Expression of recombinant proteins in attenuated bacteria

The invention concerns a vaccine comprising an attenuated Salmonella bacterium which contains a nirB promoter operably linked to a DNA sequence encoding a heterologous protein. The nirB promoter directs expression of the heterologous protein in a host it is wished to vaccinate.

This invention relates to attenuated bacteria capable of expressing a 
heterologous protein, to their preparation and to vaccines containing 
them. 
Virulent strains of Salmonella can be attenuated by introducing specific 
mutations into genes required for survival and growth in vivo. Attenuated 
variants which establish self limiting, clinically insignificant 
infections can be considered as potential live oral vaccines against 
Samonella infections. Ty21a is an attenuated variant of Salmonella typhi, 
which harbours mutations in galE and other unknown attenuating lesions, 
and is licensed for use in many countries as a live oral typhoid vaccine. 
More recently genetically defined Salmonella strains harbouring individual 
specific mutants in different genes have been tested as experimental oral 
vaccines in several target species. For example, Salmonella aro mutants, 
which have an auxotrophic requirement for several aromatic compounds, have 
been shown to be effective oral vaccines in mice, sheep, cattle, chickens 
and more recently they have been shown to be attenuated and immunogenic in 
volunteers. Salmonella double aro mutants are disclosed in EP-A-0322237. 
Salmonella cya crp double mutants are also effective oral vaccines. 
As well as being vaccines in their own right against salmonellosis, 
attenuated Salmonellae can be considered as carriers of heterologous 
antigens to the immune oral system. This is because Salmonellae can be 
delivered via the oral route and are potent immunogens being able to 
stimulate systemic and local cellular and antibody responses. Heterologous 
antigens from bacteria, viruses and parasites can be delivered to the host 
using Salmonella vaccines. 
One potentially serious drawback in using these live vaccines for antigen 
delivery relates to problems with the stability of the foreign antigen 
expression in vivo. Unregulated expression of high levels of a foreign 
protein in bacteria from multiple copy plasmids usually results in rapid 
loss of the plasmid or expressed gene from the cells. This problem can be 
controlled in fermenters by using inducible promoter systems such as trp 
or lac to allow the controlled induction of gene expression when the 
appropriate biomass has been achieved. Obviously these promoters can not 
be induced by exogenously applied inducers such as PP or IPTG when 
bacteria are growing in host tissues during the self-limited growth 
following vaccination. 
In vivo plasmid instability during vaccination with live bacterial vectors 
has in fact been reported by many workers (Maskell et al, Microb.Path 2, 
295-305, 1987; Nakayama et al, Bio/technology 6, 693-697, 1988; Tire et 
al, Immunology 70, 540-546, 1990). A number of approaches have been taken 
to overcome the problem including the use of integration systems for 
expression of the heterologous antigen from the bacterial chromosome (Hone 
et al, Microbiol. Path. 5, 407-418, 1988; Strugnell et al, Gene 88, 57-63, 
1990). However, this approach is only suitable for use with some antigens 
since expression levels are often quite low (Maskel et al, 1987). Nakayama 
et al described the use of linking an essential gene to the expression 
plasmid for stabilizing in vivo expression. Although this is a highly 
effective approach, it does not prevent the generation of plasmid free 
variants but simply ensures they do not survive. Further stable but 
constitutive high level expression of a foreign antigen in a Salmonella 
vaccine strain could slow down the growth rate and hence potentially 
effect the immunogenicity of the live vaccine. 
According to the present invention, there is provided an attenuated 
bacterium which is capable of expressing a heterologous protein, the 
expression of the heterologous protein being under the control of a 
promoter whose activity is induced by anaerobic conditions. 
Stable expression of the heterologous protein can be obtained in vivo. The 
attenuated bacterium can therefore be used as a vaccine. Any suitable 
bacterium may be employed, for example a Gram-negative bacterium. Some 
Gram-negative bacteria such as Salmonella invade and grow within 
eucaryotic cells and colonise mucosal surfaces. 
The attenuated bacterium may therefore be selected from the genera 
Salmonella, Bordetella, 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. typhimurium--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. Such bacteria are disclosed in EP-A-0322237. Double 
aro mutants which are suitable are aroA aroC, aroA aroD and aroA aroE 
mutant bacteria. Other bacteria having mutations in other combinations of 
the aroA, aroC, aroD and aeoE genes are however useful. Particularly 
preferred are Salmonella double aro mutants, for example double aro 
mutants of S.typhi or Syphimurium, in particular aroA aroC, aroA aroD and 
aroA aroE mutants. 
