gcttcaaggc actgaggtcc tggagcctcc 13320 ctaagcccct gtcaggacgg cagccaccgt ttctgggcta cccctgcccc caaccctgct 13380 ctcatcaaga ccggggctac gcgtccctcc tggctggatt cacccactcc gacagttctc 13440 tttccagcca ataaagaatt taagatgcag gttgacacac agcgcacctc ataattctaa 13500 agaaaatatt tcacgattcg ctgctgtgca gcgatcttgc agtcctacag acaccgctcc 13560 tgagacacat tcctcagcca tcactaagac ccctggtttg ttcaggcatc tcgtccaaat 13620 gtggctcccc aagcccccag gctcagttac tccatcagac gcacccaacc tgagtcccat 13680 tttccaaagg catcggaaaa tccacagagg ctcccactcg ag 13722 <210> 92 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 92 ccgctcgaga tgtatccttc catcaaggaa acc 33 <210> 93 <211> 69 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 93 tctagattat tatttatcat catcatcttt ataatcttta tcatcatcat ctttataatc 60 agcggccgc 69 <210> 94 <211> 31 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 94 ctagtctaga ttattattta tcatcatcat c 31 <210> 95 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> 'Oligonucleotide primer <400> 95 gttatgtcct aatttcgaag gcacttggga gta 33 <210> 96 <211> 30 <212> PRT
<213> Homo sapiens <400> 96 Met Lys Thr Pro Trp Lys Val Leu Leu Gly Leu Leu Gly Ala Ala Ala Leu Val Thr Ile Ile Thr Val Pro Val Val Leu Leu Asn Lys <210> 97 <211> 90 <212> DNA
<213> Homo sapiens <400> 97 atgaagacac cgtggaaggt tctcctggga ctgctgggtg ctgctgcgct tgtcaccatc 60 atcaccgtgc ccgtggttct gctgaacaaa 90 <210> 98 <211> 50 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 98 cgtgcccgtg gttctgctga acaaaatgta tccttccatc aaggaaacca 50 <210> 99 <211> 51 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 99 ccgtgcccgt ggttctgctg aacaaagtgt atccttccat caaggaaacc a 51 <210> 100 <211> 52 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 100 tgctgctgcg cttgtcacca tcatcaccgt gcccgtggtt ctgctgaaca aa 52 <210> 101 <211> 51 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 101 ggaaggttct cctgggactg ctgggtgctg ctgcgcttgt caccatcatc a 51 <210> 102 <211> 47 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 102 ccggaattca tgaagacacc gtggaaggtt ctcctgggac tgctggg 47 <210> 103 <211> 49 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 103 gactagttta ttatttatca tcatcatctt tataatcttt atcatcatc 49 <210> 104 <211> 3735 <212> DNA
<213> Artificial Sequence <220>
<223> Plasmid pME18sCD26asfp499 <400> 104 aagcttggct gtggaatgtg tgtcagttag gg.tgtggaaa gtccccaggc tccccagcag 60 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc 120 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa 180 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt 240 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cctcgatcga ggggctcgca 300 tctctccttc acgcgcccgc cgccctacct gaggccgcca tccacgccgg ttgagtcgcg 360 ttctgccgcc tcccgcctgt ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa 420 agctcaggtc gagaccgggc ctttgtccgg cgctcccttg gagcctacct agactcagcc 480 ggctctccac gctttgcctg accctgcttg ctcaactcta cgtctttgtt tcgttttctg 540 ttctgcgccg ttacagatcc aagctctgaa aaaccagaaa gttaactggt aagtttagtc 600 tttttgtctt ttatttcagg tcccggatcc ggtggtggtg caaatcaaag aactgctcct 660 cagtggatgt tgcctttact tctaggcctg tacggaagtg ttacttctgc tctaaaagct 720 gcggaattca tgaagacacc gtggaaggtt ctcctgggac tgctgggtgc tgctgcgctt 780 gtcaccatca tcaccgtgcc cgtggttctg ctgaacaaaa tgtatccttc catcaaggaa 840 accatgcgcg ttcagctttc tatggagggt agtgttaact accacgcctt caagtgcact 900 ggaaaaggag agggaaaacc atacgaaggc acccaaagcc tgaatattac aataactgaa 960 ggaggtcctc tgccatttgc ttttgacatt ctgtcacacg cctttcagta tggcatcaag 1020 gtcttcgcca agtaccccaa agaaattcct gacttcttta agcagtctct acctggtggt 1080 ttttcttggg aaagagtaag cacctatgaa gatggaggag tgctttcagc tacccaagaa 1140 acaagtttgc