Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
- 1 - 26361-74
New recombinant herpes viruses, a vaccine based on these
recombinants, their preparation ~rocess, ~enes, vectors and
plasmlds used in this process.
The present invention concerns recombinant herpes
viruses, in particular, for producing vaccines, their process of
preparation and the plasmids produced during this process. More-
over, it concerns a part of the chromosome of Marek's disease
virus (MDV) which can be used for preparing such vaccines.
Different types of viruses have been used as expression
vectors of foreign genes, in particular of genes coding for anti-
genic proteins, and have proven their potential for immunizing
animals. The vaccinia virus has, to a great extent, been used for
constructing recombinant viruses. The herpes viruses have also
been used: the herpes simplex virus (HSV) (M. Shih et al., Proc.
Natl. Acad. Sci., USA. 1984, 81, 5867-5870), the varicella virus
(VZV) (R. Lowe et al., Proc. Natl. Acad. Sci., USA. 1987, 84,
3896-3900). The foreign gene is inserted into a fragment of the
genomic DNA of the herpes virus, corresponding to a non-essential
region for the viral replication, cloned in a plasmid. This gene
is transferred into the viral genome by homologous recombination.
This latter step is carried out by cotransfection of the herpes
genomic DNA and plasmid since this genomic DNA is by nature infec-
tious.
Different genes of herpes viruses have been identified
as non-essential to viral growth, certain of these genes being
associated with virulence.
- 2 - 26~1~7~
The gene of the thymidine kinase of the herpes simplex
virus (D. Dubbs et al., Virology, 1465, 22, 493-502), of
the Aujeszky virus ~G. Tatarov, Zentralbl. Vet. Med.,
1968, 15 B! 848853), of the rhinotracheal infectious bovine
virus (S. Kit et al., Virology, 1983, 130, 381-389).
- The gene gIII of the Aujeszky virus (A. Robins e-t al., J.
Virol., 1986, 59, 635-645).
- The gene gX of the Aujeszky virus (D. Thomsen et al., J.
Virol., 1987, 61, 229-232).
- The gene gI of the Aujeszky virus (C. Mettenleiter et al.,
J. Virol., 1987, 61, 4030-4032).
Viruses in which one or other of these genes has been
deleted nonetheless retain the capacity to produce a latent infec-
tion in mice.
Studies pertaining to the unique short region of the
genome of the herpes simplex virus HSV-l have been conducted (B.
Megnier et al., Virology, 1988, 162, 251-254) and have shown that
the viruses HSV-l in the short region of which a gene has been
deleted, have undergone an attenuation.
For their part, F'.C. Purves et al. (Journal oE Virology,
1987, vol. 61, No. 9, 2896-2901) have demonstrated that the open
reading frame US3 of the short fragment of the HSV-l virus genome
codes for a virus enzyme, the kinase protein, and is not essential
to the replication of said virus.
Studies have been undertaken on Marek's disease virus
which belongs to the subfamily of gamma herpes viruses. This is
an enveloped virus having a double stranded linear genomic DNA of
- 3 - 263~
about 175 kilobases. Its genome is composed of a long segment
(UL) and of a short segment (Us) framed by repeated inversed ter-
minal sequences.
Marek's disease virus causes paralysis and a lympho-
proliferative disease in chickens, usually, at the age of 2 to 5
months. This disease results in very significant economic losses
(L. Payne, Biology of Marek's Disease Virus and the Herpes Virus
of Turkeys, in The Herpes Virus, vol. 1, pp. 347-431, edited by B.
Roizman, Plenum Press).
The strains of Marek's disease virus have been
classified into three serotypes:
- serotype 1 comprises the pathogenic strains and attenuated
strains derived therefrom.
- serotype 2 comprises the naturally attenuated strains.
- serotype 3 comprises the herpes virus of turkeys (HVT) and
its variants.
Consequently, the term Marek's attenuated disease virus
will designate serotypes 1, 2 and 3 at the same time.
Marek's disease virus (MDV) and herpes virus of turkeys
2~ (HVT) have similar genomic arrangements (A. Buckmaster et al., J.
Gen. Virol., 1988, 69, 2033-2042) and numerous homologies of
sequence all along their genome (C. Gibbs et al., Proc. Acad.
Natl. Sci. USA, 1984, 81, 3365-3369).
The chicks are vaccinated at the age of one day and are
then protected against Marek's disease for their entire life. For
numerous years, vaccination with the herpes virus of live turkeys
(HVT) has been very effective for controlling the disease.
4 ~ 2 6 ~j~6~ ,7,~ r
Nevertheless. -the emergence of new viral strains which
are highly virulent has led to the use of strains of attenuated
Marek's disease viruses of another serotype, to vaccinate and thus
increase the level of protection, either, for example, the strain
CVI 988, MDV serotype 1 attenuated by passing over cells (B.
Rispens et al., Avian Dis., 1972, 16, 1108-125), or, for example,
the association of the HVT MDV serotype 3 and SBl strains, MDV
serotype 2 (K. Schat et al., J. Natl. Cancer Inst., 1987, 60,
1075-1082).
The genome of Marek's virus has certain similarities
with the genome of alpha viruses, herpes simplex (~SV) and
chickenpox (VZV). Most of the genes localized in the long region
UL of the genome are approximately colinear between the herpes
simplex virus, chickenpox (D. McGeogh, J. Gen. Virol., 1988, 69,
1531-1574) and Marek's disease virus (A. Buckmaster, 1988). Thus.
in the international patent application WO 90/02802, it was pro-
posed that the genes be inserted into this UL region of HVT and
MDV.
On the other hand, the loca]ization of genes in the Us
segment shows a laryer divergence between herpes viruses. Also
among the dozen open reading frames identified for the herpes
simplex virus, only four have a homology with the chickenpox virus
(McGeogh, 1988, cited above).
In fact, the prior art does not suggest that there is an
interest in proceeding with a homology study of open reading
frames of the unique short region of the genome of Marek's disease
virus with the HSV-l genes.
- 5 -- 263~6~7~
( J
The present invention divulges, for the first time, the
sequence coding for the kinase protein of the genome of Marek's
disease virus (MDV) and allows one to establish, in a surprising
manner, that this gene can be deleted without blocking the viral
replication and allowing the insertion of heterologous sequences
opening the way for development of a series of viral expression
vectors.
