EP0587676A1 - A mouse immunoglobulin heavy-chain locus enhancer which crosshybridizes with the 3'-enhancer element of rat - Google Patents

Abstract

4kb / Xba I fragment of DNA which is a 3 'activator of IgH, said fragment having been obtained by cloning a partial Sau 3AI condensate of BALB / c DNA from mouse liver in the lambda 2001 vector and hybridizing crossed with the activating element 3 'of the rat. The present invention further relates to a new DNA sequence having activation properties of specific lymphoid transcription as well as to an expression vector of the eukaryotic immunoglobulin which contains said activator 3 ′ of IgH.

Description

A mouse immurioglobulin heavy-chain locus enhancer which crosshybridizes with the 3' -enhancer element of rat.

The present invention concerns a novel DNA 4kb/Xba I fragment and novel DNA seguence with lymphoid-specific transcription enhancing properties. The present invention further concerns an eukaryotic immunoglobulin expression vector comprising the novel enhancer.

The expression of immunoglobulin gene is controlled by lympho- id-specific enhancer elements acting in concert with lymphoid- specific promoters. In the case of the cappa (χ) locus, a moderately weak enhancer was initially identified in the J_χ-C_χ intron [1, 2]; subseguently, a second, stronger enhancer was discovered 3' of the C_x exon [3]. It transpires that it is the 3'-enhancer which plays a critical role in ensuring the high level expression of rearranged χ genes [4, 5]. The intron- enhancer appears to be involved in the regulation of V_χ-J_χ joining.

In the case of the H chain locus, there is also a lymphoid- specific intron-enhancer and lymphoid-specific V_H gene pro¬ moters, although the IgH intron-enhancer appears considerably stronger than the χ intron-enhancer. It has been demonstrated that rearranged mouse IgH genes which have deleted their IgH intron-enhancer can nevertheless retain full transcriptional activity [6-9]. It is also known that many IgH transgenes are not expressed at the same high level as the endogenous IgH gene ([10] and references therein). Further the c-myc proto-oncogene can become activated on translocation into the IgH locus without necessarily becoming linked to the IgH intro-enhancer

[11]. It was in the rat that the IgH 3'-enhancer originally was identified [12]. However, unlike the situation in the mouse χ locus, it has not yet been ascertained whether the IgH 3'- enhancer does in fact play a role in IgH gene transcription, or it could be involved in ^■'positioning" effect as described for the human β-globin locus [25].

The mouse IgH3'-enhancer has now been isolated the and it is

BSTITUTESHEET homologous to that of the rat both as regards sequence and organization although the enhancer is present in the two species in opposite orientations. We further show that its inclusion stimulates expression of rearranged μ genes that have been stably transfected into a plasmacytoma host.

According to one aspect of the present invention there is provided a recombinant DNA molecule comprising the novel enhancer described above, having at least part or parts of the sequence given in Figure 3.

The molecule may additionally comprise other elements, and will generally further comprise a promoter and a gene or genes coding for a protein or proteins of interest.

The novel enhancer may be used to enhance expression of genes in host cells, in vivo and in vitro, particularly in certain lymphoid cell lines and in transgenic animals. The novel enhancer may further be used for the production of monoclonal antibodies.

Because the novel.enhancer is B-cell specific it may be used to target tissue specific expression of genes. This property means the enhancer potentially finds particular use in certain therapeutic applications, for targeting production of proteins, and in hybridoma technology.

The sequence of the novel enhancer may be derived by recom¬ binant DNA techniques from a naturally occurring gene system or may correspond to a naturally occurring gene system in the sense of being manufactured using known techniques of poly- nucleotide synthesis from sequence data relating to a naturally occurring gene system. Alterations of the sequence may be made which do not alter the function of the enhancer.

The invention also provides a vector for the integration of a gene into the genetic material of a hosT cell such that the gene may be expressed by the hosT cell, the vector comprising

SUBSTITUTE SHEET the gene, a promoter and the novel enhancer.