agggtgattg catcatctgc aaagttaaag tccttggcac caattttccc 1200 gcaaacggtc cagtgatgca aaagaagacc tgtggatggg agccatcaac tgaaacagtc 1260 atcccacgag atggtggact tctgcttcgc gatacccccg cacttatgct ggctgacgga 1320 ggtcatcttt cttgcttcat ggaaacaact tacaagtcga agaaagaggt aaagcttcca 1380 gaacttcact ttcatcattt gcgtatggaa aagctgaaca taagtgacga ttggaagacc 1440 gttgagcagc acgagtctgt ggtggctagc tactcccaag tgccttcgaa attaggacat 1500 aacgcggccg ctgattataa agatgatgat gataaagatt ataaagatga tgatgataaa 1560 taataaacta gtctagagaa aaaacctccc acacctcccc ctgaacctga aacataaaat 1620 gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa 1680 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc 1740 caaactcatc aatgtatctt atcatgtctg gatccccggg taccgagctc gaattaattc 1800 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 1860 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 1920 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 1980 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 2040 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 2100 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 2160 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 2220 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 2280 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 2340 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 2400 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt 2460 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 2520 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 2580 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 2640 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 2700 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 2760 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 2820 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 2880 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 2940 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 3000 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 3060 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 3120 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 3180 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 3240 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 3300 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 3360 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 3420 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 3480 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 3540 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 3600 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 3660 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 3720 aaaagtgcca cctgc 3735 <210> 105 <211> 28 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 105 atggacagcc tcttgatgaa ccggagga 28 <210> 106 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 106 caaagtccca aagtacgaaa tgcgt 25 <210> 107 <211> 596 <212> DNA
<213> Homo sapiens <400> 107 atggacagcc tcttgatgaa ccggaggaag tttctttacc aattcaaaaa tgtccgctgg 60 gctaagggtc ggcgtgagac ctacctgtgc tacgtagtga agaggcgtga cagtgctaca 120 tccttttcac tggactttgg ttatcttcgc aataagaacg gctgccacgt ggaattgctc 180 ttcctccgct acatctcgga ctgggaccta ga:ccctggcc gctgctaccg cgtcacctgg 240 ttcacctcct ggagcccctg ctacgactgt gcccgacatg tggccgactt tctgcgaggg 300 aaccccaacc tcagtctgag gatcttcacc gcgcgcctct acttctgtga ggaccgcaag 360 gctgagcccg aggggctgcg gcggctgcac cgcgccgggg tgcaaatagc catcatgacc 420 ttcaaagatt atttttactg ctggaatact tttgtagaaa accatgaaag aactttcaaa 480 gcctgggaag ggctgcatga aaattcagtt cgtctctcca gacagcttcg gcgcatcctt 540 ttgcccctgt atgaggttga tgacttacga gacgcatttc gtactttggg actttg 596 =

