U.S. patent application number 08/860844 was filed with the patent office on 2003-06-05 for method of detection of nucleic acids with a specific sequence composition.
Invention is credited to WEININGER, ARTHUR M., WEININGER, SUSAN.
Application Number | 20030104361 08/860844 |
Document ID | / |
Family ID | 25334160 |
Filed Date | 2003-06-05 |
United States Patent
Application |
20030104361 |
Kind Code |
A1 |
WEININGER, SUSAN ; et
al. |
June 5, 2003 |
METHOD OF DETECTION OF NUCLEIC ACIDS WITH A SPECIFIC SEQUENCE
COMPOSITION
Abstract
This invention is a novel method for detecting and localizing
specific nucleic acid sequences in a sample with a high degree of
sensitivity and specificity. The method and novel compositions used
in the method involve the use of Probe Nucleic Acids, the
production of nucleic acid binding regions and the use of nucleic
acid Target Binding Assemblies to detect and localize specific
Target Nucleic Acids. The detection and localization of the Target
Nucleic Acid is accomplished even in the presence of nucleic acids
which have similar sequences. The method provides for a high degree
of amplification of the signal produced by each specific binding
event. In particular, methods and compositions are presented for
the detection of HIV and HPV nucleic acid in samples. These methods
and compositions find use in diagnosis of disease, genetic
monitoring, forensics, and analysis of nucleic acid mixtures. Some
of the novel compositions used in the detection method are useful
in preventing or treating pathogenic conditions.
Inventors: |
WEININGER, SUSAN; (SEATTLE,
WA) ; WEININGER, ARTHUR M.; (SEATTLE, WA) |
Correspondence
Address: |
DAVID R SALIWANCHIK
2421 NW 41ST STREET
SUITE A1
GAINESVILLE
FL
326066669
|
Family ID: |
25334160 |
Appl. No.: |
08/860844 |
Filed: |
September 29, 1997 |
PCT Filed: |
December 7, 1995 |
PCT NO: |
PCT/US95/15944 |
Current U.S.
Class: |
435/6.15 ;
514/44R |
Current CPC
Class: |
C12Q 1/6811 20130101;
C12Q 1/682 20130101; C12Q 1/6804 20130101; A61P 31/14 20180101;
C12Q 1/6813 20130101; C12Q 1/708 20130101; C12Q 1/6827 20130101;
C12Q 1/6811 20130101; A61P 31/18 20180101; C12Q 1/6827 20130101;
C12Q 1/6853 20130101; C12Q 1/703 20130101; C12Q 2537/149 20130101;
C12Q 2537/161 20130101; C12Q 2525/301 20130101; C12Q 2525/301
20130101; C12Q 2537/143 20130101; C12Q 2537/161 20130101; C12Q
2537/143 20130101; C12Q 2537/149 20130101 |
Class at
Publication: |
435/6 ;
514/44 |
International
Class: |
C12Q 001/68; A01N
043/04 |
Claims
What is claimed is:
1. A probe nucleic acid (PNA) comprising: (a) a single-stranded
sequence, 1/2 TBR, which is capable of forming under hybridizing
conditions, a hybrid, TBR, with a 1/2 TBR present in a target
nucleic acid (TNA); (b) a single stranded sequence, 1/2 BBR, which
is capable of forming, under hybridizing conditions, a hybrid BBR,
with about 0-10 1/2 BBR present in a booster nucleic acid (BNA);
and (c) an OSA, which is no attached support and/or indicator, or
an attached support or other means of localization, including, but
not limited to, attachment to beads, polymers, and surfaces, and/or
indicators; wherein said TBR is capable of binding with high
affinity to a TBA, said TBA being a substance capable of
discriminating between a paired TBR and a TBR having unpaired
nucleotides, and further, wherein said BBR is capable of binding
with high affinity to a BBA, said BBA being a substance capable of
discriminating between a paired BBR and a BBR having unpaired
nucleotides.
2. A booster nucleic acid (DNA) comprising: (a) a 1/2 BBR which has
a sequence which is complementary to a 1/2 BBR sequence in a PNA or
another BNA and which is capable of forming, under hybridizing
conditions a hybrid, BBR, with the PNA; (b) an OSA, which is no
attached support or indicator or an attached support or other means
of localization, including, but not limited to, attachment to
beads, polymers, and surfaces, and/or indicators; and (c)
additional hybridization sites, 1/2 BBRs, for hybridization with
additional BNAs; wherein said BBR is capable of binding with high
affinity to a BBA, said BBA being a substance capable of
discriminating between a paired BBR and a BBR having unpaired
nucleotides.
3. A Hairpin Nucleic Acid (HNA) comprising a single-stranded
sequence, 1/2 BBR, which under hybridizing condition is capable of
forming a hairpin while at the same time binding to a BNA to form a
BBR capable of binding a BBA, wherein said BBR is capable of
binding with high affinity to a BBA, said being a substance capable
of discriminating between a perfect BBR and a BBR having impaired
nucleotides.
4. The PNA of claim 1 wherein the TBR is comprised of one or more
recognition sites for a nucleic acid binding protein, a DNA binding
protein, a DNA-RNA hybrid binding protein or an RNA binding
protein.
5. The PNA of claim 4 wherein the TBR is a nucleic acid binding
protein recognition site present in the genome of a pathogen or is
a binding site associated with a pathogenic condition in a
vertebrate genome is a nucleic acid binding protein recognition
site present in the genome of an organism which contaminates a
fermentation process.
6. The PNA of claim 4 wherein the TBR is the HIV-LTR or a portion
thereof.
7. A method for detecting or localizing a specific TNA sequence,
comprising the steps of: (a) hybridizing said TNA with the PNA of
claim 1; (b) hybridizing said PNA with a BNA containing a 1/2 BBR
whose sequence is complementary to a 1/2 BBR sequence in the PNA;
(c) adding the products of steps (a) and (b) containing a TBR and a
BBR, to a surface, liquid or other medium containing a TBA; (d)
adding BBAs to the mixture in step (c) wherein said BBA comprises:
(i) molecule or a portion of a molecule which is capable of
selectively binding to a BBR; (ii) a detectible indicator; and (e)
detecting signal produced by the indicator attached to the BBA.
8. The method of claim 7 wherein said indicator is a protein,
including enzymes capable of catalyzing reactions leading to
production of colored reaction products; a radionuclide; colored
beads.
9. A method of detecting the presence in a sample of a specific
Target Nucleic Acid, TNA, which comprises: (a) contacting said
sample with a Probe Nucleic Acid, PNA, which, upon hybridization
with said TNA if present in said sample, forms a Target Binding
Region, TBR, which is capable of binding a Target Binding Assembly,
TBA; and (b) contacting said sample, already in contact with said
PNA, with a TBA capable of binding to any TBRs formed by the
hybridization of said PNA and said TNA in the sample.
10. A method for detecting or localizing specific nucleic acid
sequences with a high degree of sensitivity and specificity which
comprises: (a) adding PNAs containing a 1/2 BBR and a 1/2 TBR to a
sample containing or suspected of containing TNAs containing 1/2
TBR sequences, to form a complex having target binding regions,
TBRs, formed by the hybridization of complementary 1/2 TBRs present
in the PNAs and TNAs respectively; (b) binding the TBRs formed in
step (a) to an immobilized TBA to form a TBA-TNA-PNA complex; (c)
adding Booster Nucleic Acids, BNAs, containing booster binding
regions, 1/2 BBRs, to the complex formed in step (b) such that the
1/2 BBRs in the BNAs hybridize with the 1/2 BBR present in the PNAs
or to 1/2 BBRs present in BNAs already bound to the PNA, to form
BBRs, such that TBA-TNA-PNA-(BNA).sub.n complexes are formed; (d)
adding Hairpin Nucleic Acids, HNAs, containing 1/2 BBR sequences,
to the complex formed in step (c) such that the 1/2 BBRs in the
HNAs hybridize with any available 1/2 BBR sequences present in the
BNAs of the complex of step (c), thereby capping the extension of
the BNAs onto the TBA-TNA-PNA-(BNA).sub.n complexes of step (c) to
form TBA-TNA-PNA-(BNA).sub.n-HNA complexes; (e) adding Booster
Binding Assemblies, BBAs, linked to indicator moieties, to the
TBA-TNA-PNA-(BNA).sub.n-HNA complexes formed in step (d) to form
TBA-TNA-PNA-(BNA-BBA).sub.n-HNA complexes; and (f) detecting the
signals produced by the indicator moieties linked to the TBAs,
PNAs, BNAs, BBAs or HNAs in the TBA-TNA-PNA-(BNA-BBA).sub.n-HNA
complexes of step (e); wherein the TNA comprises: (i) one or more
specific 1/2 TBR nucleic acid sequences, the presence or absence of
which in a particular sample is to be confirmed; the PNA comprises:
(i) a single/stranded sequence, 1/2 TBR, which is capable of
forming, under hybridizing conditions, a hybrid, TBR, with a 1/2
TBR present in a target nucleic acid (TNA); (ii) a single stranded
sequence, 1/2 BBR, which is capable of forming, under hybridizing
conditions, a hybrid BBR with a 1/2 BBR present in a booster
nucleic acid (BNA); and (iii) an OSA, which is no attached support
and/or indicator, or an attached support or other means of
localization, including, but not limit to, attachment to beads,
polymers, and surfaces, and/or indicators; the BNA comprises: (i) a
1/2 BBR, as shown in FIG. 1(IIb), which has a sequence which is
complementary to a 1/2 BBR sequence in a PNA and which is capable
of forming, under hybridizing conditions, a hybrid, BBR, with the
PNA; (ii) an OSA, which is no attached support or indicator or an
attached support or other means of localization, including, but not
limited to, attachment to beads, polymers, and surfaces, and/or
indicators; (iii) additional hybridization sites, 1/2 BBRs, for
other BNAs; and (iv) sequences, 1/2 BBRs, which can hybridize to
BNAs already hybridized to the PNA; the BBA comprises: (i) a
molecule or a portion of a molecule which is capable of selectively
binding to a BBR; and (ii) an OSA, which is no attached support
and/or indicator, or an attached support or other means of
localization, including, but not limited to, attachment to beads,
polymers, and surfaces, and/or indicators; and the TBA comprises:
(i) a molecule or a portion of a molecule which is capable of
selectively binding to a TBR; and (ii) no attached support and/or
indicator, or an attached support or other means of localization,
including, but not limited to, attachment to beads, polymers, and
surfaces, and/or indicators.
11. In a solid phase hybridization method for detecting the
presence of a target polynucleotide involving: immobilizing a
target polynucleotide, if present in a test sample, directly or via
an intermediate capture structure, on a solid phase at a capture
site; before, during or after said immobilization, attaching a
detectable label to said target polynucleotide, if present; and
detecting said label, if any, at said capture site; the improvement
comprising: (a) using a Target Binding Assembly, TBA, as the means
for achieving immobilization of said target polynucleotide, wherein
said TBA binds only to a unique hybrid formed between a specific
Probe Nucleic Acid, PNA, and said target nucleic acid such that a
perfect Target Binding Region, TBR, recognizable by said TBA is
formed; and (b) including in the PNA a single stranded sequence,
1/2 BBR, capable of binding a Booster Nucleic Acid, BNA, containing
a single stranded complementary 1/2 BBR which, upon hybridization
with the 1/2 BBR in the PNA, forms a BBR capable of binding labeled
Booster Binding Assemblies, BBAs.
12. A target binding assembly, TBA, or a booster binding assembly,
BBA, comprising at least one nucleic acid recognition unit, and
optional one or all of the sequences selected from the group
consisting of a linker sequence, an assembly sequence, an asymmetry
sequence, a nuclear localization signal sequence (NLS) and an
OSA.
13. The TBA of claim 12 wherein the nucleic acid recognition unit
is selected from the group consisting of an NF-kB binding unit, an
SP1 binding unit, a TATA binding unit, a human papillomavirus E2
binding unit, an HPV LTR binding unit, an HIV LTR binding unit, and
Tat.
14. The TBA of claim 13 wherein the nucleic acid recognition unit
has the sequence selected from the group consisting of SEQ ID NO.
63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ
ID NO. 68 SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71, SEQ ID NO.
72, SEQ ID NO. 73, SEQ ID NO. 74, SEQ ID NO. 75, SEQ ID NO. 76, SEQ
ID NO. 77, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 80, SEQ ID NO.
81, SEQ ID NO. 82, SEQ ID NO. 83, SEQ ID NO. 84, SEQ ID NO. 93, SEQ
ID NO. 94, SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. 97,SEQ ID NO.
98, and SEQ ID NO. 118.
15. The TBA of claim 12 wherein the linker sequence is an
oligopeptide which does not interfere with the nucleic acid
recognition function of the nucleic acid recognition unit and which
provides stability and control over the spacing of the nucleic acid
recognition unit from the remainder of the TBA.
16. The TBA of claim 15 wherein the linker sequence is an
oligopeptide sequence from the interdomain primary of a structural
protein.
17. The TBA of claim 12 wherein the assembly sequence is an
oligopeptide sequence which directs the folding and association of
nucleic acid recognition units.
18. The TBA of claim 17 wherein the assembly sequence is derived
from the bacteriophage lambda cro protein or the CI protein and is
selected from the group consisting of SEQ ID NO. 104, SEQ ID NO.
105, SEQ IN NO. 106 , SEQ ID NO. 107, and SEQ ID NO. 108.
19. The TBA of claim 12 wherein the asymmetry sequence directs the
association of nucleic acid recognition and assembly sequences in a
predetermined order.
20. The TBA of claim 19 wherein the asymmetry sequence is derived
from insulin, gonadotropic hormone, FSH, HCG, LH, ACTH, or
relaxin.
21. The TBA of claim 20 wherein the asymmetry sequence is selected
from the group consisting of SEQ ID NO. 85, SEQ ID NO. 86, SEQ ID
NO. 87, SEQ ID NO. 88, SEQ ID NO. 89, SEQ ID NO. 90, SEQ ID NO. 91,
and SEQ ID NO. 92.
22. The TBA of claim 12 wherein the NLS is an oligopeptide which
directs the migration and uptake of a protein or complex associated
with said NLS into the nucleus of a cell.
23. The TBA of claim 22 wherein the NLS is selected from the group
consisting of SEQ ID NO. 72 and SEQ ID NO. 103.
24. The TBA of claim 12 which is HIV Detect I-IV or HPV Detect
I-IV.
25. The TBA of claim 12 having a sequence selected from the group
consisting of SEQ ID NO. 109, SEQ ID NO. 110, SEQ ID NO. 111, SEQ
ID NO. 112, SEQ ID NO. 113, SEQ ID NO. 114, SEQ ID NO. 115, SEQ ID
NO. 116.
26. A method of using the TBA of claim 12 to bind a particular
nucleic acid sequence in a target nucleic acid sample which
comprises: (a) fragmenting the nucleic acid in the target nucleic
acid sample; (b) contacting, under hybridizing conditions, the
fragmented nucleic acid with a probe nucleic acid complementary to
the particular nucleic acid sequence of interest, wherein said
probe nucleic acid, upon hybridization with said particular nucleic
acid sequence of interest forms a target binding region to which
said TBA specifically binds.
27. The method of claim 26 wherein said probe nucleic acid, in
addition to sequences complementary to said particular nucleic acid
sequence of interest, also has additional sequences to which a
booster nucleic acid can bind to form a booster binding site to
which a labeled booster binding assembly can bind to provide a
signal showing and amplifying the binding of the probe nucleic acid
to the target nucleic acid sequence of interest.
28. A method of using the TBA of claim 12 wherein said TBA is
administered to patient in need of such treatment a therapeutically
or prophylactically effective amount of said TBA, which comprises
administering the TBA, either in the form of a purified protein
complex or in the form of a recombinant vector which, upon entry
into the patient is able to express the TBA, such that the TBA
binds the particular nucleic acid sequence to achieve the desired
prophylactic or therapeutic result.
29. The method of claim 28 wherein said TBA is selected from the
group consisting of SEQ ID NO. 109, SEQ ID NO. 110, SEQ ID NO. 111,
SEQ ID NO. 112, SEQ ID NO. 113, SEQ ID NO. 114, SEQ ID NO. 115, and
SEQ ID NO. 116, and the patient is infected with HIV or HPV.
30. The method of claim 26 further comprising the step of: (c)
monitoring the shift in mobility of nucleic acids in the target
nucleic acid sample as a function of the size such that binding of
the TBA to a particular fragment in the sample modifies the
mobility of the fragment.
31. A diagnostic or forensic test kit for the detection in a sample
of nucleic acid having a specific sequence composition, which
comprises: (a) a first nucleic acid probe complementary to nucleic
acid with specific sequence composition, the presence of which is
to be ascertained in a test sample, wherein said first nucleic acid
probe and said nucleic acid with specific sequence composition
forming, upon hybridization, a binding site for a first nucleic
acid binding protein, and wherein said first nucleic acid probe
further comprises additional sequence complementary to a second
nucleic acid probe; (b) a first nucleic acid binding protein
specific for the duplex formed by hybridization of said first
nucleic acid probe and said nucleic acid with specific sequence
composition; (c) a second nucleic acid probe complementary to said
additional sequence in said first nucleic acid probe, wherein, upon
hybridization of said first and second nucleic acid probes, a
binding site for a second nucleic acid binding protein is formed;
(d) a second nucleic acid binding protein which binds specifically
to the duplex formed upon hybridization of said first nucleic acid
probe and said second nucleic acid probe, wherein said second
nucleic acid binding protein is labeled with a detetable label.
32. The diagnostic or forensic test kit of claim 31 wherein said
first nucleic acid probe is complementary to the HIV LTR, such that
upon hybridization of said first nucleic acid probe with an HIV
LTR, a binding site is formed for NF-kB or a subunit thereof, SP1,
TATA binding protein, HIV-Detect I, II, III, or IV, or
HIV-Lock.
33. The diagnostic or forensic test kit of claim 32 wherein said
first nucleic acid binding protein is NF-kB or a subunit thereon
SP1, TATA binding protein HIV-Detect I, II, III, or IV, or
HIV-Lock.
34. The diagnostic or forensic test kit of claim 33 wherein said
first nucleic acid probe, in addition to being complementary to the
HIV LTR, comprises a sequence encoding the bacteriophage lambda
left or right operator and said second nucleic acid probe comprises
sequences complementary to said bacteriophage lambda left or right
operator sequences in said first nucleic acid probe, such that upon
hybridization of said first and second nucleic acid probes, a
binding site for the bacteriophage lambda CI repressor protein, the
bacteriophage lambda cro protein or a derivative or homology
thereof, is formed.
35. The diagnostic or forensic test kit of claim 34 wherein said
second nucleic acid binding protein is the bacteriophage lambda CI
repressor protein, the bacteriophage lambda cro protein or a
derivative or homology thereof.
36. A composition comprising HIV-Lock or a recombinant vector
encoding HIV-Lock and a pharmaceutically acceptable carrier.
37. A method of differentially binding a nucleic acid binding
protein to a nucleic acid sequence correlated with a pathogenic
condition which comprises: (a) selecting a particular configuration
of nucleic acid binding protein sequences present in the nucleic
acid sequence correlated with a pathogenic condition as a target
sequence for designing a probe nucleic acid which will hybridize to
that particular configuration of nucleic acid sequences if present
in a test sample, and further, ensuring that a binding site for an
available nucleic acid binding protein is formed upon hybridization
of said probe nucleic acid and said particular configuration of
nucleic acid sequences chosen as a target; (b) selecting a nucleic
acid binding protein which specifically binds to the selected
particular configuration of nucleic acid binding protein sequences
correlated with a pathogenic condition, but which does not bind to
sequences not correlated with said pathogenic condition; (c)
hybridizing said probe nucleic acid with a test sample suspected of
containing said particular configuration of nucleic acid binding
protein sequences present in nucleic acid sequences correlated with
a pathogenic condition; (d) contacting said nucleic acid binding
protein with any hybrids formed in step (b); and (e) detecting any
binding of said nucleic acid binding protein with said hybrids.
38. The method of claim 37 wherein said particular configuration of
nucleic acid binding protein sequences is chosen from a necessary
step or control point in the development of a pathogenic
condition.
39. The method of claim 9 wherein said method is carried out in an
automated fashion.
40. The method of claim 39 wherein the method is carried out in the
Abbott Laboratories IMx machine.
41. The method of claim 9 carried out in a microtiter plate.
42. A method of amplifying the signal obtained through binding the
PNA of claim 1 to a TNA which comprises binding BNAs to the PNA-TNA
hybrid and binding labeled BBAs to the BNAs.
43. A method of assembling a nucleic acid binding complex which
comprises using asymmetry sequences to direct the association or
non-association of components of the nucleic acid binding
complex.
44. A method of assembling a nucleic acid binding complex which
comprises using assembly sequences derived from bacteriophage
lambda cro or CI to assemble associated components of the nucleic
acid binding complex.
45. A method of using assembly, asymmetry, or piloting sequences to
assemble a multimeric protein complex which comprises linking
subunits to be incorporated into the multimeric protein complex to
said assembly, asymmetry, or piloting sequence, and recovering said
multimeric complex.
46. A composition comprising SEQ ID NO. 105, SEQ ID NO. 106, or SEQ
ID NO. 108.
47. A nucleic acid encoding the TBA or BBA of claim 12.
48. The TBA of claim 12 or a nucleic acid encoding said TBA,
wherein the amino acid sequence of said TBA is selected from the
group consisting of set A, set B and set C, wherein said sets are
comprised as follows:
7 Set Link Sequences from Groups A I + II + II B IV + V + III C IV
+ III
wherein groups I-V consist of sequences selected from: Group
Selected from Sequences I Any of SEQ ID NOS. 85-92 II Met Ser,
linked to any of SEQ ID NOS. 104-106, each of which is linked to
SEQ ID NO. 99 III SEQ ID NO. 100 linked to any of SEQ ID NOS. 75-84
or 94-98; SEQ ID NO. 101 linked to either SEQ ID NO. 74 of SEQ ID
NO. 93; or SEQ ID NO. 102 linked to SEQ ID NO. 74 or SEQ ID NO. 93;
or any of SEQ ID NOS. 72, 103, 73, or 63-71 IV Any of SEQ ID NOS.
104-108 V SEQ ID NO. 99.
49. A method of assembling multimeric TBAs in vivo or in situ which
comprises introducing component TBAs into a cell utilizing a
covalently or non-covalently attached protein or bi-layer vesicle
or by introducing nucleic acids encoding component TBAs into a
cell, said component TBAs each comprising a DNA recognition unit,
assembly sequences, asymmetry sequences, nuclear localization
signal sequences, and optional linker sequences, such that upon
proximal binding via the DNA recognition unit of each component TBA
to nucleic acid sequences encountered in the nucleus or elsewhere
in the cell, component expressed TBAs assemble via said assembly
and asymmetry sequences into multimeric TBAs.
50. A method for identifying nucleic acid binding molecules for
preparation of a target binding assembly or a booster binding
assembly comprising: a. Obtaining a sample containing the target
nucleic acid; b. Fragmenting the sample so as to expose the nucleic
acids and to reduce the size complexity of the nucleic acids
contained in the sample; c. Contacting a first aliquot of the
fragmented nucleic acids with a control buffer medium and
contacting a second aliquot of the fragmented nucleic acids with
the control buffer medium containing a known profile of nucleic
acid binding molecules; d. Analyzing the two aliquots to identify
fragments which have altered behavior in the aliquot contacted with
the target binding molecules as opposed to the aliquot contacted
with the control buffer medium; e. Identifying and isolating
fragments which do exhibit altered behavior when contacted with the
nucleic acid binding molecule and either sequencing the nucleic
acid fragment to determine whether known nucleic acid binding
molecule motifs are present, or directly identifying the nucleic
acid binding molecule bound to the nucleic acid; and f.
synthesizing TBAs comprising the nucleic acid binding molecules
which produced the altered behavior using assembly, asymmetry
nuclear localization and, optionally, linker sequences.
51. A method for identifying specific nucleic acid sequences in a
sample comprising: a. Fragmenting the nucleic acids in said sample
to expose the nucleic acids and reduce the size complexity of the
nucleic acids; b. Contacting a TBA with the sample, said TBA
comprising two or more nucleic acid binding components each of
which has a relatively weak binding for its nucleic acid
recognition unit within the TBR but which in combination provides
strong binding for the complete TBR; and c. Eliminating any
"cross-talk" produced by binding of the TBA to cousin nucleic acids
that contain individual recognition units, which comprises
contacting the sample with excess nucleic acid binding components
with relatively strong binding affinity for cousin nucleic acids
that contain the individual recognition units but relatively weak
binding relative to the TBA's affinity for binding to the complete
TBR having said two or more nucleic acid binding components.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention provides a method and compositions for use in
binding, detecting, and amplifying the detection of specific Target
Nucleic Acid sequences in a sample with fidelity and accuracy, even
in the presence of closely related but different nucleic acids. The
binding may involve the chaperoning and assembly of specific
molecules into Target Binding Assemblies which specifically bind
Target Binding Regions formed by the hybridization of Probe Nucleic
Acids and Target Nucleic Acid sequences. The amplifying may involve
the chaperoning and/or assembly of specific molecules into Booster
Binding Assemblies which specifically bind Booster Binding Regions
formed by the hybridization of Booster Nucleic Acids with Probe
Nucleic Acids, Target Nucleic Acids, or other Booster Nucleic
Acids. A method, and compositions, involving Hairpin Nucleic Acids
is also provided to enable control of the size of specifically or
non-specifically elongated Booster Nucleic Acids and Booster
Binding Assemblies used in the amplification. The detecting
involves providing one or more detectable labels, including
radioactive, light- or fluorescent-emitting, enzymatic, or other
detectable or signal-generating molecules, in association with the
Probe Nucleic Acid, the Target Binding Assembly, the Booster
Nucleic Acid, the Booster Binding Assembly, or the Hairpin Nucleic
Acid. A method is presented for isolating nucleic acid fragments
from an organism which has TBA component binding sites in order to
create a probe nucleic acid and a TBA which is unique for that
fragment and/or organism. Therapeutic and prophylactic uses of the
Target Binding Assemblies and compositions for such use are also
provided.
[0003] 2. Background and Description of Related Art
[0004] There are an increasing number of cases in which it is
important to be able to detect nucleic acids containing a specific
sequence, hereinafter named Target Nucleic Acids (TNAs), in a
sample. It is desirable to be able to detect the TNAs with the
smallest number of processing steps, with the simplest components
and to the exclusion of other similar but different nucleic acids,
hereinafter named Cousin Nucleic Acids (CNAs). It is desirable to
be able to detect specific TNAs to the exclusion of any and all
CNAs in the detection sample without the necessity of amplification
or other post-detection processing.
[0005] There are numerous methods which use immobilized or tagged
nucleic acids as probes for TNAs. However, using known methods, it
is difficult to discriminate between a TNA bound to the Probe
Nucleic Acid (PNA) as opposed to a CNA bound to the PNA. For
example, one or more base mismatches between the PNA and a CNA can
still result in a CNA-PNA hybridization which is almost
indistinguishable from a TNA-PNA hybridization. Thus, hybridization
alone is not an optimal indicator that a PNA has hybridized to a
unique TNA.
[0006] There are many situations in which a PNA would be used to
try to determine whether a TNA was present in a sample which may
contain CNAs. Hybridization of the PNA to any CNA in this situation
would limit the diagnostic value that the PNA might have for the
detection of a TNA, absent additional verification. Furthermore, it
is desirable to be able to detect and localize TNAs with low copy
numbers in samples which may contain many copies of CNAs, without
the necessity of creating additional copies of the TNA. It would
also be desirable to be able to confirm the presence of CNAs,
independent of the TNAs, without the necessity of separating the
CNAs and TNAs in the sample.
[0007] Furthermore, it would be desirable to be able to amplify the
signal of even a low frequency hybridization of a particular
TNA-PNA. For this purpose, a method of polymers multiple copies of
a label, hereinafter referred to as a Booster Nucleic Acid (BNA)
onto the TNA-PNA would be desirable.
[0008] The instant invention provides methods and compositions for
achieving the foregoing desired objectives. As revealed by the
following review, the instant compositions and methods have not
been reported or suggested in the art. A general and comprehensive
review of the state of art of nucleic acid detection is provided in
Keller, H., M. M. Manak (1989) DNA Probes, Stockton Press.
[0009] A method has been reported for detecting base pair
mismatches by chemical means in order to determine whether a PNA
has hybridized to a CNA rather than to a TNA. In U.S. Pat. No.