This type of a recombinant and at-tenuated virus of
Marek's disease constitutes a choice candidate for developing a
viral vector expressing foreign genes to be used for polyvalent
vaccination of poultry since it has the advantage of being able to
be used for its own vaccinal properties and as a vaccine against
other viral, bacterial and parasitic diseases as, for example,
infectious avian bronchitis, Newcastle's disease, fowl plague,
egg-drop syndrome, Gumboro's disease, chicken anaemia, coccidio-
sis, fowl pox, infectious laryngotracheitis, avian colibacillos,
is pasteurellosis, haemophilosis.
SUMMARY OF THE INVENTION
The object of the invention is to provide recombinant
herpes viruses, including a recombinant attenuated Marek's disease
virus (serotypes 1, 2 or 3), recombinant which can be used as a
vaccine, the method for constructing such a recombinant virus as
well as a vector virus allowing the multivalent vaccination
against viral, bacterial or parasitic aviall diseases.
One object of the invention is the nucleotide sequence
- 6 - 263~
and its variants corresponding to the US3 gene which is homologous
to the kinase protein gene of the herpes simplex virus and the
surrounding regions. The term variants of the nucleotide se-
quence, as commonly used, means any equivalent sequence such as
obtained, for example, by degeneration of the code, minor modifi-
cations, mutations or corresponding to viral variants.
Another object of the invention is a recombinant virus
selected from the herpes viruses, including the viruses of
pseudorabies disease, infectious bovine rhinotracheitis, equine
rhinopneumonitis, feline rhinotracheitis, canine herpes.
Another object of the invention is also a Marek's
disease recombinant virus comprising one or more heterologous
genes inserted in the region of its genome corresponding to US3
gene in such a way so as to be expressed.
By "heterologous gene", one means, in particular, a gene
coding for a protein or an immunogenic glycoprotein of a viral,
bacterial or parasitic pathogenic agent, in particular, an agent
associated with an avian pathology. This also relates to the
construction of hybrid viruses, for example, by introducing, into
the genome of a turkey herpes virus, genes coding for immunogenes
of a Marek's disease virus of serotype 1 and/or serotype 2.
"Heterologous gene" is also intended to mean a gene
coding for a peptide or a protein, for example, hormone, growth
factor, immunomodulator.
The heterologous gene is preferably expressed under the
control of regulating sequences of US3 gene transcription. One
can, however, see to it that this expression is either controlled
- 7 - 26361-74
by a promoting sequence coming from another gene of the virus in
question, for example, the promoter of TK gene, of gA gene, of gB gene
or from another herpes virus, for example, the promoter of gI gene of
the infectious bovine rhinotracheitis virus or from gene II or gene
III of pseudorabies virus.
Preferably, the start and stop codons of US3 gene are
substituted by those of the gene to be expressed.
DESCRIPTION OF THE FIGURES
Figure 1 shows the construction of the plasmid pMDV 53L
which contains the lacZ gene inserted instead of US3 gene.
Figure 2 shows the construction of the plasmid pMDV 53 CL
whlch contains the lacZ gene under the control of the promoter iE of
the human cytomegalovirus instead of US3 gene.
Figure 3 shows the plasmid pMDV 53F which contains the gene
of the fusion protein of Newcastle's disease virus instead of US3
gene.
DESCRIPTION OF THE INVENTION: MATERIALS AND METHODS
Viral Strain
The serotype 1 strain RBlB of Marek's disease virus (MDV)
was used (Schat K.A. et al., 1982, Avian Pathol. 11, 593-605).
The virus culture methods and methods for extraction of
viral DNA having a high molecular weight have been described (C. Lee
et aL., 1980, J. Gen. Virol., 51, 235-253; N. Ross et al., 1989, 70,
1789-1804).
'~ , . , ~ . ', .
- 8 ~ ? ~!J 2~361-74
Cell Culture
The fibroblasts of chicken embryo (CEP) were cultivated
in 199 F10, medium supplemented by penicillin, streptomycin,
fungizone and fetal calf serum, as described (N. Ross, 1975, J.
Gen. Virol., 28, 37-47).
Cloning of Viral DNA
Generally, the techniques used for the construction of
recombinant plasmids are those described by T. Maniatis et al.
(T. Maniatis et al., 1982, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor. NY).
For all clonlng and subcloning steps, the vector
linearized by the appropriate restriction enzymes is
dephosphorized before ligation. The purification of the DNA
fragments starting from an agarose gel is done according to the
technique described by the manufacturer: "Geneclean" (Bio 101,
San Diego, California, USA).
Sequencing
The cloned fragments are sequenced according to the
classic technique described by Sanger (G. Sanyer, S. Nicklen. A.
Coulson, 1977, Proc. Natl. Acad., USA 74, 5463-5467). The
sequences aEter translation have been compared to -the published
sequences of herpes simplex virus and chickenpox (McGeogh, 1985,
J. Mol. Biol. 181, 1, 13; K. Davison et al., 1986, J. Gen. Virol.
67, 1759-1816).
Contro]led Mutagenesis
The DNA fragments, subcloned in the Blue Script vector
(Stratagene, La Jolla, California, USA) are mutagenesized after
- 9 - 2 ~ ~ fJ ~ 26361-74
separation of the DNAs simple fragment with the help of the R408
helper phage (Stratagene, La Jolla, California, USA) (M. Russel,
S. Kidd. M. Kelley, 1986, Gene 45, 333-338~.
The mutagenesis procedure and selection of the mutants
by using the strain CJ 236 dut-, ung- of E. coli (In Vitrogen, San
Diego, California, USA) is described by T. Kunkel, (T. Kunkel
1985, Proc. Natl. Acad. Sci., 82, 488-492 and T. Kunkel et al.,
1987, Methods of Enzymology 154, 367-382, Acad. Press).
In vivo Recombination
The recombinant viruses are obtained according to the
conventional techniques of transfection of the sensitive cells,
such as the calcium phosphate method or the one using the Lipo-
fectine reactive described by the manufacturer BRL (P.L. Felgner
et al., 1987. Proc. Natl. Acad. Sci., USA, 84, 7413). For this,
the chicken embryo fibroblasts, cultivated to confluence, are
cotransfected with the genomic DNA and the plasmid carrier of the
DNA fragment to be inserted, flanked in 5' and 3' by the genome
sequences which allows the recombination.
The recombinant viruses can then be screened by hybridi-
zation with an appropriate probe or by plaque colouring. When agene marker, the lacZ gene of ~-galactosidase, is inserted into
the gene of Marek's disease virus, the expression of this gene can
be followed by adding, to the cell covering, an agarose overcoat
enclosing the chromogenic subætrate for the ~-galactosidase, e.g.
Xgal (5-bromo-4-chloro-3-indolyl, B.D. galactopyranoside).