Alternatively, the attenuated bacterium may harbour a non-reverting 
mutation in a gene concerned with the regulation of one or more other 
genes (EP-A-0400958). Preferably the mutation occurs in the ompR gene or 
another gene involved in regulation. There are a large number of other 
genes which are concerned with regulation and are known to respond to 
environmental stimuli (Ronson et al, Cell 49, 579-581). 
This type of attenuated bacterium may harbour a second mutation in a second 
gene. Preferably the second gene is a gene encoding for an enzyme involved 
in an essential biosynthetic pathway, in particular genes involved in the 
pre-chorismate pathway involved in the biosynthesis of aromatic compounds. 
The second mutation is therefore preferably in the aroA, aroC or aroD 
gene. 
Another type of attenuated bacterium is one in which attenuation is brought 
about by the presence of a non-reverting mutation in DNA of the bacterium 
which encodes, or which regulates the expression of DNA encoding, a 
protein that is produced in response to environmental stress. Such 
bacteria are disclosed in WO 91/15572. The non-reverting mutation may be a 
deletion, insertion, inversion or substitution. A deletion mutation may be 
generated using a transposon. 
Examples of proteins that are produced in response to environmental stress 
include heat shock proteins (which are produced in response to a 
temperature increase above 42.degree. C.); nutrient deprivation proteins 
(which are produced in response to levels of essential nutrients such as 
phosphates or nitrogen which are below that which the microorganism 
requires to survive); toxic stress proteins (which are produced in 
response to toxic compounds such as dyes, acids or possibly plant 
exudates); or metabolic disruption proteins (which are produced in 
response to fluctuations in for example ion levels affecting the 
microorganisms ability to osmoregulate, or vitamin or co-factor levels 
such as to disrupt metabolism). 
Preferably a heat shock protein is the one encoded by the htrA gone, also 
characterised as degP. Other proteins are encoded by genes known to be 
involved in the stress response such as grpE, groEL, (moPA), dnaK, groES, 
lon and dnaJ. There are many other proteins encoded by genes which are 
known to be induced in response to environmental stress (Ronson et al, 
Cell 49, 579-581). Amongst these the following can be mentioned: the 
ntrB/ntrC system of E. coli, which is induced in response to nitrogen 
deprivation and positively regulates glnA and nifLA (Buck et al., Nature 
320, 374-378, 1986; Hirschman et al., Proc. Natl. Acad. Sci. USA, 82, 
7525, 1985; Nixon et al., Proc. Natl. Acad. Sci. USA 83, 7850-7854, 1986, 
Reitzer and Magansanik, Cell 45, 785, 1986); the phoR/phoB system of E. 
coli which is induced in response to phosphate deprivation (Makino et al., 
J. Mol. Biol. 192, 549-556, 1986b); the cpxA/sfrA system of E. coli which 
is induced in response to dyes and other toxic compounds (Albin et al., J. 
Biol. Chem. 261 4698, 1986; Drury et al., J. Biol. Chem. 260, 4236-4272, 
1985). An analogous system in Rhizobium is dctB/dctD, which is responsive 
to 4C-discarboxylic acids (Ronson et al., J. Bacteriol. 169, 2424 and Cell 
49, 579-581, 1987). A virulence system of this type has been described in 
Agrobacterium. This is the virA/virG system, which is induced in response 
to plant exudates (le Roux et al., EMBO J. 6, 849-856, 1987; Stachel and 
Zambryski., Am. J. Vet. Res 45, 59-66, 1986; Winans et al., Proc. Natl. 
Acad. Sci. USA, 83, 8278, 1986). Similarly the bvgC-bvgA system in 
Bordetela pertussis (previously known as vir) regulates the production of 
virulence determinants in response to fluctuations in Mg2+ and nicotinic 
acid levels (Arico et al, 1989, Proc. Natl. Acad. Sci. USA 86, 6671-6675). 
For use in the form of a live vaccine, an attenuated bacterium should not 
revert back to the virulent state. The probability of this happening with 
a mutation in a single DNA sequence is considered to be small. However, 
the risk of reversion occurring with a bacterium attenuated by the 
presence of mutations in each of two discrete DNA sequences is considered 
to be insignificant. A preferred attenuated bacterium is therefore one in 
which attenuation is brought about by the presence of a mutation in a DNA 
sequence which encodes, or which regulates the expression of DNA encoding, 
a protein that is produced in response to environmental stress and by the 
presence of a mutation in a second DNA sequence. 