4,794,075 to Ford et al., a method for distinguishing fragments of
DNA which contain single base mismatches from their perfectly
paired homologs is discussed. Single stranded regions within a
duplex fragment are modified with carbodiimide, which reacts with
unpaired guanine (G) and thymine (T) residues in DNA. Linear duplex
DNA molecules do not react, while DNA molecules with single base
mismatches react quantitatively. Following reaction with
carbodiimide, the DNA molecules are fractionated on high percentage
polyacrylamide gels such that modified and unmodified fragments can
be distinguished. Ford et al. applied this technique in order to
locate and purify DNA sequence differences responsible for
phenotype variation and inherited disease. Although this method is
useful for following variations in genetic material, it has a large
number of steps, it requires costly components, and it does not
offer a direct means of determining whether a PNA has hybridized to
the TNA exclusive of CNAs in the sample.
[0010] There have been some attempts to assure that at least a
portion of the hybridization between the PNA and another nucleic
acid is complementary. One method involves the monitoring of
transcription products which are produced if the PNA hybridizes to
a nucleic acid sufficiently to be transcribed from a promoter site
contained in the probe. U.S. Pat. No. 5,215,899 to Dattagupta
discloses how specific nucleic acid sequences are amplified through
the use of a hairpin probe which, upon hybridization with and
ligation to a target sequence, is capable of being transcribed. The
probe comprises a single self-complementary sequence which, under
hybridizing conditions, forms a hairpin structure having a
functional promoter region, and further comprises a single stranded
probe sequence extending from the 3' end of the hairpin sequence.
Upon hybridization with a target sequence complementary to the
probe sequence and ligation of the 3' end of the hybridized target
sequence to the 5' end of the hairpin probe, the target sequence is
rendered transcribable in the presence of a suitable RNA polymerase
and appropriate ribonucleoside triphosphates (rNTPs). Amplification
is accomplished by hybridizing the desired TNA sequence with the
probe, ligating the TNA to the PNA, adding the RNA polymerase and
the rNTPs to the separated hybrids, and allowing transcription to
proceed until a desired amount of RNA transcription product has
accumulated. That method generally and specifically involves the
use of hairpin DNA formed with a single stranded unpaired end to
anneal a target sequence. When the target sequence is bound, the
production of RNA transcription products is enabled. Thus, the
method involves the detection of secondary transcription products
rather than the use of a nucleic acid binding assembly to directly
immobilize and/or localize a target sequence. A CNA could easily
bind to the probe, and the lack of complementarity would not
necessarily interfere with the formation of a CNA-PNA hybrid which
could then support the production of unwanted transcription
products.
[0011] A CNA bound to the PNA might be detected if the lack of
complementarity interferes with the susceptibility of the hybrid
CNA-PNA pair to be cut by a restriction endonuclease. In U.S. Pat.
No. 5,118,605 to Udea and U.S. Pat. No. 4,775,619 to Urdea, novel
methods for assaying a nucleic acid analyte were provided, which
employ polynucleotides having oligonucleotide sequences
substantially homologous to a sequence of interest in the analyte,
where the presence or absence of hybridization at a predetermined
stringency provides for the release of a label from a support.
Various techniques are employed for binding a label to a support,
whereupon cleavage of either a single or double strand a label may
be released from a support, and the release of the label can be
detected as indicative of the presence of a particular
polynucleotide sequence in a sample. However, this technique has
the shortcoming that a CNA-PNA pair could be cut by the restriction
endonuclease, even if there is a mismatch, so long as the mismatch
was outside of the endonuclease recognition region. This would lead
to failure of the assay to identify a CNA-PNA hybrid.
[0012] Another method uses a branched DNA probe to detect nucleic
acids. U.S. Pat. No. 5,124,246 to Urdea et al. discloses liner or
branched oligonucleotide multimers useful as amplifiers in
biochemical assays which comprise (1) at least one first
single-stranded oligonucleotide unit (PNA) that is complementary to
a single-stranded oligomucleotide sequence of interest (TNA), and
(2) a multiplicity of second single-stranded, oligonucleotide units
that are complementary to a single-stranded labeled
oligonucleotide. Although amplified sandwich nucleic acid
hybridizations and immunoassays using the multimers are described,
the method has the limitation that PNA-CNA hybridization could
occur and would reset in production of unwanted signal.
[0013] In addition to methods for identification of TNAs, methods
have been disclosed for the amplification of this DNA. In U.S. Pat.
No. 5,200,314 to Urdea, an analyte polynucleotide strand having an
analyte sequence (TNA) is detected within a sample containing
polynucleotides by contacting the analyte polynucleotide with a
capture probe (PNA) under hybridizing conditions, where the capture
probe has a first binding partner specific for the TNA, and a
second binding sequence specific for a solid phase third binding
partner. The resulting duplex is then immobilized by specific
binding between the binding partners, and non-bound polynucleotides
are separated from the bound species. The analyte polynucleotide is
optionally displaced from the solid phase, then amplified by PCR.
The PCR primers each have a polynucleotide region capable of
hybridizing to a region of the analyte polynucleotide, and at least
one of the primers further has an additional binding partner
capable of binding a solid-phase binding partner. The amplified
product is then separated from the reaction mixture by specific
binding between the binding partners, and the amplified product is
detected. Although it is possible to confirm (by PCR) that a
particular nucleic acid has hybridized with the PNA, confirmation
is expensive and involves multiple steps.
[0014] As for reports that involve the interaction of a double
stranded nucleic acid and a DNA-binding protein, a method has been
described whereby a sequence of immobilized DNA which contains
binding sites for a single protein is used to purify that protein.
U.S. Pat. No. 5,122,600 to Kawaguchi et al. discloses a
DNA-immobilized microsphere comprising DNA chains having base
sequences which specifically bind a particular protein, and a
carrier having a particle size of not more than 50 .mu.m and not
less tan 0.01 .mu.m which does not adsorb any protein, said carrier
and said DNA chains being bound to each other by a chemical bond,
and a process for purifying a protein using said microsphere. As
this is a purification method for a protein, it does not disclose a
method of detection of a TNA nor a method whereby more than one
protein is bound to a double stranded nucleic acid for the purposes
of detection and localization of specific TNA sequences.
[0015] In EP 0 453 301, a method for detecting a polynucleotide
target sequence in a sample was described wherein sequences in a
TNA are detected by hybridizing a first and a second PNA to the
TNA. Each of said first and second PNAs contained a pre-formed
duplex sequence, or a duplex that is formed through chin extension,
capable of binding a nucleotide sequence specific binding protein.
A method for binding a nucleotide specific binding protein to a
duplex formed between a TNA and a PNA only upon formation of a
duplex between the PNA and TNA is neither disclosed nor
suggested.
[0016] In US. Pat. No. 4,556,643, a method was disclosed for the
non-radioactive detection of specific nucleotide sequences in a
sample which involved hybridization of a probe containing DNA
binding protein specific sequences. However, this disclosure
neither taught nor suggested a method for binding a nucleotide
specific biding protein to a duplex formed between a TNA and a PNA
only upon formation of a duplex between sequences present in the
PNA and sequences present in the TNA.
BRIEF SUMMARY OF THE INVENTION
[0017] Disclosed are methods by which specific Target Nucleic Acid
(TNA) sequences are detected through the use of Probe Nucleic Acids
(PNAs) which, upon hybridization with TNAs, are capable of binding
Target Binding Assemblies (TBAs). Each TBA binds at least one
specific region of the PNA-TNA hybrid pair, the Target Binding
Region (TBR). The TBA is comprised of one or more molecules, one or
more of which can bind to TBR sequences in a specific and sequence
or conformation dependent manner. The TBA may comprise one or more
piloting sequences, called "PILOTS" or "Asymmetry Sequences," which
assemble and constrain the nucleotide binding components of the TBA
to specific geometries. The PILOTS act to assemble specific nucleic
acid recognition units or other pilots to which specific nucleic
acid recognition units are attached into the TBAs in a
predetermined fashion. The TBA may also contain one or more
molecules which anchor or localize the TBA. Novel TBAs having
unique discriminating characteristics which surprisingly render the
TBAs useful not only as diagnostic tools but also as prophylactic
or therapeutic compounds, are also disclosed. Disclosed are methods
and compositions for utilization of the PNAs, TBRs, TBAs, and TBA
PILOTS, including their utilization as components of diagnostic and
forensic test kits and the utilization of the novel TBAs as
prophylactic or therapeutic agents.
[0018] The PNAs, in addition to TNA-specific sequences, may also
contain one or more sequences, 1/2 BBRs, capable of hybridizing
with complementary 1/2 BBRs in Booster Nucleic Acids (BNAs).
Through hybridization of added BNAs to the starter 1/2 BBRs present
in the PNAs, extensions of the PNAs are made in the form of PNA-BNA
and then BNA-BNA hybrids. These extensions can contain one or more
Booster Binding Regions (BBRs). Each BBR is capable of being a
Booster Binding Assembly (BBA). The BBA is comprised of molecules,
one or more of which can bind to a BBR in a specific and sequence
or conformation dependent manner. The BBA may comprise one or more
piloting sequences, called "PILOTS" or "Asymmetry Sequences," which
assemble and constrain the nucleotide binding components of the TBA
to specific geometries. The PILOTS act to assemble specific nucleic
acid recognition units or other pilots to which specific nucleic
acid recognition units are attached into the BBAs in a
predetermined fashion. The BBA may contain molecules which anchor
or localize the BBA or which allow for detection of the bound BBAs
and thereby of the TBA-TNA-PNA complexes to which they, in turn,
are bound. Disclosed are methods and compositions for utilization
of the 1/2 BBRs, BNAs, BBRs, BBAs, and BBA PILOTS, including their
utilization as components of diagnostic and forensic test kits.
[0019] Methods and compositions are disclosed for the use of
Hairpin Nucleic Acids (HNAs) as capping structures. The HNAs
contain a self-hybridizing region and a single stranded 1/2 BBR
which, under hybridizing conditions, can hybridize directly to the
1/2 BBRs in the PNAs or the 1/2 BBRs in BNAs already bound to the
PNAs, to terminate the extension of BNAs onto the PNA or onto other
BNAs.
[0020] Methods and compositions are disclosed for test procedures
and the production of a test kit containing PNAs, TBAs, TBRs, BNAs,
BBRs, BBAs and HNAs for the detection, localization and
differentiation of specific nucleic acid sequences, including
nucleic acid sequences which are found in human cells, in the Human
Immunodeficiency Virus (HIV), Human Papillomavirus (HPV), and in
other nucleic acid containing systems including viruses and
bacteria.
[0021] Accordingly, it is an object of this invention to provide
methods and compositions for use in binding, detecting, and
amplifying the detection of specific Target Nucleic Acid sequences
in a sample with fidelity and accuracy, even in the presence of
closely related but different nucleic acid sequences.
[0022] Accordingly, it is an object of this invention to provide
methods and compositions for the creation of Target Binding
Assemblies which specifically bind Target Binding Regions formed by
the hybridization of Probe Nucleic Acids and Target Nucleic Acid
sequences.
[0023] Another object of this invention is to provide a method and
compositions for the creation of Booster Binding Assemblies which
specifically bind Booster Binding Regions formed by the
hybridization of Booster Nucleic Acid sequences with Probe Nucleic
Acids, Booster Nucleic Acids and Hairpin Nucleic Acids.
[0024] Another object of this invention is to provide a method and
compositions containing Hairpin Nucleic Acids which enable the
control of the size of specifically or non-specifically elongated
Booster Nucleic Acids and Booster Binding Assemblies used in
amplification of PNA-TNA hybridization events.
[0025] Another object of this invention is to provide a method and
compositions for use in the selection, assembly and or chaperoning
of specific molecules, each with nucleic acid binding
discriminating capabilities, into Target and Booster Binding
Assemblies.
[0026] Another object of this invention is to provide a method and
compositions for use in amplifying the detection of Target Binding
assemblies bound to Target Binding Regions using Booster Binding
Assemblies and Booster Nucleic Acids.
[0027] Another object of this invention is to provide a method and
compositions which allow the use of one or more detectable labels,
including but limited to radioactive labels, light emitting,
fluorescent, enzymatic or other signal generating molecules. These
labels are used in association with Probe Nucleic Acids, Target
Binding Assemblies, Booster Binding Assemblies, Booster Nucleic
Acids or Hairpin Nucleic Acids.
[0028] Another object of this invention is to provide a method for
isolating nucleic acid fragments form an organism which has TBA
component binding sites in order to create Probe Nucleic Acids and
TBAs which are unique for that fragment or organism.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] The following illustrations are contained in FIG. 1: FIG.
1-I is a PNA containing a 1/2 TBR, which is a single-stranded
sequence which is complementary to a TNA and a 1/2 BBR sequence.
FIG. 1-IIa is a TNA to which is added the components of FIG. 1-I,
and, under hybridizing conditions, binds the PNA to form the
components of FIG. 1-IIIa, a PNA-TNA hybrid containing at least one
TBR FIG. 1-IVa is a BNA which is a to the components of FIG. 1-IIIa
and, under hybridizing conditions, binds the 1/2 BBR of FIG. 1-IIIa
to form a PNA-BNA hybrid containing a BBR shown in FIG. 1-Va.
[0030] FIG. I-IIb is a BNA which is added the components of FIG.
1-I, and which, under hybridizing conditions, binds the PNA to form
the components of FIG. 1-IIIb, a PNA-TNA hybrid containing a BBR.
FIG. I-IVb is a TNA to which is added the components of FIG. 1-IIIb
and which, under hybridizing conditions, binds the 1/2 TBR of FIG.
1-IIIb to form a PNA-BNA hybrid containing a TBR shown in FIG.
I-Vb.
[0031] FIG. 1-IIc is a HNA which is added to the components of FIG.
1-I and which, under hybridizing conditions, binds the PNA to form
the components of FIG. 1-IIIc, a PNA-HNA hybrid containing a BBR.
FIG. 1-IVc is a TNA which is added to the components of FIG. 1-IIIc
and which, under hybridizing conditions, binds the 1/2 TBR of FIG.
1-IIIc to form a PNA-BNA hybrid containing a BBR shown in FIG.
I-Vc.
[0032] The hybrids which form the TBRs and BBRs are useful in the
present invention. The PNAs and BNAs, as indicated in FIG. 1, may
contain no attached support and/or indicator (OSA), or an attached
support or other means of localization, including, but not limited
to, attachment to heads, polymers, and surfaces, and/or
indicators.
[0033] FIG. 2a is a diagram of strategies for polymerization of
BNAs onto PNAs and capping by HNAs.
[0034] FIG. 2b is a diagram of additional strategies for amplifying
PNA-TNA signals via polymerization of BNAs and capping by HNAs.
[0035] FIG. 3 is a diagram showing the use of BNAs containing
multiple 1/2 BBRs per BNA.
[0036] FIG. 4a is a diagram showing the binding of TBAs and BBAs to
TBRs and BBRs, and the ability of the TBA to discriminate between
TNAs and CNAs. According to this embodiment, if the TBA is
immobilized, either on a bead, microtiter plate surface, or any
other such surface, only complexes such as complex X would be red
and detected, while complexes such as complex XI would not.
[0037] FIG. 4b is a diagram exemplifying events similar to those
shown in FIG. 4a but in a slightly different order of
occurrence.
[0038] FIG. 5 is a diagram exemplifying PNAs containing between one
1/2 TBR and no 1/2 BBR to PNAs containing up to five 1/2 TBRs and
one 1/2 BBR. The (a) and (b) members of each numeral (I, II, III,
IV, V) form a set which, upon hybridization to a TNA, provide TBRs
either with ((a) members) or without ((b) members) an available 1/2
BBR for amplification via hybridization to BNAs having
complementary 1/2 BBRs.
[0039] FIG. 6a is a diagram exemplifying a particular TNA having
two 1/2 TBRs which, upon binding an appropriate PNA, forms two
closely associated TBRs capable of binding two TBAs. A 1/2 BER is
also provided for amplification.
[0040] FIG. 6b is a diagram showing the same events as in FIG. 6a
except here, a double TBA is used so that discrimination between
single TBRs that occur in normal cellular samples may be
discriminated from abnormal, double TBRs.
[0041] FIG. 6c is a diagram showing the same scenario as in FIG. 6a
except that here, five TBRs are identified in the TNA. Each TBR may
be bound to a TBA same or different, and each TBA may be
differentially labeled, allowing for confirmation that all five
sites are present in the TNA.
[0042] FIG. 6d is a diagram of the same events as in FIG. 6c except
here, a double TBA is shown, extending what is shown in FIG. 6b to
the use of the double TBA. An example of the TNA shown in item II
in FIGS. 6a, 6b, 6c and 6d is HIV single stranded DNA or RNA.
[0043] FIG. 7 shows the HIV LTR as a TNA, and two PNAs, and a
strategy for detection of the TNA using the PNAs.
[0044] FIG. 8 is a schematic of one embodiment of the invention
wherein a target binding assembly is used to bind a hybrid TNA-PNA,
and booster binding assemblies are used to bind polymerized
BNAs.
[0045] FIG. 9 is a schematic of a modular TBA in which assembly
sequences, linker sequences, and asymmetry sequences are used to
chaperone desired nucleic acid recognition units together to form a
TBA.
[0046] FIG. 10 shows modular TBAs useful in detection of
HIV-specific sequences.
[0047] FIG. 11 shows modular TBAs useful in the detection of human
papillomavirus sequences. Each unit of E2 is actually a dimer of
the DNA binding portion of E2.
[0048] FIG. 12a is a schematic of TNA fractionation and shift in
mobility due to binding of a TBA.
[0049] FIG. 12b is a schematic of TNA fractionation and enhanced
shift in mobility due to binding of BBAs in addition to TBAs.
[0050] FIG. 13 shows a detection strategy for deletion sequences;
an example of use of this strategy is for a human papillomavirus
integration assay.
[0051] FIG. 14 shows assembly of higher order TBAs through use of
nucleic acid recognition units, linker, assembly, and asymmetry
sequences such that various Target Binding Assemblies specific to
binding sites in the HIV LTR are formed.
[0052] FIG. 15 shows assembly of higher order TBAs through use of
DNA recognition units, linker, assembly, and asymmetry sequences
such that various Target Binding Assemblies specific to binding
sites in the HPV genome are formed.
[0053] FIG. 16 shows the discrimination achieved by using a complex
TBA and the ability of endogenous competitor target binding
molecules to eliminate binding of the TBA to a cousin nucleic acid
but not from the TNA which contains the appropriate orientation of
more than one site recognized by the TBA.
[0054] FIG. 17 shows the ability of a TBA to specifically be
targeted to bind to sites of sequence mismatch and to
preferentially bind those sites over cousin sites which do not
contain all of the targeted mismatches.
BRIEF DESCRIPTION OF THE SEQUENCES
[0055] SEQ ID NO. 1 corresponds to FIG. 5-Ia-1 and shows the class
I MHC NF-kB binding site.
[0056] SEQ ID NO. 2 corresponds to FIG. 5 (Ia) and shows the
B2-microglobulin NF-kB binding site.
[0057] SEQ ID NO. 3 corresponds to FIG. 5 (Ia) and shows the kappa
immunoglobulin NF-kB binding site.
[0058] SEQ ED NO. 4 corresponds to FIG. 5 (Ia) and shows one of the
HIV NF-kB binding sites.
[0059] SEQ ID NO. 5 corresponds to FIG. 5 (Ia) and shows one of the
HIV NF-kB binding sites.
[0060] SEQ ID NO. 6 corresponds to FIG. 5 (Ia) and shows the c-myc
NF-kB binding site.
[0061] SEQ ID NO. 7 corresponds to FIG. 5 (IIa) and shows a double
HIV NF-kB binding site.
[0062] SEQ ID NO. 8 corresponds to FIG. 5 (IIa) and shows a double
HIV NF-kB binding site.
[0063] SEQ ID NOS. 9-16 correspond to FIG. 5 (IIa) and show a
double binding site with one site being an HIV NF-kB binding site,
and the other site being an HIV SP1 binding site.
[0064] SEQ ID NOS. 17-18 correspond to FIG. 5 (IIa) and show a
double HIV SP1 binding site.
[0065] SEQ ID NOS. 19-31 correspond to FIG. 5 (IIIa) and show a
double HIV NF-kB binding site and an HIV SP1 binding site.
[0066] SEQ ID NOS. 32-33 correspond to FIG. 5 (IVa) and show a
quadruple binding site where two sites are HIV NF-kB binding sites
and two sites are HIV SP1 binding sites.
[0067] SEQ ID NO. 34 corresponds to FIG. 5 VIa) and show a
quintuple binding site where two sites are HIV NF-kB binding sites
and three sites are HIV SP1 binding sites.
[0068] SEQ ID NO. 35 is an example of a 1/2 BBR, in this case the
OL1, OL2 and OL3 elements of the bacteriophage lambda left
operator, including intervening sequences.
[0069] SEQ ID NO. 36 is an example of a 1/2 BBR, in this case the
OR3, OR2 and OR1 elements of the bacteriophage lambda right
operator, including intervening sequences.
[0070] SEQ ID NO. 37 is the HIV LTR.
[0071] SEQ ID NO. 38 is a PNA complementary to PNA of the HIV
LTR.
[0072] SEQ ID NO. 39 is a PNA complementary to a different PNA of
the HIV LTR than SEQ ID NO. 38.
[0073] SEQ ID NO. 40 is a PNA complementary to part of the HIV LTR
and it also contains a 1/2 BBR and an overhang sequence for
polymerizing BNAs onto the PNA.
[0074] SEQ ID NO. 41 is a BNA complementary to the SEQ ID NO. 40
1/2 BBR.
[0075] SEQ ID NO. 42 is a BNA that will polymerize onto the SEQ ID
NO. 41 BNA and which, with SEQ ID NOS. 40 and 41, creates a PstI
recognition site.
[0076] SEQ ID NO. 43 is a BNA that is complementary to the SEQ ID
NO. 42 BNA and which completes a BamHI recognition site.
[0077] SEQ ID NO. 44 is an HNA which has a BamHI recognition site
that will hybridize with the BamHI recognition site created by SEQ
ID NOS. 42 and 43 to the growing polymer.
[0078] SEQ ID NO. 45 is a second PNA which, like SEQ ID NO. 40, is
complementary to part of the HIV LTR, but not to the same sequences
as SEQ ID NO. 40. SEQ ID NO. 45 also encodes a 1/2 BBR and an
overhang which will allow polymerization of BNAs starting with a
Sph1 recognition site.
[0079] SEQ ID NOS. 46-62 are human papillomavirus (HPV) specific
PNAs which, upon hybridization with HPV sequences, form TBRs which
bind HPV DNA binding proteins.
[0080] SEQ ID NOS. 63-71 are NF-kB DNA recognition units for
incorporation into TBAs.
[0081] SEQ ID NO. 72 is a nuclear localization sequence.
[0082] SEQ ID NO. 73 is a SP1sequence recognition unit.
[0083] SEQ ID NO. 74 is a TATA binding protein recognition
unit.
[0084] SEQ ID NOS. 75-84 are papillomavirus E2 DNA recognition
units.
[0085] SEQ ID NOS. 85-92 are asymmetry sequences.
[0086] SEQ ID NO. 93 is an arabidopsis TATA binding protein
recognition unit.
[0087] SEQ ID NO. 94 is an HPV-16-E2-1 DNA binding protein
recognition unit.
[0088] SEQ ID NO. 95 is an HPV-16E2-2 DNA binding protein
recognition unit.
[0089] SEQ ID NO. 96 is an HPV-18-E2 DNA binding protein
recognition unit.
[0090] SEQ ID NO. 97 is an HPV-33-E2 DNA binding protein
recognition unit.
[0091] SEQ ID NO. 98 is a bovine papillomavirus E2 DNA binding
protein recognition unit.
[0092] SEQ ID NOS. 99-102 are exemplary linker sequences.
[0093] SEQ ID NO. 103 is an exemplary nuclear localization signal
sequence (NLS).
[0094] SEQ ID NOS. 104-108 are exemplary chaperone sequences.
[0095] SEQ ID NOS. 109-116 are exemplary assembled TBA
sequences
[0096] SEQ ID NO. 117 is a consensus NF-kB binding site.
[0097] SEQ ID NO. 118 an HIV Tat amino acid sequence.
1 Abbreviations 1 single stranded nucleic acid 2 double-stranded
nucleic acid 3 binding region on nucleic acid 4 no support or
indicators, or solid support, or other means of localization, in-
cluding, but not limited to, attachment to beads, polymers, and
surfaces, or indicators = OSA BBA booster binding assembly BBR
booster binding region BNA booster nucleic acid CNA cousin nucleic
acid 1/2 BBR single-stranded region which, when hybridized to the
complementary sequence from an HNA or a BNA, can bind a BBA 1/2 TBR
single-stranded region of the PNA which, when hybridized to the
complementary sequence from a TNA, can bind a TBA OSA optional
support or attch- ment, circle with box PNA probe nucleic acid TBA
target binding assembly TBR target binding region TNA target
nucleic acid HNA Hairpin Nucleic Acid
DEFINITIONS
[0098] It should also be understood from the disclosure which
follows that when mention is made of such terms as target binding
assemblies (TBAs), booster binding assemblies (BBAs), DNA binding
proteins, nucleic acid binding proteins or RNA binding proteins,
what is intended are compositions comprised of molecules which bind
to DNA or RNA target nucleic acid sequences (TNAs) irrespective of
the specificity of the category of binding molecules from which
they are derived. Thus, for example, a TBA adapted to bind to human
immunodeficiency virus sequences may be most similar to an NF-kB
transcriptional factor which typically binds DNA sequences.
However, as used herein, it will be understood that the TBA may be
adapted for optimal use to bind to RNA sequences of a particular
sequence composition or conformation.
[0099] The fidelity of the detection method disclosed herein
depends in large measure on the selective binding of TBAs and BBAs
to particular nucleic acid motifs. It should be understood
throughout this disclosure that the basis of TBA and BBA
discrimination of TNAs from related sequences (cousin nucleic acids
or CNAs) may be the formation of precise probe nucleic acid
(PNA)-target nucleic acid (TNA) hybrid segments (PNA-TNA hybrids).
However, the basis of discrimination may just as well be the
formation of a particular conformation, and may not require the
complete absence of mismatched-base pairing in the TNA-PNA hybrid.
Accordingly, the basis of TBA or BBA operation should be understood
throughout to depend on discrimination of any property unique to
the TNA-PNA hybrid as opposed to any properties displayed by any
PNA-CNA hybrids that may be formed in a test sample contacted with
a given PNA.
DETAILED DISCLOSURE OF THE INVENTION
[0100] The present invention provides a method for specifically
identifying a target nucleic acid (TNA) in a sample through the use
of target binding assemblies (TBAs) which incorporate specific
nucleic acid binding proteins. By using probe nucleic acids (PNAs)
specific to a given TNA sequence, and a TBA which is specific to
the duplex target binding region (TBR) formed upon formation of
hybrid TNA-PNA sequences, a stable TBA-TNA-PNA complex is formed.
By additionally providing specific amplifiable sequences in the
PNA, in addition to sequences which specifically contribute to the
formation of the TBR recognized by the TBA, the binding of the PNA
to the TNA is detected and the detection amplified. For this
purpose, any of a number of nucleic acid amplification systems,
including polymerase chain reaction, or the use of branched DNA,
each branch of which contains a detetable label, may be used. In
particular, a novel method of amplification is described herein
where the amplifiable portion of the PNA contains sequences onto
which booster nucleic acids (BNAs) may be polymerized. Upon
formation of each BNA-PNA hybrid, a booster binding region (BBR) is
formed to which a booster binding assembly (BBA) binds
specifically. If detectably labeled, the BBAs or BNAs provide
essentially unlimited amplification of the original TNA-PNA binding
event.
[0101] According to this invention, the TNA will be understood to
include specific nucleic acid sequences. The TBA will be understood
to be any molecular assembly which can specifically and tightly
bind to a formed TNA-PNA hybrid. The TBA will contain one or more
molecules whose sequences are sufficient to bind to the TBR.
Nucleic acid binding domains which are known can either be used
directly as components of the TBA or modified according to the
teachings provided herein. The most readily available molecules
with such sequences are the DNA-binding domains of DNA-binding
proteins. Specifically, many DNA or RNA binding proteins are known
which can either used directly as the known, unmodified protein, or
the TBA may be a nucleic acid binding protein, modified according
to the specific teachings provided herein. In the later case,
specific modifications that are desirable would include
optimization of binding affinities, removal of unwanted activities
(such as nuclease activity and reorganization of the TBA in the
presence of other molecules with an affinity for components of the
TBA), optimization of selectivity of a target sequence over closely
related sequences, and optimization of stability.