<210> 108 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer -<400> 108 ccctcgagat ggacagcctc ttgatgaacc gga 33 <210> 109 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 109 gctctagaca aagtcccaaa gtacgaaatg cgt 33 <210> 110 <211> 12990 <212> DNA
<213> Artificial Sequence <220> .
<223> Plasmid KW3 <400> 110 gtcgacggta ccatgactct cagaaagcac ttcccatgtg agctggaccc tgaatttaag 60 gaaatgtgta gtcatttcct gtgggtgcct aagtgaggat ttgcatgtgg gtggtgcctt 120 tgtatggata ggtaaaaagg gatgagggag gccccagtct tttgggctca ccctgggagg 180 tgtatgctgg ctgtgccctc tgagaactca gttctcttcc tgtggcctcc cctcaccaaa 240 cccagagtcc tcttcttcca ggtaggaaat gtgctgaagg agctggtctg ggagacaagt 300 gtgatcatgg atcaaagaca gattttggaa tacagttaat actgttctac atttaaagat 360 tcatataaca ccaaccatac acccaggtca cctaaattgt catttacccc ttcagacata 420 ttgaaacagc tgctgagtgt aataatcaca gtgaattgag acaaacctgg atccatgcaa 480 tgtgtactgt agttcagaac atccatcatg gttagaagga tgctacctgt cccaggaagt 540 gggttatttt taaatagtac ctgagagctg cccttctgag accttttgaa atttgagatt 600 gtgtgtgaga tctcaggaga aggtagtaga atatatctcc atccttctca atgtgtaacc 660 ctgagaatat ggcctgacct ctaaacattt ctgtgtgaaa agatgtacat tggggatagc 720 agtgacagct tcagatgaaa actctatagt acatcagcac tggaggatag tctcatcacc 780 aagattagtg aaattacctt tcctgggaac cagagaggac ctctgtgagc tctaccctct 840 gagagaacaa ggaactctgg ttcttccctg acaggtcaca cctgtgaaac atggctggac 900 aatgacactc aaacccagaa ttcgtaccca catatttacc aattcagatc catctgtctc 960 tgaaagaatt tctcctccgc tgaattgcaa gaacataccc tagggtgtgc agtattgcaa 1020 cttgggcatt tcacattagt ttggtgaatt atataataca caaaatatct ccatggatgt 1080 tgtaacagga gagtcatcag aagctggttg tgttgtataa tctggataaa cctgggctct 1140 cttcttagga gctgaacaag tgggctggcc ttctatgaga cgacagaggg aaagagacag 1200 actcaatatc cagagcgagg tgagctcctt acctacctac caggtggtct ctgggccatt 1260 tgtttgagca gacccagaag taccttcctc accctcagga gaattatgaa cattgagaga 1320 aactgagata ctttttttat ttacagggaa tatttcatcg gcgtgtttac atctacctgg 1380 gtgtgtacag ggatgctagg atgtgctcat acacagaaga gcaagaatta tatttcgtgg 1440 aaagaaaacc aaagagcttc tgaatttgta ggtattgttt gctgcaaatg tgtcaggtca 1500 ctagatcatg ttatgctgct agaagaaaaa cttcccaaca ttgtcatgga gacaaaatgc 1560 aaaacagtaa agattcaact gagattccct tgaaaatcac cagtaatgaa caggccaaaa 1620 gaaatcaacc attgtggaaa gagtggtcat taagtgaaat agcaaattcc atgttgcagt 1680 gagaaggaag atccatctga cagctcattt tcacctctac aaagacttca gaacatagac 1740 taagagcaga gcgtgaactt agggcaaaca gaggccagat gtttgaggag gttggagagt 1800 gagctggagt cattgtgagc cattcagaaa agcagagtgt tccagggtgt attgagtcct 1860 cctgagttaa gaggtgctga atatacgcaa gtttcactgc cctcattgcg ttttattctc 1920 tagactctct tggatgtcca gatttgaaca tgtggagtgt tgatggaact caacataact 1980 aggaactttt cagtgaaggt gtaggtaaca atgtgggtat aattaaattc ggtttatgaa 2040 aatattatta tccgaaatgt caaagtcagt atctattaat ttatctttct tttgtatttt 2100 acagacaaga ttattctgtt gcccaggctg gagtgcagac tcacctatta atttaacagc 2160 ataaaaacga tcagtccaat ttacgtagtc ctgtaatctc catcaaggat ttaggtccat 2220 gtggcctgtg acagagctct agtcacagag ggaagagagt ggtttgttgg gttgatgctg 2280 cttcttcaga ggggaattta aacaactcct cacctcatca agtctatttt tataactgtg 2340 caagaccctt gggaatgcac tcaccatttc ttacataatg ggagttgact gtgtcatgaa 2400 ggtaacacga agatgtgcaa atttaaagcc tggttacata acctgttgaa tttaaaatgc 2460 ctggagccaa tcacattctg gcatcttgtt taattagttc tgaatgtatt ttcagttggt 2520 tagttcaagt tcccataatt