EXPRESSION OF HETEROLOGOUS PROTEINS IN ATTENUATED BACTERIA USING THE HTRA- PROMOTERS.

This invention relates to DNA constructs, replicable expression vectors containing the constructs, attenuated bacteria containing the constructs and vaccines containing the said bacteria.

In recent years, there has emerged a new generation of live oral salmonella vaccines based upon strains of Salmonella which have been attenuated by the introduction of a non-reverting mutation in a gene in the aromatic biosynthetic pathway of the bacterium. Such strains are disclosed, for example, in ^' EP-A-0322237. The aforesaid live oral salmonella vaccines are showing promise as vaccines for salmonellosis in man and animals, and they can also be used effectively as carriers for the delivery of heterologous antigens to the immune system. Combined salmonella vaccines have been used to deliver antigens from viruses, bacteria, and parasites, eliciting secretory, humoral and cell-mediated immune responses to the recombinant antigens. Combined salmonella vaccines show great potential as single dose oral

multivaccine delivery systems [C. Hormaeche et al, FEMS Symposium No. 63, Plenum,New York; pp 71-83, 1992].

There are problems to be overcome in the development of combined salmonella vaccines. A major consideration is obtaining a high level of expression of the recombinant antigen in the salmonella vaccine so that it will be sufficient to trigger an immune response. However, unregulated high level expression of foreign antigens can be toxic and affect cell viability [I. Charles and G. Dougan, TIBTECH 8 , pp 117-21, 1990], rendering the vaccine ineffective or causing loss of the recombinant DNA. Several possible solutions to this problem have been described, such as expression from plasmids carrying essential genes, "on-off" promoters or incorporation of the foreign genes into the salmonella chromosome.

An alternative approach to overcoming the aforesaid problem would be to use a promoter which is inducible _in vivo, and one such promoter is the E.coli nitrite reductase promoter nirB which is induced under anaerobiosis. Vaccine compositions containing bacteria transformed with constructs comprising the nirB promoter are described in our earlier International Patent Application PCT/GB93/01617.

The present invention relates to the preparation of DNA constructs containing a different inducible promoter, namely the promoter for the htrA gene which encodes a stress induced protein.

The htrA gene is described in K. Johnson et al Mol. Microbiol 1991; 5:401-7 and references cited therein and is an example of a gene encoding a heatshock protein which is produced in response to a temperature increase above 42°C.

Accordingly, in a first aspect, the invention provides a DNA construct comprising the htrA promoter sequence operably linked to a DNA sequence encoding one or more heterologσus proteins.

In one embodiment, the invention provides a DNA construct as hereinbefore defined wherein the htrA promoter sequence is operably linked to a DNA sequence encoding a fusion protein of two or more heterologous proteins.

The proteins making up the fusion may be linked by means of a flexible hinge region.

In a further aspect, the invention provides a DNA construct comprising the htrA promoter sequence operably linked to a DNA sequence encoding first and second heterologous proteins wherein the first heterologous protein is an antigenic sequence comprising tetanus toxin fragment C or one or more epitopes thereof.

In a further aspect, the invention provides a replicable expression factor, suitable for use in bacteria, containing a DNA construct as hereinbefore defined.

In another aspect, the invention provides a fusion protein, preferably in substantially pure form, the fusion protein being expressed by a construct as hereinbefore

defined .

In a further aspect, the invention provides a process for the preparation of an attenuated bacterium which comprises transforming an attenuated bacterium with a DNA construct as hereinbefore defined.

In a still further aspect, the invention provides a host cell, such as a bacterial cell, containing a DNA construct as hereinbefore defined. The DNA construct may be present in extra-chromosomal form, e.g. in a plasmid, or may be integrated into the host (e.g. bacterial) chromosome by methods known per se.

The invention also provides a vaccine composition comprising an attenuated bacterium as hereinbefore defined, or a fusion protein expressed therefrom, and a pharmaceutically acceptable carrier.

The first and second proteins are preferably heterologous proteins and in particular can be polypeptide immunogens; for example they may be antigenic sequences derived from a virus, bacterium, fungus, yeast or parasite. In particular, it is preferred that the first said protein is an antigenic sequence comprising tetanus toxin fragment C or epitopes thereof.

The second protein is preferably an antigenic determinant of a pathogenic organism. For example, the antigenic determinant may be an antigenic sequence derived from a virus, bacterium, fungus, yeast or parasite.

Examples of viral antigenic sequences for the first and/or second heterologous proteins are sequences derived from a type of human immunodeficiency virus (HIV) such as HIV-1 or HIV-2, the CD4 receptor binding site from HIV, for example from HIV-1 or -2, hepatitis A, B or C virus, human rhinovirus such as type 2 or type 14, Herpes simplex virus, poliovirus type 2 or 3, foot-and-mouth disease virus (FMDV), rabies virus, rotavirus, influenza virus, coxsackie virus, human papilloma virus (HPV), for example the type 16 papilloma virus, the E7 protein thereof, and fragments containing the E7 protein or its epitopes; and simian immunodeficiency virus (SIV).

Examples of antigens derived from bacteria are those derived from Bordetella pertussis (e.g. P69 protein and filamentous hae agglutinin (FHA) antigens), Vibrio cholerae, Bacillus anthracis, and E.coli antigens such as E.coli heat Labile toxin B subunit (LT-B), E.coli K88 antigens, and enterotoxigenic E.coli antigens. Other examples of antigens include the cell surface antigen CD4, Schistosoma mansoni P28 glutathione S-transferase antigens (P28 antigens) and antigens of flukes, mycoplasma, roundworms, tapeworms, Chlamydia trachomatis, and malaria parasites, eg. parasites of the genus plasmodium or babesia, for example Plasmodium falciparum, and peptides encoding immunogenic epitopes from the aforementioned antigens.

Particular antigens include the full length Schistosoma

mansoni P28, and oligomers (e.g. 2, 4 and δmers) of the immunogenic P28 aa 115-131 peptide (which contains both a B and T cell epitope), and human papilloma virus E7 protein, Herpes simplex antigens, foot and mouth disease virus antigens, simian immunodeficiency virus antigens, and the diphtheria toxin antigens, e.g. the diphtheria toxin ganglioside binding region.

As used herein, references to the htrA promoter refer to the promoter itself or a part or derivative thereof which is capable of promoting expression of a coding sequence. The preferred sequence, and which contains the htrA promoter is: AATTCTATTCCGGAACTTCGCGTTATAAAATGAATCTGACGTACACAGCAATTTA (SEQ.ID.N0.1)

In the constructs of the present invention, the DNA sequence may encode a fusion protein of two or more proteins in which adjacent proteins are separated by a hinge region. The hinge region is a region designed to promote the independent folding of both the first and second proteins by providing both spatial and temporal separation between the domains.