[0102] Examples of DNA binding proteins which could be used
according to this invention are the DNA-binding portions of the
transcription factor NF-kB (p50 and p65), NF-IL6, NF-AT, rel, TBP,
the papilloma virus'E2 protein, sp1, the repressors cro and CI from
bacteriophage lambda, and like proteins are well known proton whose
DNA binding portion has been isolated, cloned, sequenced, and
characterized. In addition, any other DNA-binding protein or
portion of a protein that is necessary and sufficient to bind to a
TBR hybrid or a BBR is included. This includes proteins or portions
of wild-type proteins with altered DNA binding activity as well as
protein created with altered DNA-binding specificity, such as the
exchange of a DNA-binding recognition helix from one protein to
another. In addition, proteins which exhibit nucleic acid biding
and other nucleic acid functions, such as restriction
endonucleases, could be used as the nucleic acid binding function.
Proteins which bind to target regions in DNA-RNA hybrids as well as
RNA-RNA hybrids are included. (See, for example, Shi 1995,
DeStefano 1993, Zhu 1995, Gonzales 1994, Salazar 1993, Jaishree
1993, Wang 1992, Roberts 1992, Kainz 1992, Salazar 1993(b)). The
binding assemblies may be constructed with the use of a molecule
which chaperones portions of the binding assembly so that specific
component combinations and geometries can be achieved. This
molecule is designated here as a PILOT. Pilots can be comprised of
proteins or any combination of organic and inorganic materials
which achieve the combinatorial selection and/or to induce specific
geometries between members of the TBA or BBAs. A chaperone is a
stable scaffold upon which a TBA or BBA may be constructed such
that the correct conformation of the TBA or BBA is provided while
at the same time eliminating undesirable properties of a naturally
occurring nucleic acid binding protein. As a specific example of
this embodiment, a modified version of the pleiotropic
transcription factor, NF-kB, is provided using a modified
bacteriophage lambda cro protein as the chaperone. Each NF-kB
binding dimer retains the picomolar binding affinity for the NF-kB
binding site while at the same time the binding assembly presents
several advantageous manufacturing, stability, and specificity
characteristics.
[0103] In view of the foregoing, the various aspects and
embodiments of this invention are described below in detail.
[0104] 1. The Probe Nucleic Acids (PNAs) and Their Preparation.
[0105] The PNAs of the present invention comprise at least three
principal parts joined together. With reference to FIG. 1(I) of the
drawings, the first part of the PNA is one or more sequences of
bases, designated "1/2 TBR". With reference to FIG. 1(I and IIa) of
the drawings, the 1/2 TBR in the PNA is complementary to a sequence
of interest in a sample, the TNA containing a 1/2 TBR. With
reference to FIG. I(IIIa) of the drawings, the TNA, when added to
the PNA under hybridizing conditions, forms a PNA-TNA hybrid
containing a TBR. With reference to FIG. 1(I) of the drawings, the
second part of the PNA is a sequence of bases, designated "1/2
BBR". With reference to FIG. 1(I, IIb, IIc, and IVa) of the
drawings, the 1/2 BBR in the PNA is complementary to a 1/2 BBR
contained in a BNA or a HNA. With reference to FIG. 1(IIIb, IIIc,
and Va) of the drawings, the BNA or HNA, when added to the PNA
under hybridizing conditions, forms a PNA-BNA hybrid or PNA-HNA
hybrid, respectively, containing a BBR. With reference to FIG. 1(I)
of the drawings, the third part of the PNA is the OSA, designated
by a circle with a box around it. The OSA is no support and/or an
indicator, or solid support, or other means of localization,
including but not limited to, attachment to beads, polymers, and
surfaces and/or indicators which is/are covalently attached to, or
non-covalently, but specifically, associated with the PNA. The OSA
may be an atom or molecule which aids in the separation and/or
localization such as a solid support binding group or label which
can be detected by various physical means including, but not
limited to, adsorption or imaging of emitted particles or light.
Methods for attaching indicators to oligonucleotides or for
immobilizing oligonucleotides to solid supports are well known in
the art (see Keller and Manak, supra, herein incorporated by
reference).
[0106] The PNA of the present invention can be prepared by any
suitable method. Such methods, in general, will include
oligonucleotide synthesis and cloning in a replicable vector.
Methods for nucleic acid synthesis are well-known in the art. When
cloned or synthesized, strand purification and separation may be
necessary to use the product as a pure PNA. Methods of preparing
RNA probes are well known (see for example Blais 1993, Blais 1994,
which uses in vitro transcription from a PCR reaction incorporating
a T7 RNA polymerase promoter).
[0107] The length and specific sequence of the PNA will be
understood by those skilled in the art to depend on the length and
sequence to be detected in a TNA, and the strictures for achieving
tight and specific binding of the particular TBA to be used (see
discussion on TBAs below). In general, PNAs of sequence lengths
between about 10 and about 300 nucleotides in length are adequate,
with lengths of about 15-100 nucleotides being desirable for many
of the embodiments specifically exemplified herein.
[0108] It should also be understood that the PNA may be constructed
so as to contain more than one 1/2 TBR and to produce more than one
TBR for one or more TBAs, same or different, as well as complex
TBRs recognized by novel duplex and multiplex TBAs (see description
below regarding these novel TBAs) upon hybridization of the PNAs
and TNAs. FIG. 5 illustrates specific PNAs which contain one or
more 1/2 TBRs. Specific sequences which correspond to the 1/2 TBR
sees illustrated in FIG. 5 (Ia, IIa, IIIa, IVa, and Va) are SEQ ID
NOS. 1-34 (see Description of Sequences above).
[0109] As shown in FIGS. 2a and 2b, the PNA, containing a 1/2 TBR,
may be hybridized with one or more BNAs (see description below) and
the chain of BNAs polymerized to any desired potential length for
amplification of the TNA-PNA hybridization evens. Preferably,
between about 0 and about 10 1/2 BBRs will be present in the
PNA.
[0110] As shown in FIGS. 6a and 6b, the PNA may contain several 1/2
TBRs, same or different, which can hybridize with several 1/2 TBRs
in a TNA. Each time a 1/2 TBR in the PNA matches a 1/2 TBR in a
TNA, a Target Binding Region, TBR, is formed which can bind a TBA.
Furthermore, it is not essential that all of the TBRs be on a
single, contiguous PNA. Thus, in one embodiment of the invention,
two different PNAs are used to detect sequences on a particular
TNA. As an illustration of this aspect of the invention, FIG. 7
shows one representation of the human immunodeficiency virus (HIV)
long terminal repeat (LTR). As is known in the art, the HIV LTR
comprises two NF-kB binding sites and three SP1 binding sites, in
close proximity, wherein NF-kB and SP1 are known DNA binding
proteins. FIG. 7 provides two PNAs, PNA1 (SEQ ID NO. 38) and PNA2
(SEQ ID NO. 39), each of which is complementary to the opposite
strand shown as a TNA (SEQ ID NO. 37), which shows the two NF-kB
binding sites and the three SP1 binding sites of the HIV LTR.
According to this aspect of the invention, PNA1 specifically
hybridizes with that section of the TNA shown in FIG. 7 with bases
underscored with a "+" symbol, while PNA2 specifically hybridizes
with that section of the TNA shown in FIG. 7 with bases underscored
with an "=" symbol. Each of PNA1 or PNA2 may also contain sequences
(indicated by the symbols "#" or "*") which will hybridize with a
BNA's 1/2 BBR sequences (see below). In addition, each of PNA1 and
PNA2 may be differentially tagged with an OSA, such as a
fluorophore such as a fluorescein or a rhodamine label, which would
allow confirmation that both probes have become bound to the TNA.
If only one label or neither label is detected, it is concluded
that the TNA is not present in the sample being tested.
[0111] In a further aspect of the embodiment shown in FIG. 7, a
method for altering the specificity of the instant assay method is
shown. By changing the length of the gap between PNA1 and PNA2,
such that the region of TNA remaining unhybridized is altered, one
practicing this invention is able to alter the discrimination of
the assay.
[0112] In order to more clearly exemplify this aspect of the
invention, it is necessary to emphasize that the TBR may have a
helical structure. Thus, while PNA1 creates TBRs on one "face" of
the helix, PNA2 creates a TBR on either the same or a different
face of the helix, depending on the distance between the middle of
each TBR (underlined in FIG. 7). If the middle of each binding site
is an integral product of 10.5 bases apart, the TBRs will be on the
same side of the helix, while non-integer products of 10.5 bases
apart would place the TBRs on opposite sides of the helix. In this
fashion, any cooperativity in binding by the TBA recognizing the
PNA1 TBR and the TBA recognizing the PNA2 TBR can be manipulated
(see Hochschild, A., M. Ptashne [1986] Cell 44:681-687, showing
this effect for the binding of bacteriophage lambda repressor to
two different operator sites located at different distances from
each other in a DNA helix). As described by Perkins et al. ([1993]
EMBO J. 12:3551-3558), cooperativity between NF-kB and the SP1
sites is required to achieve activation of the HIV LTR. However,
for the purpose of the instant invention, the double NF-kB-triple
SP1 binding site motif in the HIV LTR may be taken advantage of by
providing a single, novel binding protein capable of binding both
sites simultaneously, but only if the spacing between the sites is
geometrically feasible. This is controlled both by the structure of
the selected TBA and by the PNAs used. Thus, in the embodiment
exemplified in FIG. 7, the two probes may be used with a large
enough interprobe region of single-stranded DNA remaining such
that, even if the NF-kB and SP1 binding sites are on opposite sides
of the helix, the single-stranded region between the probes
provides a sufficiently flexible "hinge" so that the DNA can both
bend and twist to accommodate the geometry of the TBA.
Alternatively, a more stringent assay may be designed by narrowing
the interprobe distance such that the DNA may only bend, but not
twist. Finally, the probes may be so closely spaced or a single PNA
used, such that the DNA can only bend but not twist. Thus, this
figure exemplifies and enables the production of detection systems
with any given desired degree of discrimination between target
nucleic acids having similar sequences, but different
juxtapositions of these sequences.
[0113] In terms of a diagnostic or forensic kit for HIV, those
skilled in the art would understand that the aforementioned aspects
of this invention allow for the tailoring of the components of the
diagnostic or forensic kit to match what is known at any given time
about the prevalent strains of HIV or another pathogen or disease
condition. It will also be appreciated by those skilled in the art
that, while detection of HIV infection is not the only utility of
the instant invention, due to the mutability of the HIV genome, it
is probably one of the most complex test environments for such a
diagnostic. It is precisely in such a mutable environment, however,
where the flexibility of the instant method, coupled with its
ability to discriminate between very closely related sequences, may
be most clearly appreciated. In less mutable environments, some of
the sophistication to which this invention is amenable need not be
utilized. Thus, in a diagnostic kit for papillomavirus infection,
all of the discrimination characteristics of the TBA-TBR
interaction are available, along with the ability to amplify the
signal using the BNAs and BBAs, but a single, simple PNA, such as
any one of SEQ ID NOS. 46-62, may be used which identifies unique
papillomavirus sequences, which also are known to bind to a TBA
such as the papillomavirus E2 protein or truncated DNA binding
portions thereof (see Hegde et al. [1992] Nature 359:505-512;
Monini et al. [1991] J. Virol. 65:2124-2130).
[0114] In applying the instant method to the detection of a
particular TNA for the purposes of assessing whether certain
nucleic acids are present which are associated with the progression
of melanoma, hepatoma, breast, cervical, lung, colon, prostate,
pancreatic or ovarian cancers, the TNA may be obtained from biopsy
materials taken from organs and fluids suspected of containing the
cancerous cells. For the detection of genetic deficiencies, the TNA
may be obtained from patient samples containing the affected cells.
For detection of fermentation contaminants and products in the
manufacture of food, chemical or biotechnology products or in the
bioremediation of wastes, the TNA may be obtained from samples
taken at various stages in the fermentation or treatment process.
For detection of food or drug pathogens or contaminants the TNA
sample may be obtained from the food or drug, swabs of food or
surfaces in contact with the food, fluids in contact with the food,
processing materials, fluids and the like associated with the
manufacture of or in contact with the food, drug, or biological
samples taken from those in contact with the food or drug or the
like.
[0115] 2. The Booster Nucleic Acids (BNAs). Booster Binding Regions
(BBRs) and Their Preparation.
[0116] The BNAs of the present invention are comprised of at least
one or more 1/2 BBRs coupled to an OSA. The 1/2 BBRs can hybridize
to complementary 1/2 BBRs contained in the PNA, other BNAs or an
HNA.
[0117] With referee to FIG. 1(I, IIb and IIIb) of the drawings, the
simplest BNA is comprised of two parts. With reference to FIG. I
(IIb) of the drawings, the first part of the simplest BNA is a
sequence of bases which is complementary to the sequence in the PNA
which is designated "1/2 BBR". With reference to FIG. 1(IIb) of the
drawings, the second part of the simplest BNA is the OSA, designate
by a circle with a box around it. The OSA is no support and/or
indicator, or solid support, or other means of localization,
including but not limited to, attachment to beads, polymers, and
surfaces and/or indicators which are covalently attached to, or
non-covalently, but specifically, associated with the BNA.
[0118] With reference to FIG. 2a(II and III) of the drawings, the
BNA may contain more than one 1/2 BBR sequence. The BNA illustrated
in FIG. 3(II) contains a sequence which is complementary to the PNA
illustrated in FIG. 3(I) and two other 1/2 BBR sequences. The BNA
illustrated in FIG. 3(III) contains two 1/2 BBR sequences which are
complementary to two of the 1/2 BBR sequences in the BNA
illustrated in FIG. 3(II), plus up to "n" additional 1/2 BBRs for
polymerization of additional BNAs.
[0119] Under hybridizing conditions, the BNA illustrated in FIG.
3(II), when combined with the PNA illustrated in FIG. 3(I), creates
the PNA-BNA hybrid illustrated in FIG. 3(IVa) containing a BBR and
an unhybridized extension with two additional 1/2 BBR sequences or
"booster" sequences. The BBRs created by said hybridization can be
identical, similar or dissimilar in sequences. The BBRs created by
said hybridization can bind identical, similar or dissimilar BBAs
(see below). The BNAs may have prepared analogously to the
PNAs.
[0120] Under hybridizing conditions, the BNA-BNA hybrid illustrated
in FIG. 3(IVb), when combined with the PNA illustrated in FIG.
3(Vb), creates the PNA-BNA hybrid illustrated in FIG. 3(VI)
containing a BBR, two additional BNA-BNA hybrids containing BBRs,
and an unhybridized extension with an additional 1/2 BBR sequence,
a "booster" sequence. The BBRs created by said hybridization can be
identical, similar or dissimilar in sequence. The BBRs created by
said hybridization can bind identical, similar or dissimilar BBAs
(see below). The BNAs may be prepared in a fashion analogous to
preparation of the PNTAs.
[0121] 3. The Target Nucleic Acids (TNAs) and Their
Preparation.
[0122] The first step in detecting and amplifying signals produced
through detection of a particular TNA according to the present
method is the hybridization of such target with the PNA in a
suitable mixture. Such hybridization is achieved under suitable
conditions well known in the art.
[0123] The sample suspected or known to contain the intended TNA
may be obtained from a variety of sources. It can be a biological
sample, a food or agricultural sample, an environmental sample and
so forth In applying the instant method to the detection of a
particular TNA for the purposes of medical diagnostics or
forensics, the TNA may be obtained from a biopsy sample, a body
fluid or exudate such as urine, blood, mill; cerebrospinal fluid,
sputum, saliva, stool lung aspirates, throat or genital swabs and
the like. In addition, detection may be in situ (see for example
Embretson 1993; Patterson 1993; Adams 1994).
[0124] Accordingly, PNAs specific to vertebrates (including mammals
and including humans) or to any or all of the following
microorganisms of interest may be envisioned and used according to
the instant method:
2 Corynebacteria Corynebactenum diphtheria Bacillus Bacillus
thuringiensis Pneumococci Diplococcus pneumoniae uz,1/6
Streptococci Streptococcus pyogenes Streptococcus salivarius
Staphylococcus Staphylococcus aureus Staphylococcus albus
Pseudomonas Pseudomonas stutzen Neisseria Neisseria meningitidis
Neisseria gonorrhea Enterobacteriaceae Escherichia coli
Aerobacteria aerogenes Klebsiella pneumoniae The coliform bacteria
Salmonella typhosa Salmonella choleroesuis The Salmonellae
Salmonella typhimurium Shigellae dysenteriae Shigellae schmzitzii
Shigellae arabinotardo Shigellae flexneri The Shigellae Shigellae
boydii Shigellae sonnei Other enteric bacilli Proteus vulgaris
Proteus mirabilis Proteus species Proteus morgani Pseudomonas
aeruginosa Alcaligenes faecalis Vibrio cholerae
Hemophilus-Bordetella group Hemophilus influenza, H ducryi
Hemophilus hemophilus Hemophilus aegypticus Hemophilus
parainfluenzae Bordetella pertussis Pasteurellae Pasteurella pestis
Pasteurella tulareusis Brucellae Brucella melitensis Brucella
abortus Brucella suis Aerobic Spore-Forming Bacilli Bacillus
anthracis Bacillus subtilis Bacillus megaterium Bacillus cereus
Anaerobic Spore-Forming Bacilli Clostridium botulinum Clostridium
tetani Clostridium perfringens Clostridium novyi Clostridium
septicum Clostridium histolyticum Clostridium tertium Clostridium
bifermentans Clostridium sporogenes Mycobacteria Mycobacterium
tuberculosis hominis Mycobacterium bovis Mycobacterium avium
Mycobacterium leprae Mycobacterium paratuberculosis. Actinomycetes
(fungus-like bacteria) Actinomyces isaeli Actinomyces bovis
Actinomyces naeslundii Nocardia asteroides Nocardia brasiliensis
The Spirochetes Treponema pallidum Treponema pertenue Treponema
carateum Spirillum minus Streptobacillus moniliformis Borrelia
recurrens Leptospira icterohemorrhagiae Leptospira canicola
Trypanasomes Mycoplasmas Mycoplasma pneumoniae Other pathogens
Listeria monocytogenes Erysipelothrix rhusiopathiae Streptobacillus
moniliformis Donvania granulomatis Bartonella bacillformis
Rickettsiae (bacteria-like parasites) Rickettsia prowazekii
Rickettsia mooseri Rickeasia rickettsii Rickettsia conori
Rickettsia australis Rickettsia sibiricus Rickettsia akari
Rickettsia tsutsugamushi Rickettsia burnetti Rickettsia quintana
Chlamydia (unclassifiable parasites bacterial/viral) Chlamydia
agents (naming uncertain) Fungi Cryptococcus neoformans Blastomyces
dermatidis Histoplasma capsulatum Coccidioides immitis
Paracoccidioides brasiliensis Candida albicans Aspergillus
fumigatus Mucor corymbifera (Absidia corymbifera) Rhizopus oryzae
Rhizopus arrhizus Phycomycetes Rhizopus nigricans Sporotrichum
schenkii Flonsecaea pedrosoi Fonsecaea compact Fonsecacae
dermatidis Cladosporium carrioni Phialophora verrucosa Aspergillus
nidulans Madurella mycetomi Madurella grisea Allescheria boydii
Phialophora jeanselmei Microsporum gypsum Trichophyton
mentagrophytes Keratinomyces ajelloi Microsporum canis Trichophyton
rubrum Microsporum adouini Viruses Adenoviruses Herpes Viruses
Herpes simplex Varicella (Chicken pox) Herpes zosrer (Shingles)
Virus B Cytomegalovirus Pox Viruses Variola (smallpox) Vaccinia
Poxvirus bovis Paravaccinia Molluscum contagiosum Picornaviruses
Poliovirus Coxsackievirus Echoviruses Rhinoviruses Myxoviruses
Influenza (A, B, and C) Parainfluenza (1-4) Mumps virus Newcastle
disease virus Measles virus Rinderpest virus Canine distemper virus
Respiratory syncytial virus Rubella virus Arboviruses Eastern
equine encephalitis virus Western equine encephalitis virus Sindbis
virus Chikugunya virus Semliki forest virus Mayora virus St. Louis
encephalitis virus California encephalitis virus Colorado tick
fever virus Yellow fever virus Dengue virus Reoviruses Reovirus
types 1-3 Retroviruses Human immunodeficiency viruses (HIV) Human
T-cell lymphotrophic virus I & II (HTLV) Hepatitis Hepatitis A
virus Hepatitis B virus Hepatitis nonA-nonB virus Hepatitis, C, D,
E Tumor viruses Rauscher leukemia virus Gross virus Maloney
leukemia virus Human papilloma viruses
[0125] It would be understood by one of skill in the art that it is
generally required to treat samples suspected of containing a
particular TNA in such a fashion as to produce fragments that can
easily hybridize with the PNA. It may be necessary to treat the
test sample to effect release of or to extract the TNA for
hybridization, such as by exposing blood or other cells to a
hypotonic environment, or otherwise disrupting the sample using
more vigorous means. When the TNA is thought to be present in
double stranded form, it would naturally be desirable to separate
the stands to render the TNA hybridizable in single stranded form
by methods well known in the art, including but not limited to
heating or limited exposure to alkaline conditions which may be
neutralized upon addition of the single stranded PNA to allow
hybridization to occur. Methods for preparing RNA targets are well
known (see Waterhouse 1993, Mitchell 1992).
[0126] Fragmentation of nucleic acid samples containing TNAs is
usually required to decrease the sample viscosity and to increase
the accessibility of the TNAs to the PNAs. Such fragmentation is
accomplished by random or specific means known in the art Thus, for
example, specific nucleases known to cut with a particular
frequency in the particular genome being analyzed, may be used to
produce fragments of a known average molecular size. In addition,
other nucleases, phosphodiesteses, exonucleases and endonucleases,
physical shear and sonication are all methods amenable for this
purpose. These processes are well known in the art. The use of
restriction enzymes for the purpose of DNA fragmentation is
generally preferred. However, DNA can also be fragmented by a
variety of chemical means such as the use of the following types of
reagents: EDTA-Fe(II) (according to Stroebel et al. [1988] J. Am.
Chem. Soc. 110:7927; Dervan [1986] Science 232:464);
Cu(II)-phenanthroline (according to Chen and Sigman [1987] Science
237:1197); class IIS restriction enzyme (according to Kim et al.
[1988] Science 240:504); hybrid DNAse (according to Corey et al.
[1989] Biochem. 28:8277); bleomycin (according to Umezawa et al.
[1986] J. Antibiot. (Tokyo) Ser. A, 19:200); neocarzinostatin
(Goldberg et al. [1981] Second Annual Bristol-Myers Symposium in
Cancer Research, Academic Press, New York, p. 163); and
methidiumpropyl-EDTA-Fe(II) (according to Hertzberg et al. [1982]J.
Am. Chem. Soc. 104:313). Removal of proteins, as by treatment with
a protease, is also generally desirable and methods for effecting
protein removal from nucleic acid samples, without appreciable loss
of nucleic acid, are well known in the art.
[0127] The TNAs of the present invention should be long enough so
that there is a sufficient amount of double-stranded hybrid
flanking the TBR so that a TBA can bind unpertrbed by the unligated
fragment ends. Typically, fragments in the range of about 10
nucleotides to about 100,000 nucleotides, and preferably in the
range of about 20 nucleotides to about 1,000 nucleotides are used
as the average size for TNA fragments. Examples of specific TNA
sequences that could be detected are sequences complementary to the
PNA sequences described herein for detection of normal cellular,
abnormal cellular (as in activated oncogenes, integrated foreign
genes, genetically defective genes), and pathogen-specific nucleic
acid sequences, for which specific nucleic acid binding proteins
are known, or which can be produced according to methods described
in this disclosure. With reference to FIG. 7, a specific
HIV-related TNA is shown as SEQ ID) NO. 37.
[0128] 4. Extensions to the PNA Using BNAs, Their Preparation, and
Signal Amplification.
[0129] Under hybridizing conditions, BNAs can be added that
hybridize to the PNAs, PNA-BNA hybrids, BNAs and/or BNA-BNA
hybrids. The aforementioned additions can be made in a
non-vectorial polymeric fashion or in a vectorial fashion, with a
known order of BNAs.
[0130] With reference to FIG. 2a, a simple booster is presented. A
booster polymer is produced by adding two BNAs, illustrated in FIG.
2a(Ib and Ic), which when combined under hybridizing conditions
with the PNA, form PNA-BNA-BNA hybrids, comprised of the PNA and
"booster extensions", illustrated in FIG. 2a(IIa,IIb,IIc and IId)
leaving at least one unpaired 1/2 BBR sequence. Each unpaired 1/2
BBR sequence, illustrated in FIG. 2a(IIa, IIb, IIc, IId) can
hybridize with additional BNAs to form additional "booster"
extensions. Each unpaired 1/2 BBR sequence, illustrated in FIG.
2a(IIa,IIb,IIc and IId) can hybridize with added HNAs, illustrated
in FIG. 2a(IIIa and IIIb). The hybridization of the HNAs, which
cannot hybridize additional BNAs, acts to "cap" the addition of the
BNAs onto the PNA, as illustrated in FIG. 2a(IVa, IVb, IVc and
IVd).
[0131] With reference to FIG. 2b, it is possible to control and
specify the order and components of extensions to the PNA. If a
single BBR is required, a HNA containing the complementary sequence
to the 1/2 BBR in the PMA is added to the PNA to produce a single
BBR and to "cap" any "booster" extensions to the PNA. If additional
BBRs are to be added to the PNA, a controlled extension of the PNA
can be accomplished.
[0132] With reference to FIG. 2b, a simple booster is presented.
Vectorial polymer extension is accomplished by adding a BNA which
is specific for the PNA, as illustrated in FIG. 2b(Ia and IIa),
which when combined under hybridizing conditions with the PNA, form
PNA-BNA-BNA hybrids, comprised of the PNA and "booster" extensions.
These extensions, if labeled with an OSA, provide a method for
greatly amplifying any signal produced upon binding of a PNA to a
TNA in the sample. Furthermore, by binding labeled BBAs to the BBRs
in the polymer, additional amplification is achieved.
[0133] Any of a number of methods may be used to prepare the BNAs,
including, e.g., synthesis via known chemistry or via recombinant
DNA production methods. In the latter method, an essentially
unlimited number of BNAs may be produced simply and inexpensively,
for example, by production in prokaryotes (E. coli for example) of
a plasmid DNA having multiple repeats of the specific BNA sequences
flanked by restriction sites having overhanging ends. In this
fashion, for example, the bacteriophage lambda left or right
operator sites, or any other DNA or other nucleic acid sequence
known to specifically and tightly bind a particular BBA, such as a
DNA or RNA binding protein, may be produced in an essentially
unlimited number of copies, with each copy flanked by an EcoRI,
PstI, BamHI or any of a number of other common restriction nuclease
sites. Alternatively, a polymer at repeated sites may be excised by
unique restriction sites not present within the polymer. Large
quantities of pBR322, pUC plasmid or other plasmid having multiple
copies of these sequences are produced by methods well known in the
art, the plasmid cut with the restriction enzyme flanking the
polymerized site, and the liberated multiple copies of the
operators isolated either by chromatography or any other convenient
means known in the art. The BNA, prior to use, is then strand
separated and is then amenable for polymerization onto a PNA
encoding a single stranded complementary copy of the operator as a
1/2 BBR. The BNAs may be polymerized vectorially onto the PNA by
using different restriction enzymes to flank each repeat of the
polymer in the plasmid used to produce multiple copies of the BNA.
Alternatively, the BNA polymer may be hybridized to the PNA via
overhangs at one or both ends of the BNA polymer, without the need
to strand separate and anneal each BNA segment. Examples of
specific BNA sequences are provided above in the section entitled
Description of Sequences, as SEQ ID NOS. 35-36. To stabilize the
BNA polymer, DNA ligase may be used to covalently link the
hybridized BNAs.
[0134] 5. The Hairpin Nucleic Acids (HNAs) and Their
Preparation.
[0135] The HNAs of the present invention comprise at least two
principal parts joined together: A single-stranded sequence, which
is complementary to a 1/2 BBR, and a double-stranded nucleic acid
region formed, under hybridizing conditions, by the
self-association of self-complementary sequences within the HNA.
With reference to FIG. 1(IIc) of the drawings, the 1/2 BBR in the
HNA may be constructed so as to be complementary to the 1/2 BBR
sequence in the PNA. With reference to FIG. 1(I, IIc and IIIc) of
the drawings, the aforementioned HNA, when added to the PNA under
hybridizing conditions, forms a PNA-HNA hybrid containing a BBR
With reference to FIG. 1(IIIc, IVc and Vc) of the drawings, a
PNA-HNA hybrid, under hybridizing conditions, upon addition of the
TNA, can form a TNA-PNA-HNA hybrid containing a TBR and a BBR.