: :'
~ . .
3 ~ ~
- 10 - 26361-74
Example 1: Isolating an EcoRI fragment of 5.25 kilobases.
The viral genomic DNA was digested by the restriction enzyme EcoRI
and the fragments cloned in the vector pUC 13 (Pharmacia)
(Yannisch, Perron et al., 1985, Gene 33, 103-119).
Among the cloned fragments, a fragment of 5.25 kilo-
bases, localized at the level of the small fragment Us, has been,
more particularly, analyzed by sequencing (pMDV 05; sequence ID
no. 1).
The sequence comprises six open reading frames (ORF).
The translated sequences of 4 of these ORF have a homology with
the type I HSV virus proteins, localized in the Us fragment. In
particular, the US3 gene of Marek's disease virus has a homology
with the gene of the kinase protein of the herpes simplex virus.
Surprisingly, the study of this region has shown that
the US3 gene can be deleted without blocking the viral replica-
tion.
Example 2: Construction of a plasmid pMDV 53L for which the US3
ene has been replaced by the lacZ gene (Figure 1).
The EcoRI fragment of 5.25 kilobases stemming from the
clone pMDV 05 was digested by the NcoI enzyme and the extremities
thus generated restored by the DNA polymerase Klenow fragment. It
was then digested by the KpnI enzyme and the fragment of 1989
pairs of bases thus liberated was cloned in the Blue Script vector
linearized by the enzymes EcoRV and KpnI to give the plasmid pMDV
~" :''"' .
r; ~ j~
~ 26361 - 74
52 having 4947 pairs of bases.
The NcoI and Sa]I sites were respectively introduced to
the extremities 5' and 3' of -the cloned fragment by controlled
mutagenesis using the oligonucleotides designated by Seq ID no. 2
and 3, in the list of attached sequences, which generates the pMDV
53 plasmid. The lacZ gene was purified from the pMC 1871 plasmid
(Pharmacia LKB, Uppsala, Sweden) (S.K. Shamira et al. 1983, Gene
25, 71-82) by digestion by the enzymes SmaI and SalI.
It was then inserted into the pMDV 53 plasmid, partially
digested by the NcoI enzyme, treated by the DNA polymerase Klenow
fragment and then controlled by the enzyme SalI, which generates
the plasmide pMDV 53L of about 6687 pairs of bases~
Example 3: Preparation of a Marek's disease virus comprising the
lacZ ~ene.
The chicken embryo fibroblasts were cotransfected with
the total chromosomal DNA of the virus and the DNA of the
linearized plasmid pMDV 53L (10 to 50 ~(~). I'he cultures were
observed for 4 to 6 days until infectious centres appeared.
Alternatively, the cells were trypsinated after 72 hours, then
20 reinoculated (1:1 or 1:2) in a secondary passage, until lysis
segments were ob-tained.
The medium was then replaced by the new medium
comprising 1% agarose and 0.5~ Xgal.
The plaques which are due to the recombination viruses
are distinguished by their blue colour.
- 12 ~ 26361-74
The viruses can thus be purified by plaque purification
and, after inoculation with healthy cells, give cytopathogenic
effect shapes coloured in blue in the presence of Xgal.
Example 4: Construction of the plasmid pMDV 53 CL which comprises
the lacZ gene under the control of the immediate early promoter of
the human cytomegalovirus (CMV) (Figure 2).
The lacZ gene of ~-galactosidase was placed under the
control of the immediate early promoter of the human
cytomegalovirus (IECMV) in the vector pCMV-lacZ.
The fragment of about 4500 pairs of bases comprising the
whole, the promoter IE of the cytomegalovirus and the lacZ gene,
were digested by the EcoRI enzyme and the extremities filled by
the DNA polymerase Klenow fragment. It was then digested by the
SalI enzyme.
The fragment thus liberated was cloned in the partially
digested pMDV 53 vector, by the NcoI enzyme, treated by the DNA
polymerase Klenow fragment and then digested by the SalI enzyme.
This plasmid composed of about 8200 pairs of bases is called pMDV
53 CL.
Example 5: Construction of a Marek recombinant virus for which
the lacZ gene was introduced under control of the immediate early
promoter of the cytomegalovirus, inst ad of the US3 gene.
The CEP were cotransfected with the genomic D~A of the
virus and from 10 to 50 ~g of DNA of the linearized plasmid pMDV
53 CL.
~, r - - ~J ~
- 13 - ~ - i 26361-74
One can thus obtain the recombinant viruses which are
distinguished by the appearance of blue-coloured infectious
plaques in the presence of the chromogen substrate Xgal. These
viruses were purified according to the plaque purification
technique and allowed to infect the secondary chicken embryo
fibroblasts.
The blue-coloured infectious pLaques can be obtained in
the presence of Xgal which shows that the lacZ gene is inserted
at the locus of the US3 gene.
Ex~le ~. Construction of the pMDV 53F plasmid which comprises
the ~ene of the fusion protein of Newcastle's disease virus
instead of the US3 gene (Figure 3) .
The fusion gene (J. Taylor et al., 1990, J. Virol.,
64, 1441-1450) was introduced in the form of a fragment at the
blunt ends in the Blue Script vector at the SmaI site to give the
pNFl plasmid having 5300 pairs of bases.
NcoI and SalI sites were introduced by controlled
mutagenesis at the level of the ~TG and stop codons of the fusion
gene, due to the oligonucleotides indexed SEQ ID no. 4 and SEQ ID
no. 5 respectively in the list of the attached sequences.
The fragment of 1682 pairs of bases NcoI/SalI coming
from the pNF2 plasmid was inserted into the pMDV 53 vector
partially digested by the NcoI enzyme and digested by the SalI
enzyme to give the pMDV 53F plasmid having 5369 bases pairs.
- 14 ~ ! 26361-74
Example 7: Construction of a Marek's disease virus comprising the
fusion protein gene of Newcastle's disease virus.
_ _ _
The chicken embryo fibroblasts were cotransfected with
the total genomic DNA of the virus and 10 to 50~ g of linearized
DNA of the pMDV 53F plasmid. The cultures were observed for the
appearance of infectious plaques.
The recombinant viruses were then screened by
hybridization with a probe including the fusion gene.
Similar procedures were used for the construction of the
non-avian recombinant herpes viruses, by inserting a heterologous
gene in the Us region and, in particular, in the homologous gene
at US3 gene of Marek's virus.
The invention also concerns vaccines, live or not, made
up of or containing recombinant viruses constructed according to
the invention, or containing immunogenes expressed by these
viruses.