cactttctgc taaaatagtc acatacaata atcttgagat 2580 attaaaataa aaaaactaat ttgaaaatga acccaacttc caggagagat gaaagttcct 2640 ttgtggtgaa gggttgaaat atggttgaac actgaggttg tcttcacaat tgttttaatt 2700 aggggaactt ctatacatcc cttatattta ttagaaactc ccattgagaa ccttgaacta 2760 acgtaattag ttgatggagc acacaacaac aatgctgaag gttattaagc aggaattgct 2820 attacaacgt tagccttgct ttaaagcaca tatttctacc tgatgtgaaa ttagcccagg 2880 tgcgctgatg agagtttgtc agttaatcaa agaccaggaa atggatacac gtgtttctgg 2940 agcagggcat ggctttggga tgctttgcga aaaaagtggc ttctcacgtc tttgggaaaa 3000 cccatcaaaa tgggcaagtt aaggatctct taggagcacc cgtctatccc atattcttgg 3060 ctaatattaa agcggaactc aatgcaaaat gagatactat ggaagttcag aaaactgctg 3120 taccactgcc tcagctcagc acagctgcct ccttcctcag gctttctgaa actctcggga 3180 tgtgggtttc cacactgtgt acttcgcaca gtaatactcg gccgtgtcct cagcctttag 3240 gctgctgatc tgaagacacg ccgtgctgac agacgtgtcc atggagaaga caaaccatcc 3300 tgtgaagccg tgggtatacg ttgggttccc agtgtaggtg atgatccatc acatccactc 3360 aaagccctgt ccaggggtct atcataccca attcatacca tagatggtga aggtgtaacc 3420 agaagactta taggagacct tcactgaggc cccaggcttc ttcacctcag gcccagactg 3480 caccagctgc agggagtggg cacctgtgga gtggacacaa gagtgggtga agtctcacat 3540 gactggcctg gtttcttcct cagccctgag actggggagc cccttacctg ttgctgctgc 3600 catcaagaag aggatccttc aggtccagtc catggtgagg agctgtgatc taggggcttc 3660 tccagaggag gggtgtggtt gttgggtgag gctctcaggg aacggagata ctatagtcac 3720 ctcagttaat tgcatattca tgaaggatgc tatttaatag cccaattcct gacccagtat 3780 gagaaacaga cacatgggtg acacaactgt agaagctgag ggttcaagcc gtaatcctgt 3840 tagaggccat gtgtccccta cacatccctg aactctgtgt tgacagagct tcccccactg 3900 gagaacaagc tcctcaagga cagcacctca ctttgaaacc acatttgact gtctcagggt 3960 caacttgcat catttctaga ccataatatg tgaatgcgtt atttagggaa tgactgtgtt 4020 tatccaaaaa ttgcatttat ttataagaaa ggatctcttc ctgacctcca gcagagtttg 4080 aaatccccat tgtaaaagtg gttctcatta caacatccag tttgataaat gctcacaatt 4140 gaatagatat ttatacaaac ttcagcagtc tttgtgaaat acttatttta gatattttta 4200 aaggaagtcc caggccctgg gaggaacctc tccccagcct cctgtgcacc tgctctgggg 4260 cgggagcctg tgctgggtgt atccggagcg ccccctgcag cccagcccca accatgcagg 4320 51(59 gaggtttcta tctgagctga cagagtatat tcctaccagt gtatccagct cagtataaag 4380 tggttgtgcc ctggctcaga attctccttt agggacacca catgctcctc acaccatctt 4440 ttgaaatagt gaattgcctt taggaaaccc agtgaactct gcagagagac tccaagaaaa 4500 gatctcatgc atcaccaggg agccctttcc tggagctcaa gaggcacaga atcattggac 4560 acacggtgaa cccaaacact cttcaggggt tgaggggaga ctcttatttc ctttagggtc 4620 ctacagttga ttatggcacc tgagaatacc tgcaggtgca ggtacatgtg gatagaaacc 4680 cactccaact ctgctattca actcacacat gcgcgcgcgt gtgtacacac acacacacac 4740 acacacacac atatatatat atacatcgtg gctaattttt atattaacgg atcccatgtt 4800 tgccattttt ttctggtatc catctcatgg aaagcgctcc ctacactggt actaagactg 4860 aatatgtgtc tactttctgt aaacagaagt aaagaaacag aatacaagtg gacacttggg 4920 aagtgcatgc acattgaatt cacctggtct cactttggaa ccctgcagat gccccgtgaa 4980 aactaaatta tcgattgcca gccctgggac cgaaccccgc gtttatgaac aaacgaccca 5040 acacccgtgc gttttattct gtctttttat tgccgtcata gcgcgggttc cttccggtat 5100 tgtctccttc cgtgtttcag ttagcctccc ccatctcccg ggcaaacgtg cgcgccaggt 5160 cgcagatcgt cggtatggag cctggggtgg tgacgtgggt ctggaccatc ccggaggtaa 5220 gttgcagcag ggcgtcccgg tagccggcgg gcgattggtc gtaatccagg ataaagacgt 5280 gcatgggacg gaggcgtttg gccaagacgt ccaaggccca ggcaaacacg