[0136] With reference to FIGS. 2a and 2b, the HNAs can be used to
"cap" or terminate the addition of BNA extensions to the PNA. The
two BNAs in FIG. 2a(Ib and Ic) can associate to form the hybrid
shown in FIG. 3(IVb) or can hybridize directly and individually to
the PNA as illustrated in FIG. 2a(Ia-c, IIa-d). The two HNAs (shown
in FIG. 2a(IIIa and IIIb)) can terminate the hybridization of the
BNA to other BNAs which extend from the PNA, as illustrated in FIG.
2a(IVa-d). The HNA in FIG. 2a(IIIa) can terminate the PNA-BNA
hybrids shown in FIG. 2a(IIb and IId) and any PNA-BNA hybrid with a
single stranded 1/2 BBR which is complementary to the 1/2 BBR in
the HNA illustrated in FIG. 2a(IIIa). Similarly, the HNA in FIG.
2a(IIIb) can terminate the PNA-BNA hybrids shown in FIG. 2a(IIa and
IIc) and any PNA-BNA hybrid with two single stranded 1/2 BBRs which
are complementary to the 1/2 BBRs in the HNA illustrated in FIG.
2a(IIIb).
[0137] HNAs are constructed that will terminate PNA-BNA hybrids
which are constructed from the sequential addition of BNAs to the
PNA as illustrated in FIG. (2b). The single stranded 1/2 BBR
sequences illustrated in FIG. 2b(Ia, IIIa, Va, and VIIa) are
specifically complementary to the single stranded 1/2 BBR sequences
illustrated in FIG. 2b(Ib,IIIb,Vb and VIIb) and produce the unique
capped PNA-BNA-HNA hybrids illustrated in FIG. 2b(Ic,IIIc,Vc and
VIIc).
[0138] The self-complementary sequences in the HNA and the loop
sequence which links the self-complementary hairpin sequences can
be of any composition and length, as long as they do not
substantially impede or inhibit the presentation of the
single-stranded 1/2 BBR that comprises part of the HNA by the HNA
or selectively bind the BBA or the TBA. The loop sequences should
be selected so that formation of the loop does not impede formation
of the hairpin. An examples of an HNA useful in this application is
provided as SEQ ID NO. 44 (see Description of Sequences above).
[0139] 6. The Target Binding Assemblies (TBAs) and Their
Preparation.
[0140] A TBA may be any substance which binds a particular TBR
formed by hybridization of particular TNAs and PNAs, provided that
the TBA must have at least the following attributes:
[0141] (a) The TBA must bind the TBR(s) in a fashion that is highly
specific to the TBR(s) of interest. That is, the TBA must
discriminate between TBRs present in the TNA-PNA hybrid and similar
duplex sequences formed by PNA-CNA hybrids. The TBA must bind the
PNA-CNA hybrid with a sufficiently low avidity that upon washing
the TBA-TNA-PNA complex, the PNA-CNA hybrid is displaced and the
PNA-TNA hybrid is not displaced;
[0142] (b) The TBA must avidly bind the TBR(s) created by the
hybridization of the TNA with the PNA. Binding affinities in the
range of 10.sup.-5 to about 10.sup.-12 or higher are generally
considered sufficient. As noted below, in some instances, it might
be desirable to utilize a particular TBA which has a very low
avidity for a particular TBR, but which has a greatly increased
affinity when a particular configuration of multiple TBRs is
provided so that the square of the affinity of the TBA for each TBR
becomes the affinity of relevance to that particular TBA.
[0143] Examples of the DNA binding components useful in the
formation of TBAs include, but are not limited to NF-kB,
papillomavirus E2 protein, transcription factor SP1, inactive
restriction enzymes, antibodies, etc. Each of these proteins has
been recognized in the art to contain sequences which bind to
particular nucleic acid sequences and the affinities of these
interactions are known. Naturally, the method of the instant
invention is not limited to the use of these known DNA binding
proteins or fragments thereof. From the instant disclosure, it
would be apparent to one of ordinary skill that the instant method
could easily be applied to the use of novel TBAs exhibiting at
least the required attributes noted above. Thus, for example, in WO
92/20698, a sequence specific DNA binding molecule comprising an
oligonucleotide conjugate formed by the covalent attachment of a
DNA binding drug to a triplex forming oligonucleotide was
described. The method of that disclosure could be used to produce
novel TBAs for use according to the instant disclosure, provided
that the TBAs thus formed meet the criteria described above. In
addition, the methods of U.S. Pat. Nos. 5,096,815, 5,198,346, and
WO88/06601, herein incorporated by reference, may be used to
generate novel TBAs for use according to the method of this
invention. Specific antibodies or portions thereof could be used
(see for example Blais 1994).
[0144] Where the TBA is a protein, or a complex of proteins, it
will be recognized that any of a number of methods routine in the
art may be used to produce the TBA. The TBA may be isolated from
its naturally occurring environment in nature, or if this is
impractical, produced by the standard techniques of molecular
biology. Thus, using NF-kB as an example, using the DNA binding
portions of p50 or p65 subunits, this binding assembly could be
produced according to recombinant methods known in the art (see for
example Ghosh [1990] Cell 62:1019-1029, describing the cloning of
the p50 DNA binding subunit of NF-kB and the homology of that
protein to rel and dorsal).
[0145] Many DNA and other nucleic acid binding proteins are known
which can be used as or in TBAs according to this invention. Once
the amino acid sequence of any DNA, RNA:DNA, RNA or other nucleic
acid binding protein is known, an appropriate DNA sequence encoding
the protein can either be prepared by synthetic means, or a cDNA
copy of the mRNA encoding the protein from an appropriate tissue
source can be used. Furthermore, genomic copies encoding the
protein may be obtained and introns spliced out according to
methods known in the art. Furthermore, the TBAs may be chemically
synthesized.
[0146] Once an appropriate coding sequence has been obtained,
site-directed mutagenesis may be used to alter the amino acid
sequence encoded to produce mutant nucleic acid binding proteins
exhibiting more desirable binding characteristics than those of the
original nucleic acid binding protein. As an example of this
process, the amino acid sequence of the DNA binding portions of
NF-kB can be altered so as to produce an NP-kB' molecule which more
tightly binds the NF-kB binding site (see examples
below--HIV-Detect and HIV-Lock).
[0147] To provide further insight into this aspect of the
invention, the following considerations are to be noted. Using
NF-kB as an example, a TBA may be prepared using the naturally
occurring NF-kB molecule. However, because this molecule is present
in vanishingly small quantities in cells, and because the subunits
of this DNA binding protein have been cloned, it would be more
reasonable to prepare large quantities of the complex via
recombinant DNA means as has already been accomplished for this
protein (see for example Ghosh [1990] Cell 62:1019-1029). NF-kB is
a pleiotropic inducer of genes involved in immune, inflammatory and
growth regulatory responses to primary pathogenic (viral, bacterial
or stress) challenges or secondary pathogenic (inflammatory
cytokine) challenges. NF-kB is a dimeric DNA binding protein
comprising a p50 and a p65 subunit, both of which contact and bind
to specific DNA sequences. In an inactivated state, NF-kB resides
in the cellular cytoplasm, complexed with a specific inhibitor,
I-kB, to form a cytoplasmic heterotrimer. Upon activation, the
inhibitor is decomplexed, and the p50-p65 dimer relocate via a
specific nuclear localization signal (NLS) to the cell's nucleus
where it can bind DNA and effect its role as a transcriptional
activator of numerous genes (see Grimm and Baeuerle [1993] Biochem.
J. 290:297-308, for a review of the state of the art regarding
NF-kB).
[0148] The p50-p65 dimer binds with picomolar affinity to sequences
matching the consensus GGGAMTNYCC (SEQ ID NO. 117), with slightly
different affinities depending on the exact sequence. It is worth
noting that homodimers of p50 and p65 have also been observed to
occur. These homodimers display different biochemical properties as
well as slightly different affinities of binding sequences within
and similar to the above consensus. Thus, depending on the desired
binding characteristics of the TBA, a p50-p65 heterodimer, a
p50-p50 homodimer, or a p65-p65 homodimer or fragments of the
aforementioned dimers may be used.
[0149] One way in which various novel TBAs may be produced for use
according to this invention is shown schematically in FIG. 9. The
nucleic acid recognition units of the TBA may be assembled and
associated with similar or dissimilar TBA nucleic acid recognition
units via a "chaperone." The chaperone is a structure on which the
various TBA recognition elements are built and which confers
desirable properties on the nucleic acid recognition units. The
chaperone is comprised of any sequence which provides assembly
sequences such that same or different nucleic acid recognition
units are brought into close and stable association with each
other. Thus, for example, in the case of a TBA designed to tightly
bind NF-kB TBRs, a TBA is assembled by providing lambda cro
sequences as assembly sequences, linked to the nucleic acid binding
sequences for either NF-kB p50 or p65. The p50 or p65 nucleic acid
binding sequences are linked to the cro sequences at either the
carboxy or amino terminus of cro and either the carboxy or amino
terminus of the nucleic acid recognition unit of the p50 or p65.
Linking sequences are optionally provided to allow appropriate
spacing of the nucleic acid recognition units for optimal TBR
binding.
[0150] The assembly sequences, exemplified above by cro and CI
sequences (SEQ ID NOS. 104-108), comprise any stable oligopeptides
which naturally and strongly bond to like sequences. Thus, in the
case of cro, it is well known that a dimer of cro binds to the
bacteriophage lambda operator sites (Anderson et al. [1981] Nature
290:754-758; Harrison and Aggarwal [1990] Ann. Rev. Biochem.
59:933-969). The monomer units of cro tightly and specifically
associate with each other. Thus, by linking DNA recognition unit
sequences to the cro sequences, close and tight association is
achieved.
[0151] The optional linker sequences comprise any amino acid
sequence which does not interfere with TBA assembly or nucleic acid
binding, and which is not labile so as to liberate the nucleic acid
recognition unit from the complete TBA. It is desirable but not
necessary that the linker sequences be covalently linked to other
binding assembly components. The association should be specific so
as to aid in the assembly and manufacture of the binding
assemblies. Examples of such sequences include, but are not limited
to, such well known sequences as are found linking various domains
in structural proteins. Thus, for example, in the lambda repressor
protein, there is a linking sequence between the DNA binding domain
and the dimerization domain which is useful for this purpose. Many
other such sequences are known and the precise sequence thereof is
not critical to this invention, provided hat routine
experimentation is conducted to ensure stability and
non-interference with target nucleic acid binding. Examples of such
sequences are provided herein as Met Ser and SEQ IN NOS. 99-102.
Insertion of specific, known proteolysis sites into these linkers
is also an integral part of this invention. The presence of such
sites in the linker sequences would provide manufacturing
advantages, allowing different molecules to be assembled on the
chaperone scaffold.
[0152] In addition to the nucleic acid recognition units, optional
linking sequences, and assembly sequences, the novel TBAs of this
invention optionally have asymmetry or PILOT TNA sequences and one
or more OSA units. The asymmetry sequences are provided to
encourage or prevent certain desirable or undesirable associations.
For example, in the event that a TBA having homodimeric p50 DNA
recognition units is desired, the asymmetry sequences are provided
to disrupt the naturally stronger association of NF-B p50 subunits
and p65 subunits, while not disrupting the assembly sequences from
bringing together p50 subunits. Examples of such sequences are
provided herein as SEQ ID NOS. 85-92 and SEQ ID NOS. 105 and
106.
[0153] In a different configuration, NF-kB p50 subunit sequences
are brought into close association with transcription factor SP1
DNA recognition unit sequences. This is desirable in the event that
an NF-kB/SP1 binding motif is of significance, as in the HIV LTR
where a motif of at least six DNA binding protein recognition
sites, two NF-kB, three SP1, and a TATA site are mown to exist.
Since it is also known that the second NF-kB and first SP1 site are
significant to regulation of HIV transcription (Perkins et al.
[1993] Embo J. 12:3551-3558), this particular configuration of TBA
is useful not only the detection of HIV, but as a therapeutic or
prophylactic against HIV infection (see below). In a similar
fashion, the long control region (LCR) of human papillomavirus may
be used as a key control region for probing according to this
method.
[0154] In view of the different elements that can be associated,
cassette fashion, according to this method of TBA formation, an
essentially unlimited variety of TBAs are produced. In FIG. 10, a
series of different molecules, referred to as "HIV-detect I-IV" are
exemplified wherein "CHAP" denotes the chaperone, "nfkb" denotes
NF-kB subunits, "sp1" denotes the nucleic acid recognition unit of
the SP1 transcription factor, and "TATA" denotes a dimer of the DNA
recognition unit of a TATA sequence DNA binding protein (TBP), also
known as a TATA binding protein, or TBP. These configurations are
further exemplified below and are all integral parts of the instant
invention.
[0155] In yet another configuration, the modular structure shown in
FIG. 9 is adapted to detection and or treatment or prophylaxis of a
completely different pathogen. In FIG. 11, in a similar fashion to
the above described "HIV-detect I-IV" molecules, a series of
"HPV-Detect I-IV" molecules is produced. In this embodiment,
advantage is taken of the DNA binding properties of the E2 protein
of human papillomavirus (HPV). In addition, the roles of SP1 and
TBP are taken advantage of by providing specific DNA recognition
units adapted to bind to these sequences in the HPV genome. In the
formation of the E2-specific TBAs for use in detecting HPV
infection, it may be desirable to use any of SEQ ID NOS. 75-84 or
93-98 as the E2 DNA recognition units. A TBA containing a bovine E2
dimer and a human E2 dimer DNA binding domain may be particularly
useful.
[0156] The various sequences described above may either be
chemically linked using pure oligopeptide starting materials, or
they may be line through provision of recombinant nucleic acids
encoding, via the well known genetic code, the various subelements.
In the event of recombinant production, liking cro coding sequences
to sequences of nucleic acid recognition units to form TBAs is
advantagous because not only does cro act as assembly sequences in
the chaperone, it also acts to direct the proper folding of the
nucleic acid recognition elements. Exemplary sequences for
chaperones are provided herein as SEQ ID NOS. 104-108. Furthermore,
in the event that higher order structures comprising multiple
binding sites is desired, as in a pentameric
NF-kB/NF-kB/SP1/SP1/SP1 TBA, proper design of the asymmetry
sequences allows such structures to be made.
[0157] In the foregoing fashion, TBAs are prepared which bind to
their cognate binding sites with high affinity. For example, the
NF-kB DNA binding components of the TBAs of FIG. 10 are expected to
bind to the HIV-LTR with an affinity of between about 10.sup.-8 and
10.sup.-12 molar. Sequences useful as the DNA recognition units are
provided as SEQ ID NOS. 63-71, 73-84, 93-98, and 104-108 and
exemplified further below.
[0158] In view of the foregoing description of directed assembly of
nucleic acid binding proteins using assembly and asymmetry (or
piloting) sequences, those skilled in the art will recognize that a
generally applicable method for assembling protein structures is
provided by this invention. The generality of this method is
demonstrated further by consideration, by way of further example,
of the use of an antibody-epitope interaction in the assembly of
desired structures. By way of specificity, a DNA binding protein
structure may be assembled by linking an NF-kB p50 subunit to an
antigen, such as a circulated (through disulfide bonds) melanocyte
stimulating hormone (MSH). This pro-MSH molecule may then be bound
by an anti-MSH antibody to provide a novel nucleic acid binding
assembly, with the antigen and antibody acting as assembly
sequences.
[0159] The modular structure provided by FIG. 9 reveals that a
great variety of TBAs may be assembled using different combinations
of components. Thus, representative embodiments of this general
structure are provided as SEQ ID NOS. 109-116.
[0160] 7. The Booster Binding Assemblies (BBAs) and Their
Preparation.
[0161] A BBA may be any substance which binds a particular BBR
formed by hybridization of particular PNAs and BNAs, including when
multiple BNAs (up to and including "n" BNAs, i.e., BNA.sub.n,
wherein "n" is theoretically 0-.infin., but practically is between
about 0 and 100) are polymerized onto the PNA for signal
amplification, provided that the BBA must have at least the
following attributes:
[0162] (a) The BRA must bind the BBRs in a fashion that is highly
specific to the BBR of interest. That is, the BBA must discriminate
between BBRs present in the PNA-BNA hybrid and similar duplex
sequences in BNA-CNA hybrids or other CNAs. Thus, where even a
single base mismatch or conformational differences with or without
base mismatches occur in the production of the PNA-BNA.sub.n or
PNA-BNA.sub.n-HNA hybrid, the BBA must bind the hybrid with a
sufficiently low avidity that upon washing the TBA-TNA-PNA-BNA
complex, the BBA is displaced from the CNA sequences but not the
BBR sequences.
[0163] (b) The BBA must avidly bind the BBR(s). Binding affinities
in the range of 10.sup.-5 to about 10.sup.-9 or higher are
generally considered sufficient.
[0164] Examples of BBAs include, but are not limited to cro, and
the bacteriophage lambda repressor protein, CI. In addition, see
U.S. Pat. No. 4,556,643, herein incorporated by reference, which
suggests other DNA sequences and specific binding proteins such as
repressors, histones, DNA modifying enzymes, and catabolite gene
activator protein. See also EP 0 453 301, herein incorporated by
reference, which suggests a multitude of nucleotide sequence
specific binding proteins (NSSBPs) such as the tetacycline
repressor, the lac repressor, and the tryptophan repressor. Each of
these BBAs has been recognized in the art to bind to particular,
known nucleic acid sequences and the affinities of these
interactions are known. Naturally, the method of the instant
invention is not limited to the use of these known BBAs. From the
instant disclosure, one of ordinary skill could easily apply the
use of novel BBAs exhibiting at least the required attributes noted
above to the instant method.
[0165] Examples of novel BBAs useful according to this aspect of
the invention include novel proteins based on the motif of a known
DNA or RNA or DNA:RNA binding protein such as cro or the .lambda.
CI repressor protein. Preferably, such modifications are made to
improve the handling of these components of the invention. Thus, it
may be desirable to add a high concentration of cro to an assay.
One of the negative qualities of cro is that at high
concentrations, the binding of cro to its DNA target comes into
competition with cro-cro interactions. Thus, for example, a
chaperoned or mutated cro may be produced which does not have this
shortcoming. Examples of such altered chaperones are SEQ ID NOS.
105-106 and 108. Methods known in the art, such as production of
novel target binding proteins using variegated populations of
nucleic acids and selection of bacteriophage binding to particular,
pre-selected targets (i.e., so-called phage-display technology, see
discussion above for production of novel TBAs) may be used to
produce such novel BBAs as well as the aforementioned novel
TBAs.
[0166] Where the BBA is a protein, or a complex of proteins, it
will be recognized that any of a number of methods routine in the
art may be used to produce the BBA. The BBA may be isolated from
its naturally occurring environment in nature, or if this is
impractical, produced by the standard techniques of molecular
biology. Thus, for example, the sequence of the cro protein is
known and any molecular clone of bacteriophage lambda may be used
to obtain appropriate nucleic acids encoding cro for recombinant
production thereof. In addition, the TBAs described herein may be
used as BRAs, provided that different TBAs are used to bind TBRs
and BBRs.
[0167] 8. The Use of BBAs and BBRs to Localize and Amplify the
Localization of the PNA-TNA-TBA Complexes (See FIG. 8).
[0168] In one embodiment of this invention, the highly specific and
extremely tight binding of TBAs comprised of nucleic acid binding
components is used to produce an amplifiable nucleic acid sandwich
assay. According to one aspect of this embodiment, a solid support
is coated with a first TBA creating an immobilized TBA. In
solution, a PNA and TNA are contacted under hybridizing conditions
and then contacted with the immobilized TBA. Only those PNA-TNA
interactions which form the specific TBR recognized by the
immobilized TBA are retained upon wash-out of the solid surface
which binds the TBA-TBR complex
[0169] Detection of the bound TBR is accomplished through binding
of Booster Nucleic Acids, BNAs, to the 1/2 BBRs present on the PNAs
under hybridizing conditions. In this manner, even if only a single
TBA-TBR complex is bound to the immobilized TBA, a large, amplified
signal may be produced by polymerizing multiple BNAs to the
immobilized TNA. Each BNA which binds to the TNA forms a BBR which
can be bound by BBAs which, like the TBAs immobilized on the solid
surface, may be chosen for their very tight and specific binding to
particular nucleic acid structures. Thus, according to this
embodiment, the immobilized TBA may contain the DNA binding portion
of NF-kB, which very specifically and tightly binds to NF-kB
binding sites formed upon hybridization of the TNA and PNA to form
such a site.
[0170] Because it is well known that there are NF-kB binding sites
both in the normal human genome and in the long terminal repeats of
human immunodeficiency virus (HIV), this invention provides a
method of discriminating between the "normal" human sites and the
sites present in cells due to HIV infection. Therefore, in a test
designed to determine the presence or absence of HIV DNA in a
sample of human DNA, the HIV NF-kB binding sites may be viewed as
the TNA, and the normal human NF-kB binding sites may be viewed as
CNAs. According to the method of this invention, discrimination
between these TNAs and CNAs is accomplished by taking advantage of
the fact that in the HIV LTR, there are two NF-kB binding sites,
followed by three SP1 sites (see, for example, Koken et al. (1992)
Virology 191:968-972), while cellular NF-kB binding sites with the
same sequences are not found in tandem.
[0171] In cases where the TNA contains more than one 1/2 TBR and it
is desirable to pursue the therapeutic and prophylactic
applications of the TBAs, it may be desirable to use more than one
TBA, each with the capacity to bind a TBR in the TNA-PNA complex.
In this case, it may be advantageous to select, as components of
the TBAs, DNA-binding or RNA-binding domains with lesser affinity
for its TBR than the wild-type DNA-binding or RNA-binding domain.
Given that the TBAs which are involved in the binding to the
multiple TBRs can either assemble together before binding to their
TBRs or assemble together after binding to their TBRs, the
individual TBAs will not block the corresponding TBRs in the other
genomes than the target genome unless the TBRs are spatially
capable of binding the assembled TBA complex. One feature of the
multimeric assembly of TBAs which is specifically claimed here as
part of this invention is that such a multimeric assembly is
expected to have a much reduced affinity for a single site within
the TNA. However, since the binding is dramatically increased
relative to any one TBA, the TBA complex would be expected to not
compete for the binding of any single TBR with the corresponding
native proteins in situ but bind tightly to sequences in the
PNA-TNA hybrid containing the TBRs for each of the nucleic
acid-binding components assembled in the TBA. The TBA complex
should be assembled and linkers adjusted in the individual TBAs so
as to allow the nucleic acid-binding regions contained in the TBA
complex to simultaneously reach and bind to these targets.
[0172] Once the TNA-PNA hybrids have formed and been contacted with
the immobilized TBA, unbound nucleic acid is washed from the
immobilized surface and the immobilize hybrids detected. This is
accomplished in any one of several ways. In one aspect of this
invention, the PNA is labeled with an OSA, such as a radionuclide,
colored beads, or an enzyme capable of forming a colored reaction
product. Furthermore, in addition to having one or more 1/2 TBRs,
the PNA also may contain at least one 1/2 BBR. The 1/2 BBR
sequences are chosen so as to be complementary to unique 1/2 BBR
sequences in BNAs. In the embodiment described above, for example,
where the TBA is NF-kB and the TBR formed upon TNA-PNA
hybridization is one or more NF-kB binding sites, the 1/2 BBRs may
provide hybridizable (that is, single-stranded, complementary)
sequences of the left or right bacteriophage lambda operators (see,
for example, Ptashne [1982] Scientific American 247:128-140, and
references cited therein for sequences of these operators). These
may be polymerized onto the PNA 1/2 BBRs in a vectorial fashion
(see FIGS. 2 and 3) providing up to "n" BBRs, and each BBR forms a
cro binding site, Enzymatically, radioactively, or otherwise
labeled cro, is contacted with the TBA-TNA-PNA-(BNA). complex. In
this fashion, a highly selective and amplified signal is produced.
Signal produced using a PNA having a single 1/2 TBR indicates
success of the assay in achieving TBA-TBR binding and
polymerization of the BNAs to produce signal from cellular sites
(i.e. from CNAs). Absence of signal when a dimerized TBA is used
indicates that in the TNA, there were no HIV LTRs as no double
NF-kB binding sites were present. On the other hand, presence of
signal using the dimer NF-kB indicates HIV infection. As a specific
example of the foregoing description of this embodiment of the
invention, see Example 6 describing an HIV test kit.
[0173] Naturally, those skilled in the art will recognize that the
foregoing description is subject to several modifications in the
choice of PNAs, TNAs, TBAs, BNAs, and BBAs. Furthermore, in systems
other than HIV, those skilled in the art will recognize that the
general method described above could be likewise applied. However,
these other applications may be simpler than the above described as
the TBAs used may not recognize any normal cellular sites and
therefore resort to dimerization or other methods of discriminating
between TNAs and CNAs may be less critical. In designing probes and
binding assemblies for these other systems, the skilled artisan
will be guided by the following principles and considerations.
[0174] In the above-described embodiment, the appeal of using the
DNA-binding portions of NF-kB protein as the TBA and the NF-kB
recognition binding elements as the TBRs is that these elements
form an important "control point" for the replication of HIV. That
is, it is known that HIV is required to use NF-kB as a critical
feature in its replicative life cycle. Similar control points for
other pathogens are chosen and used as a basis for detection
according to the methods described herein.
[0175] From the foregoing description of general features of this
invention and the mode of its operation, one skilled in the art
will recognize that there are a multiplicity of specific modes for
practicing this invention. By way of example, the method of this
invention is adaptable to a method and devices using
chromatographic test kits described in U.S. Pat. Nos. 4,690,691 and
5,310,650 (the '691 and '650 patents). In those patents, a porous
medium was used to immobilize either a TNA or a capture probe, and
a solvent was used to transport a mobile phase containing either a
labeled PNA, if the TNA was immobilized, or the TNA, if a capture
probe was immobile, into the "capture zone". Once the TNA was bound
in the capture zone, either by directly immobilizing it or through
capture, a labeled PNA was chromatographed through the capture zone
and any bound label was detected.
[0176] Adapting the instant invention to such a system provides the
improvement of using a Target Binding Assembly in the capture zone
and therefore, the capture of only perfectly matched TBR sequences
or other TBRs representing nucleic acid confirmations specifically
bound by the TBA within the TNA-PNA duplexes by virtue of the
previously described sensitive discrimination by the TBA between
TNAs and CNAs.
[0177] Once the TNA-PNA hybrids become bound to the immobilized
TBA, the signal is amplified by adding BNAs or chromatographing
BNAs through the capture zone. Finally, the signal may be further
amplified by adding BBAs or chromatographing labeled BBAs through
the capture zone. In this fashion, the ease of performing the
analysis steps described in the '691 and '650 patents is improved
upon herein by providing the additional ability to increase the
specificity and, through amplification, the sensitivity of the
method described in those patents. The disclosure of the '691 and
'650 patents is herein incorporated by reference for the purpose of
showing the details of that method and for the teachings provided
therein of specific operating conditions to which the compositions
and methods of the instant invention are adaptable.
[0178] Those skilled in the art will also recognize that the method
of the instant invention is amenable to being run in microtiter
plates or to automation. The use of machines incorporating the
method of this invention therefore naturally falls within the scope
of the instant disclosure and the claims appended hereto. Thus, for
example, this invention is adaptable for use in such instruments as
Abbott Laboratories' (Abbott Park, Ill.) IMx tabletop analyzer. The
IMx is currently designed to run both fluorescent polarization
immunoassay (FPZA, see Kier [1983] KCLA 3:13-15) and microparticle
enzyme immunoassay (MEZA, see Laboratory Medicine, Vol. 20, No. 1,
January 1989, pp. 47-49). The MEZA method is easily transformed
into a nucleic acid detection method using the instant invention by
using a TBA as a capture molecule coated onto a submicron (<0.5
.mu.m on average) sized microparticle suspended in solution. The
microparticles coated with TBA are pipetted into a reaction cell.
The IMx then pipettes sample (Hybridized PNA-TNA) into the reaction
cell, forming a complex with the TBA. After an appropriate
incubation period, the solution is transferred to an inert glass
fiber matrix for which the particles have a strong affinity and to
which the microparticles adhere. Either prior to or after filtering
the reaction mixture through the glass fiber matrix, BNAs and BBAs
are added, or another signal amplification and detection means is
used which depends on specific formation of TNA-PNA hybrids. The
immobilized complex is washed and the unbound material flows
through the glass fiber matrix.
[0179] The bound complexes are detected by means of alkaline
phosphatase labeled BBAs or otherwise (radioactively,
enzymatically, fluorescently) labeled BBAs. In the case of alkaline
phosphatase labeled BBAs, the fluorescent substrate 4-methyl
umbelliferyl phosphate or like reagent may be added. Alternatively,
the enzyme may be bypassed by directly labeling BBAs with this or a
like reagent. In any event, fluorescence or other signal is
proportional to the amount of PNA-TNA hybrids present.