The hinge region typically is a sequence encoding a high proportion of proline and/or glycine amino acids. The hinge region may be composed entirely of proline and/or glycine amino acids. The hinge region may comprise one or more glycine-proline dipeptide units.

The hinge region may, for example, contain up to about

fifteen amino acids, for example at least 4 and preferably 6- 14 amino acids, the number of amino acids being such as to impart flexibility between the first and second proteins.

In one embodiment, the hinge region can correspond substantially to the hinge domain of an antibody immunoglobulin. The hinge regions of IgG antibodies in particular are rich in prolines [T.E. Michaelson et al . J. Biol. Chem. 252, 883-9 1977], which are thought to provide a flexible joint between the antigen binding and tail domains.

Without wishing to be bound by any theory, the prolines are thought to form the rigid part of the hinge as the ring structure characteristic of this amino acid hinders rotation around the peptide bond that connects the proline residue with an adjacent amino acid. This property is thought to prevent proline, and adjacent residues, from adopting the ordered structure of an alpha helix or beta strand. Flexibility is thought to be imparted by glycine, the simplest amino acid, with very limited steric demands. Glycine is thought to function as a flexible elbow in the hinge. Other amino acids may be substituted for glycine, particularly those without bulky side-chains, such as alanine, serine, asparagine and threonine.

In one preferred embodiment, the hinge region is a chain of four or more amino acids defining the sequence

-[X] _? -Pro-[Y] _ς -Pro-[Z] _r - wherein Pro is proline, X and Y are each glycine, or an amino

acid having a non-bulky side chain; Z is any amino acid; p is a positive integer; q is a positive integer of from one to ten; and r is zero or a positive integer greater than zero.

The hinge region can be a discrete region heterologous to ^• both the first and second proteins or can be defined by a carboxy-end portion of the first protein or an amino-end portion of the second protein.

In a most preferred aspect, the present invention provides a DNA molecule comprising the htrA promoter operably linked to a DNA sequence encoding first and second polypeptide iramunogens linked by a hinge region, wherein the first polypeptide immunogen comprises tetanus toxin fragment C or epitopes thereof.

In another preferred aspect of the invention, there is provided a replicable expression vector, suitable for use in bacteria, containing the htrA promoter sequence operably linked to a DNA sequence encoding first and second polypeptide immunogens linked by a hinge region, wherein the first polypeptide immunogen comprises tetanus toxin fragment C or epitopes thereof.

In a further aspect, the invention provides a DNA construct comprising the htrA promoter operably linked to DNA encoding a first protein and, extending from the 3' end thereof a DNA sequence encoding the hinge region, and downstream thereof, one or more restriction endonuclease sites.

The said protein is preferably an antigenic protein as hereinbefore defined, and in particular is the TetC fragment or epitopes thereof.

Stable expression of the first and second heterologous proteins linked by the hinge region can be obtained in vivo. The heterologous proteins can be expressed in an attenuated bacterium which can thus be used as a vaccine.

The attenuated bacterium may be selected from the genera Salmonella, Bσrdetella, Vibrio, Haemophilus, Neisseria and Yersinia. Alternatively, the attenuated bacterium may be an attenuated strain of enterotoxigenic Escherichia coli. In particular the following species can be mentioned: S.typhi - the cause of human typhoid; S.typhimuriu - the cause of salmonellosis in several animal species; S.enteritidis - a cause of food poisoning in humans; S.choleraesuis - a cause of salmonellosis in pigs; Bordetella pertussis - the cause of whooping cough; Haemophilus influenzae a cause of meningitis; Neisseria gonorrhoeae - the cause of gonorrhoea; and Yersinia - a cause of food poisoning.

Attenuation of the bacterium may be attributable to a non-reverting mutation in a gene in the aromatic amino acid biosynthetic pathway of the bacterium- There are at least ten genes involved in the synthesis of chorismate, the branch point compound in the aromatic amino acid biosynthetic pathway. Several of these map at widely differing locations on the bacterial genome, for example aroA (5-

enolpyruvylshikimate-3-phosphate synthase), aroC (chorismate synthase), aroD (3-dihydroquinate dehydratase) and aroE (shikimate dehydrogenase) . A mutation may therefore occur in the aroA, aroC, aroD, or aroE gene.

Preferably, however, an attenuated bacterium harbours a non-reverting mutation in each of two discrete genes in its aromatic amino acid biosynthetic pathway; or harbours a non- reverting mutation in its aromatic biosynthetic pathway and a non-reverting mutation in a regulatory gene such as htrA, OmpR or OsmC. Examples of suitable attenuated bacteria are disclosed in, for example, EP-A-0322237, and EP-A-0400958.

An attenuated bacterium containing a DNA construct according to the invention can be used as a vaccine. Fusion proteins (preferably in substantially pure form) expressed by the bacteria can also be used in the preparation of vaccines. For example, a purified TetC-P28 fusion protein has been found to be immunogenic on its own. In a further aspect therefore, the invention provides a vaccine composition comprising a pharmaceutically acceptable carrier or diluent and, as active ingredient, an attenuated bacterium or fusion protein as hereinbefore defined.

The vaccine may comprise one or more suitable adjuvants.

The vaccine is advantageously presented in a lyophilised form, for example in a capsular form, for oral administration to a patient. Such capsules may be provided with an enteric coating comprising, for example, Eudragit "S", Eudragit "L",

Cellulose acetate, Cellulose acetate phthalate or Hydrσxypropyl ethyl Cellulose. These capsules may be used as such, or alternatively, the lyophilised material may be reconstituted prior to administration, e.g. as a suspension. Reconstitution is advantageously effected in buffer at a suitable pH to ensure the viability of the organisms. In order to protect the attenuated bacteria and the vaccine from gastric acidity, a sodium bicarbonate preparation is advantageously administered before each administration of the vaccine. Alternatively, the vaccine may be prepared for parenteral administration, intranasal administration or intramammary administration.

Preferably, the vaccine composition is adapted for mucosal delivery, eg by oral administration, by intranasal administration or by intrabronchial administration.

The attenuated bacterium containing the DNA construct of the invention may be used in prophylaxis or treatment of a host, particularly a human host but also possibly an animal host. An infection caused by a micro-organism, especially a pathogen, may therefore be prevented by administering an effective dose of an attenuated bacterium according to the invention. The bacterium then expresses a heterologous protein or proteins capable of raising antibody to the micro-organism. The dosage employed will be dependent on various factors including the size and weight of the host, the type of vaccine formulated and the nature of the heterologous protein.

An attenuated bacterium according to the present invention may be prepared by transforming an attenuated bacterium with a DNA construct as hereinbefore defined. Any suitable transformation technique may be employed, such as electroporation. In this way, an attenuated bacterium capable of expressing a protein or proteins heterologous to the bacterium may be obtained. A culture of the attenuated bacterium may be grown under aerobic conditions. A sufficient amount of the bacterium is thus prepared for formulation as a vaccine, with minimal expression of the heterologous protein occurring.