The second DNA sequence preferably encodes an enzyme involved in an 
essential auxotrophic pathway or is a sequence whose product controls the 
regulation of osmotically responsive genes, i.e. ompR, (Infect and Immun 
1989 2136-2140). Most preferably, the mutation is in a DNA sequence 
involved in the aromatic amino acid biosynthetic pathway, more 
particularly the DNA sequences encoding aroA, aroC or aroD. 
Attenuated bacteria may be constructed by the introduction of a mutation 
into the DNA sequence by methods known to those skilled in the art 
(Maniatis, Molecular Cloning and Laboratory Manual, 1982). Non-reverting 
mutations can be generated by introducing a hybrid transposon TnphoA into, 
for example, S.typhimurium strains. TnphoA can generate enzymatically 
active protein fusions of alkaline phosphatase to periplasmic or membrane 
proteins. The TnDhoA transposon carries a gene encoding kanamycin 
resistance. Transductants are selected that are kanamycin resistant by 
growing colonies on an appropriate selection medium. 
Alternative methods include cloning the DNA sequence into a vector, e.g. a 
plasmid or cosmid, inserting a selectable marker gene into the cloned DNA 
sequence, resulting in its inactivation. A plasmid carrying the 
inactivated DNA sequence and a different selectable marker can be 
introduced into the organism by known techniques (Maniatis, Molecular 
Cloning and Laboratory Manual, 1982). It is then possible by suitable 
selection to identify a mutant wherein the inactivated DNA sequence has 
recombined into the chromosome of the microorganism and the wild-type DNA 
sequence has been rendered non-functional in a process known as allelic 
exchange. In particular, the vector used is preferably unstable in the 
microorganism and will be spontaneously lost. The mutated DNA sequence on 
the plasmid and the wild-type DNA sequence may be exchanged by a genetic 
cross-over event. Additional methods eliminate the introduction of foreign 
DNA into vaccine strains at the site of mutations and the introduction of 
antibiotic resistant markers into the strains. 
The heterologous antigen which an attenuated bacterium is capable of 
expressing may for example comprise an antigenic determinant of a 
pathogenic organism. The antigen may be derived from a virus, bacterium, 
fungus, yeast or parasite. The heterologous protein therefore typically 
comprises an antigenic sequence derived from a virus, bacterium, fungus, 
yeast or parasite. More especially, the antigenic sequence may be derived 
from a type of human immunodeficiency virus (HIV) such as HIV-1 or HIV-2, 
hepatitis A or B virus, human rhinovirus such as type 2 or type 14, herpes 
simplex virus, poliovirus type 2 or 3, foot-and-mouth disease virus, 
influenza virus, coxsackie virus, the cell surface antigen CD4 and 
Chlamydia trachomatis. The antigen may comprise the CD4 receptor binding 
site from HIV, for example from HIV-1 or -2. Other useful antigens include 
E. coli heat labile toxin B subunit (LT-B), E. coli K88 antigens, P.69 
protein from B. pertussis, tetanus toxin fragment C and antigens of 
flukes, mycoplasma, roundworms, tapeworms, rabies virus and rotavirus. 
A preferred promoter for use in controlling the expression of the 
heterologous protein is the nirB promoter. The nirB promoter has been 
isolated from E. coli, where it directs expression of an operon which 
includes the nitrite reductase gene nirB (Jayaraman et al, J. Mol. Biol. 
196, 781-788, 1987), and nirD, nirC and cvsG (Peakman et al, Eur. J. 
Biochem. 191, 315-323, 1990). It is regulated both by nitrite and by 
changes in the oxygen tension of the environment, becoming active when 
deprived of oxygen (Cole, Biochim. Biophys. Acta, 162, 356-368, 1968). 
Response to anaerobiosis is mediated through the protein FNR, acting as a 
transcriptional activator, in a mechanism common to many anaerobic 
respiratory genes. 