[0180] The fluorescence is detected on the surface of the matrix by
means of a front surface fluorometer as described by the
manufacturer of the IMx. With minor adjustments that can be made
through routine experimentation to optimize an instrument such as
the IMx for nucleic acid hybridization and nucleic acid-TBA
interactions, the instant invention is completely adaptable to
automated analyses of TNA samples.
[0181] 9. Other Diagnostic Applications of this Invention.
[0182] While the foregoing description enables the use of the
instant invention in a number of different modes, many additional
utilities of this invention are readily appreciated, for example,
in a mobility retardation system.
[0183] In this embodiment of the invention, an improvement of the
well known electrophoretic mobility shift assay (EMSA) is conducted
as follows (See FIGS. 12a and 12b):
[0184] A sample of DNA is fragmented, either through random
cleavage or through specific restriction endonuclease treatment.
The DNA in the sample is then split into two equal aliquots and a
specific TNA is added to the first aliquot but not to the second.
The first and second aliquot are then electrophoresed in an
acrylamide or agarose gel, and the pattern of DNA bands (either
visualized through ethidium bromide binding or through being
radioactively labeled prior to electrophoresis is then compared for
the two aliquots. Fragments of DNA having binding sites to which
the TBA is specific are retarded in their migration through the
electrophoretic medium. By using an appropriate TBA, any number of
DNA or other nucleic acid sequences may be tracked in this
fashion.
[0185] In a modification of the EMSA described above, fragmented
TNA is hybridized with a PNA and fractionated in a fist dimension.
The fractionated DNA is then reacted with an appropriate TBA and
the change in mobility of the DNA fragments is noted. Enhancement
of the retardation is possible by adding BBAs as described above.
(See, for example, Vijg and references cited therein for known
techniques of two (2) dimensional nucleic acid electrophoresis, to
which the instant method may be applied).
[0186] 10. Therapeutic Applications.
[0187] Because of the very tight and selective nucleic acid binding
characteristics of the novel TBAs described herein, therapeutic
utilities are contemplated in addition to the diagnostic utilities
of these compounds. Thus, a TBA comprising tight and specific
binding for the HIV-LTR, by virtue of having an NF-kB p50 and an
SP1 DNA recognition unit in close association (see FIG. 10,
HIV-Detect II) is useful to bind up the HIV-LTR and thereby prevent
transcription from this key element of the HIV genome. The unique
features of the assembly sequences of the TBA allow recombinant
vectors to induce DNA encoding such a TMA into a cell and the
proper folding of the expressed sequences. Once inside the cell,
the nuclear localization signals of the p50 subunit directs the
transport of the TBA to the nucleus where it binds tightly to the
LTR of any integrated HIV, effectively shutting the pathogen down.
In a prophylactic mode, one that is concerned about potential HIV
exposure is administered a sufficient dose of a TBA or a
recombinant vector able to express the TBA, so as to lock up any
HIV that might have entered the person. In this mode, the use of
the TBA is analogous to passive protection with a specific immune
globulin. In the therapeutic or prophylactic mode, NLS sequences
are used in place of the OSAs used in the diagnostic mode.
Exemplary NLS sequences are provided as SEQ ID NOS. 72 and 103 (see
also Heinzinger 1994 and Bukinsky 1993, describing NLS sequences of
the HIV Vpr and gag proteins respectively). In any event, the TBA
is administered in a pharmaceutically-acceptable carrier, known in
the art such as a sterile salt solution or associated with a
lysosome or in the form of a recombinant vector, preferably one
which directs expression of the TBA in a chosen cell type, or by a
protein delivery system.
[0188] II. Embodiments of the Invention
[0189] In view of the foregoing description and the examples which
follow, those skilled in the art will appreciate that this
disclosure describes and enables various embodiments of this
invention, including:
[0190] 1. A probe nucleic acid (PNA) comprising:
[0191] (a) a single-stranded sequence, 1/2 TBR, which is capable of
forming, under hybridizing conditions, a hybrid, TBR, with a 1/2
TBR present in a target nucleic acid (TNA);
[0192] (b) zero, one or more, and preferably one to ten single
stranded sequences, 1/2 BBR, which is capable of forming, under
hybridizing conditions, a hybrid BBR, with a 1/2 BBR present in a
booster nucleic acid (BNA); and
[0193] (c) an OSA, which is no attached support and/or indicator,
or an attached support or other means of localization, including,
but not limited to, attachment to beads, polymers, and surfaces,
and/or indicators;
[0194] wherein said TBR is capable of binding with high affinity to
a TBA, said TBA being a substance capable of discriminating between
a paired TBR and a TBR having unpaired nucleotides, and further,
wherein said BBR is capable of binding with high affinity to a BBA,
said BBA being a substance capable of discriminating between a
paired BBR and a BBR having unpaired nucleotides. This embodiment
includes TBRs which are nucleic acid binding protein recognition
sites, such as the HIV LTR, and other nucleic acid binding protein
recognition sites in other pathogens, some of which are noted
above. The PNA of this embodiment of the invention may produce a
TBR which is a nucleic acid binding protein recognition site
present in the genome of a pathogen or is a binding site associated
with a pathogenic condition in the human genome or a contaminant in
a fermentation process.
[0195] 2. A booster nucleic acid (BNA) comprising:
[0196] (a) a 1/2 BBR which has a sequence which is complementary to
a 1/2 BBR sequence in a PNA or another BNA already hybridized to
the PNA and which is capable of forming, under hybridizing
conditions, a hybrid, BBR, with the PNA;
[0197] (b) an OSA attached support or other means of localization,
including, but not limited to, attachment to beads, polymers, and
surfaces, and/or indicators; and
[0198] (c) additional hybridization sites, 1/2 BBRs, for
hybridization with additional BNAs;
[0199] wherein said BBR is capable of binding with high affinity to
a BBA, said BBA being a substance capable of discriminating between
a paired BBR and a BBR having unpaired nucleotides.
[0200] 3. A Hairpin Nucleic Acid (HNA) comprising a single-stranded
sequence, 1/2 BBR, which under hybridizing conditions is capable of
forming a hairpin while at the same time binding to a BNA to form a
BBR capable of binding a BBA, wherein said BBR is capable of
binding with high affinity to a BBA, said BBA being a substance
capable of discriminating between a paired BBR and a BBR having
unpaired nucleotides.
[0201] 4. A method for detecting a specific TNA sequence,
comprising the steps of:
[0202] (a) hybridizing said TNA with a PNA as described above;
[0203] (b) hybridizing said PNA with a BNA containing a 1/2 BBR
whose sequence is complementary to a 1/2 BBR sequence in the
PNA;
[0204] (c) adding the products of steps (a) and (b) containing a
TBR and a BBR, to a surface, liquid or other medium containing a
TBA;
[0205] (d) adding BBAs to the mixture in step (c) wherein said BBA
comprises:
[0206] (i) a molecule or a portion of a molecule which is capable
of selectively binding to a BBR; and
[0207] (ii) a detectible indicator; and
[0208] (e) detecting signal produced by the indicator attached to
the BBA. This method includes the use of a protein indicator,
including enzymes capable of catalyzing reactions leading to
production of colored reaction products. It also includes
indicators such as a radionuclide or colored beads.
[0209] 5. A method for detecting the presence in a sample of a
specific Target Nucleic Acid, TNA, which comprises:
[0210] (a) contacting said sample a Probe Nucleic Acid, PNA, which,
upon hybridization with said TNA if present in said sample, forms a
Target Binding Region, TBR, which is capable of binding a Target
Binding Assembly, TBA;
[0211] (b) contacting said sample, already in contact with said
PNA, with a TBA capable of binding to any TBRs formed by the
hybridization of said PNA and said TNA in the sample.
[0212] 6. A method for detecting or localizing specific nucleic
acid sequences with a high degree of sensitivity and specificity
which comprises:
[0213] (a) adding PNAs containing a 1/2 BBR and a 1/2 TBR to a
sample containing or suspected of containing TNAs containing 1/2
TBR sequences, to form a complex having target binding regions,
TBRs, formed by the hybridization of complementary 1/2 TBRs present
in the PNAs and TNAs respectively;
[0214] (b) binding the TBRs formed in step (a) to an immobilized
TBA to form a TBA-TNA-PNA complex;
[0215] (c) adding Booster Nucleic Acids, BNAs, containing booster
binding regions, 1/2 BBRs, to the complex formed in step (b) such
that the 1/2 BBRs in the BNAs hybridize with the 1/2 BBR sequences
present in the PNAs or to 1/2 BBRs present in BNAs already bound to
the PNA, to form BBRs, such that TBA-TNA-PNA-(BNA).sub.n complexes
are formed;
[0216] (d) adding Hairpin Nucleic Acids, HNAs, containing 1/2 BBR
sequences, to the complex formed in step (c) such that the 1/2 BBRs
in the HNAs hybridize with any available 1/2 BBR sequences present
in the BNAs of the complex of step (c), thereby capping the
extension of the BNAs onto the TBA-TNA-PNA-(BNA).sub.n complexes of
step (c) to form TBA-TNA-PNA-(BNA).sub.n-HNA complexes;
[0217] (e) adding Booster Binding Assemblies, BBAs, linked to
indicator moieties, to the TBA-TNA-PNA-(BNA).sub.n-HNA complexes
formed in step (d) to form TBA-TNA-PNA-(BNA-BBA).sub.n-HNA
complexes; and
[0218] (f) detecting the signals produced by the indicator moieties
inked to the TBAs, PNAs, BNAs, BBAs or HNAs in the
TBA-TNA-PNA(BNA-BBA).sub.n-H- NA complexes of step (e);
[0219] wherein:
[0220] the TNA comprises:
[0221] (i) one or more specific 1/2 TBR nucleic acid sequences, the
presence or absence of which in a particular sample is to be
confirmed;
[0222] the PNA comprises:
[0223] (i) a single stranded sequence, 1/2 TBR, which is capable of
forming, under hybridizing conditions, a hybrid, TBR, with a 1/2
TBR present in a target nucleic acid (TNA);
[0224] (ii) a single stranded sequence, 1/2 BBR, which is capable
of forming, under hybridizing conditions, a hybrid BBR with a 1/2
BBR present in a booster nucleic acid (BNA); and
[0225] (iii) an OSA, which is no attached support and/or indicator,
or an attached support or other means of localization, including,
but not limited to, attachment to beads, polymers, and surfaces,
and/or indicators;
[0226] the BNA comprises:
[0227] (i) a 1/2 BBR, as shown in FIG. 1(IIb), which has a sequence
which is complementary to a 1/2 BBR sequence in a PNA and which is
capable of forming, under hybridizing conditions, a hybrid, BBR,
with the PNA;
[0228] (ii) an OSA attached support or other means of localization,
including, but not limited to, attachment to beads, polymers, and
surfaces, and/or indicators;
[0229] (iii) additional hybridization sites, 1/2 BBRs, for other
BNAs; and
[0230] (iv) sequences, 1/2 BBRs, which can hybridize to BNAs
already hybridized to the PNA;
[0231] the BBA comprises:
[0232] (i) a molecule or a portion of a molecule which is capable
of selectively binding to a BBR, and
[0233] (ii) no attached support and/or indicator, or an attached
support or other means of localization, including, but not limited
to, attachment to beads, polymers, and surfaces, and/or
indicators;
[0234] and the TBA comprises:
[0235] (i) a molecule or a portion of a molecule which is capable
of selectively binding to a TBR; and
[0236] (ii) no attached support and/or indicator, or an attached
support or other means of localization, including, but not limited
to, attachment to beads, polymers, and surfaces, and/or
indicators.
[0237] 7. An improvement to a solid phase hybridization method for
detecting the presence of a target polynucleotide involving:
immobilizing a target polynucleotide, if present in a test sample,
directly or via an intermediate capture structure, on a solid phase
at a capture site; before, during or after said immobilization,
attaching a detectable label to said target polynucleotide, if
present; and detecting said label, if any, at said capture site;
the improvement comprising:
[0238] (a) using a Target Binding Assembly, TBA, as the means for
achieving immobilization of said target polynucleotide, wherein
said TBA binds only to a perfect hybrid formed between a specific
Probe Nucleic Acid, PNA, and said target nucleic acid such that a
perfect Target Binding Region, TBR, recognizable by said TBA is
formed; and
[0239] (b) including in the PNA a single stranded sequence, 1/2
BBR, capable of binding a Booster Nucleic Acid, BNA, containing a
single stranded complementary 1/2 BBR which, upon hybridization
with the 1/2 BBR in the PNA, forms a BBR capable of binding labeled
Booster Binding Assemblies, BBAs.
[0240] 8. A target binding assembly, TBA, comprising one or more
nucleic acid recognition units, linker sequence(s), assembly
sequence(s), asymmetry sequence(s), nuclear localization sign
sequence(s) (NLS) and OSA(s). The nucleic acid recognition unit may
be an NF-kB binding unit, an SP1 binding unit, a TATA binding unit,
a human papillomavirus binding unit, an HIV LTR binding unit, or a
binding unit for any other fragment of specific sequence the
detection of which is desirable and which can be achieved through
specific association with the TBA. Such recognition units include,
but are not limited to those exemplified herein as SEQ ID NO. 63,
SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID
NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71, SEQ ID NO. 72,
and SEQ ID NO. 73. Linker sequences such as oligopeptides which do
not interfere with the nucleic acid recognition function of the
nucleic acid recognition unit and which provide stability and
control over the spacing of the nucleic acid recognition unit from
the remainder of the TBA. Examples of such link sequences are well
known in the art and include, but are not limited to oligopeptide
sequences from the interdomain primary sequence of a structural
protein. Assembly sequences include oligopeptide sequences which
direct the folding and association of nucleic acid recognition
units. A preferred example of such sequences are oligopeptides
derived from the bacteriophage lambda cro protein. The asymmetry
sequence directs the association of nucleic acid recognition and
assembly sequences in a predetermined order. Such asymmetry
sequences are exemplified by sequences derived from insulin,
relaxin, gonadotropic hormone, FSH, HCG, LH, ACTH, including but
not limited to SEQ ID NOS. 85-92. With reference to FIGS. 14 and
15, SEQ ID NO. 85 is an "A" and SEQ ID NO. 86 is a "B" sequence;
SEQ ID NO. 87 is an "A" and SEQ ID NO. 88 is a "B" sequence SEQ ID
NO. 89 is a human relaxin "A" and SEQ ID NO. 90 is a human relaxin
"B" sequence; SEQ ID NO. 91 is a skate relaxin "A" and SEQ ID NO.
92 is a skate relaxin "B" sequence. In addition, the TBA may
contain nuclear localization signal sequences, NLS, which direct
the migration and uptake of a protein or complex associated with
said NLS into the nucleus of a cell. Examples of such NLS sequences
are provided as SEQ ID NOS. 72 and 103. Preferred embodiments of
the TBA include but are not limited to HIV Detect I-IV or HPV
Detect I-IV, and SEQ ID NOS. 109-116.
[0241] 9. Methods of using the novel TBAs of this invention
include, but are not limited to a method of using the TBA to bind a
particular nucleic acid sequence in a target nucleic acid sample
which comprises:
[0242] (a) fragmenting the nucleic acid in the target nucleic acid
sample;
[0243] (b) contacting, under hybridizing conditions, the fragmented
nucleic acid with a probe nucleic acid complementary to the
particular nucleic acid sequence of interest, wherein said probe
nucleic acid, upon hybridization with said particular nucleic acid
sequence of interest forms a target binding region to which said
TBA specifically binds.
[0244] In this method, the probe nucleic acid, in addition to
sequences complementary to said particular nucleic acid sequence of
interest, also may have additional sequences to which a booster
nucleic acid can bind to form a booster binding site to which a
labeled booster binding assembly can bind to provide a signal
showing and amplifying the binding of the probe nucleic acid to the
target nucleic acid sequence of interest.
[0245] An additional aspect of this invention not requiring
fragmentation of Target Nucleic Acid, involves administration of
the TBA to a patient in need of such treatment of a therapeutically
or prophylactically effective amount of said TBA, which comprises
administering the TBA, either in the form of a purified protein
complex or in the form of a recombinant vector which, upon entry
into the patient is able to express the TBA, such that the TBA
binds the particular nucleic acid sequence to achieve the desired
prophylactic or therapeutic result. This may include providing a
dosage which can be determined by routine experimentation to be
sufficient to prevent establishment of an active infection by a
pathogen. Dosages of purified TBAs may be in the range of about
0.001 to 100 mg/kg. When provided as a recombinant expression
vector which will direct the in vivo expression and folding of the
TBA, dosages of the recombinant nucleic acid may be substantially
lower, particularly if provided in the form of non-pathogenic viral
vector. The methods of using the TBAs also include monitoring the
shift in mobility of nucleic acids in target nucleic acid samples
as a function of the size such that binding of the TBA to a
particular fragment in the sample modifies the mobility of the
fragment. This aspect of the method provides a useful method of
analyzing nucleic acid fragments for particular aberrations, such
as might be found associated with metastases.
[0246] 10. Diagnostic or forensic kits useful in determining the
presence of an infection, the susceptibility to a disease, or the
origin of a particular nucleic acid containing sample.
[0247] 11. A method of assembling multimeric TBAs in vivo which
comprises introducing nucleic acids encoding component TBAs into a
cell. The component TBAs should each contain a nucleic acid
recognition unit, assembly sequences, asymmetry sequences, and
nuclear localization signal sequences. Linker sequences, optionally
included if TBA footprinting experiments indicate the need for such
linkers to attain optimal geometry of the multimeric TBA. Upon in
vivo expression of each component TBA and primal binding, via the
nucleic acid recognition unit of each component TBA to nucleic acid
sequences encountered in the nucleus or elsewhere in the cell,
component expressed TBAs are directed to assemble via the included
assembly and asymmetry sequences into multimeric TBAs. As described
above, such multimeric TBAs will have the advantage of binding
specifically with high affinity to TBRs in a specific target
sequence, but not at all or with very low affinity to cousin
nucleic acids.
[0248] The forgoing description of the invention will be
appreciated by those skilled in the art to enable preferred
embodiments as well as the best mode of this invention. Without
limiting the subject matter to the specifics of the examples
provided hereinafter, the following examples are provided to
further guide those skilled in the art on methods of practicing
this invention. Standard recombinant DNA techniques as disclosed in
Sambrook, Fritsch, and Maniatis (1989) Molecular Cloning: A
Laboratory Manual, 2nd Ed, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., and more recent texts are not disclosed
as these are now well within the skill of the ordinary artisan.
EXAMPLE 1
Preparation of PNAs and Labeling of PNAs
[0249] Probe nucleic acids, PNAs, may be prepared by means well
known in the art. Thus, single stranded polynucleotide PNAs of
defined sequence may be prepared via solid phase chemical synthesis
according to Merrifield. PNAs may be prepared by automated
synthesis using commercially available technology, such as resins
and machines produced or marketed by Applied Biosystems, ABI, or
other manufacturers. Alternatively, through known recombinant DNA
methods, particular PNA sequences are synthesized in vivo, for
example by cloning a duplex PNA into a vector which can replicate
in E. coli, large quantities of the duplex PNA may be prepared.
Multimers of the PNA may be cloned into the vector such that for
each mole of vector, several moles of PNA is liberated upon
digestion of the vector with a restriction fragment flanking the
PNA sequence. Subsequent to synthesis or recombinant production,
the PNAs are purified by methods well known in the art such as by
gel electrophoresis or high pressure liquid chromatography (HPLC).
If the PNA is produced as a duplex, prior to use in a hybridization
assay for detection of target nucleic acid sequences the strands of
the PNA are separated by heating or other methods known in the
art.
[0250] The specific sequence of bases in the PNA is chosen to
reflect the sequence to be detected in a TNA, with the proviso
that, according to this invention, the PNA contains a 1/2 TBR
sequence, which is one that upon hybridization of the PNA and TNA,
a TBR is formed. As there are an essentially unlimited number of
such sequences known in the art, the choice of the PNA sequence is
amenable to selection by the skilled researcher for any given
application. The sequence of the HIV LTR is one such sequence,
which upon hybridization of a PNA encoding portions of the LTR with
TNAs encoding the HIV LTR, TBRs capable of binding the NP-kB or SP1
DNA binding proteins are formed.
[0251] In addition to sequences which will form a TBR upon
hybridization, the PNA also may contain a 1/2 BBR. This sequence is
one which, upon hybridization with a booster nucleic acid, BNA,
forms a BBR which is capable of binding a BBA. The BBA is
preferably a DNA binding protein having high affinity for the BBR
sequence.
[0252] In this particular example, hybridization between a PNA
having as a 1/2 TBR, SEQ ID NO. 4 and, at the 3' end of that
sequence, a 1/2 BBR sequence shown as SEQ ID NO. 35. The PNA
encoding these sequences is either used without labeling or is
labeled with a radioactive isotope such as P.sup.32, S.sup.35, or a
similar isotope, according to methods known in the art.
Alternatively, the PNA is bound to a bead of between 0.01 to 10
.mu.m, which may be colored for easy visual detection. This label
forms the OSA as described in the specification. This probe
hybridizes with HIV LTR sequences to form a TBR that binds NF-kB.
In addition, the PNA hybridizes with BNAs having a complementary
1/2 BBR to form a bacteriophage lambda left operator that binds
either cro or lambda repressor proteins.
[0253] In a manner similar to that described above, PNAs are used
wherein the 1/2 TBR is any one of SEQ ID NO. 5 or SEQ ID NOS. 7-34,
and a 1/2 BBR, such as SEQ ID NO. 35 or SEQ ID NO. 36 is either at
the 3' end or 5' end of the 1/2 TBR.
EXAMPLE 2
Preparation and Labeling of BNAs
[0254] Similar to the methods described in Example 1 for
preparation and labeling of PNAs, BNAs are prepared and labeled
according to methods known in the art. As described in U.S. Pat.
No. 4,556,643, herein incorporated by reference (see particularly
Example 1), nucleic acid sequences encoding particular nucleic acid
binding sequences may be mass produced by cloning into a replicable
vector. Furthermore, similar to that disclosure, the 1/2 TBR and
1/2 BBR sequences may be co-linearly produced in this fashion, with
the distinction, however, that according to the instant invention,
the 1/2 TBR sequence itself forms a nucleic acid binding component
recognition site and the 1/2 BBR, while forming a nucleic acid
binding component recognition site, also provides a means of
amplifying the signal produced upon binding of the 1/2 TBR to
complementary sequences in the TNA by providing for polymerization
of BNAs onto the TNA bound PNA. To enable this, a sequence such as
SEQ ID NO. 35, which encodes the left operator of bacteriophage
lambda, is provided with additional sequences such that an overhang
sequence is created on one or both ends of the BNA upon
hybridization with the PNA.
[0255] As a specific example, vectorial polymerization of BNAs onto
a TNA is provided by SEQ ID NOS. 40-43. In this example, SEQ ID NO.
40 encodes two 1/2 TBRs which will hybridize with two 1/2 TBRs in a
TNA to form two NT-kB binding sites, while at the same time
providing a bacteriophage lambda left operator 1/2 BBR, which
additionally is terminated at the 3' end with the recognition site
for the restriction enzyme PstI. Addition of the BNA, SEQ ID NO.
41, with the 1/2 BBR complementary to the 1/2 BBR on the PNA, SEQ
ID NO. 40, completes the BBR while at the same time completing the
PstI recognition site, leaving a four base overhang for
hybridization with additional BNAs. Accordingly, SEQ ID NO. 42 is
added which has a four base pair sequence at the 3' end which is
complementary to the four-base overhang remaining from the
hybridization of SEQ ID NOS. 40 and41. In addition, SEQ ID NO. 42
is provided with a five base sequence at its 5' end which forms
part of a BamHI recognition site. The growing polymer of BNAs is
extended further by addition of the BNA SEQ ID NO. 43, which is
complementary to SEQ ID NO. 42, completing the BBR while at the
same time completing the BamHI recognition site and leaving a four
base overhang which may be further hybridized with BNAs having
complementary sequences. In this fashion, the BNAs may be
hybridized extensively so as to greatly amplify the signal of a
single PNA-TNA hybridization event.
[0256] As with the PNAs described in Example 1, the BNAs may be
used in an unlabeled form or may be labeled according to methods
known in the art and described in Example 1. It will also be
appreciated that, rather than produce the BNA polymer by sequential
addition of BNAs to the PNA-TNA complex, the BNA polymer may be
performed and added directly to the PNA-TNA complex. One simple
method for performing such a BNA polymer includes the recombinant
production of a vector in which multimers of the BNA are provided
with a unique restriction site at either end of the polymer. This
polymer of BNAs containing multiple BBRs is cut out of the vector
and hybridizes to a single stranded 1/2 BBR remaining in the PNA
upon hybridization of the PNA and the TNA. This is accomplished by
providing a single stranded sequence in the PNA complementary to an
overhang produced in the BNA polymer when it is excised from the
production vector.
EXAMPLE 3
Production of HNAs and Their Use for Capping BNA Polymers
[0257] The HNAs of this invention are produced according to methods
known in the art for polynucleotide production as described in
Examples 1 and 2 for PNAs and BNAs. In the production of the HNAs,
however, the sequence of the HNA is specifically designed so that a
substantial portion of the HNA forms a self-complementary
palindrome to form a hairpin, while at the same time, leaving in
single stranded form enough bases to be able to hybridize with
single stranded sequences in the growing chain of BNAs described in
Example 2.
[0258] In this Example, a HNA of SEQ ID NO. 44 is provided to cap
the extension of BNAs onto the PNA in Example 2 after the addition
of the BNA, SEQ ID NO. 43. This is accomplished because SEQ ID NO.
44, while having a palindromic sequence that forms a stable
hairpin, also has a sequence at the 5' end of the HNA which
completes the BamHI sequence formed by the hybridization of SEQ ID
NO. 42 and SEQ ID NO. 43. Naturally, termination of the polymer
after addition of only 3 BNAs is for the purpose of simplicity in
demonstrating the invention. As described above, his polymerization
may be continued essentially indefinitely to amplify the signal of
the PNA-TNA hybridization event. Once the HNA hybridizes to the
growing chain of BNAs, the polymer is capped and no further
extension of the polymer is possible.
EXAMPLE 4
Preparation of TBAs and BBAs, Labeling, and Immobilization
Thereof
[0259] The TBAs and BBAs which may be used according to the instant
invention include any substance which can specifically bind to the
TBRs and BBRs formed by hybridization of the PNAs, TNAs and BNAs.
Use of DNA binding proteins forms one example of such
substances.
[0260] For this example, the TBA is the dimer of the DNA binding
portion of p50, and the BBA is the lambda cro protein. These
proteins may be produced according to methods known in the art. The
genes for both of these proteins have been cloned. Thus, these
proteins are recombinantly produced and purified according to
methods known in the art. Furthermore, these proteins are labeled,
either with a radioisotope, such as radioactive iodine, or with an
enzyme, such as beta-galactosidase or horseradish peroidase, or
with a fluorescent dye such as fluorescein or rhodamine, according
to methods well known in the art. In addition, either or both of
the TBA and BBA may be immobilized on a solid surface such as the
surface of a microfiter plate or the surface of a bead, such as a
colored bead of diameter anywhere from 0.01 to 10 .mu.m. The labels
on the TBAs and BBAs may be the same or different.
[0261] In this example, the TBA containing the dimeric p50 DNA
binding domain is labeled with rhodamine, while the BBA, cro, is
labeled with fluorescein. Accordingly, upon hybridization of the
PNAs, TNAs, BNAs and HNAs as described in this patent disclosure
and the foregoing and following examples, the nucleic acid hybrids,
if formed, are contacted with excess labeled TBA and cro. The
fluorescence of these labels is measured according to known methods
and, detection of both signals is indicative of the presence of 1/2
TBR sequences in the TNA. The differential signal produced by the
fluorescence of the NF-kB and cro is a measure of the degree to
which the polymerization of BNAs onto the PNA-TBA hybrid has
resulted in amplification of the signal. Amplification from one to
over a thousand fold is contemplated according to the method of
this invention.
EXAMPLE 5
Hybridization of Two PNAs with a TNA and Discrimination Between a
TNA and a CNA
[0262] The PNAs, PNA1, SEQ ID NO. 40 and PNA2, SEQ ID NO. 45, are
used in about ten-fold molar excess over the concentration of TNAs
in a test sample. For this example, an isolated duplex HIV LTR,
wherein one strand of which has the sequence SEQ ID NO. 37, shown
in FIG. 7, and the other stand of which is complementary to the
sequence shown in FIG. 7, is used as the TNA. A duplex isolated CNA
is also used in this example, one strand of which has the same
sequence as SEQ ID NO. 37, except that, in the first NF-kB binding
site shown in FIG. 7, at the center of the binding site, position 1
in FIG. 7, instead of a "T," there is an "A," the complementary
strand of which therefore mismatches with the SEQ ID NO. 40 PNA at
that location
[0263] SEQ ID NO. 40 and SEQ ID NO. 45 are both added to separate
reactions, the first containing the above described TNA and the
second containing the above described CNA. The samples are
solubilized in an appropriate hybridization buffer, such as 10 mM
Tris (pH 7.5), 1 mM EDTA. The samples reheated to about 90.degree.