ttatacaggt 5340 cgccgttggg ggccagcaac tcgggggccc gaaacagggt aaataacgtg tccccgatat 5400 ggggtcgtgg gcccgcgttg ctctggggct cggcaccctg gggcggcacg gccgtccccg 5460 aaagctgtcc ccaatcctcc cgccacgacc cgccgccctg cagataccgc accgtattgg 5520 caagcagccc gtaaacgcgg cgaatcgcgg ccagcatagc caggtcaagc cgctcgccgg 5580 ggcgctggcg tttggccagg cggtcgatgt gtctgtcctc cggaagggcc cccaacacga 5640 tgtttgtgcc gggcaaggtc ggcgggatga gggccacgaa cgccagcacg gcctgggggg 5700 tcatgctgcc cataaggtat cgcgcggccg ggtagcacag gagggcggcg atgggatggc 5760 ggtcgaagat gagggtgagg gccgggggcg gggcatgtga gctcccagcc tcccccccga 5820 tatgaggagc cagaacggcg tcggtcacgg cataaggcat gcccattgtt atctgggcgc 5880 ttgtcattac caccgccgcg tccccggccg atatctcacc ctggtcgagg cggtgttgtg 5940 tggtgtagat gttcgcgatt gtctcggaag cccccagcac ctgccagtaa gtcatcggct 6000 cgggtacgta gacgatatcg tcgcgcgaac ccagggccac cagcagttgc gtggtggtgg 6060 ttttccccat cccgtgagga ccgtctatat aaacccgcag tagcgtgggc attttctgct 6120 ccaggcggac ttccgtggct tcttgctgcc ggcgagggcg caacgccgta cgtcggttgc 6180 tatggccgcg agaacgcgca gcctggtcga acgcagacgc gtgttgatgg caggggtacg 6240 aagccatacg cgcttctaca aggcgcttgc cgaagaggtg cgggagtttc acgccaccaa 6300 gatctgcggc acgctgttga cgctgttaag cgggtcgctg caggtcgaaa ggcccggaga 6360 tgaggaagag gagaacagcg cggcagacgt gcgcttttga agcgtgcaga atgccgggcc 6420 tccggaggac cttcgggcgc ccgccccgcc cctgagcccg cccctgagcc cgcccccgga 6480 cccacccctt cccagcctct gagcccagaa agcgaaggag caaagctgct attggccgct 6540 gccccaaagg cctacccgct tccattgctc agcggtgctg tccatctgca cgagactagt 6600 gagacgtgct acttccattt gtcacgtcct gcacgacgcg agctgcgggg cgggggggaa 6660 cttcctgact aggggaggag tagaaggtgg cgcgaagggg ccaccaaaga acggagccgg 6720 ttggcgccta ccggtggatg tggaatgtgt gcgaggccag aggccacttg tgtagcgcca 6780 agtgcccagc ggggctgcta aagcgcatgc tccagactgc aagcttggcg taatcatggt 6840 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg 6900 gaagcataaa gtgtaaagcc tggggtgcct aa'tgagtgag gtaactcaca ttaattgcgt 6960 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg 7020 gccaacgcgc ggggagaggc ggtttgcgta ttggcgctct tccgcttcct cgctcactga 7080 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 7140 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 7200 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 7260 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 7320 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 7380 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc 7440 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 7500 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 7560 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 7620 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 7680 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 7740 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 7800 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 7860 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 7920 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 7980 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 8040 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 8100 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 8160 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 8220 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 8280 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 8340 