By deletion and mutational analysis the part of the promoter which responds 
solely to anaerobiosis has been isolated and by comparison with other 
anaerobically-regulated promoters a consensus FNR-binding site was 
identified (Bell et al, Nucl. Acids. Res. 17, 3865-3874, 1989; Jayaraman 
et al, Nucl. Acids Res. 17, 135-145, 1989). It was also shown that the 
distance between the putative FNR-binding site and the -10 homology region 
is critical (Bell et al, Molec. Microbiol. 4, 1753-1763, 1990). It is 
therefore preferred to use only that part of the nirB promoter which 
responds solely to anaerobiosis. As used herein references to the nirB 
promoter refer to the promoter itself or a part or derivative thereof 
which is capable of promoting expression of a coding sequence under 
anaerobic conditions. The sequence which we have in fact used and which 
contains the nirB promoter is: 
AATTCAGGTAAATTTGATGTACATCAAATGGTACCCCTTGCTGAATCGTTAAGGTA GGCGGTAGGGCC (SEQ 
ID NO: 1) 
An attenuated bacterium according to the present invention may be prepared 
by transforming an attenuated bacterium with a DNA construct comprising a 
promoter whose activity is induced by anaerobic conditions, such as the 
nirB promoter, operably linked to a DNA sequence encoding a heterologous 
protein. Any suitable transformation technique may be employed, such as 
electroporation. In this way, an attenuated bacterium capable of 
expressing a protein 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 DNA construct is typically a replicable expression vector comprising 
the nirB promoter operably linked to a DNA sequence encoding the 
heterologous protein. The nirB promoter may be inserted in an expression 
vector, which already incorporates a gene encoding the heterologous 
protein, in place of the existing promoter controlling expression of the 
protein. The expression vector should of course be compatible with the 
attenuated bacterium into which the vector is to be inserted. 
The expression vector is provided with appropriate transcriptional and 
translational control elements including, besides the nirB promoter, a 
transcriptional termination site and translational start and stop codons. 
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 a plasmid. 
An attenuated bacterium of the invention can be used as a vaccine. The 
vaccine comprises a pharmaceutically acceptable carrier or diluent and, as 
active ingredient, the attenuated bacterium. 
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, Eudragate 
"S", Eudragate "L", Cellulose acetate, cellulose phthalate or 
hydroxypropylmethyl 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 a 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. 
The attenuated bacterium of the invention may be used in the prophylactic 
treatment of a host, particularly a human host but also possibly an animal 
host. An infection caused by a microcrganism, 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 capable of raising antibody to the microorganism. 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. However, for attenuated S.typhi a dosage 
comprising the oral administration of from 10.sup.9 to 10.sup.11 S.typhi 
organisms per dose is generally convenient for a 70 kg adult human host. 
The following Example illustrates the invention.

EXAMPLE 
Construction of pTETnir15 
Expression plasmid pTETnir15 was constructed from pTETtac115 (Makoff et al, 
Nucl. Acids Res. 17 10191-10202, 1989) by replacing the EcoRI-ADaI region 
(1354bp) containing the lacI gene and tac promoter with the following pair 
of oligos 1 and 2: 
##STR1## 
The oligonucleotides were synthesized on a Pharmacia Gene Assembler and the 
resulting plasmids confirmed by sequencing (Makoff et al, Bio/Technology 
7, 1043-1046, 1989). 
Construction of SL1334 aroA aroD harbouring pTETnir15 
In order to construct a Salmonella vaccine strain expressing tetanus toxin 
fragment C under the control of the nirB promoter, an intermediate strain, 
S.typhimurium LB5010 (r.sup.-m+) (Bullas and Ryo, J. Bact. 156, 471-474, 
1983), was transformed with pTETnir15. Colonies expressing fragment C were 
detected by antibiotic selection followed by colony immunoblotting with 
anti-tetanus toxin fragment C sera. Colonies were grown overnight on 
nitrocellulose filters aerobically and then induced by incubating under 
anaerobic conditions for four hours prior to immunoblotting. One strain 
that was stably expressing fragment C was used to prepare plasmid DNA. 
This was used to transform an isolate of S.typhimurium SL1344 aroA aroD 
designated BRD509 by electroporation. A strain that was stably expressing 
fragment C (checked by immunoblotting as described above) was chosen for 
the in vivo studies and was designated BRD847. 
Comparison of in vivo kinetics of BRD743 and BRD847 in BALB/c mice 
The ability of BRD743 (BRD509 harbouring pTET85) and of BRD847 to grow in 
vivo was compared after oral administration to BALB/c mice. pTET85 was 
constructed from pTETtac115 (Makoff et al, Nucl. Acids Res. 17, 
10191-10202, 1989) by deleting the 1.2 kb EcoRI fragment carrying the lacI 
gene. This resulted in the constitutive expression of fragment C in 
Salmonella strains. Numbers of bacteria were enumerated in livers, 
spleens, Peyers patches and mesenteric lymph nodes. The bacteria isolated 
from mice were also assessed for their ability to grow on plates 
containing ampicillin as an indicator of the percentage of organisms still 
retaining the plasmid expressing fragment C. The results are shown in 
FIGS. 1 to 4. 