A phage library generated by cloning a partial Sau 3AI digest of BALB/c mouse liver DNA into the vector λ2001. A 4 kb/Xba I fragment which cross-hybridizes with the rat 3'-enhancer[12]- element, was inserted into the plasmid pIC20H for further investigation (figure 4 A) . Filters of a cosmid library were generated by cloning sheared BALB/c mouse liver DNA into the Bam HI site of cosmid pWE15. DNA of cosmid Cos37-l-l which includes rat C_a and the rat IgH 3'-enhancer [13]. DNA sequen- cing was carried out by the chain termination method using Sequenase^* (USB, Cleveland, OH) .

The cell line PC 11 3U4 (obtained from B. Wasylyk, LGME, Strasbourg), NSO [14], HeLa (from our laboratory collection), HOPC-1 [15], A20 and M12 cells [16] were cultured in DMEM/10% FCS containing 50 μM 2-ME.

Transient transfection of MPCll BU4 plasmacytoma or HeLa cells was performed by calcium phosphate coprecipitation [17] using 20 μg of test plasmid and 5 μg of internal reference. For HOPC- 1, A20 and M12, DEAE-dextran was used to mediate transfection followed by a shopk with 10% DMSO as previously described [18]. The test plasmids were constructed from pUC12 derivatives that contained the human β-globin gene from 128 nucleotides upstream of the transcription start to the Pst I site downstream of the coding sequence cloned between the Xba I and Pst I sites of the polylinker. Enhancer fragments to be assayed were introduced between the Sac I and Xba I sites upstream of the gene. The internal reference was provided by plasmid πSVHPα2 [19] which contains the human α₂~gl°-^D;Ln gene. Total cytoplasmic RNA was prepared from cells 30-36 h after transfection and assayed by ribonuclease protection assays as previously described [3].

Transfectants of NSO that carry a long or short transfected χ gene in a pSV2neo-based vector (Lχ or Sχ; constructs 1 and 4) have been described previously [5]; these cells were super- transfected with pSV2gpt-based plasmids that contained a rearranged mouse μ gene (pSV-Vμl; [20]) or a derivative of pSV- Vμl which includes the Stu I fragment of the mouse IgH 3'- en ancer cloned into the plasmid's unique Xho I site.

In figure 1 (A) shows alignment of the 5'-repeat with the invert of the 3'-repeat of the rat enhancer. Nucleotide posi¬ tions are numbered as in Fig. 3 and positions of identity are indicated by an asterisk. (B) shows invert repeats in the rat IgH 3'-enhancer. Sequences of at least 12 bases of invert identity are joined by a line. The enhancer core lies within the Stu I-Eco RV fragment (indicated by a line) whose sequence was determined previously [5]. (C) shows invert repeats in the mouse IgH 3'-enhancer. (D) shows southern blot of Ba un¬ digested genomic mouse DNA hybridized with a probe that in- eludes the repeat flanking the mouse IgH 3'-enhancer (the Eco RI-Apa LI fragment depicted as probe A in Fig. 4; nucleotides 55-748 in the mouse IgH 3'-enhancer sequence). DNA was either from mouse liver or from the MOPC315 or J558L plasmacytomas. The filter was washed at 65°C in 2 x SSC.

Figure 2 shows the map of the mouse IgH locus and of regions present in cosmid and phage clones.

Figure 3 shows the sequences of mouse (A) and rat (B) IgH 3'- enhancers of the invention.

In Figure 4. is shown that the mouse and rat IgH 3'-enhancers are in opposite orientations. (A) is the restriction map of the mouse C_β/IgH 3'-enhancer region is used to show the derivation of the probes used for probing an Eco RI (R) digest of BALB/c mouse liver DNA. Probes A, B, C and F are Eco RI-Apa LI, Stu I- Xba I, Xba I-Pst I and Sac I subfragments of phage λM2 whereas probes D and E were obtained as Eco RI-Bgl I (nucleotides 565- 740) and Ace I-Eco RI (nucleotides 431-565) subfragments of the rat IgH 3'-enhancer over a region where the mouse and rat enhancers are highly homologues. (B) is the map of the rat C_α/IgH 3'-enhancer region. Cosmid 37-1-1 DNA was digested with

SUBSTITUTESHEET various restriction endonucleases and hybridized as indicated. Restriction sites are abbreviated C. Cla I; N, Nru I; R, Eco RI; S, Sac I; X, Xho I.