The expression vector is provided with appropriate transcriptional and translational control elements including, besides the htrA promoter, a transcriptional termination site and translational start and stop codons and an appropriate ribosome binding site is provided. The vector typically comprises an . origin of replication and, if desired, a selectable marker gene such as an antibiotic resistance gene. The vector may be, for example, a plasmid.

The invention will now be illustrated, but not limited, by reference to the following examples, and the accompanying drawings, in which:

Figure 1 is a schematic illustration of the construction of a plasmid pHTRAl containing the htrA promoter in accordance with one aspect of the invention;

Figure 2 is a schematic illustration of the construction

of a plasmid pHTRA2 containing the htrA promoter and DNA encoding the tetanus toxin C-fragment linked to a hinge region;

Figure 3 illustrates the structure of the plasmid pTECH2;

Figure 4 illustrates the structure of the intermediate plasmid pBD907;

Figure 5 shows the structure of the plasmid pHTRAl prepared in accordance with the scheme shown in Figure 1;

Figure 6 shows the structure of the product plasmid pHTRA2 prepared in accordance with the scheme shown in Figure 2;

Figures 7A to 7B illustrate the influence of temperature shifts on the promoters nirB, groE and hrtA; and

Figure 8 shows the expression of lacZ from htrA, nirB and groE in macrophages.

EXAMPLE 1

Preparation of htrA-TetC-Hinge Construct

As can be seen from Figure 1, the starting material for the preparation of a vector containing the htrA promoter and genes coding for the tetanus toxin C fragment was the plasmid pTETnirl5, the structure and preparation of which is disclosed in our earlier application PCT/GB93/01617 (Publication No. WO 94/03615) and references cited therein, e.g. WO-A-92159689.

The pTETnirl5 plasmid contains the nirB promoter linked

95/20665 - '

14 to the gene encoding the C-fragment of tetanus toxin (TetC). As shown in Figure 1, pTETnirl5 was digested with SacII and Ba HI and the resulting 2.9kb and 813bρ fragments were gel- purified. The 2.9kb fragment was ligated with a 1.74kb fragment derived from the B. pertussis filamentous haemagglutinin (FHA) gene, the fragment having the sequence shown in SEQ.ID.N0.7. The resulting plasmid was designated pBD907 and the restriction map of the plasmid is shown in Figure 5. The purpose of preparing the intermediate plasmid pBD907 was to remove the EcoRI site present in the TetC fragment in order that the nirB promoter sequence could be replaced by the htrA promoter sequence. This was achieved by digesting plasmid pBD907 with EcoRI and Bglll. The resulting 4535bp fragment was gel-purified and ligated with the following 55bp oligonucleotides containing the htrA promoter:

Oligo-1 5' AATTCTATTCCGGAACTTCGCGTTATAAAATGAATGTGACGTACACAGCAATTTA

(SEQ.ID.N0.2)

01igo-2 3' GATAAGGCCTTGAAGCGCAATATTTTACTTACACTGCATGTGTCGTTAAATCTAG

(SEQ.ID.N0.3)

The presence of the promoter in the resulting intermediate plasmid pINT was confirmed by DNA sequencing. The plasmid pINT was then digested with SacII and BamHII and ligated to the 813bρ fragment from pTETnirlδ to form plasmid pHTRAl. The DNA sequence of pHTRAl is shown in SEQ.ID.N0.4;

the htrA region which is defined by the first 55 base pairs, has the sequence

AATTCTATTCCGGAACTTCGCGTTATAAAATGAATCTGACGTACACAGCAATTTA (SEQ.ID.NO.l) .

In relation to SEQ.ID.NO.4, GAACTT is -35 box, and TCTGA is -10 box. At 513 and 2235 base pairs respectively are SacII and AlwN 1 restriction sites.

Plasmid pHTRAl was used to transform Salmonella typhimurium strain BRD509 (deposited under accession number NCTC .... ) and the resulting strain, designated BRD935, was checked for expression of TetC fragment by standard methods. Strain BRD935 has been deposited at the National Collection of Type Cultures, Colindale, United Kingdom on... under the accession number ) .

As shown in Figure 2, plasmid pHTRAl was used to prepare a modified construct in which a "hinge" region is present at the C-terminal of the TetC fragment. The nucleotide sequence representing the "hinge" region was obtained from plasmid pTECH2 which has the DNA sequence set forth in SEQ.ID.NO.5, and possesses SacII and AlwNI restriction sites at positions 533 and 2304 respectively. The preparation of this plasmid is disclosed in our earlier Application PCT/GB93/01617 (Publication No )

The pTECH2 plasmid comprises the nirB promoter region

linked to the tetanus toxin C fragment which, at its 3 ' terminal, is linked via a BamHI restriction site to a hinge region encoded a Gly-Prσ-Gly-Pro repeat motif along with a number of restriction sites allowing the insertion of genes encoding further polypeptides. A 1.7kb fragment encoding the hinge region and part of the tetanus toxin C fragment region was removed from pTECH2 through digestion with SacII and AlwNI and purified. The DNA sequence of the resulting fragment is shown in SEQ.ID.N0.6.

Plasmid pHTRAl, which encodes the htrA promoter and the tetanus toxin C fragment, but includes no hinge, was digested with SacII and AlwNI and the resulting 2kb fragment was gel- purified.

The 1.7kb fragment (SEQ.ID.NO.6) from pTECH2 and the 2kb fragment from the pHTRAl were ligated to form plasmid pHTRA2 which incorporates a htrA promoter operably linked to the gene for tetanus toxin C fragment, having at the 3* terminal thereof the hinge region.

An attenuated Salmonella typhimurium strain was transformed with vector pHTRA2 and after selection by means of standard techniques, the salmonella strain BRD1062, harbouring the plasmid pHTRA2 was isolated.

Plasmid pHTRA2 serves as an intermediate for the preparation of constructs coding for a fusion protein linked by the hinge region. Thus, in accordance with the techniques described in our earlier Application No. PCT/GB93/01617,

further proteins can be cloned into the restriction endonuclease sites on the hinge region.

MATERIALS AND METHODS

Bacterial Strains

E.coli HBlOl and BRD509 (an attenuated S. typhimurium aroA aroD strain (Deposited under accession number NCTC .... ) were used throughout the experiments. The bacteria were grown in Luria broth (LB) or LB solidified with 1.6% w/v agar supplemented with appropriate antibiotics.

DNA Manipulations

Plasmid DNA was purified by the alkaline lysis method (R. Maniatis, et al., 1982 Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, New York). Restricted DNA fragments were purified from agar gels by the method of Tautz and Rentz (1983, "An optimised freeze-squeeze method for the recovery of DNA Fragments from agarose gels". Analytical Biσchem. , 132, 14-19). Restriction enzymes were supplied by Boehringer Mannheim, Germany and New England Biolabs, USA and were used according to the manufacturer's instructions.