When similar initial numbers of organisms (5.times.10.sup.9) were used to 
infect mice it was found that both BRD743 and 847 were able to invade into 
and persist in all the murine tissues examined but at a lower level than 
BRD509. However, the interesting feature is that the number of ampicillin 
resistant organisms obtained from mice infected with BRD743 decreases 
rapidly and all organisms recovered were ampicillin sensitive by day 14. 
This indicates that in vivo selection rapidly results in the loss of 
pTET85 from the Salmonella vaccine strain. In contrast, counts with and 
without ampicillin for BRD847 were essentially the same for the time the 
infections were monitored. This demonstrates the added advantage of 
pTETnir15 in the S.typhimurium vaccine strain resulting in organisms with 
the potential to express fragment C in vivo for a longer period of time 
with obvious advantages in terms of immunogenicity. 
Immunisation of BALB/c mice using Salmonella strains harbouring pTET85 
(BRD743) or pTETnir15 (BRD847) 
Groups of twenty mice were incubated orally with 5.times.10.sup.9 cells per 
mouse of either BRD743, BRD847 or BRD509. On day 25 sera were collected 
from all mice and analysed by ELISA for anti-tetanus antibodies. All mice 
vaccinated with BRD847 had detectable anti-fragment C antibody at 25 days 
whereas those vaccinated with BRD743 or BRD509 did not (FIG. 5). On day 25 
ten mice from each group were boosted by oral inoculation with a similar 
amount of homologous organisms. ELISA analysis of the serum taken from 
these mice at day 46 showed that the anti-fragment C responses had been 
boosted for groups inoculated with BRD743 and BRD847. The titres for those 
mice boosted with BRD847 was significantly higher than for those mice 
boosted with BRD743. Mice boosted orally with BRD509 failed to produce a 
detectable antibody response to fragment C. 
Tetanus toxin challenge of mice orally immunised with BRD847 and 743 
The mice vaccinated orally with BRD743, 847 and 509 were tested for 
immunity against tetanus toxin challenge after one or two doses of the 
immunising strain. Groups of twenty mice received one single oral dose of 
5.times.10.sup.9 organisms and groups of ten mice were challenged on day 
25 with 500 50% lethal doses of tetanus toxin (see Table 1). Mice 
vaccinated with BRD847 were completely protected against challenge after a 
single oral dose whereas those vaccinated with BRD743 were only partially 
protected (2/10 survivors). The remaining groups of 10 mice received a 
second dose of organisms (5.times.10.sup.9) on day 25 and were challenged 
on day 46 (after the 1st dose). Again mice immunised with BRD847 were 
completely protected after challenge with tetanus toxin whereas those 
immunised with BRD743 were only partially protected (5/10). Mice immunised 
with 1 or 2 doses of BRD509 and challenged with tetanus toxin all died. 
BRD847 is an effective single dose oral vaccine against tetanus toxin 
challenge in mice. Groups of mice were also challenged with tetanus toxin 
after receiving 1 and 2 intravenous doses of 10.sup.5 organisms of BRD847 
and BRD743. All mice were fully protected against challenge with tetanus 
toxin after 1 or 2 doses of vaccine strain. 
TABLE 1 
______________________________________ 
Oral immunisation of mice against tetanus using 
S. typhimurium SL1344 aroA aroD pTET85 and S. typhimurium 
SL1344 aroA aroD pTETnirl5 
No. of mice 
No. surviving tetanus 
Vaccine Dose Doses challenge 
______________________________________ 
SL1344 aroA aroD 
8.6 .times. 10.sup.9 
1 0/10 
(BRD509) 7.4 .times. 10.sup.9 
2 0/10 
SL1344 aroA aroD 
6.4 .times. 10.sup.9 
1 2/10 
pTET85(BRD743) 
8.2 .times. 10.sup.9 
2 5/10 
SL1344aroA aroD 
9.5 .times. 10.sup.9 
1 10/10 
pTETnir15(BRD847) 
7.5 .times. 10.sup.9 
2 9/9 
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SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 2 
(2) INFORMATION FOR SEQ ID NO: 1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 68 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: 
AATTCAGGTAAATTTGATGTACATCAAATGGTACCCCTTGCTGAATCGTTAAGGTAGGCG60 
GTAGGGCC68 
(2) INFORMATION FOR SEQ ID NO: 2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 60 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 
CTACCGCCTACCTTAACGATTCAGCAAGGGGTACCATTTGATGTACATCAAATTTACCTG60 
__________________________________________________________________________