Figure 5 shows that the mouse IgH 3'-enhancer exhibits lympho¬ id-specific transcriptional activity. Cells were transfed with a test plasmid containing the human 0-globin gene linked to the SV40, rat IgH-intron, mouse IgH-intron, mouse IgH-3' or no enhancer as indicated along with and α₂-globin plasmid as internal reference. Markers (M) were provided by a Hpa II digest of pBR322.

In Figure 6 the effect of the 3'-enhancer on the expression of stably transfected IgH genes is shown. RNA was extracted from pools of NSO transfectants carrying either a short (Sχ) or long X (1>X) gene which had been supertransfected with either pSV-V_μl (denoted V_μ) . E]) or no (-) plasmid. A Northern blot of this RNA was then hybridized with either C_μ or V_χ-Ox-l probes. The transfected Sχ and Lχ genes contain a V_χ-0χ-l variable segment [5].

The IgH 3'-enhancer is flanked by an invert repeat. The sequen¬ ce of the Stu I-Eco RV fragment of the rat 3'-enhancer has been determined previously [12]. The sequence of the invention (see Fig. 3) does indeed confirm the existence of flanking invert repeats (Fig. 1) . These repeats show 16 mismatches over a 357- bp stretch (> 95% identity) .

A mouse liver library in bacteriophage λ was therefore screened using as a probe an Ace I-Bgl I subfragment from the core of the rat 3'-enhancer (nucleotides 432-748 in the rat sequence; see Fig. 4) . Two overlapping groups of clones (λM2 and λM3) were isolated. The restriction map of λM2 is depicted in Fig. 2; phage λM3 extends 3' of λM2.

The Xba I fragment of λM2 that contained the enhancer homology was subcloned into plasmid pIC20H; a processive set of dele-

SUBSTITUTE SHEET tions was made using exonuclease III and subcloned into M13. The sequence of the mouse IgH 3'-enhancer was determined as is shown in Fig. 3 where it is aligned with that of the rat. The enhancer of the two species show good homology although the [GT]₇[ (G or C)A]₈₉] repeat of the rat is not observed in the mouse. Furthermore, while the invert repeats that flank the ra IgH 3'-enhancer show a 96% identity, the internal 250 nucleoti des of the equivalent mouse repeats manifest only 89% identity (Fig. 1C) . The octanucleotide element ATTTGCAT is conserved in rat and mouse.

A fragment spanning one of the flanking repeats was used to probe the mouse genome to see if the repeats are part of a larger family of repetitive sequences. The results (Fig. ID) show that probe A hybridizes strongly to a 2-kb Eco RI fragment (from which it is derived) and more weakly to a 12-kb fragment (which includes the homologous flanking repeat) and a third band of 4.8 kb. However, the absence of further bands suggests that the repeats flanking the mouse 3'-enhancer are not part of a larger family of highly conserved repeats.

Neither λM2 nor λM3 hybridized to an immunoglobulin α_s H chain cDNA probe. In order to determine whether the mouse enhancer eas indeed linked to the IgH locus, it was necessary to es- tablish an overlap between phage λM2 and IgH locus DNA. We screened a mouse cosmid library for clones containing the C_a region; the probe was an 1100-nt fragment generated by polyme- rase chain reaction that spans the coding region of the membrane exon. The restriction map of the cosmid isolated (CosMlOB) is depicted in Fig. 2. Cross-hybridization of sub¬ fragments of CosMlOB with those of λM2 as well as restriction mapping allowed the overlap to be established. Phage Ch28.M.Iaα: (described in [22]) was also found to overlap both CosMlOB and λM2s. The restriction map reveals that the mouse IgH 3'-enhancer is located 16 kb downstream of the Cαl exon.