- 15 - ~ 26361-74
APPE~DIX I
LIST OF SEQUE~CES
SEQ ID no. 1
Length of sequence: 5.255 pairs of bases
Type of molecule sequenced: genomic DNA
origin of the molecule: Marek's disease virus,
strain RBl B
Experimental source: pMDV 05 plasmid
Characteristics:
from 1 to 324 pairs of bases: non-coding region
from 325 to 1,135 pairs of bases: USl gene. The gene is
coded by the complementary DNA segment at
the indicated sequence and is transcribed
from right to left (SEQ ID no. l.B)
from623 to 1,214 pairs of bases: US2 gene
from1,215 to 1,245 pairs of bases: non-coding region
from1,246 to 2,451 pairs of bases: US3 gene
from2,452 to 2,563 pairs of bases: non-coding region
from2,564 to 3,004 pairs of bases: US4 gene
from3,005 to 3,190 pairs of bases: non-coding region
from3,191 to 4,384 pairs of baRes: US5 gene
from4,385 to 4,494 pairs of bases: non-coding region
from4,495 to 5,253 pairs of bases: US6 gene
16
.
CAAAAATTTACATTAGTAATCTTTCTCGGTGGCTTACCAAATCGTCCTCTTGGTATATCCATArCATCGAAC 72
ATTGTAGcATTGAcTcTGcTcATcGTTGTcTTTcAAATGcGcTcGATTGTTGAATcTcTccTGATGTTAGAA 14
GTATATGGAAGATAGCCTGGATACATAAGTGATCTAGAAGGGTTTGTTATTGCAGTAATATACAAATTATAC 21
GTGACACTATAGCGACGGTTGTAGCGATGCACCTAATCGTAATGTGTATACGCCCCATCATGTAATTATATC 28
TAATTGGTAGCAAGTAGGTCTGTCGAATAACAGCTAATGACTACCGGCTCTACATTTTTTCTGTATTCGTGA 360
CTTTCCTGTCGCAGTGTAACGAACCGGAATTGCAATCGCATCTCTATCTTCTTTCTTGCAACATTTTCCACA 432
ACAGAATAATCTGCCGGGTGTACTACTCATTTGAGGTGGTTCGATTTCCGGAGGTTTTAGAGGATTGGGTGG 504
GGACCCGAGGATTTTGTATACACATACCATATCACTGTCGCAAAAATGCGCTCTATCTTCTGGGGTGTCGAA 576
CTTCGGTTCCCATGTAGATGTCAAGAGAGTTTGAATATTGTCGGGA ATG GCC CAC GGC ATA CCG 640
~et Ala His GIY lle Pro 6
GAC CAG GTC CCA GAC ACT TTG ATT GCA AGT AAC CTT TTT GGC AAA GGA ATA CAT 694
Asp Gln Val Pra AsP Thr Leu lle Ala Ser Asn Leu Phe GIY LY5 GIY Ile His 24
TCG AGC GCA ATG CGA CAT ATA TCT GCC GCC CCA ACT ATC CAC AAG CTA TGT GGA 74
Ser Ser Ala ~et Arg His Ile Ser Ala Ala Pro Thr Ilo His LY5 LeU CYS Gly 42
GCA TTA CCA GAA ACT TCA GAT TCC AAC ATC AAA TAT CCA GAT AGA ACA TCC TGC P0~
Ala Leu Pro Glu Thr Sar AsP Ser Asn lle LYS Tyr Pro AsP Arg Thr Ser CYS 60
CAT TCT GTG GAA CAT CCT GCA ACA TCT TCA AAT AGC CGC ACT ATA AAC GAA TCC 856
His Ser Val Glu His Pro Ala Thr Ser Ser Asn Ser Arg Thr lle Asn Glu Ser 78
CTA GTT CCG GCC AAT CCG GTA CCA CGA ACT CCA GTT CCA TCT GGT GGC TTT GTC 910
Leu Val Pro Ala Asn Pro Val Pro Arg Thr Pro Val Pro Sqr Gly GIY Phe Val 96
CTT ACT ATC GGT CGA TGT TGC CGA GGA AGA ATT AAC ATG GGT TTG GCA AAA CGG 964
Leu Thr Ile GlY Arg Cys CY5 Arg Gly Arg Ile Asn ~et GlY Leu Ala Lys Arg 114
AAT AGG TCT GCA GCT CTG ACG ATT ATG GGC ACA CCC ACA TCA TCC TGT ATT TGT 1018
Asn Arg Ser Ala Al4 L-u Thr lle ~et GIY Thr Pro Thr Ser Ser CYS Il~ CY5 132
TCC ATA CAT TGC TTT ATA AGG AAT ATC CAT AAA GTA GAT GCA GCA TCJ CTA GAT 1072
Ser lle His CYS Phe lle Arg Asn Ile His LYS Val AsP Ala Ala Ser LeU Asp 150
CTT CCT GGC AAT CGA TCG CAT TCA TCT AGA AGT GTG ACT ATA GTT ATC ATG GAC 1126
LeU Pro GlY Asn Arg S-r His Ser S-r Arg Ser Val Thr lle Val lle ~et Asp 168
ACA CCC ATC TTC ACT CCA CCA ATA ATC TTT TTT ATT GTT AAT AAC TGG GCC GGT 1180
Thr Pro ll~ Ph~ Thr Pro Pro lle 11- Phe Ph- lle Val Asn Asn Trp Al4 GIY 186
CTG ATC TCC AAA TCT TAT ACC TCT GGT AGA ATA TGAAACAGGGTTAAAACTAGGTAATAG 1240
Leu lle Ser LYS Ser Tyr Thr S~r Gly Arg Il- 197
ACTGGATG TCT TCG AGT CCG GAG GCA GAA ACG ATG GAA TGC GGC ATT TCT TCG TCG 1296
~et Ser Ser S~r Pro Glu Ala Glu Thr ~et Glu CYS Gly lle Ser S~r Ser 214
AAA GTA CAC GAC TCT AAA ACT AAT ACT ACC TAC GGA ATT ATA CAT AAC AGC ATC 135~0
Lys Val His Asp Ser LYS Thr Asn Thr Thr Tyr Gly lle Ila His Asn Ser 11- 23~