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 8400 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 8460 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 8520 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 8580 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 8640 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 8700 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 8760 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 8820 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 8880 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 8940 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgccca 9000 gcccagccta gctcagccca gcccagctca attcagccca gccctgccca gctcagccca 9060 ccttaatgca gccaagccca gctcaactca gctcacctgg tgcaacttag cccagctcag 9120 ctcagctcag ctcagcccag ttcaactcag cccagttcag ctcagctcag cccagttcgg 9180 ccttgtttag tctaggtcaa cttaggtcag ttttgcccat ctgagtccat ttctgaaaac 9240 tggatggagt tgtcatggcc agaaatggtc agcccaccag acctgcttgt ctcagctaaa 9300 gccatctcat tgccgggttc ctgcacagcc aggctggctt ccatcttttg tctccctcta 9360 cttgataccc cagttccctg cagtcctgcc ccagcgccac ctgggttttg gttccaaagc 9420 attaccaatc attaccaccc tccactacct gggtggaata tttctttgct gctttaaagt 9480 cattaaaaca tcttgagaat gagaccaaga atttaggagc ctgtgctgtg ataaaaatga 9540 gcaggtcccc ttgctctaga agtggcagca tatcttctgc accaagagga gggtattgag 9600 atgctcagag cctccacctt cccggagcat cccctccctt ctgagtctgc agtaaacccc 9660 tgcctttaaa ttccctctag ataacagtca tcattggaaa caaccaagaa atgcatttta 9720 tctgaatttg ccacttaaaa ttctgccatt taccataaat cgctttggaa ggcatgggct 9780 actttcaagg gtgcgatgat gacctacagt caatgactta gacaagggcg atgccagtgg 9840 gacttggtat gttctcaagc atcattaccc atgccatccc cattcagagg ttgtggagca 9900 gctcgtgcga cctctccttc aaatgggctt tagggaaagt taaatgggag tgacccagac 9960 aatggtcact caaaagactc acataaatga gtctcctgct cttcatcaag caattaagac 10020 cagttcccct tctagtggaa ataagacgtc aaatacaaag ttttaagaga agcaaatgca 10080 gcagcggcgg ctgcctgtct cttaccatgt cgggcgcctg gtcactgcga gccttgcaaa 10140 gctttggcat ggaatcattc ctccaagtcc attaacaagg gctggggcct gagcagccag 10200 tcggcccggc agcagaagcc acgcatccca gctctgggta gtccggggag acccagagcc 10260 caggccgggc ctggcagcca ccctcccaga gcctccgcta ggccagtcct gctgacgccg 10320 catcggtgat tcggaacaga atctgtcctt ctaaggtgtc tccacagtcc tgtcttcagc 10380 actatctgat tgagttttct cttatgccac caactaacat gcttaactga aataattcag 10440 gataatgatg cacatttttc ctaaaactta tcctaaagtg agtagttgaa aagtggtctt 10500 gaaaaatact aaaatgaagg ccactctatc agaatatcaa agtgtttctc cttaatcaca 10560 aagagaaaac gagttaacct aaaaagattg tgaacacagt cattatgaaa ataatgctct 10620 gaggtatcga aaaagtattt gagattaatt atcacatgaa gggataacaa gctaatttaa 10680 aaaacttttt gaatacagtc ataaactctc cctaagactg tttaatttct taaacatctt 10740 actttaaaaa tgaatgcagt ttagaagttg atatgctgtt tgcacaaact agcagttgat 10800 aagctaagat tggaaatgaa attcagatag ttaaaaaaag ccttttcagt ttcggtcagc 10860 ctcgccttat tttagaaacg caaattgtcc aggtgttgtt ttgctcagta gagcactttc 10920 agatctgggc ctgggcaaaa ccacctcttc acaaccagaa gtgataaatt taccaattgt 10980 gtttttttgc ttcctaaaat agactctcgc ggtgacctgc ttcctgccac ctgctgtggg 11040 tgccggagac ccccatgcag ccatcttgac tctaattcat catctgcttc cagcttcgct 11100 caattaatta aaaaaataaa cttgatttat gatggtcaaa acgcagtccc gcatcggggc 11160 cgacagcact gtgctagtat ttcttagctg agcttgcttt ggcctcaatt ccagacacat 11220 atcactcatg