Hybridization of subclones of the mouse IgH 3'-enhancer to

SUBSTITUTE SHEET phage λM2 shows that the 3'-enhancers of mouse and rat were present in the genome in opposite orientations with respect to the IgH C-regions. To confirm that this was not a cloning artefact, a Southern blot analysis of genomic mouse DNA was carried out. The core region of the enhancer in rat and mouse contains a conserved Eco RI site. In genomic mouse plots, probes F and C hybridize to a 12-kb Eco RI fragment whereas probes B and E hybridize to a 2-kb Eco RI fragment (Fig. 4A) . All these probes light up the same sized fragments in phage λM2 and, moreover, probe F hybridizes to a region just 3'of C_a in cosmid M10B (not shown) . Thus, probe E lights up the C_^-distal side of the Eco RI site in the mouse 3'-enhancer. However, probe E is an Ace I-Eco RI subclone of the rat 3'-enhancer and derives from a region (nucleotides 432-565) which is located on the Cα-proximal side of the Eco RI site within the rat 3'- enhancer [12]. This is confirmed in Fig. 4B which shows that, in the rat C_a cosmid 37-1-1, probe E and a C_α-distal side of the rat IgH 3'-enhancer. Thus, the rat and mouse-membrane probe light up a common 19-kb Eco RI fragment whereas probe D hybri- dizes to the enhancers are in opposite orientations.

To confirm that the putative mouse 3'-enhancer does indeed exhibit enhancer activity, the 4 kb/Xba I fragment was linked to an enhancerless human β-globin gene and introduced into MPCll mouse plasmacytoma cells along with an α₂-globin gene as internal reference. As shown in Fig. 5, the IgH 3'-enhancer (like the SV40 and IgH intron-enhancers) does indeed potentiate 3-globin expression in the transfected myeloma cells. Further¬ more, like the IgH intron-enhancer but in contrast to the SV40 enhancer, the IgH 3'-enhancer is inactive in HeLa cells. Thus, the mouse IgH 3'-enhancer appears lymphoid-specific.

One of the reasons for looking for the IgH 3'-enhancer was the fact that while IgH genes introduced into plasmacytomas or into the mouse germ line are usually well expressed, the level of this expression often falls short of that of the endogenous IgH gene. Could the absence of the 3'-enhancer on the transfected gene be responsible for this shortfall? To test this a mouse plasmacytoma cells was transfected with plasmid pSV-Vl, which contains a rearranged μ gene including the IgH intron-enhancer, and a derivative, pSV-Vl[3'E] , which also includes the IgH 3'- enhancer cloned downstream of the transcription unit. As a plasmacytoma host we used NSO cells that had been previously transfected with one of two plasmids encoding a mouse χ chain. The NSO[Sχ] cells carry a transfected χ gene that includes the χ-intron but not the χ3' -enhancer whereas the transfected gene in the NS0[Lχ] cells includes both the χ-intron and the χ3 ' - enhancers [5]. Northern blot analysis of pools of transfected cells (Fig. 6) reveals that inclusion of the IgH 3'-enhancer on plasmid pSV-V^l has about a sixfold effect on the level, of μ gene expression in the transfected cells.

Thus, like the rat, the mouse also contains a lymphoid-specific enhancer located downstream of the C_H regions. The enhancer in both species is flanked by an invert repeat. Interestingly, certain differences between rat and mouse in the sequences of the upstream copy of their repeats are also found in the downstream copy. Thus, nucleotides 574-578 (CATGA) in the mouse upstream repeat are not present in the rat; similarly, the invert of this sequence (TCATG) is retained in the downstream copy of the mouse repeat but is nevertheless still absent in the rat. Thus, the flanking repeats appear to have co-evolved with information transfer between the two such that some changes that occur in the upstream repeat also become fixed in the downstream copy.

The term "vector" as used herein connotes in its broadest sense any recombinant DNA material capable of transferring DNA from one cell to another.

The vector may be a single piece of DNA in linear or circular form an may, in addition to the essential functional elements of the invention, include such other sequences as are necessary for particular applications. For example, the vector may

SUBSTITUTE SHEET contain additional features such as a selectable marker gene o genes, and/or features which assist translation or other aspects of the production of a cloned product.

The term "gene" as used herein connotes a DNA sequence, prefe¬ rably a structural gene encoding a polypeptide. The polypeptid may be a commercially useful polypeptide, such as a pharmaceu¬ tical and may be entirely heterologous to the host cell. Alternatively the gene may encode a polypeptide which is deficient, absent or mutated in the host cell.

The host cell may be any host cell susceptible to uptake of th vector of the invention. The vector DNA may be transferred to the host cell by transfection, infection, microinjection, cell fusion, or protoplast fusion.

The host cell may be a cell of a living human or animal. In particular, the host cell may be a cell of a transgenic animal such as a mouse. The host cell may be a human stem cell such a bone marrow cell.

The promoter may be any promoter capable of functioning^' in the host cell and may be for example a mammalian or viral promoter.