AAT GGT ACG GAT ACG ACG TTG TTT GAT ACT TTT CCC GAC AGT ACC GAT AAC GCG 1404
Asn Gly Thr AsP Thr Thr Leu Phe Asp Thr Phe Pro Asp Ser Thr AsP Asn Ala 250
GAA GTG ACG GGG GAT GTG GAC GAT GTG AAG ACT GAG AGC TCT CCC GAG TCC CAA 14S8
Glu Val Thr Gly AsP Val Asp Asp Val Lys Thr Glu Ser Ser Pro Glu Ser Gln 268
TCT GAA GAT TTG TCA CCT TTT GGG AAC GAT GGA AAT GAA TCC CCC GAA ACG GTG 1512
Ser Glu Asp Leu Ser Pro Phe Gly Asr\ Asp GIY Asn Glu Ser Pro Glu Thr Val 286
ACG GAC ATT GAT GCA GTT TCA GCT GTG CGA ATG CAG TAT AAC AAT GTT TCA TCG 156~
Thr AsP Ile Asp Ala ~al Ser Ala Val Arg ~et Gln Tyr Asn Asn Val Ser Ser 304
TTA TCG CCC GGA TCT GAA GGG TAT ATC TAT GTT TGT ACA AAG CGT GGG GAT AAT 1620
Leu Ser Pro G1Y Ser Glu Gly Tyr Ile Tyr Val Cys Thr LYS Arg GIY Asp Asn 322
ACC AAG AGA AAA GTC ATT GTG AAA GCT GTG ACT GGT GAC AAA ACC CTT GGG AGT 1674
Thr LYS Arg LY5 Val Ile Val Lys Ala Val Thr G1Y Asp LYS Thr Leu GIY Ser 340
GAA ATT GAT ATA TTA AAA AAA ATG TCT CAC CGC TCC ATA ATT AGA TTA GTT CAT 1728
Glu Il- Asp Ile Leu LYS LYS ~et Ser His Arg Ser Ile Ile Arg Leu Val His 358
GCT TAT AGA TGG AAA TCG ACA GTT TGT ATG GTA ATG CCT AAA TAC AAA TGC GAC 1782
Ala Tyr Arg Trp LYS Ser Thr Val Cys ~et Val ~et Pro LY5 Tyr Lys Cys Asp 376
TTG TTT ACG TAC ATA GAT ATC ATG GGA CCA TTG CCA CTA AAT CAA ATA ATT ACG 1836
Leu Ph- Thr Tyr Ile Asp Ile ~et Gly Pro Leu Pro Leu Asn Gln ~le Il- Thr 394
ATA GAA CGG GGT TTG CTT GGA GCA TTG GCA TAT ATC CAC GAA AAG GGT ATA ATA t890
Ile Glu Arg Gly Leu Leu Gly Ala Leu Ala Tyr Ile His Glu Lys Gly Ile Ile 412
CAT CGT GAT GTA AAA ACT GAA AAT ATA TTT TTG GAC AAA CCT GAA AAT GTA GTA 194q
His Arg Asp Val Lys Thr Glu Asn Ile Phe Leu Asp Lys Pro Glu Asn Val Val 430
TT5 GGG GAC TTT GGG GCA GCA TGT AAA TTA GAT GAA CAT ACA GAT AAA CCC AAA 1998
Leu GlY Asp Phe GIY Ala Ala Cy5 Lys Leu Asp Glu His Thr Asp LYS Pro LYS 44e
TGT TAT GGA TGG AGT GGA ACT CTG GAA ACC AAT TCG CCT GAA CTG CTT GCA CTT 2052
Cys Tyr Gly Trp Ser GlY Thr Leu Glu Thr Asn Ser Pro 61u Leu L-U Ala LeU 466
GAT CCA TAC TGT ACA AAA ACT GAT ATA TGG AGT GCA GGA TTA GTT CTG TTT GAG 2106
Asp Pro Tyr Cys Thr LYS Thr Asp Ile Trp Ser Ala Gly Leu Val Leu Phe Glu 484
ATG TCA GTA AAA AAT ATA ACC TTT TTT GGC AAA CAA GTA AAC GGC TCA GGT TCT 2160
~et Ser Val LYS Asn Ile Thr Phe Phe Gly LYS Gln Val Asn GIY Ser Gly Ser 502
CAG CTG AGA TCC ATA ATT AGA TGC CTG CAA GTC CAT CCG TTG GAA TTT CCA CAG 2214
Gln Leu Arg Ser lle Ilo Arg CYS Leu Gln Val His Pro Leu Glu Phe Pro Gln 520
AAC AAT TCT ACA AAC TTA TGC AAA CAC TTC AAG CAG TAC GCG ATT CAG TTA CGA 2268
Asn Asn Ser Thr Asn Leu Cys Lys His Phe LYS Gln Tyr Ala lle Gln Leu Arg 538
CAT CCA TAT GCA ATC CCT CAG ATT ATA CGA AAG AGT GGT ATG ACG ATG GAT CTT 2322
His Pro Tyr Ala Ile Pro Gln Ile Ile Arg Lys Ser Gly ~et Thr ~et Asp Leu 556
GAA TAT GCT ATT GCA AAA ATG CTC ACA TTC GAT CAG GAG TTT AGA CCA TCT GCC 2376
Glu Tyr Ala lle Ala LYS ~st Leu Thr Phe Asp Gln Glu Phe Arg Pro Ser Ala 574
CAA GAT ATT TTA ATG TTG CCT CTT TTT ACT AAA GAA CCC GCT GAC GCA TTA TAC 2430
Gln Asp Ilo Leu ~st L-u Pro Leu Phe Thr Lys Glu Pro Ala Asp Al~ L-u Tyr S92
ACG ATA ACT GCC GCT CAT ATG TAAACACCCGTCAAAAATAACTTCAATGATTCATTTTATAATA 2494
Thr Ile Thr Ala Ala His ~et 599
TATAcTAcGcGTTAccTGcAATAATGAcAAcATTcGAAGTcTTTGAAGATTcGcAGAccTTTTTTGcGAATG 2566
~-t 600
GCA CCT TCG GGA CCT ACG CCA TAT TCC CAC AGA CCG CAA ATA AAG CAT TAT GGA 2620
Ala Pro Ser GlY Pro Thr Pro Tyr Ser His Arg Pro Gln Ile LYS His Tyr Gl~ 618
ACA TTT TTG GAT TGC ATG AGA TAT ACT CTA AAC GAT GAG AGT AAG GTA GAT GAT 2674