C. for about five minutes to strand separate the duplex TNAs and
CNAs in the samples, and then the samples are allowed to cool to
allow strands of PNAs, TNAs and CNAs to anneal.
[0264] Once the hybridization has gone to completion, which can be
determined according to known methods such as by calculating the
t1/2 based on base compositions and annealing temperature according
to known methods, the SEQ ID NO. 40 PNA is polymerized by addition
of BNAs as in Example 2 and the SEQ ID NO. 45 PNA2 probe is
polymerized with BNAs staring with Sph1 recognition site overhang.
Following addition of the BNAs and a brief hybridization period,
the separate samples are added to beads coated with covalently
immobilized NF-kB, and the NF-kB is allowed to bind to any TBRs
formed in the TNA and CNA samples. After about 15 minutes of
binding, the samples are washed twice with about three volumes of
an appropriate washing buffer, such as 10 mM Tris, pH 7.5, 100 mM
NaCl, or another buffer pre-determined not to interfere with NF-kB,
or bacteriophage lambda CI repressor protein binding activity.
After each wash, the beads are allowed to settle under gravity or
by brief centrifugation. This removes any nucleic acids which do
not have a perfect NF-kB binding site formed by hybridization of
the PNA1 and TNA sequences.
[0265] After the final wash, bacteriophage lambda CI repressor
protein labeled with a radioactive isotope, such as with
radioactive iodine, or labeled with an enzyme, such as horseradish
peroxidase, with colored beads, or with a fluorescent label is
added to each sample. The samples are then washed several times
(about 3) with several volumes (about 2) of an appropriate washing
buffer such as 10 mM Tris, pH 7.5, 100 mM NaCl, or another buffer
pre-determined not to interfere with NF-kB, or bacteriophage lambda
CI repressor protein binding activity. After each wash, the beads
are allowed to settle under gravity or by brief centrifugation.
Following the last settling or centrifugation, the bound label is
quantitated by detecting the bound radioactivity, liberated color
in an enzymatic assay, color of bound beads, or fluorescence
detection. Alternatively, an anti-CI antibody can be added and a
standard sandwich enzyme linked immunoassay or radioimmunoassay
performed to detect bound repressor. In addition, as a negative
control (background), all of the foregoing manipulations are
carried out in tandem with a sample in which beads are used having
no immobilized NF-kB.
[0266] As a result of the foregoing assay, the control and CNA
containing samples have similarly low signals while the TNA
containing sample has a signal well above background.
EXAMPLE 6
A Test Kit for the Detection of HIV
[0267] A. Kit contents:
[0268] 1. Microtiter plate.
[0269] 2. 1 mg/mL solution of recombinantly produced NF-kB in
tris-buffered saline.
[0270] 3. Tube containing single stranded HIV PNAs (a mixture of
pre-mixed oligonucleotides encoding two NF-10 1/2 binding sites,
i.e. a mixture of SEQ. ID. Nos. 7 and 8).
[0271] 4. Tube containing single stranded human genomic PNA, SEQ ID
NO. 1.
[0272] 5. Tube of nuclease (PstI).
[0273] 6. Tube of protease.
[0274] 7. Tube containing pre-polymerized BNAs, 100 repeat units of
bacteriophage lambda O.sub.R, capped with an HNA but with free 1/2
BBRs available for binding to PNA-TNA hybrids.
[0275] 8. Tube of horseradish peroxidase (hrp) conjugated cro.
[0276] 9. Tube of hrp colored substrate.
[0277] 10. Tris buffered saline, 100 mL.
[0278] 11. Lancet.
[0279] 12. Reaction tubes A, B, C, each containing 250 .mu.L of
distilled water.
[0280] 13. Medicine dropper.
[0281] B. Assay Method:
[0282] (a) The microtiter plate (item 1) is coated with the
solution of recombinantly produced NF-kB (item 2) at a
concentration of 1 mg/mL in tris buffered saline overnight at
4.degree. C. with rocking.
[0283] (b) Three drops of blood of the test taker is obtained by
pricking a finger with the lancet (reagent 11), and a drop of blood
is dispensed into each of reaction tubes A, B, and C (reagent
12).
[0284] (c) Into each tube is dispensed one drop of protease
solution (reagent 6) with the medicine dropper (item 12) and the
tube agitated and allowed to sit for 5 minutes.
[0285] (d) One drop of nuclease (item 5) is added to each of tubes
A-C using the medicine dropper and the tubes agitated and allowed
to sit for 10 minutes.
[0286] (e) One drop of item 3 is added to tube A (test sample); one
drop of item 4 is added to tube B (positive control); and one drop
of saline (item 12) is added to tube C as a negative control. The
tubes are heated to 50.degree. C. in hot water and allowed to cool
to room temperature over one hour.
[0287] (f) While the hybridization is allowed to occur in step (d),
the excess protein is drained from the surface and the microtiter
plate, from step (a), and the plate is rinsed with tris buffered
saline (tube 10).
[0288] (g) The contents of tubes A-C from step (e) are transferred
to three wells of the microtiter plate and allowed to stand for 1
hour with rocking.
[0289] (h) The microtiter wells containing the contents of tubes
A-C are rinsed with tris buffered saline and emptied.
[0290] (i) One drop of item 7 is added to each well and allowed to
hybridize with any 1/2 BBR sites bound to the plate, over one hour,
followed by three rinses with tris buffered saline.
[0291] (j) One drop of item 8 is added to each well and cro is
allowed to bind to any bound BNA's over 10 minutes, followed by
five, one mL washes with tris-buffered saline.
[0292] (k) One drop of hrp substrate is added to each well and
color allowed to develop.
[0293] C. Results:
[0294] If wells A and B both show color development, and well C
does not, the test is valid and the subject has been infected wit
HIV. If only well A shows color development, or if well C shows
color development, the test has been performed incorrectly, and is
invalid. If wells A and C show no color development but well B
does, the test is valid and the individual has not been infected
with HIV.
EXAMPLE 7
Production of Various Novel TBAs
[0295] Novel TBAs for use according to the instant invention are
prepared as follows:
[0296] (a) NFkB/NF-kB (HIV-Detect I). A nucleic acid encoding any
one of SEQ ID NOS. 63-71 or a like NF-kB DNA binding protein, is
fused, in frame, to a nucleotide sequence encoding an assembly
sequence, such as cro, such that the NE-kB DNA recognition sequence
is encoded at amino or carboxy terminus of the cro sequence.
Optionally, a linker sequence is provided between the NF-kB
sequence and the cro sequence. At the other terminus of cro, a
nuclear localization signal sequence, such as SEQ ID NO. 72, is
optionally provided. Further, asymmetry sequences are optionally
provided at the cro terminus unused by the NF-kB recognition
sequence. Examples of complete TBAs are shown below.
[0297] (b) NF-kB/SP1 (HIV-Detect II). In a similar fashion to that
described in (a) above, a recombinant coding sequence encoding an
NF-kB recognition domain is prepared. In a separate construct,
instead of SEQ ID NOS. 63-72, the coding sequence for the DNA
recognition portion of SP1 is included. Such a sequence should
encode all or a functional part of SEQ ID NO. 73, which is that
portion of the SP1 transcription factor exhibiting DNA binding (see
Kadonaga et al. [1987] Cell 51:1079-1090). The NF-kB-encoding
vector and the SP1-encoding vector are then co-transfected into an
appropriate expression system such as is well known in the art. A
monomeric NF-kB recognition unit is added to complete the NF-kB
recognition dimer after the assembly of the SP1 and NF-kB
recognition units by the chaperone. The asymmetry sequences prevent
the formation of NF-kB or SP1 dimers and direct, instead, the
formation of NFkB-SP1 heterodimers (i.e., HIV-Detect II), which are
then isolated from the expression system (mammalian or bacterial
cells) by known methods.
[0298] (c) SP1/SP1 TBAs (HIV-Detect III). As described in (b)
above, an SP1-encoding TBA construct is prepared. However, only
this construct is transfected into the expression system, and
asymmetry sequences allowing the formation of SP1-SP 1 dimers are
included.
[0299] (d) SP1-TATA (HIV-Detect IV). As described in (b) above, an
SP1-encoding TBA recombinant is produced. In addition, a
recombinant encoding a TBA having the binding sequence, SEQ ID NO.
74, or like sequence encoding a TATA recognition unit is prepared
with asymmetry sequences complementary to those included in the SP1
TBA-encoding construct. These constructs are co-transfected and the
heterodimers isolated by standard methods, including affinity
purification on a DNA column having the appropriate SP1-TATA target
binding regions.
[0300] (e) SP1-E2 (HPV-Detect I). An SP1-encoding construct is
prepared as in (b) above. An E2 TBA-encoding construct is prepared
by using a sequence encoding any one of SEQ ID NOS. 75-84 and 94-98
which are papillomavirus E2 DNA recognition units (see Hegde et al.
[1992] Nature 359:505-512) or like recognition units, is prepared
and co-transformed or co-transfected with the SP1 TBA-encoding
construct. Monomeric E2 recognition unit is added to the complete
E2 recognition dimer after the assembly of the E2-SP1 recognition
unit by the chaperone. The heterodimer HPV-Detect I is isolated
according to known methods.
[0301] (f) E2-E2 (HPV-Detect II). As described above in (e), an E2
TBA-encoding construct is prepared except that asymmetry sequences
are included which permit the formation of E2 dimers. The expressed
dimers are then isolated by known methods including affinity for a
dimeric E2 binding site on a DNA affinity column.
[0302] (g) E2-TATA (HPV-Detect III). As described above in (e) and
(d), E2 and TATA binding TBAs are prepared (respectively), except
that asymmetry sequences are included which enhance the formation
of heterodimers rather than homodimers. These constructs are then
co-expressed and the heterodimers are isolated.
[0303] (h) TATA-TATA (HPV-Detect IV). As described above in (a) and
(d), a TATA binding TBA-encoding construct is prepared using
asymmetry sequences that encourage this homodimer formation and the
homodimer is isolated.
[0304] (i) Other TBAs. As described above for HIV and HPV TBAs,
TBAs for any given pathogen or disease state may be produced by
identifying specific DNA binding proteins and forming an expression
construct using appropriate linker, assembly, and asymmetry
sequences.
EXAMPLE 8
[0305] In a similar fashion to the assay described in Example 5, a
more stringent assay is produced by using the duplex NF-kB-SP1
binding protein prepared according to Example 6. Accordingly, the
probes shown in FIG. 7 and used in Example 5 may be lengthened to
reduce the interprobe distance and thereby reduce the flexibility
of the DNA in the TNA.
EXAMPLE 9
Production of "High-Order" TBAs
[0306] By the appropriate use of asymmetry sequences, TBAs are
produced which are dimers, trimers, tetameres, pentamers, or
hexamers of particular DNA recognition units. In this fashion, a
hexameric TBA is produced by making a first NF-kB p50 dimeric TBA
using asymmetry sequences which enable dimer formation. In
addition, the asymmetry sequences enable the tetramerization of the
p50 dimer with an SP1-SP1 dimer. Finally, additional asymmetry
sequences direct the hexamerization with a dimer exhibiting nuclear
localization sequences. This is accomplished by incorporating, for
example, asymmetry sequences from insulin, which in nature forms
hexamers. This hexamer formation is directed by the sequences, SEQ
ID NOS. 85 (A) and 86 (B), 87 (A) and 88 (B), 89 (A) and 90 (B),
and 91 (A) and 92 (B) (see FIGS. 13 and 14).
[0307] Because of the extremely high affinity for the HIV-LTR that
can be generated using a multimeric TBA, the compounds having this
structure and which can be used for this purpose are referred to
herein as "HIV-Lock."
[0308] An optimal HIV-Lock is defined by footprinting (according to
methods well known in the art) TBAs bound to TBRs in the HIV LTR to
confirm that the binding affinity of each DNA binding protein
contributing to the formation of the multimeric TBA complex is
downshifted relative to the affinity for any natural target
sequence (i.e. CNAs) from which the DNA binding recognition unit of
the TBA is derived. Any concomitant loss in binding affinity for
the HIV TBRs is more than compensated for upon formation of the
multimer as described below.
[0309] There may be competition between the binding of each
component TBA for its TBR and assembly, via asymmetry sequences to
form the multimer. This is obviated by adjusting the linkers
between the chaperone and asymmetry sequences in each TBA component
such that these competing events are uncoupled. The resultant
reduction in the dimensionality of diffision (effective
concentration increase) for the TBA asymmetry and assembly
components results in efficient formation of the multimeric
complex.
[0310] On the basis of the footprinting, the length and composition
of linkers is adjusted to achieve optimal discrimination between
target HIV sequences and natural sequences. In this fashion,
although each component TBA will have a low affinity for CNA and
TBR sequences, the multimeric complex will have an extremely high
affinity for the now expanded TBR recognized by the multimeric
complex (the square of the affinity of each TBR recognized by each
component TBA of the multimeric TBA), while still having a low
affinity for CNAs. In the same fashion, other multimeric TBA
complexes, aside from HIV-Lock, are prepared.
[0311] TBAs which can be formed in this fashion include the
following sequences, which are assembled by linking either the
protein subunits or nucleic acid sequences encoding these subunits,
as follows:
3 Set Link Sequences from Groups A I + II + III B IV + V + III C IV
+ III
[0312] wherein groups I-V consist of sequences selected from:
4 Group Selected from Sequences I Any of SEQ ID NOS. 85-92 II Met
Ser, linked to any of SEQ ID NOS 104-106, each of which is linked
to SEQ ID NO. 99. III SEQ ID NO. 100 linked to any of SEQ ID NOS.
75-84 or 94-98; SEQ ID NO. 101 linked to either SEQ ID NO. 74 or
SEQ ID NO. 93; or SEQ 1D) NO. 102 linkedto SEQ ID NO. 74 or SEQ ID
NO. 93; or any of SEQ ID NO. 72, 103, 73, or 63-71. IV Any of SEQ
ID NOS. 104-106. V SEQ ID NO. 99.
[0313] Specific examples of such TBAs are SEQ ID NOS. 109-116,
assembled as follows:
5 Set SEQ ID NO. Link SEQ IDS A 109 85 + MetSer + 104 + 99 + 100 +
94 A 110 85 + Met Ser + 104 + 99 + 72 A 111 86 + MetSer + 105 + 99
+ 102 + 74 A 112 86 + MetSer + 106 + 99 + 73 A 113 89 + MetSer +
106 + 99 + 63 C 114 106 + 64 C 115 105 + 64 B 116 106 + 99 + 73
[0314] In this fashion, choosing between appropriate asymmetry
sequences, assembly sequences, and DNA recognition units, many
different TBAs may be formed. Furthermore, sets of these, such as
SEQ ID NOS. 114 and 115,will associate with each other but dimers
of SEQ ID NO. 114 or 115 will not form due to charge repulsion in
the mutated assembly sequences (SEQ ID NO. 104 is cro; SEQ ID NO.
105 is a novel mutated, negatively charged cro, and SEQ ID NO. 106
is a novel mutated, positively charged cro).
[0315] Naturally, given the amino acid sequence of these TBAs, one
of ordinary skill could produce recombinant nucleic acid clones
encoding these, and such recombinant clones naturally form an
integral part of this invention.
EXAMPLE 10
HIV Test Using "HIV-LOCK"
[0316] In much the same method as used in Example 6, the "HIV-LOCK"
produced according to Example 9 is used as the TBA, reagent 2, with
similar results.
Example 11
HIV Test Using "HIV-LOCK" When Testing Blood for Donation
[0317] When the quantity of blood to be tested is not limiting, as
when samples of blood for donation are to be tested for HIV
contamination, tests similar to Example 6 are run, but for each of
tubes A-C, about 5 mL of blood is pelleted in a tabletop
centrifuge. Other reagents are scaled up as necessary to handle the
larger quantity of TNA present in the sample.
[0318] EXAMPLE 12
"HIV-LOCK" as an Anti-HIV Therapeutic Agent
[0319] "HIV-LOCK" produced according to Example 9 is formulated as
a 1 mg/mL solution in liposomes and injected intravenously into a
subject who has been tested and confirmed to be infected with HIV.
A dose of it 0.1 mg to 100 mg of "HIV-LOCK"/kilogram body mass is
infused over a twenty-four hour period and the concentration of HIV
p24 in the patient's serum monitored. The treatment is repeated as
often as necessary, such as when elevations in the serum p24
occur.
EXAMPLE 13
Use of an HIV-TBA Construct as a Therapeutic
[0320] A recombinant retroviral or like vector is used to deliver a
construct encoding an HIV-LTR binding TBA to an infected patient.
The vector encodes a chaperone, such as cro, and sequences DNA for
binding portions of p50. The same vector also encodes a chaperone
on which an SP1 TBA folds. Asymmetry sequences are provided such
that upon co-expression of the p50-TBA and the SP1-TBA in a single
HIV infected cell in vivo, an immediate association occurs between
these TBAs, while at the same time preventing any association
between the DNA binding portion of p50 and endogenous p50 or p65
monomers. NLS sequences are also provided in the TBAs so that, upon
dimer formation, the TBA immediately relocates to the nucleus of
the cell and binds specifically to integrated HIV sequences, thus
preventing any transcription from that locus.
[0321] For this purpose, it is desirable to select sequences
encoding DNA binding domains such that the expressed monomers are
assembled into a TBA which does not bind to natural human
sequences. Thus, it is only upon binding of the TBA components to
their target sequences that association between all components of
the TBA occurs to form a complex which tightly and specifically
binds the HIV LTR.
EXAMPLE 14
Diagnostic Test Kit for Human Papillomavirus
[0322] This diagnostic for human papillomavirus takes advantage of
the known differential between benign and carcinogenic HPV to
provide a test which indicates the susceptibility to malignancy in
a patient. The papillomaviruses a group of small DNA viruses
associated with benign squamous epithelial cell tumors in higher
vertebrates. At least 27 distinct human types of papillomaviruses
(HPVs) have been found; many of these have been associated with
specific clinical lesions. Four of these, HPV-6, HPV-11, HPV-16,
HPV-18, and HPV-33 have been associated with human genital tract
lesions. In general, PV-6 and HPV-11 DNAs have been found
associated with benign lesions of the genital tract. HPV-16,
HPV-18, and HPV-33 have also been found associated with
premalignant and malignant lesions and are transcribed in most cell
lines established from cervical carcinomas. HPV-16, HPV-18, and
HPV-33 are likely to be only two members of a large set of HPV DNAs
associated with malignant human cervical carcinomas.
[0323] Animal models have shown that benign papillomavirus lesions
can progress to malignant lesions in the presence of a
co-carcinogen. HPV DNA has been found in metastases of cervical
carcinomas. In malignant cervical lesions, HPV DNA is usually
integrated into the human genome, but there may also be
extrachromosomal HPV DNA present integration of HPV to form the
provirus usually results in the disruption of the viral E2 open
reading frame (ORF). Despite disruption of the E2 ORF, and
examination of cell lines from several cervical carcinomas has
shown transcriptionally active and integrated HPV-16 and HPV-18.
When HPV-16 genomes which are present in the human cervical
carcinoma cell lines SiHa and CaSki have been examined, there are
differences found in the integration of HPV-16. In the SiHa line,
the single HPV-16 genome integration occurred at bases 3132 and
3384, disrupting the E1 and E2 ORFs with a deletion of 0.3 kb. An
additional 50-basepair deletion of HPV-16 DNA resulted in the E2
and E4 OFRs being fused. The 5' portion of the HPV-16 DNA,
consisting of the disrupted E2 ORF, is ligated to continuous human
right flanking sequences. In addition, a single additional guanine
is detected at nucleotide 1138 in the middle of the E1 ORF. This
basepair addition results in the fusion of the E1a and E1b ORFs to
a single E1 ORF.
[0324] The complete genome of HPV-16 is available on GenBank as
accession number K02718; the complete genome of HPV-33 is available
on GenBank as accession number M12732; the complete genome of
HPV-18 is available on GenBank as accession number X05015.
[0325] As a preliminary screen, the fact of an HPV infection is
established for a given cervical biopsy sample by a simple "yes/no"
type of analysis using, for example, any or all of the PNAs SEQ ID
NOS. 46-53 and an E2 TBA as described above (i.e., fragment DNA,
binding the PNA, immobilize with the TBA, and detect signal with
BNAs and BBAs).
[0326] Once a biopsy sample is found to be positive for HPV,
additional information is obtained as to the malignancy potential
of the HPV by analyzing the integration status of die virus in the
human genome.
[0327] 1. Fragment the DNA in the cervical biopsy sample and
hybridize to a blocking probe having the sequence, SEQ ID NO. 60.
This probe will bind to all the fragments in the DNA which have not
spliced out the 0.3 kb fragment.
[0328] 2. Expose the DNA in the biopsy sample to a PNA having the
sequence, SEQ ID NO. 61. This probe will only bind to fragments
which have deleted the 0.3 kb fragment (the blocking probe will
prevent the looping out of the large deletion segments if
present).
[0329] 3. A PNA having SEQ ID NO. 62 is hybridized with SEQ ID NO.
41 to form a BBR which will bind to cro or .lambda. CI repressor as
a BBA, leaving a single-stranded portion capable of hybridizing
with the TATA site on SEQ ID NO. 61. This added to form a TBR on
the 5' end of the large deletion.
[0330] 4. The TBR is immobilized by a TBA having a TATA binding
protein DNA recognition unit.
[0331] 5. The bound fragments are detected by adding BNAs and BBAs
as described above.
[0332] Detection of signal in this assay indicates that the large
fragment is deleted in HPV present in the TNA. Since this deletion
is correlated with malignancy, this assay provides insight into the
malignancy potential of the HPV infection. This conclusion can be
confirmed by performing an analogous assay based on the deletion of
the 52-basepair fragment which is also correlated with HPV-induced
malignancy.
[0333] The TBP recognition unit used in the TBA for this assay may
be chosen, for example, from a sequence such as SEQ ID NO. 70 or
SEQ D NO. 93.
EXAMPLE 15
Recombinant HIV-LOCK.TM. Production
[0334] Phase One--Preparation of DNA to Produce the
HIV-Lock.TM..
[0335] In vitro mutagenesis of the coding regions of the naturally
occurring, cloned components of the HMV-Lock.TM. which need to be
modified is performed with a MutaGene Phagemid kit. The modified
protocol includes the use of a Blue-script plasmid containing each
of the binding components of MV-Lock.TM.. These are transformed
into competent cells and uracil-containing phagenids are grown.
Single stranded DNA is extracted and used as a template for the
mutagenic strand. Oligonucleotides containing the desired
mutations, including the incorporation of a novel restriction site,
are synthesized and treated with polynucleotide kinase and ATP. The
kinase treated oligonucleotides are annealed to the single-stranded
template, and a mutagenic strand is synthesized and ligated
according to the MutaGene protocol, with the exception that
Sequenase 2.0 provides the polymerase. Libraries are screened using
both g-.sup.32P end-labeled nucleotides containing sequences
complementary to the introduced mutations and by isolating the
plasmid DNA and identifying the mutants by the presence of the
introduced restriction site. The mutations are also confirmed by
sequencing with a Sequenase kit. The HIV-Lock.TM. DNA is cloned
into the baculovirus expression system with a polyhedron
promotor.
[0336] Phase Two--Production of HIV-Lock.TM. Proteins Using
Baculovirus.
[0337] Sf-9 cells are cultured to a pre-determined density (about
1.times.10.sup.6 cells/ml, log phase), infected with the
baculovirus containing the HIV-Lock.TM. instructions and harvested
to recover the recombinant proteins comprising the HIV-Lock.TM.. In
the scale-up process, cultures are expanded from flasks to spinners
and subsequently to bioreactors. Following infection the cells are
harvested at 12, 24, 36 and 48 hours for the protein. Indices of
viability are monitored throughout the entire process.
[0338] Phase Three--Purification of the HIV-Lock.TM. Proteins.
[0339] The harvested proteins are first separated from particulates
by flow-through ultracentrifugation to facilitate downstream
purification. The centrifuged product is then sterile filtered.
Extracts are then centrifuged at 40,000 rpm at 4.degree. C. for 30
minutes and aliquots are immunoprecipitated with polyclonal rabbit
antibody against one of the HIV-Lock.TM. components.
Immunoprecipitated proteins are run on an SDS-10% PAGE gel.
[0340] Phase Four--Test of HIV-Lock.TM.Proteins Against HIV DNA
[0341] Mobility shift assays are carried out using an
oligonucleotide probe comprising elements of the HIV long terminal
repeat and fragments containing NFKB binding DNA associated with
kappa light chain and microglobulin regulation. The oligonucleotide
is annealed to its complimentary strand and end-labeled with
g-.sup.32P ATP.
[0342] Footprinting is accomplished by combining small (10.sup.-15
M) of radiolabeled HIV LTR DNA with a slightly larger amount of
HIV-Lock.TM. in a buffer at room temperature for 10 minutes.
Dithiothreitol is added prior to the addition of protein Iron (II),
EDTA, hydrogen peroxide and sodium ascorbate are added and the
reaction mixture is incubated. A quenching agent is added and the
products are analyzed suing denaturing gel electrophoresis. This is
done for different concentrations of protein. The resulting gel is
imaged using a phosphoimager scanner and the resulting high
resolution image file is analyzed to abstract the binding affinity
of HIV-Lock.TM.for the HIV DNA relative to cellular DNA.
[0343] Multiple design and testing iterations may be used in order
to refind binding of HIV-Lock.TM. and other TBAs for HIV and other
organisms. This process makes it possible to design binding
assemblies such that the binding assembly is not competitive with
the wild type proteins for single binding sites in the genome
samples. The development of TBAs for other organisms and TNAs for
sequences within these organisms can be made using the
aforementioned method. This method is valid when producing binding
assemblies for all nucleic acid TBRs including DNA-DNA, DNA-RNA and
RNA-RNA hybrids and combinations of these hybrids.
EXAMPLE 16
Method for Identifying Nucleic Acid Binding Molecules for
Production of TBAs and BBAs of the Invention
[0344] In the method of this invention, target binding assemblies
and booster binding assemblies are assembled by identifying nucleic
acid binding molecules, and linking the nucleic acid binding
portions of the molecules in such a fashion as to achieve TBAs
which discriminate between particular target sequences and even
closely related sequences. One method for identifying the nucleic
acid binding molecules involves the following steps:
[0345] 1. Obtaining a biological sample containing the target
nucleic acid. This could be, for example, an organism or a tissue
extract infected with a pathogen.
[0346] 2. Fragmenting the sample so as to expose the nucleic acids
and to reduce the size complexity of the nucleic acids contained in
the sample.
[0347] 3. Contacting a first aliquot of the fragmented nucleic
acids with a control buffer medium and contacting a second aliquot
of the fragmented nucleic acids with the control buffer medium
containing a known profile of nucleic acid binding molecules.
[0348] 4. Analyzing the two aliquots to identity fragments which
have altered behavior in the aliquot contacted with the target
binding molecules as opposed to the control aliquot. This is
accomplished by single dimension gel electrophoresis, two dimension
gel electrophoresis, high performance liquid chromatography, paper
chromatography or any other means which reveals a different
behavior of the nucleic acid fragments when bound to a nucleic acid
binding molecule as opposed to when the nucleic acid fragment is
unbound.
[0349] 5. Identifying and isolating fragments which do exhibit
altered behavior when contacted with the nucleic acid binding
molecule and either sequencing the nucleic acid fragment to
determine whether known nucleic acid binding molecule motifs are
present, or directly identifying the nucleic acid binding molecule
bound to the nucleic acid. The latter can be achieved, for example,
by contacting a two dimensional grid of the electrophoresed nucleic
acids with differentially labeled antibodies which bind to the
various nucleic acid binding molecules.
[0350] In this method, preferably nucleic acid motifs are used for
either diagnostic or therapeutic purposes wherein the target
nucleic acid has more than a single utilizable nucleic acid binding
molecule target. In this way, a complex target binding assembly can
be generated which takes advantage of the proximity of different
nucleic acid binding molecular motifs to enhance the specificity of
the TBA assembled from the individual nucleic acid binding
components identified. The various nucleic acid binding portions of
the nucleic acid binding molecules are then assembled into the
complete TBAs as described above, for example, for
HIV-LOCK.TM..