DNA Sequencing

DNA for double stranded sequencing was isolated by the method of Stephen et al. (1990, A Rapid Method for Isolating High Quality Plasmid DNA Suitable for DNA Sequencing, Nucleic Acid Research, 18, No. 24, p 7463). Sequencing was carried out using a Sequenase Version 2 kit (USB) and was used according to the manufacturer's instructions.

01igonucleotides

These were synthesised on a SAM1 oligonucleotide synthesiser (Biolabs, UK).

EXAMPLE 2

Preparation of htrA-lacZ Construct

The properties of the htrA promoter were composed with two other inducible promotors, namely the nirB and groE promoters.

Sub-cloning of lacZ downstream to nirB, htrA and groE promoters

A DNA fragment encoding a promoterless lacZ gene was purified from plasmid pMAC1871 (Pharmacia) by the low melting point agarpse technique, following cleavage of the plasmid with restriction enzymes Sail and BamHI [14]. Plasmids pTETnir-15 [S.N. Chatfield et al , Bio/Technology 10, 888-892]

and pTEThtrA-1 harbouring the nirB and htrA promoters respectively, were digested with Sail and BamHI endonucleases and the purified lacZ encoding fragment was cloned, in-frame, downstream of the promoters. Plasmid pRZ-PES was used to measure expression of ^β -galactosidase ( ^β -gal) from the groE promoter. pRZ-PES contains the E. coli groE-operon promoter upstream of groES and lacZ genes. It was constructed by sub- cloning a 2.1 Kb EcoRI-Hindlll fragment carrying the operon from plasmid pOF39 [0. Fayet et al J. Bacteriol. 171, 1379- 1385 (1989)] into pUC19. A novel Bglll site was then introduced between the groES and groEL genes using site directed mutagenesis. The EcoRI-Bglll fragment carrying the groE promoter and groES gene was cloned into EcoRI-BamHI cut promoter-probe plasmid pRF5255 [P.F. Lambert et al J. Bacteriol. 162, 441-444 (1985)] to give plasmid pRZ-PES. Plasmids, prepared in S. typhimurium LB5010 (r ^" m ⁺ ) [L.R. Bullas et al J. Bacteriol. 256, 471-474 (1983)], were introduced into S. typhimurium strain BRD915 (S. typhimurium SL1344 htrA) [S.N. Chatfield et al Microbial Pathog. 12, 145-151 (1992)] using electroporation. Lac positive recombinants were screened on L agar plates containing ampicillin and X-gal.

Effect of changes in environmental conditions on lacZ expression

Bacterial strains harbouring the recombinant plasmids were grown overnight in L-broth, with skaing at 30°C. The

cultures were diluted 1:50 and growth was allowed to continue for an additional 3 hours at 30°C until an ODggg of 2.8-3.4 was reached. 0.2 ml of each culture was stored at 4°C and used to determine the base-line of 6-gal activity. The remaining portions of the cultures were then shifted to different growth conditions as described below and samples were taken at 0, 2, 4, 6 and 24 hours, unless otherwise specified. At each time point the ODggg was determined and the bacteria were stored at 4°C prior to performing a 6-gal assay.

Measuring expression in infected HEp-2, Caco-2 and THP 1- macrophage cell lines

Cells were seeded at approximately 10° cell per well in twenty four well plates and grown overnight in Dolbecco's modified Eagles medium, without phenol red (ICN Flow), supplemented with 10% (vol/vol) fetal calf serum and 2 mM glutamine at 37°C, in an atmosphere of 5% C0 ₂ . 10 ⁸ CFU bacteria of the diluted overnight culture were added to the tissue culture cells and incubated at 30°C. At various time points samples of the tissue culture medium were taken to measure 6-gal activity in the extra cellular bacteria. The. numbers of bacteria in each sample were determined by viable count and the corresponding ODgg was determined using a standard curve. Infected cells were washed with phosphate buffer saline (PBS) and incubated for an additional hour in the presence of 200 mg/ml of gentamicin to kill extra cellular

bacteria. Thereafter, the cells were lysed using sterile distilled water and vigorous pipetting. 6-gal activity was determined for each cell lysate. The numbers of bacteria in each lysate were determined by viable count and the corresponding ODgg values were determined using a standard curve.

RESULTS

Expression from each of the promoters selected for this study is sensitive to changes in environmental conditions. nirB has been shown previously to respond to changes in anaerobicity. Initial experiments were performed to assess the levels of lacZ expression from each of the promoters, resident on similar multicopy plasmids, harboured within Salmonella vaccine strain BRD915. The influence of temperature shifts on the different promoters is shown in Figure 7. Temperature shifts from 30°C to 37°C (Figure 7A) resulted in an increase in 6-gal enzyme units when lacZ was expressed from the nirB and htrA promoters. No significant increase in 6-gal units was detected from the groE promoter. A temperature shift from 30°C to 42° resulted in an increase. in the number of 6-gal units from all three promoters. The rate of the increase in the level of 6-gal was faster from htrA and nirB compared with groE (Figure 7B) . Temperature shifts from 37°C to 42°C resulted in the induction of both nirB and htrA promoters, with more moderate increase in 6-gal

units from groE promoter (Figure 7C).

Expression of ^β -gal from the different promoters was also tested by selecting for bacteria that had entered eukaryotic cells. HEp-2, Caco-2 and THP-1 macrophage cell lines were infected with 10 ⁸ bacteria and incubated at 30°C. The number of 6-gal units, determined three hours after infection of HEp- 2, showed that expression of lacZ from both htrA and nirB promoters was significantly enhanced (Figure 2). However there was no detectable increase in lacZ expression from groE promoter. Similar results were obtained in infected Caco-2 cells (not shown). In contrast, in the macrophage's intracellular environment, all three promoters were induced (Figure 8) . nirB promoter was most affected and groE promoter was least affected (Figure 8). When the number of 6-gal units in the extra-cellular medium of either cell line was determined, no increase in the enzyme activity was seen (not shown) .

Since growth within macrophages was found to influence expression from all three promoters, their sensitivity to hydrogen peroxide, commonly found within the phagosome of macrophages, was monitored. Incubating the bacteria at 30°C in 100 μM hydrogen peroxide resulted in no significant effect on the groE and nirB promoters. In contrast, the level of 6- gal was increased from the htrA promoter reaching 10 U above base-line level by 4 hours. This was followed by a rapid decrease to base-line levels by 6 hours (not shown).

Constitutive expression of lacZ from plasmid pLK [M. Szabo et al J. Bacteriol. 174, 7245-7252] was not significantly affected by any of the environmental conditions (not shown).