Thr Phe Leu Asp Cys ~et Arg Tyr Thr LeU Asn A5P Glu Ser LYS Val Asp Asp 636
I8
AGA TGT TCA GAC ATA CAT AAC rcc TTA GCA CAA TCC AAT GTT ACT TCA AGC ATG 2728
Arg CYS Ser Asp Ile His Asn Ser Leu Ala Gln Ser Asn Val Thr Ser Ser ~et 6S4
TCT GTA ATG AAC GAT TCG GAA GAA TAT CCA TTA ATA AAT GGA CCT TCG ATG CAG 2782
Ser Val ~et Asn Asp Ser Glu Glu TYr Pro Leu Ile Asn GIY Pro Ser ~et Gln 672
GCA GAG GAC CCT AAA AGT GTT TTT TAT AAA GTT CGT AAG CCT GAC CGA AGT CGT 2836
Ala Glu AsP Prr Lys Ser Val Phe Tyr Ly5 Val Arg Lys Prr. Asp Arg Ser Ar~ 69C
GAT TTT TCA TGG CAA AAT CTG AAC TCC CAT GGC AAT AGT GGT CTA CGT CGT GAA 2890
Asp Phe Ser Trp Gln Asn Leu Asn Ser His G1Y Asn Ser Gly Leu Arg Arg Glu 708
AAA TAT ATA CGT TCC TCT AAG AGG CGA TGG AAG AAT CCC GAG ATA TTT AAG GTA 2944
Lys Tyr Ile Arg Ser Ser Lys Arg Arg Trp Lys Asn Pro Glu Ile Phe Lys Val 726
TCT TTG AAA TGT GAA TCA ATT GGC GCT GGT AAC GGA ATA AAA ATT TCA TTC TCA 2998
Ssr Leu Lys Cys GIU Ser Ile GIY Ala Gly Asn Gly Ile Lys Ila Ser Phe Ser 744
TTT TTC TAACATTATAATATATCAGATCGTTTCTTATATACTTATTTTCATCGTCGGGATATGACTAAC 3067
Phe Phe 746
GTATACTAAGTTACAAGAAACAACTGCITAACGTCGAACATAACGGAAATAAAAATATATATAGCGTCTCCT 3139
TAANTGTTATATTGGCACCTTTTAGAGCTTCGGTATGAATAGATACAGATATG AAA GTA TTT TTT 3205
~et Lys Val Phe Phe 7S1
TTT AGA TAT ATC TCA TCC ACG AGA ATG ATT CTT ATA ATC TGT CTA CTT TTG GGA 3259
Phe Arg Tyr Ile Ser Ser Thr Arg ~et Ile Leu Ile Ile Cys Leu Leu Leu Gly 769
ATT GGG GAC ATG TCC GCA ATG GGA CTT AAG AAA GAC AAT TCT CCG ATC ATT CCC 3313
Ile Gly Asp ~et Ser Ala ~et Gly Leu LY5 LY5 AsP Asn Ser Pro Ile Ile Pro 787
ACA TTA CAT CCG AAA GGT AAT GAA AAC CTC CGG GCT ACT CTC AAT GAA TAC AAA 3367
Thr Leu His Pro LY5 GlY Asn Glu Asn Leu Arg Ala Thr Leu Asn Glu TYr LYS 805
ATC CCG TCT CCA CTG TTT GAT ACA CTT GAC AAT TCA TAT GAG ACA AAA CAC GTA 3421
Ile Pro Ser Pro Leu Phe AsP Thr Leu Asp Asn Ser Tyr Glu Thr LYs His Val 823
ATA TAT ACG GAT AAT TGC AGT TTT GCT GTT TTG AAT CCA TTT GGC GAT CCG AAA 3475
I1R Tyr Thr Asp Asn Cys Ser Phe Ala ~al Leu Asn Pro Phe G1Y Asp Prr. LY5 841
TAT ACG CTT CTC AGT TTA CTG TTG ATG GGA CGA CGC AAA TAT GAT GCT CTA GTC 3529
Tyr Thr Leu L~u Ser Leu Leu Leu ~et G1Y Ar3 Arg Lys Tyr AsP Ala Leu Val 8~9
GCA TGG TTT GTC TTG GGC AGA GCA TGT GGG AGA CCA ATT TAT TTA CGT GAA TAT 3583
Ala TrP Phe Val Leu GIY Arg Ala CY5 GlY Arg Pro Ile Tyr Leu Arg Glu Tyr 877
GCC AAC TGC TCT ACT AAT GAA CCA TTT GGA ACT TGT AAA lTA AAG TCC CTA GGA 3637
Ala Asn Cys Ser Thr Asn Glu Pro Phe GlY Thr Cys LY5 Leu Lys Ser Leu Gly 895
TGG TGG GAT AGA AGA TAT GCA ATG ACG AGT TAT ATC GAT CGA GAT GAA TTG AAA 3691
Trp Trp Asp Arg Arg Tyr Ala ~et Thr Ser Tyr 119 Asp Arg AsP Glu Leu Lys 913
TTG ATT ATT GCA GCA CCC AGT CGT GAG CTA AGT GGA TTA TAT ACG CGT TTA ATA 3745
Leu Ile Ile Ala Ala Pra Ser Arg Glu Leu Ser GIY Leu Tyr Thr Arg Leu 11e 931
ATA ATT AAT GGA GAA CCC ATT TCG AGT GAC ATA TTA CTG ACT GTT AAA GAA ACA 3799
Ile Ile Asn GlY Glu Pro Ile Ser Ssr Asp Ile Leu Leu Thr Val Lys Glu Thr 949
TGT AGT TTT TCG AGA CGG GGG ATA AAG GAT AAC AAA CTA TGC AAA CCG TTC AGT 3853
Cys Ser Phe Ser Arg Arg G1Y Ile Lys Asp Asn LYS Leu CYS LYS Pro Phe Ser 967
TTT TTT GTC AAT GGT ACA ACA CGG CTG TTA GAC ATG GTG GGA ACA GGA ACC CCG 39C7
Phe Phe Val Asn Gly Thr Thr Arg Leu Leu Asp ~et Val GIY Thr GIY Thr Pro 985
AGA GCT CAT GAA GAA AAT GTG AAG CAG TGG CTT GAA CGA ATT GGT GGT AAA CAT 3961
19 2
Arg Ala His Glu Glu Asn Val LYS Gln Trp Leu Glu Arg lle Gly Gly LYS His 1003
CTA CCA ATC GTC GTC GAA ACA TCT ATG CAA CAA GTC TCA AAT TTG CCG AGA AGT 4015
Leu Pro lle Val Val Glu Thr Ser ~et Gln Gln Val Ser Asn Leu Pra Ars Ser 1021