EXAMPLE 17
Method of Identifying Specific RNA Sequences in a Sample
[0351] According to the methods and compositions taught in this
invention, any nucleic acid sequence can be specifically
identified. Identification of target HIV RNA in a sample is
achieved by obtaining a sample of a patient's blood or other
biological fluid or extract which may contain the HIV RNA, and
testing for the presence of TAR binding sites. Tat is a positive
regulator of HIV replication which binds to the TAR region of the
HIV RNA. The smallest naturally occurring, fully active form of
HIV-Tat is 72 amino acids in length, SEQ Id. 118 herein. Tat
contains at least two functional domains, and transactivates gene
expression from the HIV long terminal repeat (HIV LTR). Tat binds
to an RNA stem loop structure formed from the self-hybridization of
sequences in TAR, which is just 5' to the HIV LTR HIV TAR RNA forms
a dinucleotide bulge and two stem-loop structures (Rhim et al. 1994
Virology:202, 202-211). The Tat (SEQ. Id. 118) binds to this
structure with lower avidity than does Tat variants wherein Ala58
is a threonine or where His65 is an Asp residue. (Derse et al.,
1993 Virology: 194,530-536). Utilizing these facts in the instant
method is accomplished by:
[0352] 1. Fragmenting a biological sample to the nucleic acids and
reduce the size complexity of the nucleic acids.
[0353] 2 Contacting a TBA with the sample which identifies a hybrid
TAR binding protein sequence and a proximate flanking sequence in
the HIV genome. The TBA used for this purpose is assembled on cro
as the chaperone using Tat as the HIV RNA specific binding
molecule. To provide specificity such that cross-talk between the
HIV TAR site and closely related TAR sites which may be present due
to such other pathogens as cytomegalovirus, the TBA also has an
antibody component which recognizes the DNA-RNA hybrid target
binding region formed when a probe nucleic acid binds to the HIV
LTR RNA.
[0354] 3. Eliminating any "cross-talk" produced by binding of Tat
to the TAR region of the HIV RNA due to such contaminants (cousin
RNAs) as the CMV TAR sequence by contacting the reaction with
excess Tat variant (either the Ala58 to Thr or the His65 to Asp
variants) which bind more avidly. In this way, single binding
events due to the TBA binding to a cousin RNAs are competed from
the nucleic acid sample by the Tat variant. On the other hand, by
appropriately selecting the affinity of the double binding achieved
as a result of the antibody and Tat, the TBA is not displaced from
true targets. This process is illustrated in FIG. 16. In another
aspect of this same method, the TBA could be one in which, rather
than using a variant of Tat, an antibody is used which recognizes
this nucleic acid segment, and the TBA used is a double antibody
TBA.
[0355] In an alternate version of this method, a probe nucleic acid
may be used which hybridizes with the HIV LTR RNA. Accordingly, a
duplex segment of the LTR sp1 sites can be created as part of the
target binding region. This region of the HIV RNA flanks the TAR
region which is 5' to the LTR but is in close proximity thereto. A
TBA containing Tat and two Sp1 binding units is chaperoned to
provide Tat binding to TAR and Sp1 binding to the Sp1 binding
sites. Amplification and detection is then carried out by adding
appropriate BNAs, BBAs and HNAs. In yet another alternative, PNAs
having Seq. ID. 38 and Seq. ID. 39 (see FIG. 7) could be used. A
TBA is used which contains one or more Sp1 binding units and an
antibody unit which binds to the DNA-RNA hybrid produced from
sample RNA and the Seq. Id. 38 PNA. Appropriate BNAs, BBAs and HNAs
are then added to amplify the signal.
[0356] Naturally, those skilled in the art will recognize that
other TBA and TNA combinations could be used to optimize the
methods exemplified herein.
[0357] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application and the scope of the
appended claims. It will be understood that sequences provided
herein are exemplary only and that other like sequences suggested
by these could be used in the methods of this invention. It will
also be understood that although any sequence provided herein might
be designated as linear, it could be used in a circularly or
otherwise permuted form and although designated as not being
anti-sense, it could be used in the coding or non-coding form or to
bind to coding or non-coding complementary sequences.
6 # SEQUENCE LIS #TING (1) GENERAL INFORMATION: (iii) NUMBER OF
SEQUENCES: 118 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 13 base #pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:1: TGGGGATTCC CCA # # # 13 (2) INFORMATION
FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 base
#pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: AAGGGACTTT
CCC # # # 13 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 13 base #pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:3: AGGGGACTTT CCG # # # 13 (2) INFORMATION
FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base
#pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GCTGGGGACT
TTCCA # # # 15 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 15 base #pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:5: ACAAGGGACT TTCCG # # # 15 (2) INFORMATION
FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 base
#pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CCGGGTTTTC
CCC # # # 13 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 27 base #pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:7: AAGGGACTTT CCGCTGGGGA CTTTCCA # # 27 (2)
INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 27 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:8: AAGGGACTTT CCGCTGGGGA CTTTCCG # # 27 (2) INFORMATION FOR
SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base
#pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GCTGGGGACT
TTCCAGGGAG GCGTGG # # 26 (2) INFORMATION FOR SEQ ID NO:10: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:10: GCTGGGGACT TTCCAGGGGA GGTGTG #
# 26 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 26 base #pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:11: GCTGGGGACT TTCCGGGGAG CGTGGC # # 26 (2)
INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 26 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:12: GCTGGGGACT TTCCGGGGAG GCGCGG # # 26 (2) INFORMATION FOR
SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base
#pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GCTGGGGACT
TTCCAGAGAG GCGTGG # # 26 (2) INFORMATION FOR SEQ ID NO:14: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:14: GCTGGGGACT TTCCAGGGGA GGCGTG #
# 26 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 26 base #pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:15: GCTGGGGACT TTCCAGGGAG GCGTGG # # 26 (2)
INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 26 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:16: GCTGGGGACT TTCCAGGGAG GCTGCC # # 26 (2) INFORMATION FOR
SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base
#pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: TTTCCAGGGA
GGCGTGGCCT GGGCGGGACT GGG # # 33 (2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:18: CGTGGCCTGG GCGGGACTGG
GGAGTGGCGT CCC # # 33 (2) INFORMATION FOR SEQ ID NO:19: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:19: CTACAAGGGA CTTTCCGCTG
GGGACTTTCC AGGGAGGCGT GGCCT # #45 (2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:20: CAGCAAGGGA CTTTCCGCTG
GGGACTTTCC AGGGGAGGTG TGGCCT # 46 (2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:21: CATCAAGGGA CTTTCCGCTG
GGGACTTTCC AGGGGAGGTG TGGCCT # 46 (2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:22: CAACAAGGGA CTTTCCGCTG
GGGACTTTCC AGGGGAGGTG TGGCCT # 46 (2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:23: CTACAAGGGA CTTTCCGCTG
GGGACTTTCC AGGGAGGCGT GGCAT # #45 (2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:24: CTACAAGGGA CTTTCCGCTG
GGGACTTTCC GGGGAGCGTG GCCT # # 44 (2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:25: CTACAAGGGA CTTTCCGCTG
GGGACTTTCC GGGGAGGCGC GGCT # # 44 (2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:26: CTACAAGGGA CTTTCCGCTG
GGGACTTTCC AGAGAGGCGT GGACT # #45 (2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:27: CTACAAGGGA CTTTCCGCTG
GGGACTTTCC AGGGGAGGCG TGGACT # 46 (2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 46 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:28: CTACAGGGGA CTTTCCGCTG
GGGACTTTCC AGGGAGGCGT GGGGAG # 46 (2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 43 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:29: CTACAGGGGA CTTTCCGCTG
GGGACTTTCC AGGGAGGCTG CCT # # 43 (2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 48 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:30: CTTTCCGCTG GGGACTTTCC
AGGGAGGCGT GGCCTGGGCG GGACTGGG # 48 (2) INFORMATION FOR SEQ ID
NO:31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base #pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:31: TTTCCAGGGA
GGCGTGGCCT GGGCGGGACT GGGGAGTGGC GTCCC # #45 (2) INFORMATION FOR
SEQ ID NO:32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 59 base
#pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: CTACAAGGGA
CTTTCCGCTG GGGACTTTCC AGGGAGGCGT GGCCTGGGCG GG #ACTGGGG 59 (2)
INFORMATION FOR SEQ ID NO:33: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 59 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:33: TTTCCGCTGG GGACTTTCCA GGGAGGCGTG GCCTGGGCGG GACTGGGGAG TG
#GCGTCCC 59 (2) INFORMATION FOR SEQ ID NO:34: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 70 base #pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:34: CTACAAGGGA CTTTCCGCTG GGGACTTTCC
AGGGAGGCGT GGCCTGGGCG GG #ACTGGGGA 60 GTGGCGTCCC # # # 70 (2)
INFORMATION FOR SEQ ID NO:35: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 61 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:35: TATCACCGCC AGTGGTATTT ATGTCAACAC CGCCAGAGAT AATTTATCAC CG
#CAGATGGT 60 T # # # 61 (2) INFORMATION FOR SEQ ID NO:36: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 64 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:36: TATCACCGCA AGGGATAAAT
ATCTAACACC GTGCGTGTTG ACTATTTTAC CT #CTGGCGGT 60 GATA # # # 64 (2)
INFORMATION FOR SEQ ID NO:37: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 70 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:37: CTACAAGGGA CTTTCCGCTG GGGACTTTCC AGGGAGGCGT GGCCTGGGCG GG
#ACTGGGGA 60 GTGGCGTCCC # # # 70 (2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:38: CTACAAGGGA CTTTCCGCTG
GGGACTTTCC AGGGAGG # # 37 (2) INFORMATION FOR SEQ ID NO:39: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:39: CGGGACTGGG GAGTGGCGTC CC # # 22
(2) INFORMATION FOR SEQ ID NO:40: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 103 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:40: CTACAAGGGA CTTTCCGCTG GGGACTTTCC AGGGAGGTAT CACCGCCAGT GG
#TATTTATG 60 TCAACACCGC CAGAGATAAT TTATCACCGC AGATGGTTCT GCA # #103
(2) INFORMATION FOR SEQ ID NO:41: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 62 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:41: GAACCATCTG CGGTGATAAA TTATCTCTGG CGGTGTTGAC ATAAATACCA CT
#GGCGGTGA 60 TA # # # 62 (2) INFORMATION FOR SEQ ID NO:42: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 71 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:42: GATCCAACCA TCTGCGGTGA
TAAATTATCT CTGGCGGTGT TGACATAAAT AC #CACTGGCG 60 GTGATACTGC A # # #
71 (2) INFORMATION FOR SEQ ID NO:43: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 63 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:43: GTATCACCGC CAGTGGTATT TATGTCAACA CCGCCAGAGA TAATTTATCA CC
#GCAGATGG 60 TTG # # # 63 (2) INFORMATION FOR SEQ ID NO:44: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:44: GATCCGGGGG GATACCCCCC G # # #21
(2) INFORMATION FOR SEQ ID NO:45: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 91 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:45: CGGGACTGGG GAGTGGCGTC CCTATCACCG CAAGGGATAA ATATCTAACA CC
#GTGCGTGT 60 TGACTATTTT ACCTCTGGCG GTGATAGCAT G # # 91 (2)
INFORMATION FOR SEQ ID NO:46: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 53 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:46: CTAAGGGCGT AACCGAAATC GGTTGAACCG AAACCGGTTA GTATAAAAGC AG
#A 53 (2) INFORMATION FOR SEQ ID NO:47: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 54 base #pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:47: AAAAGGGAGT AACCGAAAAC GGTCGGGACC
GAAAACGGTG TATATAAAAG AT #GT 54 (2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 54 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:48: AGTAGGGTGT AACCGAAAGC
GGTTCAACCG AAAACGGTGC ATATATAAAG CA #AA 54 (2) INFORMATION FOR SEQ
ID NO:49: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base #pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: GCTTCAACCG AATTCGGTTG CATG
# # 24 (2) INFORMATION FOR SEQ ID NO:50: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 24 base #pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:50: TGTGCAACCG ATTTCGGTTG CCTT # # 24 (2)
INFORMATION FOR SEQ ID NO:51: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 24 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:51: TATGCAACCG AAATAGGTTG GGCA # # 24 (2) INFORMATION FOR SEQ
ID NO:52: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base #pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: TGCCTAACCG TTTTCGGTTA CTTG
# # 24 (2) INFORMATION FOR SEQ ID NO:53: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 24 base #pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:53: GGACTAACCG TTTTAGGTCA TATT # # 24 (2)
INFORMATION FOR SEQ ID NO:54: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 52 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:54: GACGACTATC CAGCGACCAA GATCAGAGCC AGACACCGGA AACCCCTGCC AC
# 52 (2) INFORMATION FOR SEQ ID NO:55: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 53 base #pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:55: GACGACACGG TATCCGCTAC TCAGCTTGTT
AAACAGCTAC AGCACACCCC CT #C 53 (2) INFORMATION FOR SEQ ID NO:56:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 60 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:56: GACGACGACC TGCAGACACC
ACAGACACCG CCCAGCCCCT TACAAAGCTG TT #CTGTGCAG 60 (2) INFORMATION
FOR SEQ ID NO:57: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 68 base
#pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: CATACCAAAG
CCGTCGCCTT GGGCACCGAA GAAACACAAC CACTAAGTTG TT #GCACAGAG 60
ACTCAGTG # # # 68 (2) INFORMATION FOR SEQ ID NO:58: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 77 base #pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:58: TAATGTAATT GATTGTAATG ACTCTATGTG
CAGTACCAGT ACCGTATTCC AG #CACCGTGT 60 CCGTGGGCAC CGCAAAG # # # 77
(2) INFORMATION FOR SEQ ID NO:59: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 80 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
both (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ
ID NO:59: ACAGACAACG ATAACCGACC ACCACAAGCA GCGGCCAAAC ACCCCGCCTT GG
#ACAATAGA 60 ACAGCACGTA CTGCAACTAA # # # 80 (2) INFORMATION FOR SEQ
ID NO:60: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 266 base #pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:60: CATATGCAAT ACAATGCATT ATACAAACTG
GACACATATA TATATTTGTG AA #GAAGCATC 60 AGTAACTGTG GTAGAGGGTC
AAGTTGACTA TTATGGTTTA TATTATGTTC AT #GAAGGAAT 120 ACGAACATAT
TTTGTGCAGT TTAAAGATGA TGCAGAAAAA TATAGTAAAA AT #AAAGTATG 180
GGAAGTTCAT GCGGGTGGTC AGGTAATATT ATGTCCTACA TCTGTGTTTA GC #AGCAACGA
240 AGTATCCTCT CCTGAAATTA TTAGGC # # 266 (2) INFORMATION FOR SEQ ID
NO:61: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 95 base #pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:61: AGGATGTATA AAAAAACATG
GATATACAGT GGAAGTGCAG TTTGATGGAG AC #ATATGCTA 60 TTAGGCAGCA
CTTGGCCAAC CACCCCGCCG CGACC # # 95 (2) INFORMATION FOR SEQ ID
NO:62: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 81 base #pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:62: CATGTTTTTT TATACATCCA
TATCACCGCC AGTGGTATTT ATGTCAACAC CG #CCAGAGAT 60 AATTTATCAC
CGCAGATGGT T # # #81 (2) INFORMATION FOR SEQ ID NO:63: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 322 amino #acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: Met Ala Asp Asp Asp Pro
Tyr Gly Thr Gly Gl #n Met Phe His Leu Asn 1 5 # 10 # 15 Thr Ala Leu
Thr His Ser Ile Phe Asn Ala Gl #u Leu Tyr Ser Pro Glu 20 # 25 # 30
Ile Pro Leu Ser Thr Asp Gly Pro Tyr Leu Gl #n Ile Leu Glu Gln Pro
35 # 40 # 45 Lys Gln Arg Gly Phe Arg Phe Arg Tyr Val Cy #s Glu Gly
Pro Ser His 50 # 55 # 60 Gly Gly Leu Pro Gly Ala Ser Ser Glu Lys As
#n Lys Lys Ser Tyr Pro 65 #70 #75 #80 Gln Val Lys Ile Cys Asn Tyr
Val Gly Pro Al #a Lys Val Ile Val Gln 85 # 90 # 95 Leu Val Thr Asn
Gly Lys Asn Ile His Leu Hi #s Ala His Ser Leu Val 100 # 105 # 110
Gly Lys His Cys Glu Asp Gly Val Cys Thr Va #l Thr Ala Gly Pro Lys
115 # 120 # 125 Asp Met Val Val Gly Phe Ala Asn Leu Gly Il #e Leu
His Val Thr Lys 130 # 135 # 140 Lys Lys Val Phe Glu Thr Leu Glu Ala
Arg Me #t Thr Glu Ala Cys Ile 145 1 #50 1 #55 1 #60 Arg Gly Tyr Asn
Pro Gly Leu Leu Val His Se #r Asp Leu Ala Tyr Leu 165 # 170 # 175
Gln Ala Glu Gly Gly Gly Asp Arg Gln Leu Th #r Asp Arg Glu Lys Glu
180 # 185 # 190 Ile Ile Arg Gln Ala Ala Val Gln Gln Thr Ly #s Glu
Met Asp Leu Ser 195 # 200 # 205 Val Val Arg Leu Met Phe Thr Ala Phe
Leu Pr #o Asp Ser Thr Gly Ser 210 # 215 # 220 Phe Thr Arg Arg Leu
Glu Pro Val Val Ser As #p Ala Ile Tyr Asp Ser 225 2 #30 2 #35 2 #40
Lys Ala Pro Asn Ala Ser Asn Leu Lys Ile Va #l Arg Met Asp Arg Thr
245 # 250 # 255 Ala Gly Cys Val Thr Gly Gly Glu Glu Ile Ty #r Leu
Leu Cys Asp Lys 260 # 265 # 270 Val Gln Lys Asp Asp Ile Gln Ile Arg
Phe Ty #r Glu Glu Glu Glu Asn 275 # 280 # 285 Gly Gly Val Trp Glu
Gly Phe Gly Asp Phe Se #r Pro Thr Asp Val His 290 # 295 # 300 Arg
Gln Phe Ala Ile Val Phe Lys Thr Pro Ly #s Tyr Lys Asp Val Asn 305 3
#10 3 #15 3 #20 Ile Thr (2) INFORMATION FOR SEQ ID NO:64: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 325 amino #acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: Met Ala Glu Asp Asp Pro
Tyr Leu Gly Arg Pr #o Glu Gln Met Phe His 1 5 # 10 # 15 Leu Asp Pro
Ser Leu Thr His Thr Ile Phe As #n Pro Glu Val Phe Gln 20 # 25 # 30
Pro Gln Met Ala Leu Pro Thr Ala Asp Gly Pr #o Tyr Leu Gln Ile Leu
35 # 40 # 45 Glu Gln Pro Lys Gln Arg Gly Phe Arg Phe Ar #g Tyr Val
Cys Glu Gly 50 # 55 # 60 Pro Ser His Gly Gly Leu Pro Gly Ala Ser Se
#r Glu Lys Asn Lys Lys 65 #70 #75 #80 Ser Tyr Pro Gln Val Lys Ile
Cys Asn Tyr Va #l Gly Pro Ala Lys Val 85 # 90 # 95 Ile Val Gln Leu
Val Thr Asn Gly Lys Asn Il #e His Leu His Ala His 100 # 105 # 110
Ser Leu Val Gly Lys His Cys Glu Asp Gly Il #e Cys Thr Val Thr Ala
115 # 120 # 125 Gly Pro Glu Asp Cys Val His Gly Phe Ala As #n Leu
Gly Ile Leu His 130 # 135 # 140 Val Thr Lys Lys Lys Val Phe Glu Thr
Leu Gl #u Ala Arg Met Thr Glu 145 1 #50 1 #55 1 #60 Ala Cys Ile Arg
Gly Tyr Asn Pro Gly Leu Le #u Val His Pro Asp Leu 165 # 170 # 175
Ala Tyr Leu Gln Ala Glu Gly Gly Gly Asp Ar #g Gln Leu Gly Asp Arg
180 # 185 # 190 Glu Lys Glu Leu Ile Arg Gln Ala Ala Leu Gl #n Gln
Thr Lys Glu Met 195 # 200 # 205 Asp Leu Ser Val Val Arg Leu Met Phe
Thr Al #a Phe Leu Pro Asp Ser 210 # 215 # 220 Thr Gly Ser Phe Thr
Arg Arg Leu Glu Pro Va #l Val Ser Asp Ala Ile 225 2 #30 2 #35 2 #40
Tyr Asp Ser Lys Ala Pro Asn Ala Ser Asn Le #u Lys Ile Val Arg Met
245 # 250 # 255 Asp Arg Thr Ala Gly Cys Val Thr Gly Gly Gl #u Glu
Ile Tyr Leu Leu 260 # 265 # 270 Cys Asp Lys Val Gln Lys Asp Asp Ile
Gln Il #e Arg Phe Tyr Glu Glu 275 # 280 # 285 Glu Glu Asn Gly Gly
Val Trp Glu Gly Phe Gl #y Asp Phe Ser Pro Thr 290 # 295 # 300 Asp
Val His Arg Gln Phe Ala Ile Val Phe Ly #s Thr Pro Lys Tyr Lys 305 3
#10 3 #15 3 #20 Asp Ile Asn Ile Thr 325 (2) INFORMATION FOR SEQ ID
NO:65: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 268 amino #acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT
TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65: Met Glu Pro
Ala Asp Leu Leu Pro Leu Tyr Le #u Gln Pro Glu Trp Gly 1 5 # 10 # 15
Glu Gln Glu Pro Gly Gly Ala Thr Pro Phe Va #l Glu Ile Leu Glu Gln
20 # 25 # 30 Pro Lys Gln Arg Gly Met Arg Phe Arg Tyr Ly #s Cys Glu
Gly Arg Ser 35 # 40 # 45 Ala Gly Ser Ile Pro Gly Glu His Ser Thr As
#p Ser Ala Arg Thr His 50 # 55 # 60 Pro Thr Ile Arg Val Asn His Tyr
Arg Gly Pr #o Gly Arg Val Arg Val 65 #70 #75 #80 Ser Leu Val Thr
Lys Asp Pro Pro His Gly Pr #o His Pro His Glu Leu 85 # 90 # 95 Val
Gly Arg His Cys Gln His Gly Tyr Tyr Gl #u Ala Glu Leu Ser Pro 100 #
105 # 110 Asp Arg Ser Ile His Ser Phe Gln Asn Leu Gl #y Ile Gln Cys
Val Lys 115 # 120 # 125 Lys Arg Glu Leu Glu Ala Ala Val Ala Glu Ar
#g Ile Arg Thr Asn Asn 130 # 135 # 140 Asn Pro Phe Asn Val Pro Met
Glu Glu Arg Gl #y Ala Glu Tyr Asp Leu 145 1 #50 1 #55 1 #60 Ser Ala
Val Arg Leu Cys Phe Gln Val Trp Va #l Asn Gly Pro Gly Gly 165 # 170
# 175 Leu Cys Pro Leu Pro Pro Val Leu Ser Gln Pr #o Ile Tyr Asp Asn
Arg 180 # 185 # 190 Ala Pro Ser Thr Ala Glu Leu Arg Ile Leu Pr #o
Gly Asp Arg Asn Ser 195 # 200 # 205 Gly Ser Cys Gln Gly Gly Asp Glu
Ile Phe Le #u Leu Cys Asp Lys Val 210 # 215 # 220 Gln Lys Glu Asp
Ile Glu Val Arg Phe Trp Al #a Glu Gly Trp Glu Ala 225 2 #30 2 #35 2
#40 Lys Gly Ser Phe Ala Ala Ala Asp Val His Ar #g Gln Val Ala Ile
Val 245 # 250 # 255 Phe Arg Thr Pro Pro Phe Arg Glu Arg Ser Le #u
Arg 260 # 265 (2) INFORMATION FOR SEQ ID NO:66: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 263 amino #acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: Met Asp Asp Leu Phe Pro
Leu Ile Phe Pro Se #r Glu Pro Ala Gln Ala 1 5 # 10 # 15 Ser Gly Pro
Tyr Val Glu Ile Ile Glu Gln Pr #o Lys Gln Arg Gly Met 20 # 25 # 30
Arg Phe Arg Tyr Lys Cys Glu Gly Arg Ser Al #a Gly Ser Ile Pro Gly
35 # 40 # 45 Glu Arg Ser Thr Asp Thr Thr Lys Thr His Pr #o Thr Ile
Lys Ile Asn 50 # 55 # 60 Gly Tyr Thr Gly Pro Gly Thr Val Arg Ile Se
#r Leu Val Thr Lys Asp 65 #70 #75 #80 Pro Pro His Arg Pro His Pro
His Glu Leu Va #l Gly Lys Asp Cys Arg 85 # 90 # 95 Asp Gly Tyr Tyr
Glu Ala Asp Leu Cys Pro As #p Arg Ser Ile His Ser 100 # 105 # 110
Phe Gln Asn Leu Gly Ile Gln Cys Val Lys Ly #s Arg Asp Leu Glu Gln
115 # 120 # 125 Ala Ile Ser Gln Arg Ile Gln Thr Asn Asn As #n Pro