In this study three environmentally regulated promoters were used to express lacZ gene under different growth conditions. The promoters are representatives of three classes of inducible bacterial promoters: the anaerobically inducible E. coli nirB, the σ^ dependent htrA and cr ² -dependent groE. Expression from the nirB promoter is dependent on the transcription factor FNR which binds between positions -52 and -30 upstream from transcription start. In some cases FNR dependent transcription is modulated together with a second transcription factor NarL. However, plasmid pTETnir-15 used here contains only the FNR dependent bind site.

Bacterial respond to environmental stress conditions by rapid change in the rate of synthesis of many proteins. In many cases the transit induction rapidly adjusts the protein levels to a new steady state. In this study we tested the influence of environmental conditions on the level of 6-gal. We found that temperature shift had a greater effect on htrA promoters compared with groE. This result is in line with the fact that in vivo the htrA promoter is induced before groE (which is σ ^j2 -dependent) and together with σ ^j2 , by σ ^E containing RNA polymerase [11, 12]. Surprisingly, the nirB promoter was also enhanced by elevated temperature. Although it is possible that at high temperature the concentration of oxygen

in the growing media is reduced the fact that the temperature shift from 30°C to 37°C brings rapid increase in the 6-gal units expressed from the nirB promoter may suggest that FNR, like other stress protein modulators, responds to a number of environmental stimuli. Similarly, htrA was also induced under anaerobic growth conditions, and therefore it seems that this promoter is either being regulated by factors other than σ , or that cr is being activated also at low oxygen tension.

For S. typhimurium to retain virulence the bacteria has to be able to survive in macrophages. This survival is dependent on the ability of the bacteria to tolerate a range of toxic killing machanis s including the production of hydrogen peroxide. Unlike E. coli htrA mutants, S. typhimurium htrA mutants have been found previously not to be killed by elevated temperature, but rather to have impaired ability to survive significant levels of hydrogen peroxide. Interestingly, the htraA promoter was the only one of the test promoters whose expression was increased in the presence of hydrogen peroxide.

In order to determine the influence of the intracellular environment, the level of expression from the three different promoters was monitored after Salmonella harbouring the test plasmids had entered a number of different cultured eukaryotic cell lines. Bacteria were grown in vitro and used to infect eukaryotic cells at 30°C since a temperature shift from 30°C to 37°C dramaticallv induced both the htrA and the nirB

promoters. We found that while the level of 6-gal expression from both the nirB and htrA promoters increased in all the cell lines tested, groE promoter was induced only in infected macrophages.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: MEDEVA HOLDINGS BV

(B) STREET: CHURCHILL-LAAN 223

(C) CITY: AMSTERDAM

(E) COUNTRY: THE NETHERLANDS

(F) POSTAL CODE (ZIP): 1078 ED (ii) TITLE OF INVENTION: VACCINES

(iii) NUMBER OF SEQUENCES: 7 (iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO) (vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: GB 9401795.1

(B) FILING DATE: 31-JAN-1994

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 55 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Salmonella typhimurium

(ix) FEATURE:

(A) NAME/KEY: promoter

(B) LOCATION: 1..55

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: AATTCTATTC CGGAACTTCG CGTTATAAAA TGAATCTGAC GTACACAGCA ATTTA 55 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 55 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

AATTCTATTC CGGAACTTCG CGTTATAAAA TGAATGTGAC GTACACAGCA ATTTA 55 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 55 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: YES

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GATAAGGCCT TGAAGCGCAA TATTTTACTT ACACTGCATG TGTCGTTAAA TCTAG 55 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3712 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (ix) FEATURE:

(A) NAME/KEY: htrA promoter

(B) LOCATION: 1..55 (ix) FEATURE:

(A) NAME/KEY: SacII restriction site

(B) LOCATION: 513 (ix) FEATURE:

(A) NAME/KEY: AlwN 1 restriction site

(B) LOCATION: 2235

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: AATTCTATTC CGGAACTTCG CGTTATAAAA TGAATCTGAC GTACACAGCA ATTTAGATCT 60

TAATCATCCA CAGGAGACTT TCTGATGAAA AACCTTGATT GTTGGGTCGA CAACGAAGAA 120

GACATCGATG TTATCCTGAA AAAGTCTACC ATTCTGAACT TGGACATCAA CAACGATATT 180

ATCTCCGACA TCTCTGGTTT CAACTCCTCT GTTATCACAT ATCCAGATGC TCAATTGGTG 240

CCGGGCATCA ACGGCAAAGC TATCCACCTG GTTAACAACG AATCTTCTGA AGTTATCGTG 300

CACAAGGCCA TGGACATCGA ATACAACGAC ATGTTCAACA ACTTCACCGT TAGCTTCTGG 360

CTGCGCGTTC CGAAAGTTTC TGCTTCCCAC CTGGAACAGT ACGGCACTAA CGAGTACTCC 420

ATCATCAGCT CTATGAAGAA ACACTCCCTG TCCATCGGCT CTGGTTGGTC TGTTTCCCTG 480

AAGGGTAACA ACCTGATCTG GACTCTGAAA GACTCCGCGG GCGAAGTTCG TCAGATCACT 540

TTCCGCGACC TGCCGGACAA GTTCAACGCG TACCTGGCTA ACAAATGGGT TTTCATCACT 600

ATCACTAACG ATCGTCTGTC TTCTGCTAAC CTGTACATCA ACGGCGTTCT GATGGGCTCC 660

GCTGAAATCA CTGGTCTGGG CGCTATCCGT GAGGACAACA ACATCACTCT TAAGCTGGAC 720

CGTTGCAACA ACAACAACCA GTACGTATCC ATCGACAAGT TCCGTATCTT CTGCAAAGCA 780

CTGAACCCGA AAGAGATCGA AAAACTGTAT ACCAGCTACC TGTCTATCAC CTTCCTGCGT 840

GACTTCTGGG GTAACCCGCT GCGTTACGAC ACCGAATATT ACCTGATCCC GGTAGCTTCT 900

AGCTCTAAAG ACGTTCAGCT GAAAAACATC ACTGACTACA TGTACCTGAC CAACGCGCCG 960

TCCTACACTA ACGGTAAACT GAACATCTAC TACCGACGTC TGTACAACGG CCTGAAATTC 1020

ATCATCAAAC GCTACACTCC GAACAACGAA ATCGATTCTT TCGTTAAATC TGGTGACTTC 1080

ATCAAACTGT ACGTTTCTTA CAACAACAAC GAACACATCG TTGGTTACCC GAAAGACGGT 1140

AACGCTTTCA ACAACCTGGA CAGAATTCTG CGTGTTGGTT ACAACGCTCC GGGTATCCCG 1200

CTGTACAAAA AAATGGAAGC TGTTAAACTG CGTGACCTGA AAACCTACTC TGTTCAGCTG 1260

AAACTGTACG ACGACAAAAA CGCTTCTCTG GGTCTGGTTG GTACCCACAA CGGTCAGATC 1320.