TTT AGA GAT TCA TAT TTC AAA TCA CCT GAC GAC GAT AAA TAT GAT GAC GTC AAA 4069
Phe Arg Asp Ser Tyr Phe LYS Ser Pro Asp Asp Asp Lys Tyr Asp Asp Val LYS 1039
ATG ACA TCG GCC ACT ACT AAT AAC ATT ACC ACC TCC GTG GAT GGT TAC ACT GGA 4123
~st Thr Ser Ala Thr Thr Asn Asn Ile Thr Thr S-r Val Asp Gly Tyr Thr GIY 1057
CTC ACT AAT CGG CCC GAG GAC TTT GAG AAA GCA CCA TAC ATA ACT AAA CGA CCG 4177
Leu Thr Asn Arg Pro Glu Asp Phe Glu LYS Ala Pro Tyr Ile Thr Lys Arg Pro 1075
ATA ATC TCT GTC GAG GAG GCA TCC AGT CAA TCA CCT AAA ATA TCA ACA GAA AAA 4231
Ile Ile Ser Val Glu Glu Ala Ser Ser Gln Ser Pro LYS lle Ser Thr Glu Lys 1093
AAA TCC CGA ACG CAA ATA ATA ATT TCA CTA GTT GTT CTA TGC GTC ATG TTT TGT 4285
Lys Ser Arg Thr Gln Ile Ile Ile Ser Leu Val Ual Leu Cys Val ~et Phe CYS 1111
TTC ATT GTA ATC GGG TCT GGT ATA TGG ATC CTT CGC AAA CAC CGC AAA ACG GTG 4339
Phe Ile Val Ile G1Y Ser Gly Ile Trp Ile Leu Arg Lys His Arg Lys Thr Val 1129
ATG TAT GAT AGA CGT CGT CCA TCA AGA CGG GCA TAT TCC CGC CTA TAACACGTGTT 4395
~et Tyr Asp Arg Arg Ars Pro S-r Arq Arg Ala Tyr Ssr Ars L-u 1144
TGGTATGGGCGTGTCGCTATAGTGCATAAGAAGTTGACTACATTGCATCAATGACATTATATAGCTTCTTTG 4467
GTCAGATAGACGGCGTGTGTGATTGCGATG TAT CTA CTA CAA TTA TTA TTT TGG ATC CGC 4527
~e~ Tyr Leu Leu Gln Leu L~u Phe Trp Il~ Arg 1155
CTC TTT CGA GGC ATC TGG TCT ATA GTT TAT ACT GGA ACA TCT GTT ACG TTA TCA 4581
Leu Phe Arg Gly lle Trp Ser Ile Val Tyr Thr G1Y Thr Ser Val Thr Leu Ser 1173
AcG GAC CAA TCT GCT CTT GTT GCG TTC TGC GGA TTA GAT AAA ATG GTG AAT GTA 4635
Thr Asp Gln Ser Ala Leu Val Ala Phe CYS GIY L-u Asp LYs ~et Val Asn Val 1191
CGC GGC CAA CT r TTA TTC CTG GGC GAC CAG ACT CGG ACC AGT TCT TAT ACA GGA 4689
Arg Gly Gln Leu Leu Phe Leu Gly Asp Gln Thr Arg Thr Ser Sdr Tyr Thr GIY 1209
ACG ACG GAA ATC TTG AAA TGG GAT GAA GAA TAT AAA TGC TAT TCC GTT CTA CAT 4743
Thr Thr Glu Ile Leu Lys Trp Asp Glu Glu Tyr LYS Cys Tyr Ser Val Leu His 1227
GCG ACA TCA TAT ATG GAT TGT CCT GCT ATA GAC GCC ACG GTA TTC AGA GGC TGT 4797
Ala Thr Ser Tyr ~et Asp CYS Pro Ala Ile Asp Ala Thr Val Phe Arg Gly Cys 1245
AGA GAC GCT GTG GTA TAT GCT CAA CCT CAT GAT AGA GTA CAA CCT TTT CCC GAA 4851
Arg Asp Ala Val Val Tyr Ala Gln Pro His Asp Arg Val Gln Pro Phe Pro Glu 1263
AAG GGA ACA TTG TTG AGA ATT GTC GAA CCC AGA GTA TCA GAT ACA GGC AGC TAT 4905
Lys Glr Thr Leu Leu Arg Ile Val Glu Pro Arg Val Ser Asp Thr G1Y S~r Tyr 1281
TAC ATA CGT GTA GCT CTC GCT GGA AGA AAT ATG AGC GAT ATA TTT AGA ATG GCT 4959
Tyr Ile Arg Val Ala Leu Ala GIY Arg Asn ~et Ser Asp lle Phe Arg n.t Ala 1299
GTT ATT ATA AGG AGT AGC AAA TCT TGG GCN TGT AAT CAC TCT GCT AGT TCA TTT 5013
Val Ile Ile Arg Ser Ser LYS Ssr Trp aa cys Asn His Ser Ala Ser Ser Phe 1317
CAG GCC CAT AAA TGT ATT CGC TAT GTC GAC CGT ATG GCC TTT GAA AAT TAT CTG 5067
Gln Ala Hls LY5 Cys Ile Arg Tyr Val Asp Arg ~et Ala Phe Glu Asn Tyr Leu 1335
ATT GGA CAT GTA GGC AAT TTG CTG GAC AGT GAC TCG GAA TTG CAT GCA ATT TAT 5121
Ile Gly His Val G1Y Asn Leu Leu Asp Ser Asp Sar Glu Leu His Ala Ile Tyr 1353
AAT ATT ACT CCC CAA TCC ATT TCC ACA GAT ATT AAT ATT ATA ACG ACT CCA TTT 5175
Asn lle Thr Pro Gln Ser Ile Ser Thr Asp Ild Asn Ile lle Thr Thr Pro Phe 1371
TAC GAT AAT TCG GGA ACA ATT TAT TCA CCT ACG GTT TTT AAT TTG TTT AAT AAC 5229
Tyr ~sP Asn Ser GIY Thr lle Tyr Ser Prtl Thr Val Phe Asn Le~J Phe Asn Asn 1389
. AAT TCC CAT GTC GAT GCA ATG AAT TC 5255
Asr~ Ser His Val AsP Ala ~et Asn 1397
~ 7
- 21 - 26361-74
SEQ ID no. 1 B
. . _
Length of sequence: 1,188 pairs of bases
Type of molecule sequenced: genomic DNA
Origin of the molecule: Marek's disease virus, strain
RBl B