ggtgttaatc aaatgataag aatttcaaat acttggacag ttaaaaaaat 11280 taatatactt gaaaatctct cacattttta agtcataatt ttcttaacca tttttctcag 11340 aagccacttc aaacatatcc tgtcttttaa cagtaagcat gcctcctaag ataaacaatc 11400 cttttctcat ggaaaccagc ttcaaggcac tgaggtcctg gagcctccct aagcccctgt 11460 caggacggca gccaccgttt ctgggctacc cctgccccca accctgctct catcaagacc 11520 ggggctacgc gtccctcctg gctggattca cccactccga cagttctctt tccagccaat 11580 aaagaattta agatgcaggt tgacacacag cgcacctcat aattctaaag aaaatatttc 11640 acgattcgct gctgtgcagc gatcttgcag tcctacagac accgctcctg agacacattc 11700 ctcagccatc actaagaccc ctggtttgtt caggcatctc gtccaaatgt ggctccccaa 11760 gcccccaggc tcagttactc catcagacgc acccaacctg agtcccattt tccaaaggca 11820 tcggaaaatc cacagaggct cccactcgag cagtgtggtt ttgcaagagg aagcaaaaag 11880 cctctccacc caggcctgga atgtttccac ccaatgtcga gcagtgtggt tttgcaagag 11940 gaagcaaaaa gcctctccac ccaggcctgg aatgtttcca cccaatgtcg agcaaacccc 12000 gcccagcgtc ttgtcattgg cgaattcgaa cacgcagatg cagtcggggc ggcgcggtcc 12060 caggtccact tcgcatatta aggtgacgcg tgtggcctcg aacaccgagc gaccctgcag 12120 ccaatatggg atcggccatt gaacaagatg gattgcacgc aggttctccg gccgcttggg 12180 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg 12240 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg 12300 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc 12360 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg 12420 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca 12480 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc 12540 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg 12600 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg 12660 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata 12720 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg 12780 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat 12840 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct 12900 tctatcgcct tcttgacgag ttcttctgag gggatcggca ataaaaagac agaataaaac 12960 gcacgggtgt tgggtcgttt gttcggatcc 12990 <210> 111 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 111 ccgctcgagt gggagcctct gtggattttc cga 33 <210> 112 <211> 32 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 112 tgaccggacg tcgcccagcc cagcctagct ca 32 <210> 113 <211> 48 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 113 cccaagctta tggactggac ctggaggatc ctcttcttgg tggcagca 48 <210> 114 <211> 51 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 114 cccaagctta tggacacact ttgctccacg ctcctgctgc tgaccatccc t 51 <210> 115 <211> 48 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 115 cccaagctta tggagtttgg gctgagctgg gttttccttg ttgctatt 48 <210> 116 <211> 48 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 116 cccaagctta tgaaacacct gtggttcttc ctcctgctgg tggcagct 48 <210> 117 <211> 51 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 117 cccaagctta tggggtcaac cgccatcctc gccctcctcc tggctgttct c 51 <210> 118 <211> 51 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 118 cccaagctta tgtctgtctc cttcctcatc ttcctgcccg tgctgggcct c 51 <210> 119 <211> 51 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 119 cccaagctta tggactggac ctggaggatc ctcttcttgg tggcagcagc a 51 <210> 120 <211> 39 <212> DNA
<213> Artificial Sequence <220>
<223> Oligonucleotide primer <400> 120 cccaagcttc cccaagcttc ccaggtgcag ctacagcag 39