GGTAACGACC CGAACCGTGA CATCCTGATC GCTTCTAACT GGTACTTCAA CCACCTGAAA 1380

GACAAAATCC TGGGTTGCGA CTGGTACTTC GTTCCGACCG ATGAAGGTTG GACCAACGAC 1440

TAAGGATCCG CTAGCCCGCC TAATGAGCGG GCTTTTTTTT CTCGGGCAGC GTTGGGTCCT 1500

GGCCACGGGT GCGCATGATC GTGCTCCTGT CGTTGAGGAC CCGGCTAGGC TGGCGGGGTT 1560

GCCTTACTGG TTAGCAGAAT GAATCACCGA TACGCGAGCG AACGTGAAGC GACTGCTGCT 1620

GCAAAACGTC TGCGACCTGA GCAACAACAT GAATGGTCTT CGGTTTCCGT GTTTCGTAAA 1680

GTCTGGAAAC GCGGAAGTCA GCGCTCTTCC GCTTCCTCGC TCACTGACTC GCTGCGCTCG 1740

GTCGTTCGGC TGCGGCGAGC GGTATCAGCT CACTCAAAGG CGGTAATACG GTTATCCACA 1800

GAATCAGGGG ATAACGCAGG AAAGAACATG TGAGCAAAAG GCCAGCAAAA GGCCAGGAAC 1860

CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC CATAGGCTCC GCCCCCCTGA CGAGCATCAC 1920

AAAAATCGAC GCTCAAGTCA GAGGTGGCGA AACCCGACAG GACTATAAAG ATACCAGGCG 1980

TTTCCCCCTG GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT TACCGGATAC 2040

CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC AATGCTCACG CTGTAGGTAT 2100

CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG TGCACGAACC CCCCGTTCAG 2160

CCCGACCGCT GCGCCTTATC CGGTAACTAT CGTCTTGAGT CCAACCCGGT AAGACACGAC 2220

TTATCGCCAC TGGCAGCAGC CACTGGTAAC AGGATTAGCA GAGCGAGGTA TGTAGGCGGT 2280

GCTACAGAGT TCTTGAAGTG GTGGCCTAAC TACGGCTACA CTAGAAGGAC AGTATTTGGT 2340

ATCTGCGCTC TGCTGAAGCC AGTTACCTTC GGAAAAAGAG TTGGTAGCTC TTGATCCGGC 2400

AAACAAACCA CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT TACGCGCAGA 2460

AAAAAAGGAT CTCAAGAAGA TCCTTTGATC TTTTCTACGG GGTCTGACGC TCAGTGGAAC 2520

GAAAACTCAC GTTAAGGGAT TTTGGTCATG AGATTATCAA AAAGGATCTT CACCTAGATC 2580

CTTTTAAATT AAAAATGAAG TTTTAAATCA ATCTAAAGTA TATATGAGTA AACTTGGTCT 2640

GACAGTTACC AATGCTTAAT CAGTGAGGCA CCTATCTCAG CGATCTGTCT ATTTCGTTCA 2700

TCCATAGTTG CCTGACTCCC CGTCGTGTAG ATAACTACGA TACGGGAGGG CTTACCATCT 2760

GGCCCCAGTG CTGCAATGAT ACCGCGAGAC CCACGCTCAC CGGCTCCAGA TTTATCAGCA 2820

ATAAACCAGC CAGCCGGAAG GGCCGAGCGC AGAAGTGGTC CTGCAACTTT ATCCGCCTCC 2880

ATCCAGTCTA TTAATTGTTG CCGGGAAGCT AGAGTAAGTA GTTCGCCAGT TAATAGTTTG 2940

CGCAACGTTG TTGCCATTGC TGCAGGCATC GTGGTGTCAC GCTCGTCGTT TGGTATGGCT 3000

TCATTCAGCT CCGGTTCCCA ACGATCAAGG CGAGTTACAT GATCCCCCAT GTTGTGCAAA 3060 AAAGCGGTTA GCTCCTTCGG TCCTCCGATC GTTGTCAGAA GTAAGTTGGC CGCAGTGTTA 3120

TCACTCATGG TTATGGCAGC ACTGCATAAT TCTCTTACTG TCATGCCATC CGTAAGATGC 3180

TTTTCTGTGA CTGGTGAGTA CTCAACCAAG TCATTCTGAG AATAGTGTAT GCGGCGACCG 3240 AGTTGCTCTT GCCCGGCGTC AACACGGGAT AATACCGCGC CACATAGCAG AACTTTAAAA 3300 GTGCTCATCA TTGGAAAACG TTCTTCGGGG CGAAAACTCT CAAGGATCTT ACCGCTGTTG 3360 AGATCCAGTT CGATGTAACC CACTCGTGCA CCCAACTGAT CTTCAGCATC TTTTACTTTC 3420 ACCAGCGTTT CTGGGTGAGC AAAAACAGGA AGGCAAAATG CCGCAAAAAA GGGAATAAGG 3480 GCGACACGGA AATGTTGAAT ACTCATACTC TTCCTTTTTC AATATTATTG AAGCATTTAT 3540 CAGGGTTATT GTCTCATGAG CGGATACATA TTTGAATGTA TTTAGAAAAA TAAACAAATA 3600 GGGGTTCCGC GCACATTTCC CCGAAAAGTG CCACCTGACG TCTAAGAAAC CATTATTATC 3660 ATGACATTAA CCTATAAAAA TAGGCGTATC ACGAGGCCCT TTCGTCTTCA AG 3712

( 2 ) INFORMATION FOR SEQ ID NO: 5 : ( i ) SEQUENCE CHARACTERISTICS :

(A ) LENGTH : 3769 base pairs

( B ) TYPE : nucleic acid

( C ) STRANDEDNESS : double

( D ) TOPOLOGY : circular ( ii ) MOLECULE TYPE : DNA ( genomic )

( iii ) HYPOTHETICAL : NO

( iii ) ANTI -SENSE : NO (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 5 :