The invention also includes within its scope a host cell, e g of a cell line or a transgenic animal, transformed with a vector according to the invention.

The invention further provides a method of producing a polypep tide, comprising culturing a host cell transformed with a vector according to the invention.

The method may be applied in vitro to produce a desired poly¬ peptide. In addition, the method may be applied in vivo to produce a polypeptide having no therapeutic value to the animal.

In a further aspect of the invention the vector may be used in

SUBSTITUTE SHEET a method of treatment of the human or animal body by replacing or supplementing a defective gene.

Many diseases of the human or animal body result from deficien cies in the production of certain gene products. The charac¬ terizing features of the vectors of this invention make them amply suited to the treatment of deficiencies by gene therapy in vivo.

A method of gene therapy is thus provided comprising removing ste cells from the body of a human or an animal, killing stem cells remaining in the body, transforming the removed stem cells with a vector of the invention containing a gene-defici¬ ent, or absent, in the human or animal body, and replacing the transformed stem cells in the human or animal body. This method can be used to replace or supplement a gene deficient in a human or animal.

Bone marrow is a suitable source of stem cells and is advanta- geous in that it contains the precursors of both lymphocytes and erythroid cells. Alternatively other tissue could be removed from the body, transfected or otherwise provided with a vector of the invention and implanted back into the body.

SUBSTITUTESHEET Literature

1 Picard, D. and Schaffner, W. , Nature 1984. 307:80.

2 Queen, C. and Stafford, J. , Mol. Cell Biol. 1984. 4: 1042.

3 Meyer K. B. and Neuberger, M.S., EMBO J. 1989. 8: 1959.

4 Xu, M. , Hammer, R.E., Plasquez, V.C., Jones, S. L. and Garrard, W.T., J. Biol. Chem. 1989. 264: 21 190.

5 Meyer, K.B., Sharpe, M.J., Surani, M.A. and Neuberger, M. S. Nucleic Acids Res. 1990. 18: 5609.

6 Klein, S., Sablitzky, F. and Radbruch, A., EMBO J. 1984. 3: 2473.

7 Wabl, M. and Burrow, P.D., Proc. Natl. Acad. Sgi_ USA

1984. 81: 2452. 8 Aguilera, R.J., Hope, T.J. and Sakano, H. , EMBO J. 1985. 4: 3689.

9 Eckhardt, L.A. and Birshtein, B.K., Mol. Cell. Biol.

1985. 5: 856.

10 Pettersson, S., Sharpe, M.J. , Gilmore, D.R. , Surani, M.A. and Neuberger, M.S., Int. Immunol 1989. 1:509.

11 Neuberger, M.S. and Calabi, F. , Nature 1983. 305:240.

12 Pettersson, S., Cook, G.P., Brϋggemann, M. , Williams, G.T. and Neuberger M.S., Nature 1990. 344: 6262.

13 Brϋggemann, M. , Free, J. , Diamond, A., Howard, J. , Co- bold, S. and Waldmann, H. , Proc. Natl. Acad. Sci. USA

1986. 83: 6075.

14 Clark, M.R. and Milstein, C, Somatic Cell Genet. 1981. 7: 657.

15 Weigert, M.G., Cesari, I.M., Yonkovitch, S.J. and Cohn, M. , Nature 1970. 228: 1045.

16 Kim, K.J., Kanellopoulos-Langevin, C. , Mervin, R.W. Sachs, D.H. and Asofsky, R. , J. Immunol. 1979. 122: 549. 7 Graham, R. and Van der Eb, A., Virology 1973. 52: 456. 8 Mason, J.O., Williams, G.T. and Neuberger, M.S., Cell 1985. 41: 479. 9 Treis an, R.H., Cell 1985. 42: 889. 0 Neuberger, M.S., EMBO J. 1983. 2: 1373. 1 Blackwell, T.K. , Kretzner, L. , Blackwood, E.M. , Eisenman,

SUBSTITUTE SHEET R.N. and Weintraub, H. , Science 1990. 25: 1149. 22 Nishida, Y., Katoka, T., Ishida, N., Nakai, S., Kishimo- to, T., Bδttcher, I. and Honjo, T. , Proc. Natl. Acad. Sci. USA 1981. 78: 1581. 23 Gregor, P.D. and Morrison S.L., Mol. Cell. Biol. 1986. 6: 1903. 24 Giannini, S.K., Calvo, C.F., Martinez, N. , Ding, G.-F. and Birshtein, B.K., J. Cell. Bioehem. 1990. Suppl. 14D: Abstr. M219. 25 Grosvfeld, F. et al. 1987. Cell 57: 975.