Claims (32)

CLAIMS:

1. A method for producing and selecting a mutant gene product with desired characteristics, the method comprising (i) introducing into a hypermutating cell a target nucleic acid molecule encoding the gene product such that a single copy of the target nucleic acid molecule is integrated into an immunoglobulin locus of the genome of the hypermutating cell, wherein the target nucleic acid molecule is introduced into the cell by way of an integration vector comprising a sequence homologous to a region upstream of a rearranged V allele and a sequence homologous to a region downstream of a rearranged V allele;
(ii) culturing the hypermutating cell such that the target nucleic acid molecule undergoes hypermutation during DNA and/or RNA synthesis, giving rise to a population of cells expressing mutant gene products; and (iii) selecting the mutant gene product with desired characteristics.

2. A method as claimed in claim 1, wherein the immunoglobulin locus contains a rearranged V gene.

3. A method as claimed in claim 1, wherein the immunoglobulin locus contains a rearranged VH gene.

4. A method as claimed in claim 1, wherein the immunoglobulin locus contains the rearranged VH4-34 allele.

5. A method as claimed in any one of claims 1 to 4, wherein following integration of the target nucleic acid molecule into the immunoglobulin locus, the target nucleic acid molecule is operatively linked to a promoter.

6. A method as claimed in claim 5, wherein the promoter is an immunoglobulin heavy or light chain promoter.

7. A method as claimed in claim 5 or claim 6, wherein the promoter is endogenous to the hypermutating cell.

8. A method as claimed in claim 5 or claim 6, wherein the promoter is exogenous to the hypermutating cell.

9. A method as claimed in any one of claims 5 to 8, wherein following integration the initiation codon of the target nucleic acid molecule is located within 2 kb of the 3' end of the promoter.

10. A method as claimed in any one of claims 5 to 8, wherein following integration the initiation codon of the target nucleic acid molecule is located within 500 bp of the 3' end of the promoter.

11. A method as claimed in any one of claims 5 to 8, wherein following integration the target nucleic acid molecule is located downstream of the promoter and upstream of an intronic enhancer with or without matrix attachment regions and/or 3' enhancer.

12. A method as claimed in any one of claims 1-11, wherein the integration vector comprises a sequence homologous to a region of at least 500 bp upstream of a rearranged V
allele and a sequence homologous to a region of at least 500 bp downstream of the rearranged V gene.

13. A method as claimed in any one of claims 1 to 12, wherein steps (ii) and (iii) are repeated.

14. A method as claimed in any one of claims 1 to 13, wherein the method comprises a further step to increase the rate of mutation of the target nucleic acid molecule.

15. A method as claimed in claim 14, wherein the further step is to increase the levels of expression of activation-induced cytidine deaminase (AID) within the hypermutating cell.

16. A method as claimed in any one of claims 1 to 13, wherein the mutant gene product is selected by way of a protein-fragment complementation assay (PCA).

17. A method as claimed in any one of claims 1 to 16, wherein the target nucleic acid molecule is linked to a sequence encoding an anchor domain such that following expression, the mutant gene product is displayed on the surface of the hypermutating cell.

18. A method as claimed in claim 17, wherein the mutant gene product is selected by detecting binding of a binding partner to the mutant gene product.

19. A method as claimed in claim 18, wherein the hypermutating cells are labelled with a detectable marker and the binding partner is immobilized.

20. The method as claimed in claim 19, wherein the detectable marker is a fluorescent dye.

21. A method as claimed in claim 18, wherein the binding partner is labelled with a fluorescent tag.

22. A method as claimed in claim 21, wherein hypermutating cells displaying the mutant gene product bound to the binding partner are sorted using a flow cytometric technique.

23. A method as claimed in any one of claims 18-22, wherein the binding partner is selected from the group consisting of an antibody, receptor, transcription factor hormone, enzyme, cell surface molecule, DNA molecule and RNA molecule.

24. A method as claimed in any one of claims 1-23, which further comprises the step of recovering the target nucleic acid molecule encoding the selected mutant gene product.

25. A method as claimed in claim 24, wherein the step of recovering the target nucleic acid molecule comprises amplification of the target nucleic acid molecule by PCR or RT-PCR.

26. A method as claimed in any one of claims 1-25, wherein the hypermutating cell is a mammalian, yeast, insect or bacterial cell.

27. A method as claimed in claim 26, wherein the hypermutating cell is a mammalian cell.

28. A method as claimed in claim 27, wherein the mammalian hypermutating cell is selected from the group consisting of RAMOS, BL2, BL41, BL70 and Nalm.

29. A vector for targeted integration into an immunoglobulin locus of a hypermutating cell, the vector comprising a sequence homologous to a region upstream of a rearranged VH gene of the hypermutating cell, a sequence homologous to a region downstream of a rearranged VH gene of the hypermutating cell and a site for integration of a target nucleic acid molecule, wherein the region upstream of the rearranged VH
gene of the hypermutating cell comprises nucleotides 191 to 5190 of SEQ ID NO: 1, and wherein the region downstream of the rearranged VH gene of the hypermutating cell comprises at least 500 contiguous nucleotides of the sequence between nucleotides 5709 and 8699 of SEQ ID
NO: 1.

30. A vector as claimed in claim 29, wherein the vector further comprises a selectable marker.

31. A vector for targeted integration comprising a sequence as set out in nucleotides 1 to 12990 of SEQ ID NO: 110.

32. A vector as claimed in any one of claims 29-31, wherein the vector further comprises a sequence encoding a signal and/or anchor domain suitable for display of a gene product encoded by the target nucleic acid molecule.