TTCAGGTAAA TTTGATGTAC ATCAAATGGT ACCCCTTGCT GAATCGTTAA GGTAGGCGGT 60

AGGGCCCAGA TCTTAATCAT CCACAGGAGA CTTTCTGATG AAAAACCTTG ATTGTTGGGT 120

CGACAACGAA GAAGACATCG ATGTTATCCT GAAAAAGTCT ACCATTCTGA ACTTGGACAT 180

CAACAACGAT ATTATCTCCG ACATCTCTGG TTTCAACTCC TCTGTTATCA CATATCCAGA 240

TGCTCAATTG GTGCCGGGCA TCAACGGCAA AGCTATCCAC CTGGTTAACA ACGAATCTTC 300

TGAAGTTATC GTGCACAAGG CCATGGACAT CGAATACAAC GACATGTTCA ACAACTTCAC 360

CGTTAGCTTC TGGCTGCGCG TTCCGAAAGT TTCTGCTTCC CACCTGGAAC AGTACGGCAC 420

TAACGAGTAC TCCATCATCA GCTCTATGAA GAAACACTCC CTGTCCATCG GCTCTGGTTG 480

GTCTGTTTCC CTGAAGGGTA ACAACCTGAT CTGGACTCTG AAAGACTCCG CGGGCGAAGT 540

TCGTCAGATC ACTTTCCGCG ACCTGCCGGA CAAGTTCAAC GCGTACCTGG CTAACAAATG 600

GGTTTTCATC ACTATCACTA ACGATCGTCT GTCTTCTGCT AACCTGTACA TCAACGGCGT 660

TCTGATGGGC TCCGCTGAAA TCACTGGTCT GGGCGCTATC CGTGAGGACA ACAACATCAC 720

TCTTAAGCTG GACCGTTGCA ACAACAACAA CCAGTACGTA TCCATCGACA AGTTCCGTAT 780

CTTCTGCAAA GCACTGAACC CGAAAGAGAT CGAAAAACTG TATACCAGCT ACCTGTCTAT 840

CACCTTCCTG CGTGACTTCT GGGGTAACCC GCTGCGTTAC GACACCGAAT ATTACCTGAT 900

CCCGGTAGCT TCTAGCTCTA AAGACGTTCA GCTGAAAAAC ATCACTGACT ACATGTACCT 960

GACCAACGCG CCGTCCTACA CTAACGGTAA ACTGAACATC TACTACCGAC GTCTGTACAA 1020

CGGCCTGAAA TTCATCATCA AACGCTACAC TCCGAACAAC GAAATCGATT CTTTCGTTAA 1080

ATCTGGTGAC TTCATCAAAC TGTACGTTTC TTACAACAAC AACGAACACA TCGTTGGTTA 1140

CCCGAAAGAC GGTAACGCTT TCAACAACCT GGACAGAATT CTGCGTGTTG GTTACAACGC 1200

TCCGGGTATC CCGCTGTACA AAAAAATGGA AGCTGTTAAA CTGCGTGACC TGAAAACCTA 1260

CTCTGTTCAG CTGAAACTGT ACGACGACAA AAACGCTTCT CTGGGTCTGG TTGGTACCCA 1320

CAACGGTCAG ATCGGTAACG ACCCGAACCG TGACATCCTG ATCGCTTCTA ACTGGTACTT 1380

CAACCACCTG AAAGACAAAA TCCTGGGTTG CGACTGGTAC TTCGTTCCGA CCGATGAAGG 1440

TTGGACCAAC GACGGGCCGG GGCCCTCTAG AGGATCCGAT ATCAAGCTTA CTAGTTAATG 1500

ATCCGCTAGC CCGCCTAATG AGCGGGCTTT TTTTTCTCGG GCAGCGTTGG GTCCTGGCCA 1560

CGGGTGCGCA TGATCGTGCT CCTGTCGTTG AGGACCCGGC TAGGCTGGCG GGGTTGCCTT 1620

ACTGGTTAGC AGAATGAATC ACCGATACGC GAGCGAACGT GAAGCGACTG CTGCTGCAAA 1680

ACGTCTGCGA CCTGAGCAAC AACATGAATG GTCTTCGGTT TCCGTGTTTC GTAAAGTCTG 1740

GAAACGCGGA AGTCAGCGCT CTTCCGCTTC CTCGCTCACT GACTCGCTGC GCTCGGTCGT 1800

TCGGCTGCGG CGAGCGGTAT CAGCTCACTC AAAGGCGGTA ATACGGTTAT CCACAGAATC 1860

AGGGGATAAC GCAGGAAAGA ACATGTGAGC AAAAGGCCAG CAAAAGGCCA GGAACCGTAA 1920

AAAGGCCGCG TTGCTGGCGT TTTTCCATAG GCTCCGCCCC CCTGACGAGC ATCACAAAAA 1980

TCGACGCTCA AGTCAGAGGT GGCGAAACCC GACAGGACTA TAAAGATACC AGGCGTTTCC 2040

CCCTGGAAGC TCCCTCGTGC GCTCTCCTGT TCCGACCCTG CCGCTTACCG GATACCTGTC 2100

CGCCTTTCTC CCTTCGGGAA GCGTGGCGCT TTCTCAATGC 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 AGGACAGTAT 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 ATTGCTGCAG 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 GCGTCAACAC 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 TGACGTCTAA GAAACCATTA TTATCATGAC 3720 ATTAACCTAT AAAAATAGGC GTATCACGAG GCCCTTTCGT CTTCA.-GAA 3769

(2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1766 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(v) FRAGMENT TYPE: internal

(ix) FEATURE:

(A) NAME/KEY: hinge region

(B) LOCATION: 923.-934

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

GGGCGAAGTT CGTCAGATCA CTTTCCGCGA CCTGCCGGAC AAGTTCAACG CGTACCTGGC 60

TAACAAATGG GTTTTCATCA CTATCACTAA CGATCGTCTG TCTTCTGCTA ACCTGTACAT 120

CAACGGCGTT CTGATGGGCT CCGCTGAAAT CACTGGTCTG GGCGCTATCC GTGAGGACAA 180

CAACATCACT CTTAAGCTGG ACCGTTGCAA CAACAACAAC CAGTACGTAT CCATCGACAA 240

GTTCCGTATC TTCTGCAAAG CACTGAACCC GAAAGAGATC GAAAAACTGT ATACCAGCTA 300

CCTGTCTATC ACCTTCCTGC GTGACTTCTG GGGTAACCCG CTGCGTTACG ACACCGAATA 360

TTACCTGATC CCGGTAGCTT CTAGCTCTAA AGACGTTCAG CTGAAAAACA TCACTGACTA 420

CATGTACCTG ACCAACGCGC CGTCCTACAC TAACGGTAAA CTGAACATCT ACTACCGACG 480

TCTGTACAAC GGCCTGAAAT TCATCATCAA ACGCTACACT CCGAACAACG AAATCGATTC 540

TTTCGTTAAA TCTGGTGACT TCATCAAACT GTACGTTTCT TACAACAACA ACGAACACAT 600

CGTTGGTTAC CCGAAAGACG GTAACGCTTT CAACAACCTG GACAGAATTC TGCGTGTTGG 660

TTACAACGCT CCGGGTATCC CGCTGTACAA AAAAATGGAA GCTGTTAAAC TGCGTGACCT 720

GAAAACCTAC TCTGTTCAGC TGAAACTGTA CGACGACAAA AACGCTTCTC TGGGTCTGGT 780

TGGTACCCAC AACGGTCAGA TCGGTAACGA CCCGAACCGT GACATCCTGA TCGCTTCTAA 840

CTGGTACTTC AACCACCTGA AAGACAAAAT CCTGGGTTGC GACTGGTACT TCGTTCCGAC 900

CGATGAAGGT TGGACCAACG ACGGGCCGGG GCCCTCTAGA GGATCCGATA TCAAGCTTAC 960

TAGTTAATGA TCCGCTAGCC CGCCTAATGA GCGGGCTTTT TTTTCTCGGG CAGCGTTGGG 1020

TCCTGGCCAC GGGTGCGCAT GATCGTGCTC CTGTCGTTGA GGACCCGGCT AGGCTGGCGG 1080

GGTTGCCTTA CTGGTTAGCA GAATGAATCA CCGATACGCG AGCGAACGTG AAGCGACTGC 1140

TGCTGCAAAA CGTCTGCGAC CTGAGCAACA ACATGAATGG TCTTCGGTTT CCGTGTTTCG 1200

TAAAGTCTGG AAACGCGGAA GTCAGCGCTC TTCCGCTTCC TCGCTCACTG ACTCGCTGCG 1260

CTCGGTCGTT CGGCTGCGGC GAGCGGTATC AGCTCACTCA AAGGCGGTAA TACGGTTATC 1320

CACAGAATCA GGGGATAACG CAGGAAAGAA CATGTGAGCA AAAGGCCAGC AAAAGGCCAG 1380

GAACCGTAAA AAGGCCGCGT TGCTGGCGTT TTTCCATAGG CTCCGCCCCC CTGACGAGCA 1440

TCACAAAAAT CGACGCTCAA GTCAGAGGTG GCGAAACCCG ACAGGACTAT AAAGATACCA 1500

GGCGTTTCCC CCTGGAAGCT CCCTCGTGCG CTCTCCTGTT CCGACCCTGC CGCTTACCGG 1560

ATACCTGTCC GCCTTTCTCC CTTCGGGAAG CGTGGCGCTT TCTCAATGCT CACGCTGTAG 1620

GTATCTCAGT TCGGTGTAGG TCGTTCGCTC CAAGCTGGGC TGTGTGCACG AACCCCCCGT 1680

TCAGCCCGAC CGCTGCGCCT TATCCGGTAA CTATCGTCTT GAGTCCAACC CGGTAAGACA 1740

CGACTTATCG CCACTGGCAG CAGCCA 1766 ( 2 ) INFORMATION FOR SEQ ID NO : 7 : ( i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 1736 base pairs

(B ) TYPE : nucleic acid

( D ) TOPOLOGY : linear

( ii ) MOLECULE TYPE : DNA ( genomic) (v) FRAGMENT TYPE : internal

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 7 :

GGCGGCCTAC GCGATTGACG GCACGGCGGC GGGCGCCATG TACGGCAAGC ACATCACGCT 60

GGTGTCCAGC GATTCAGGCC TGGGCGTGCG CCAGCTCGGC AGCCTGTCCT CGCCATCGGC 120

CATCACCGTG TCGTCGCAGG GCGAAATCGC GCTGGGCGAC GCCACGGTCC AGCGCGGCCC 180

GCTCAGCCTC AAGGGCGCGG GGGTCGTGTC GGCCGGCAAA CTGGCCTCCG GGGGGGGGGC 240

GGTGAACGTC GCGGGCGGCG GGGCGGTGAA GATCGCGTCG GCCAGCAGCG TTGGAAACCT 300

CGCGGTGCAA GGCGGCGGCA AGGTACAGGC CACGCTGTTG AATGCCGGGG GGACGTTGCT 360

GGTGTCGGGC CGCCAGGCCG TCCAGCTTGG CGCGGCGAGC AGCCGTCAGG CGCTGTCCGT 420

GAACGCGGGC GGCGCCCTCA AGGCGGACAA GCTGTCGGCG ACGCGACGGG TCGACGTGGA 480

TGGCAAGCAG GCCGTCGCGC TGGGGTCGGC CAGCAGCAAT GCGCTGTCGG TGCGTGCCGG 540

CGGCGCCCTC AAGGCGGGCA AGCTGTCGGC GACGGGGCGA CTGGACGTGG ACGGCAAGCA 600

GGCCGTCACG CTGGGTTCGG TTGCGAGCGA CGGTGCGCTG TCGGTAAGCG CTGGCGGAAA 660

CCTGCGGGCG AACGAATTGG TCTCCAGTGC CCAACTTGTG GTGCGTGGGC AGCGGGAGGT 720

CGCGCTGGAT GACGCTTCGA GCGCACGCGG CATGACCGTG GTTGCCGCAG GAGCGCTGGC 780

GGCCCGCAAC CTGCAGTCCA AGGGCGCCAT CGGCGTACAG GGTGGAGAGG CGGTCAGCGT 840

GGCCAACGCG AACAGCGACG CGGAATTGCG CGTGCGCGGG CGCGGCCAGG TGGATCTGCA 900

CGACCTGAGC GCAGCGCGCG GCGCGGATAT CTCCGGCGAG GGGCGCGTCA ATATCGGCCG 960

TGCGCGCAGC GATAGCGATG TGAAGGTCTC CGGGCACGGC GCCTTGTCGA TCGATAGCAT 1020

GACGGCCCTC GGTGCGATCG GCGTCCAGGC AGGCGGCAGC GTGTCGGCCA AGGATATGCG 1080

CAGCCGTGGC GCCGTCACCG TCAGCGGCGG CGGCGCCGTC AACCTGGGCG ATGTCCAGTC 1140

GGATGGGCAG GTCCGCGCCA CCAGCGCGGG CGCCATGACG GTGCGAGACG TCGCGGCTGC 1200

CGCCGACCTT GCGCTGCAGG CGGGCGACGC GTTGCAGGCC GGGTTCCTGA AATCGGCCGG 1260

TGCCATGACC GTGAACGGCC GCGATGCCGT GCGACTGGAT GGCGCGCACG CGGGCGGGCA 1320

ATTGCGGGTT TCCAGCGACG GGCAGGCTGC GTTGGGCAGT CTCGCGGCCA AGGGCGAGCT 1380

GACGGTATCG GCCGCGCGCG CGGCGACCGT GGCCGAGTTG AAGTCGCTGG ACAACATCTC 1440

CGTGACGGGC GGCGAACGCG TGTCGGTTCA GAGCGTCAAC AGCGCGTCCA GGGTCGCCAT 1500

TTCGGCGCAC GGCGCGCTGG ATGTAGGCAA GGTTTCCGCC AAGAGCGGTA TCGGGCTCGA 1560

AGGCTGGGGC GCGGTCGGAG CGGACTCCCT CGGTTCCGAC GGCGCGATCA GCGTGTCCGG 1620

GCGCGATGCG GTCAGGGTCG ATCAAGCCCG CAGTCTTGCC GACATTTCGC TGGGGGCGGA 1680

AGGCGGCGCC ACGCTGGGCG CGGTGGAGGC CGCCGGTTCG ATCGACGTGC GCGGCG 1736