SUBSTITUTESHEET

Claims

Claims

1. A DNA 4 kb/Xba I fragment being an IgH 3'-enhancer, which fragment has been generated by cloning a partial Sau 3AI digest of BALB/c mouse liner DNA into the vector λ 2001, which fragment crosshybridizes with rat 3'-enhancer element.

2. A DNA fragment being an IgH 3'-enhancer and having the following sequence:

1 TGATAGAGAG GAGATGACAG AAGGAAGAGA CACAGATAGA CAGAGGTAGG

51 AGATGTGCAC CTAGAGAAAG GGGGCACAAG CAGAGATGAG AGAGAGAGAG

101 AGAGAGAGAG AGAGAGAGAG AGAGAGAGAG AGAGGAAGAA GAKGAΑGGAG 151 AAGGAGAAGG AGAAGGAGAA GGAGAAGGAG AAGGAGAAGA AGAAAGAGGA

201 GAGACAAGGT GTTAATAGAG ACAGCTCTTA TGTTTTGTTA GAGATGGAGA

251 GACAGTAACT GAGGGACAGA CTGAGCAAGA ATCACACTGA GAGAGAGAGA

301 AAGAGGCAGA GGCTAAGGCT GGGAAAGGCA GGGTTGGCCA GAGATGGAGC 351 CAGGGTCAGA CACCATCTTG AAAGTGCCAG TGACAAGCAG GAGGTGACAG 01 GCTGGTACAG TTTCTATCCC ACAGTCCACA GGTTCCAGTC ACAGGCCTGT

451 CTCCATGTGG CCACAGGCTA GCTCCCTGTC TGCCAAGTCT GTCTGAGAAG

501 GGGTGGGGGA GGGGGTCTGG GCTGAGGCAG GCCAAGACTT TTCCAGTGGA

551 GTGGCCAGGG ACTGGGCTGA GGGCATGATC ACTGCCCACC ATTCCCATGG

601 TTCTGGGATA CCCAGGATTT GGAGCACACC TACAGCCTTC CTGCCTCTCT

651 CACTTCCCTG GGGTGTTGAG CCACCCATCC TTGCCCATCT CCTGTCATGT

701 CCTCTTGGTG GGTCAGATTC CAGCAGTGGT GATAACTGGG AAACACGGAA

751 TTCAACATCA TCAATAGGGT CATGGACCCC CAGTCCCAGT GGCCTATGCT

801 GGGAGTCCCC CATCCCCAAG GCTGGTCAGC CTGGCCAGGT TGGGGTGAAC

851 CTGCAGCTGC AGGTGGGGGA GTCACTCATG CTATTTCTGG AAACAACCTC 901 AGAAAGTGAG AAACATGTTT TTCTGTATGA AACAAACATC CATTTGCATG

951 TGCCCTTGTG AGCCAACTCT GCACAGAATG AGGTAACTCG GCAGCGGGGG

1001 CGAGGACATG TTGGGAGTAA GCCTTGTTTC TGGTACTGAT ATCTGAGCCC

1051 CCAACCCCCA GATTGTAACC CTAGCCCATG TGGAAGAACT ACCCTTAGGG

SUBSTITUTE SHEET

3. A DNA fragment being an IgH 3'-enhancer and having the following sequence: AGGAGAGAAA GAGGAGAGAC ACGTGTTAGA GGAGAAAGCA TTATTGTTTT