Experimental source: pMDV 05 plasmid
Characteristics:
from 1 to 324 pairs of bases: non-coding region
from 325 to 1,135 pairs of bases: USl gene: the gene is
transcribed from right to left; the
indicated sequence is complementary to
the sequence SEQ ID no. 1
22 ~ r~
~let 51/ Val S-r
GAG~TC~6ACC6GCCCAGTTATTAA~AATAA~A~A6ATTATTG6T5~A,15AA6 A15 G~JT 5T6 TCC 1189
!1st Ile T~,r lle V~l Thr 'eu Lau ~;F 61U C;~ A~P Ar~ ?U Pro 61/ Ar~ ~er
AT6 ATA ACT ATA bTC ACA CTT CTA 5AT 6AA T5C 6AT GJA TT5 CCA 6GA AGA TCT 111
Ar~ A5F Al~ Ala Ser Thr Leu Tr~ il9 Phe Leu Ile LY, Glr, C~s Met 61U Glr,
hGA GAT 6CT GCA TCT ACT TTA TGG ATA TTC CTT ATA AAG CAA TGT ATG GAA CAA 1069
Ile Gln Asp AsF Val 61Y Val Pro Ile Ile ~ai ArQ Ala Ala Asp Leu Phe Ar~
: ATA CAG 6AT GAT GTG GGT GTG CCC ATA ATC GTC AGA GCT 6CA GAC CTA TTC CGT 1015
Phe Ala Ly; Pro Met Leu Ile Leo Pro ArQ 51r His Ar~ Pro Ile Val Ar~ Thr
TTT GCC AAA CCC AT6 TTA ATT CTT CCT CJG CAA CAT CGA CCG ATA GTA AGG ACA 961
LYS Pro Pro A;p G1Y Thr Gly Val ArQ 61y Thr Gly Leu Ala Gly Thr Ar~ Asp
AAG CCA CCA GAT GGA ACT 6GA GTT C5T 6GT ACC G6A TTG GCC 66A ACT A6G GAT 907
Ser Phe 11e Val Ar~ Leu Phe GIU Asp ~al Ala 61Y CYS Ser Thr Glu TrF Gln
TCG TTT.ATA GT6 CG6 CTA TTT GAA GAT 5TT 6CA ÇGA TGT TCC ACA GAA T56 CAG 8S3
Asp Val L9U Ser 61y Tyr Leu Met L91J 51U ~er Glu Val Ser 51y Asr, Ala Pro
GAT 5TT CTA TCT GGA -TAT TT6 At6 TTG GAA TCT GAA 6TT TCT G5T AAT GC CCA '99
His Ser l.el~ Trp Ile Val Gly Ala Ala AsF Ile Cys ArQ lle Ala Leu Glu C~s
CAT AGC TTG T6G ATA GTT GGG GCG GCA 6AT ATA TGT CGC ATT GC6 CTC GAA TGT 745
: Ile Pro Le~J Pro Lys ArQ Le~J Leu Ala Ile Lys ~al Ser Gly Thr 1`rF Ser 51y
ATT ÇCT TTG CCA AAA AGG TTA CTT GCA ATC AAA GT6 TCT b6G ACC -5G TCC 6GT 691
~et ~ro Trp Ala lle Pro A;p A~r, Il9 Gln Thr Leu Leu Thr Ser Thr Trp 61u
ATG CCG TGG 5CC ATT CCC GAC AAT ATr CAA ACT CTC TlG ACA TCT ACA TG6 6~A 637
Pro ~Y5 Phe As~ T~lr Pro Glu A;P ArQ Ala His Phe Cy5 AsP Ser Asp ~et Val
CCG AA6 TTC 6AC ACC CCA 6AA 6AT A6A GCG CnT TTT TGC GAC AGT 5AT ATG 6TA 583
Çys Val Tfr L~fs Ile Leu G1Y ~er Pro Pro Asr, Pro Leu Lfs Pro Pro 61u Ile
TGT 6TA TAC AAA ATC CTC GG6 TCC CA CCC AAT CCT CTA AAA CCT CCG GAA ATC 529
.~ Glu Pro Pro Glr ~et 5~r Ser Thr Pro G1Y Ar~ Lau Phe CYS C~s G1Y LY5 Cys
1~ 5AA CCA CÇT CAA AT6 A6T AGT ACA CCC 56C A6A TTA TTC T6T TGT 6GA AAA TGT 475
ÇY; LY; LYS Glu Asp Ar~ Asp Ala Ile Ala 11e Pro ~al Ar~ TYr Thr Ala Th~
T6C AAG AAA GAA 6AT A6A GAT GCG ATT GCA ATT CCG 6TT CGT TAC ACT GCG ACA ~67
Gly Ly~ S~n Ang IIQ 61n LY5 LY5 Cyi Ar~ Ala GIY Ser His
6GA~ AAG TCA CGA ATA CA6 AAA AAA TGT AGA GCC GGT AGT CAT TA6CT6TTATTCGAC 310
A6ACCTACTT6CTACCAATTA6ATATAATTACATGATGGG6C6TATACACATTACGATTAGGT6CATC6CTA 238
CAAcc6rcGcTATAGTGTcAcGTATAATTTGTATATTAcTGcAATAAcAAAcccTTcTAGATcAcTTATGTA 166
TCCA6GCTATCTTCCATATACTTCTAACATCAGGA6AGATTCAACAATCGAGC6CATTTGAAAGACAAC6Ar 94
6AGCAGA6TCAATGCTACAATGTTCGAT6ATATGGATATACCAA6AG6AC6ATTTGGTAAGCCACC6A6AAA 22
GATTAcTAATGTAAATTTTTG
:
.
,
- ~ :. . ,
. - :
,:~ ' "
~ ,~s _, d?, f' ~ ' i
~ 23 - 26361-74
SEQ I D no. 2
Type of sequence: oligonucleotide
Length of sequence:
Type of molecule: DNA
5 ' GGA CTC GAA ~CCA TGIG AGT CTA TTA CC 3 '
NCO 1
SEQ ID no. 3
Type of sequence: oligonucleotide
Length of sequence:
Type of molecule: DNA
5 ' GAC GGG TGT CGA CAT ATG AG 3 '
Sal I
SEQ I D no. 4
_
Type of sequence: oligonucleotide
Length of sequence:
Type of molecule: DNA
5 ' CTG GAG CCC ATG GFG CAC CTT TG 3'
NCOl
-SEQ ID no. 5
Type of sequence: oligonucleotide
Length of sequence:
Type of molecule: DNA
5 ' CAA ATT GCT ATT GTC GA IC ACC TCC GCC TCT C 3 '
Sal I