10 20 30 40 50

CATAGAGACA GAGATACAGC AACTGAGGAA CAGACTGAGC AGAAAGAATC

60 70 80 90 100

CCACTCAGAG AGAGACAGAG GCTGAAAGCT GGGAGAGGCA GGGAATGGCC

110 120 130 140 150

AGAGAGGGAG ACAGAGGAGA GCCTGTGGGA CAGGTCAGAC ACCATCTTGG

160 170 180 190 200

AAGACCCAGT GACAAGCCAG GGAGGTGACA GGTTAGTGCA GTTTCTACCC

210 220 230 240 250

CACAGTCCAC AGGCTGGAGC CACAGGCCTG TGTCCATGTG GCCACAGCTA

260 270 280 290 300

GCTCCCAGCC TGCCAAGCCT GTCTGTGGAG GGATTGCGGA GTGGGGCTGG

310 320 330 340 350

GCTGAGGCAG GCCAGGATTT TTCCAGTGGA GCAGCCAGTG ACTGGGCTGA

360 370 380 390 400

GGGTCACTGC CCACCATCCC CGTGATTCTG GGTAGACCCA GGACCTGGAG

410 420 430 440 450

CACACACAGC CTTCCTGTCT CTCCCACTTC CCTGGGGTGT TTGGACCTTG

460 470 480 490 500

CCTATCTCCT GTCATTCCCT CCTAGGGTCA GATTCCAGCA GTGTCGATAA

510 520 530 540 550

ATGGGAAAAC ACTGAATTCA ATGTCACCGT TAGGGGTCAT GGAACCCCCA

560 570 580 590 600

SUBSTITUTE SHEET GCCCCATCCC CAAAGCTGGT CAACCTGGCC AGGGTGGGGT GAACCTGCAG 610 620 630 640 650

CTGCAGGTGG GGGAATCATT GATGCTATTT TTGGCTCAGA GAATGAGAAA 660 670 680 690 700

CATGTTTTTC TGCATGGAAC AAACATCCAT TTGCATGTGC CCCGAGGGCC 710 720 730 740 750

AACCCTGCAC AGAGTGAGTC ACTCGGCAGG TGTGTGTGTG TGTGTGTGTG 760 770 780 790 800 TGTGTGTGAG AGAGAGAGAG AGAGAGAGAG AGAGAGAGAG AGAGACAGAG 810 820 830 840 850

ACAGAGACAG AGACAGACAG AGAGACAGAG AGACAGAGAG AGAGACAGAG 860 870 880 890 900

AGAGACACAC ACAGAGACAG AGAGACAGAG AGAGACAGAG ACAGAGAGAG 910 920 930 940 950

ACACACACAC AGAGACAGAG AGACAGAGAG AGAGATGTTG TGAGCAAGCC 960 970 980 990 1000

CTGTTTCAGG TACTGATATC TGTGCCCCCG TGCCCCAGCC CATGTGAAAA 1010 1020 1030 1040 1050 ACTACCTTAG GGCCCATTAA CTCATCACAG TGCAGGCCAT CATGGTGAGC 1060 1070 1080 1090 1100

CCTAGCTGAC CTAAGCAGGT TCCACCCTAA ACATGAAGGA AAATAATAAA 1110 1120 1130 1140 1150

GGCCATCTGT TACCCAGAAT CCTAGGGATG GTGGGCAGTG ACCCTCAGCC 1160 1170 1180 1190 1200

CACTCCTGGC CGCTCCACTG GAAGAATCCT GGCCTGCCTC AGCCCAGCCC 1210 1220 1230 1240 1250

CACTCCGCAA TCCCTCCACA GACAGGCTTG GCAGGCTGGG AGCTAGCTGT ^' 1260 1270 1280 1290 1300 GGCCACATGG ACACAGGCCT GTGGCTCCAG CCTGTGGACT GTGGGGTAGA 1310 1320 1330 1340 1350

AACTGCATAA CCTGTCACCT CCCTGGCTGT CACTGGGTCT TCCAAGATGG 1360 1370 1380 1390 1400

TGTCTGACCT GTCCCATAGG CTCTCCTCTG TCTCCCTCTC TGGCCTAGCC 1410 1420 1430 1440 1450

CTGCCTCTCC CAGCTTTAGC TCTGTCTCTC TCTGAGTGGG ATTCTCTCTG 1460 1470 1480 1490 1500

CTAGTCTGTA GCATAGTTGG AGAGAGTCCT ATCTATGTTT GCCTCTGTCT 1510 1520 1530 1540 1550 CTGCAATTCC ATAGAGGTCT CTCATAA 1560 1570

4 . Eukaryotic immunglobulin expression vector comprising the

IgH 3 ' -enhancer .

SUBSTITUTE SHEET