Phe His Val Pro 130 # 135 # 140 Ile Glu Glu Gln Arg Gly Asp Tyr Asp
Leu As #n Ala Val Arg Leu Cys 145 1 #50 1 #55 1 #60 Phe Gln Val Thr
Val Arg Asp Pro Ala Gly Ar #g Pro Leu Leu Leu Thr 165 # 170 # 175
Pro Val Leu Ser His Pro Ile Phe Asp Asn Ar #g Ala Pro Asn Thr Ala
180 # 185 # 190 Glu Leu Lys Ile Cys Arg Val Asn Arg Asn Se #r Gly
Ser Cys Leu Gly 195 # 200 # 205 Gly Asp Glu Ile Phe Leu Leu Cys Asp
Lys Va #l Gln Lys Glu Asp Ile 210 # 215 # 220 Glu Val Tyr Phe Thr
Gly Pro Gly Trp Glu Al #a Arg Gly Ser Phe Ser 225 2 #30 2 #35 2 #40
Gln Ala Asp Val His Arg Gln Val Ala Ile Va #l Phe Arg Thr Pro Pro
245 # 250 # 255 Tyr Ala Asp Pro Ser Leu Gln 260 (2) INFORMATION FOR
SEQ ID NO:67: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 263 amino
#acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE
TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v)
FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:
Met Asp Glu Leu Phe Pro Leu Ile Phe Pro Al #a Glu Pro Ala Gln Ala 1
5 # 10 # 15 Ser Gly Pro Tyr Val Glu Ile Ile Glu Gln Pr #o Lys Gln
Arg Gly Met 20 # 25 # 30 Arg Phe Arg Tyr Lys Cys Glu Gly Arg Ser Al
#a Gly Ser Ile Pro Gly 35 # 40 # 45 Glu Arg Ser Thr Asp Thr Thr Lys
Thr His Pr #o Thr Ile Lys Ile Asn 50 # 55 # 60 Gly Tyr Thr Gly Pro
Gly Thr Val Arg Ile Se #r Leu Val Thr Lys Asp 65 #70 #75 #80 Pro
Pro His Arg Pro His Pro His Glu
Leu Va #l Gly Lys Asp Cys Arg 85 # 90 # 95 Asp Gly Phe Tyr Glu Ala
Glu Leu Cys Pro As #p Arg Cys Ile His Ser 100 # 105 # 110 Phe Gln
Asn Leu Gly Ile Gln Cys Val Lys Ly #s Arg Asp Leu Glu Gln 115 # 120
# 125 Ala Ile Ser Gln Arg Ile Gln Thr Asn Asn As #n Pro Phe Gln Val
Pro 130 # 135 # 140 Ile Glu Glu Gln Arg Gly Asp Tyr Asp Leu As #n
Ala Val Arg Leu Cys 145 1 #50 1 #55 1 #60 Phe Gln Val Thr Val Arg
Asp Pro Ser Gly Ar #g Pro Leu Arg Leu Pro 165 # 170 # 175 Pro Val
Leu Pro His Pro Ile Phe Asp Asn Ar #g Ala Pro Asn Thr Ala 180 # 185
# 190 Glu Leu Lys Ile Cys Arg Val Asn Arg Asn Se #r Gly Ser Cys Leu
Gly 195 # 200 # 205 Gly Asp Glu Ile Phe Leu Leu Cys Asp Lys Va #l
Gln Lys Glu Asp Ile 210 # 215 # 220 Glu Val Tyr Phe Thr Gly Pro Gly
Trp Glu Al #a Arg Gly Ser Phe Ser 225 2 #30 2 #35 2 #40 Gln Ala Asp
Val His Arg Gln Val Ala Ile Va #l Phe Arg Thr Pro Pro 245 # 250 #
255 Tyr Ala Asp Pro Ser Leu Gln 260 (2) INFORMATION FOR SEQ ID
NO:68: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 299 amino #acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT
TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: Met Phe Pro
Asn Gln Asn Asn Gly Ala Ala Pr #o Gly Gln Gly Pro Ala 1 5 # 10 # 15
Val Asp Gly Gln Gln Ser Leu Asn Tyr Asn Gl #y Leu Pro Ala Gln Gln
20 # 25 # 30 Gln Gln Gln Leu Ala Gln Ser Thr Lys Asn Va #l Arg Lys
Lys Pro Tyr 35 # 40 # 45 Val Lys Ile Thr Glu Gln Pro Ala Gly Lys Al
#a Leu Arg Phe Arg Tyr 50 # 55 # 60 Glu Cys Glu Gly Arg Ser Ala Gly
Ser Ile Pr #o Gly Val Asn Ser Thr 65 #70 #75 #80 Pro Glu Asn Lys
Thr Tyr Pro Thr Ile Glu Il #e Val Gly Tyr Lys Gly 85 # 90 # 95 Arg
Ala Val Val Val Val Ser Cys Val Thr Ly #s Asp Thr Pro Tyr Arg 100 #
105 # 110 Pro His Pro His Asn Leu Val Gly Lys Glu Gl #y Cys Lys Lys
Gly Val 115 # 120 # 125 Cys Thr Leu Glu Ile Asn Ser Glu Thr Met Ar
#g Ala Val Phe Ser Asn 130 # 135 # 140 Leu Gly Ile Gln Cys Val Lys
Lys Lys Asp Il #e Glu Ala Ala Leu Lys 145 1 #50 1 #55 1 #60 Ala Arg
Glu Glu Ile Arg Val Asp Pro Phe Ly #s Thr Gly Phe Ser His 165 # 170
# 175 Arg Phe Gln Pro Ser Ser Ile Asp Leu Asn Se #r Val Arg Leu Cys
Phe 180 # 185 # 190 Gln Val Phe Met Glu Ser Glu Gln Lys Gly Ar #g
Phe Thr Ser Pro Leu 195 # 200 # 205 Pro Pro Val Val Ser Glu Pro Ile
Phe Asp Ly #s Lys Ala Met Ser Asp 210 # 215 # 220 Leu Val Ile Cys
Arg Leu Cys Ser Cys Ser Al #a Thr Val Phe Gly Asn 225 2 #30 2 #35 2
#40 Thr Gln Ile Ile Leu Leu Cys Glu Lys Val Al #a Lys Glu Asp Ile
Ser 245 # 250 # 255 Val Arg Phe Phe Glu Glu Lys Asn Gly Gln Se #r
Val Trp Glu Ala Phe 260 # 265 # 270 Gly Asp Phe Gln His Thr Asp Val
His Lys Gl #n Thr Ala Ile Thr Phe 275 # 280 # 285 Lys Thr Pro Arg
Tyr His Thr Leu Asp Ile Th #r 290 # 295 (2) INFORMATION FOR SEQ ID
NO:69: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 261 amino #acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT
TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: Met Asp Phe
Leu Thr Asn Leu Arg Phe Thr Gl #u Gly Ile Ser Glu Pro 1 5 # 10 # 15
Tyr Ile Glu Ile Phe Glu Gln Pro Arg Gln Ar #g Gly Thr Arg Phe Arg
20 # 25 # 30 Tyr Lys Cys Glu Gly Arg Ser Ala Gly Ser Il #e Pro Gly
Glu His Ser 35 # 40 # 45 Thr Asp Asn Asn Lys Thr Phe Pro Ser Ile Gl
#n Ile Leu Asn Tyr Phe 50 # 55 # 60 Gly Lys Val Lys Ile Arg Thr Thr
Leu Val Th #r Lys Asn Glu Pro Tyr 65 #70 #75 #80 Lys Pro His Pro
His Asp Leu Val Gly Lys Gl #y Cys Arg Asp Gly Tyr 85 # 90 # 95 Tyr
Glu Ala Glu Phe Gly Pro Glu Arg Gln Va #l Leu Ser Phe Gln Asn 100 #
105 # 110 Leu Gly Ile Gln Cys Val Lys Lys Lys Asp Le #u Lys Glu Ser
Ile Ser 115 # 120 # 125 Leu Arg Ile Ser Lys Lys Asn Pro Phe Asn Va
#l Pro Glu Glu Gln Leu 130 # 135 # 140 His Asn Ile Asp Glu Tyr Asp
Leu Asn Val Va #l Arg Leu Cys Phe Gln 145 1 #50 1 #55 1 #60 Ala Phe
Leu Pro Asp Glu His Gly Asn Tyr Th #r Leu Ala Leu Pro Pro 165 # 170
# 175 Leu Ile Ser Asn Pro Ile Tyr Asp Asn Arg Al #a Pro Asn Thr Ala
Glu 180 # 185 # 190 Leu Arg Ile Cys Arg Val Asn Lys Asn Cys Gl #y
Ser Val Lys Gly Gly 195 # 200 # 205 Asp Glu Ile Phe Leu Leu Cys Asp
Lys Val Gl #n Lys Asp Asp Ile Glu 210 # 215 # 220 Val Arg Phe Val
Leu Gly Asn Trp Glu Ala Ly #s Gly Ser Phe Ser Gln 225 2 #30 2 #35 2
#40 Ala Asp Val His Arg Gln Val Ala Ile Val Ph #e Arg Thr Pro Pro
Phe 245 # 250 # 255 Leu Gly Asp Ile Thr 260 (2) INFORMATION FOR SEQ
ID NO:70: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 262 amino
#acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE
TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v)
FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:
Met Asp Phe Leu Thr Asn Leu Arg Phe Thr Gl #u Gly Ile Ser Glu Pro 1
5 # 10 # 15 Tyr Ile Glu Ile Phe Glu Gln Pro Arg Gln Ar #g Gly Met
Arg Phe Arg 20 # 25 # 30 Tyr Lys Cys Glu Gly Arg Ser Ala Gly Ser Il
#e Pro Gly Glu His Ser 35 # 40 # 45 Thr Asp Asn Asn Lys Thr Phe Pro
Ser Ile Gl #n Ile Leu Asn Tyr Phe 50 # 55 # 60 Gly Lys Val Lys Ile
Arg Thr Thr Leu Val Th #r Lys Asn Glu Pro Tyr 65 #70 #75 #80 Lys
Pro His Pro His Asp Leu Val Gly Lys Gl #y Cys Arg Asp Gly Tyr 85 #
90 # 95 Tyr Glu Ala Glu Phe Gly Pro Glu Arg Gln Va #l Leu Ser Phe
Gln Asn 100 # 105 # 110 Leu Gly Ile Gln Cys Val Lys Lys Lys Asp Le
#u Lys Glu Ser Ile Ser 115 # 120 # 125 Leu Arg Ile Ser Lys Lys Ile
Asn Pro Phe As #n Val Pro Glu Glu Gln 130 # 135 # 140 Leu His Asn
Ile Asp Glu Tyr Asp Leu Asn Va #l Val Arg Leu Cys Phe 145 1 #50 1
#55 1 #60 Gln Ala Phe Leu Pro Asp Glu His Gly Asn Ty #r Thr Leu Ala
Leu Pro 165 # 170 # 175 Pro Leu Ile Ser Asn Pro Ile Tyr Asp Asn Ar
#g Ala Pro Asn Thr Ala 180 # 185 # 190 Glu Leu Arg Ile Cys Arg Val
Asn Lys Asn Cy #s Gly Ser Val Lys Gly 195 # 200 # 205 Gly Asp Glu
Ile Phe Leu Leu Cys Asp Lys Va #l Gln Lys Asp Asp Ile 210 # 215 #
220 Glu Val Arg Phe Val Leu Gly Asn Trp Glu Al #a Lys Gly Ser Phe
Ser 225 2 #30 2 #35 2 #40 Gln Ala Asp Val His Arg Gln Val Ala Ile
Va #l Phe Arg Thr Pro Pro 245 # 250 # 255 Phe Leu Gly Asp Ile Thr
260 (2) INFORMATION FOR SEQ ID NO:71: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 314 amino #acids (B) TYPE: amino acid (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:71: Met Ser Asn Lys Lys Gln Ser Asn Arg Leu
Th #r Glu Gln His Lys Leu 1 5 # 10 # 15 Ser Gln Gly Val Ile Gly Ile
Phe Gly Asp Ty #r Ala Lys Ala His Asp 20 # 25 # 30 Leu Ala Val Gly
Glu Val Ser Lys Leu Val Ly #s Lys Ala Leu Ser Asn 35 # 40 # 45 Glu
Tyr Pro Gln Leu Ser Phe Arg Tyr Arg As #p Ser Ile Lys Lys Thr 50 #
55 # 60 Glu Ile Asn Glu Ala Leu Lys Lys Ile Asp Pr #o Asp Leu Gly
Gly Thr 65 #70 #75 #80 Leu Phe Val Ser Asn Ser Ser Ile Lys Pro As
#p Gly Gly Ile Val Glu 85 # 90 # 95 Val Lys Asp Asp Tyr Gly Glu Trp
Arg Val Va #l Leu Val Ala Glu Ala 100 # 105 # 110 Lys His Gln Gly
Lys Asp Ile Ile Asn Ile Ar #g Asn Gly Leu Leu Val 115 # 120 # 125
Gly Lys Arg Gly Asp Gln Asp Leu Met Ala Al #a Gly Asn Ala Ile Glu
130 # 135 # 140 Arg Ser His Asn Ile Ser Glu Ile Ala Asn Ph #e Met
Leu Ser Glu Ser 145 1 #50 1 #55 1 #60 His Phe Pro Tyr Val Leu Phe
Leu Glu Gly Se #r Asn Phe Leu Thr Glu 165 # 170 # 175 Asn Ile Ser
Ile Thr Arg Pro Asp Gly Arg Va #l Val Asn Leu Glu Tyr 180 # 185 #
190 Asn Ser Gly Ser Glu Ser His Phe Pro Tyr Va #l Leu Phe Leu Glu
Gly 195 # 200 # 205 Ser Asn Phe Leu Thr Glu Asn Ile Ser Ile Th #r
Arg Pro Asp Gly Arg 210 # 215 # 220 Val Val Asn Leu Glu Tyr Asn Ser
Gly Ile Le #u Asn Arg Leu Asp Arg 225 2 #30 2 #35 2 #40 Leu Thr Ala
Ala Asn Tyr Gly Met Pro Ile As #n Ser Asn Leu Cys Ile 245 # 250 #
255 Asn Lys Phe Val Asn His Lys Asp Lys Ser Il #e Met Leu Gln Ala
Ala 260 # 265 # 270 Ser Ile Tyr Thr Gln Gly Asp Gly Arg Glu Tr #p
Asp Ser Lys Ile Met 275 # 280 # 285 Phe Glu Ile Met Phe Asp Ile Ser
Thr Thr Se #r Leu Arg Val Leu Gly 290 # 295 # 300 Arg Asp Leu Phe
Glu Gln Leu Thr Ser Lys 305 3 #10 (2) INFORMATION FOR SEQ ID NO:72:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino #acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: Cys Asp Thr Asp Asp Arg
His Arg Ile Glu Gl #u Lys Arg Lys Arg Lys 1 5 # 10 # 15 Thr (2)
INFORMATION FOR SEQ ID NO:73: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 168 amino #acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:
NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:73: Gly Asp Pro Gly Lys Lys Lys Gln His Ile Cy #s His Ile Gln
Gly Cys 1 5 # 10 # 15 Gly Lys Val Tyr Gly Lys Thr Ser His Leu Ar #g
Ala His Leu Arg Trp 20 # 25 # 30 His Thr Gly Glu Arg Pro Phe Met
Cys Thr Tr #p Ser Tyr Cys Gly Lys 35 # 40 # 45 Arg Phe Thr Arg Ser
Asp Glu Leu Gln Arg Hi #s Lys Arg Thr His Thr 50 # 55 # 60 Gly Glu
Lys Lys Phe Ala Cys Pro Glu Cys Pr #o Lys Arg Phe Met Arg 65 #70
#75 #80
Ser Asp His Leu Ser Lys His Ile Lys Thr Hi #s Gln Asn Lys Lys Gly
85 # 90 # 95 Gly Pro Gly Val Ala Leu Ser Val Gly Thr Le #u Pro Leu
Asp Ser Gly 100 # 105 # 110 Ala Gly Ser Glu Gly Ser Gly Thr Ala Thr
Pr #o Ser Ala Leu Ile Thr 115 # 120 # 125 Thr Asn Met Val Ala Met
Glu Ala Ile Cys Pr #o Glu Gly Ile Ala Arg 130 # 135 # 140 Leu Ala
Asn Ser Gly Ile Asn Val Met Gln Va #l Ala Asp Leu Gln Ser 145 1 #50
1 #55 1 #60 Ile Asn Ile Ser Gly Asn Gly Phe 165 (2) INFORMATION FOR
SEQ ID NO:74: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 181 amino
#acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE
TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v)
FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:
Ser Gly Ile Val Pro Gln Leu Gln Asn Ile Va #l Ser Thr Val Asn Leu 1
5 # 10 # 15 Gly Cys Lys Leu Asp Leu Lys Thr Ile Ala Le #u Arg Ala
Arg Asn Ala 20 # 25 # 30 Glu Tyr Asn Pro Lys Arg Phe Ala Ala Val Il
#e Met Arg Ile Arg Glu 35 # 40 # 45 Pro Arg Thr Thr Ala Leu Ile Phe
Ser Ser Gl #y Lys Met Val Cys Thr 50 # 55 # 60 Gly Ala Lys Ser Glu
Glu Gln Ser Arg Leu Al #a Ala Arg Lys Tyr Ala 65 #70 #75 #80 Arg
Val Val Gln Lys Leu Gly Phe Pro Ala Ly #s Phe Leu Asp Phe Lys 85 #
90 # 95 Ile Gln Asn Met Val Gly Ser Cys Asp Val Ly #s Phe Pro Ile
Arg Leu 100 # 105 # 110 Glu Gly Leu Val Leu Thr His Gln Gln Phe Se
#r Ser Tyr Glu Pro Glu 115 # 120 # 125 Leu Phe Pro Gly Leu Ile Tyr
Arg Met Ile Ly #s Pro Arg Ile Val Leu 130 # 135 # 140 Leu Ile Phe
Val Ser Gly Lys Val Val Leu Th #r Gly Ala Lys Val Arg 145 1 #50 1
#55 1 #60 Ala Glu Ile Tyr Glu Ala Phe Glu Asn Ile Ty #r Pro Ile Leu
Lys Gly 165 # 170 # 175 Phe Arg Lys Thr Thr 180 (2) INFORMATION FOR
SEQ ID NO:75: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 85 amino
#acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE
TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v)
FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:
Ser Cys Phe Ala Leu Ile Ser Gly Thr Ala As #n Gln Val Lys Cys Tyr 1
5 # 10 # 15 Arg Phe Arg Val Lys Lys Asn His Arg His Ar #g Tyr Glu
Asn Cys Thr 20 # 25 # 30 Thr Thr Trp Phe Thr Val Ala Asp Asn Gly Al
#a Glu Arg Gln Gly Gln 35 # 40 # 45 Ala Gln Ile Leu Ile Thr Phe Gly
Ser Pro Se #r Gln Arg Gln Asp Phe 50 # 55 # 60 Leu Lys His Val Pro
Leu Pro Pro Gly Met As #n Ile Ser Gly Phe Thr 65 #70 #75 #80 Ala
Ser Leu Asp Phe 85 (2) INFORMATION FOR SEQ ID NO:76: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 87 amino #acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: Cys Pro Cys Leu Leu Ile
Gly Thr Ser Gly As #n Gly Asn Gln Val Lys 1 5 # 10 # 15 Cys Tyr Ser
Phe Arg Val Lys Arg Trp His As #p Arg Asp Lys Tyr His 20 # 25 # 30
His Thr Thr Thr Trp Trp Ala Val Gly Gly Gl #n Gly Ser Glu Arg Pro
35 # 40 # 45 Gly Asp Ala Thr Val Ile Val Thr Phe Lys As #p Gln Ser
Gln Arg Ser 50 # 55 # 60 His Phe Leu Gln Gln Val Pro Leu Pro Pro Gl
#y Met Ser Ala His Gly 65 #70 #75 #80 Val Thr Met Thr Val Asp Phe
85 (2) INFORMATION FOR SEQ ID NO:77: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 84 amino #acids (B) TYPE: amino acid (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:77: Pro Pro Val Ile Cys Leu Lys Gly Gly His
As #n Gln Leu Lys Cys Leu 1 5 # 10 # 15 Arg Tyr Arg Leu Lys Ser Lys
His Ser Ser Le #u Phe Asp Cys Ile Ser 20 # 25 # 30 Thr Thr Trp Ser
Trp Val Asp Thr Thr Ser Th #r Cys Arg Leu Gly Ser 35 # 40 # 45 Gly
Arg Met Leu Ile Lys Phe Ala Asp Ser Gl #u Gln Arg Asp Lys Phe 50 #
55 # 60 Leu Ser Arg Val Pro Leu Pro Ser Thr Thr Gl #n Val Phe Leu
Gly Asn 65 #70 #75 #80 Phe Tyr Gly Leu (2) INFORMATION FOR SEQ ID
NO:78: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 84 amino #acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT
TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: Pro Pro Val
Ile Leu Val Arg Gly Gly Ala As #n Thr Leu Lys Cys Phe 1 5 # 10 # 15
Arg Asn Arg Ala Arg Val Arg Tyr Arg Gly Le #u Phe Lys Tyr Phe Ser
20 # 25 # 30 Thr Thr Trp Ser Trp Val Ala Gly Asp Ser Th #r Glu Arg
Leu Gly Arg 35 # 40 # 45 Ser Arg Met Leu Ile Leu Phe Thr Ser Ala Cy
#s Gln Arg Glu Lys Pro 50 # 55 # 60 Asp Glu Thr Val Lys Tyr Pro Lys
Gly Val As #p Thr Ser Tyr Gly Asn 65 #70 #75 #80 Leu Asp Ser Leu
(2) INFORMATION FOR SEQ ID NO:79: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 84 amino #acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:
NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:79: Pro Pro Val Val Cys Val Lys Gly Gly Ala As #n Gln Leu Lys
Cys Leu 1 5 # 10 # 15 Arg Tyr Arg Leu Lys Ala Ser Thr Gln Val As #p
Phe Asp Ser Ile Ser 20 # 25 # 30 Thr Thr Trp His Trp Thr Asp Arg
Lys Asn Th #r Glu Arg Ile Gly Ser 35 # 40 # 45 Ala Arg Met Leu Val
Lys Phe Ile Asp Glu Al #a Gln Arg Glu Lys Phe 50 # 55 # 60 Leu Glu
Arg Val Ala Leu Pro Arg Ser Val Se #r Val Phe Leu Gly Gln 65 #70
#75 #80 Phe Asn Gly Ser (2) INFORMATION FOR SEQ ID NO:80: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 84 amino #acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:80: Thr Pro Ile Val Gln Leu
Gln Gly Asp Ser As #n Cys Leu Lys Cys Phe 1 5 # 10 # 15 Arg Tyr Arg
Leu Asn Asp Lys Tyr Lys His Le #u Phe Glu Leu Ala Ser 20 # 25 # 30
Ser Thr Trp His Trp Ala Ser Pro Glu Ala Pr #o His Lys Asn Ala Ile
35 # 40 # 45 Val Thr Leu Thr Tyr Ser Ser Glu Glu Gln Ar #g Gln Gln
Phe Leu Asn 50 # 55 # 60 Ser Val Lys Ile Pro Pro Thr Ile Arg His Ly
#s Val Gly Phe Met Ser 65 #70 #75 #80 Leu His Leu Leu (2)
INFORMATION FOR SEQ ID NO:81: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 84 amino #acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:
NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:81: Thr Pro Ile Val Gln Phe Gln Gly Glu Ser As #n Cys Leu Lys
Cys Phe 1 5 # 10 # 15 Arg Tyr Arg Leu Asn Arg Asp His Arg His Le #u
Phe Asp Leu Ile Ser 20 # 25 # 30 Ser Thr Trp His Trp Ala Ser Ser
Lys Ala Pr #o His Lys His Ala Ile 35 # 40 # 45 Val Thr Val Thr Tyr
Asp Ser Glu Glu Gln Ar #g Gln Gln Phe Leu Asp 50 # 55 # 60 Val Val
Lys Ile Pro Pro Thr Ile Ser His Ly #s Leu Gly Phe Met Ser 65 #70
#75 #80 Leu His Leu Leu (2) INFORMATION FOR SEQ ID NO:82: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 80 amino #acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: Thr Pro Ile Ile His Leu
Lys Gly Asp Arg As #n Ser Leu Lys Cys Leu 1 5 # 10 # 15 Arg Tyr Arg
Leu Arg Lys His Ser Asp His Ty #r Arg Asp Ile Ser Ser 20 # 25 # 30
Thr Trp His Trp Thr Gly Ala Gly Asn Glu Ly #s Thr Gly Ile Leu Thr
35 # 40 # 45 Val Thr Tyr His Ser Glu Thr Gln Arg Thr Ly #s Phe Leu
Asn Thr Val 50 # 55 # 60 Ala Ile Pro Asp Ser Val Gln Ile Leu Val Gl
#y Tyr Asn Thr Met Tyr 65 #70 #75 #80 (2) INFORMATION FOR SEQ ID
NO:83: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 80 amino #acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT
TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: Thr Pro Ile
Val His Leu Lys Gly Asp Ala As #n Thr Leu Lys Cys Leu 1 5 # 10 # 15
Arg Tyr Arg Phe Lys Lys His Cys Thr Leu Ty #r Thr Ala Val Ser Ser
20 # 25 # 30 Thr Trp His Trp Thr Gly His Asn Tyr Lys Hi #s Lys Ser
Ala Ile Val 35 # 40 # 45 Thr Leu Thr Tyr Asp Ser Glu Trp Gln Arg As
#p Gln Phe Leu Ser Gln 50 # 55 # 60 Val Lys Ile Pro Lys Thr Ile Thr
Val Ser Th #r Gly Phe Met Ser Ile 65 #70 #75 #80 (2) INFORMATION
FOR SEQ ID NO:84: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 81
amino #acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:84: Ala Pro Ile Val His Leu Lys Gly Glu Ser As #n Ser Leu Lys
Cys Leu 1 5 # 10 # 15 Arg Tyr Arg Leu Lys Pro Tyr Asn Glu Leu Ty #r
Ser Ser Met Ser Ser 20 # 25 # 30 Thr Trp His Trp Thr Ser Asp Asn
Lys Asn Se #r Lys Asn Gly Ile Val 35 # 40 # 45 Thr Val Thr Phe Val
Thr Gly Gln Gln Gln Gl #n Met Phe Leu Gly Thr 50 # 55 # 60 Val Lys
Ile Pro Pro Thr Val Gln Ile Ser Th #r Gly Phe Met Thr Leu 65 #70
#75 #80 Val (2) INFORMATION FOR SEQ ID NO:85: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 21 amino #acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:85: Gly Ile Val Glu Gln Cys
Cys Thr Ser Ile Cy #s Ser Leu Tyr Gln Leu 1 5 # 10 # 15 Glu Asn Tyr
Cys Asn 20 (2) INFORMATION FOR SEQ ID NO:86: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 30 amino #acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE:
internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86: Phe Val Asn Gln
His Leu Cys Gly Ser His Le #u Val Glu Ala Leu Tyr 1 5 # 10 # 15 Leu
Val Cys Gly Glu Arg Gly Phe Phe Tyr Th #r Pro Lys Thr 20 # 25 # 30
(2) INFORMATION FOR SEQ ID NO:87: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 21 amino #acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:
NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:87: Gly Ile Val Glu Gln Cys Cys Ala Ser Val Cy #s Ser Leu Tyr
Gln Leu 1 5 # 10 # 15 Glu Asn Tyr Cys Asn 20 (2) INFORMATION FOR
SEQ ID NO:88: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 amino
#acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE
TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v)
FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:
Phe Val Asn Gln His Leu Cys Gly Ser His Le #u Val Glu Ala Leu Tyr 1
5 # 10 # 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Th #r Pro Lys
Thr 20 # 25 # 30 (2) INFORMATION FOR SEQ ID NO:89: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 24 amino #acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:89: Gln Leu Tyr Ser Ala Leu
Ala Asn Lys Cys Cy #s His Val Gly Cys Ile 1 5 # 10 # 15 Lys Arg Ser
Leu Ala Arg Phe Cys 20 (2) INFORMATION FOR SEQ ID NO:90: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 amino #acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:90: Asp Ser Trp Met Glu Glu
Val Ile Lys Ile Cy #s Gly Arg Glu Leu Val 1 5 # 10 # 15 Arg Ala Gln
Ile Ala Ile Cys Gly Met Ser Th #r Trp Ser Lys Arg Ser 20 # 25 # 30
Leu (2) INFORMATION FOR SEQ ID NO:91: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino #acids (B) TYPE: amino acid (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:91: Glu Glu Lys Met Gly Thr Ala Lys Lys Cys
Cy #s Ala Ile Gly Cys Ser 1 5 # 10 # 15 Thr Glu Asp Phe Arg Met Val
Cys 20 (2) INFORMATION FOR SEQ ID NO:92: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 40 amino #acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:92: Arg Pro Asn Trp Glu Glu
Arg Ser Arg Leu Cy #s Gly Arg Asp Leu Ile 1 5 # 10 # 15 Arg Ala Phe
Ile Tyr Leu Cys Gly Gly Thr Ar #g Trp Thr Arg Leu Pro 20 # 25 # 30
Asn Phe Gly Asn Tyr Pro Ile Met 35 # 40 (2) INFORMATION FOR SEQ ID
NO:93: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 182 amino #acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT
TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93: Ser Gly Ile
Val Pro Thr Leu Gln Asn Ile Va #l Ser Thr Val Asn Leu 1 5 # 10 # 15
Asp Cys Lys Leu Asp Leu Lys Ala Ile Ala Le #u Gln Ala Arg Asn Ala
20 # 25 # 30 Glu Tyr Asn Pro Lys Arg Phe Ala Ala Val Il #e Met Arg
Ile Arg Glu 35 # 40 # 45 Pro Lys Thr Thr Ala Leu Ile Phe Ala Ser Gl
#y Lys Met Val Cys Thr 50 # 55 # 60 Gly Ala Lys Ser Glu Asp Phe Ser
Lys Met Al #a Ala Arg Lys Tyr Ala 65 #70 #75 #80 Arg Ile Val Gln
Lys Leu Gly Phe Pro Ala Ly #s Phe Lys Asp Phe Lys 85 # 90 # 95 Ile
Gln Asn Ile Val Gly Ser Cys Asp Val Ly #s Phe Pro Ile Arg Leu 100 #
105 # 110 Glu Gly Leu Ala Tyr Ser His Ala Ala Phe Se #r Ser Tyr Glu
Pro Glu 115 # 120 # 125 Leu Phe Pro Gly Leu Ile Tyr Arg Met Lys Va
#l Pro Lys Ile Val Leu 130 # 135 # 140 Leu Ile Phe Val Ser Gly Lys
Ile Val Ile Th #r Gly Ala Lys Met Arg 145 1 #50 1 #55 1 #60 Asp Glu
Thr Tyr Lys Ala Phe Glu Asn Ile Ty #r Pro Val Leu Ser Glu 165 # 170
# 175 Phe Arg Lys Ile Gln Gln 180 (2) INFORMATION FOR SEQ ID NO:94:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 84 amino #acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:94: Asn Ser Asn Ser Thr Pro
Ile Val His Leu Ly #s Gly Asp Ala Asn Thr 1 5 # 10 # 15 Leu Lys Cys
Leu Arg Tyr Arg Phe Lys Lys Hi #s Cys Thr Leu Tyr Thr 20 # 25 # 30
Ala Val Ser Ser Thr Trp His Trp Thr Gly Hi #s Asn Val Lys His Lys
35 # 40 # 45 Ser Ala Ile Val Thr Leu Thr Tyr Asp Ser Gl #u Trp Gln
Arg Asp Gln 50 # 55 # 60 Phe Leu Ser Gln Val Lys Ile Pro Lys Thr Il
#e Thr Val Ser Thr Gly 65 #70 #75 #80 Phe Met Ser Ile (2)
INFORMATION FOR SEQ ID NO:95: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 84 amino #acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:
NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:95: Asn Ser Asn Thr Thr Pro Ile Val His Leu Ly #s Gly Asp Ala
Asn Thr 1 5 # 10 # 15 Leu Lys Cys Leu Arg Tyr Arg Phe Lys Lys Hi #s
Cys Thr Leu Tyr Thr 20 # 25 # 30 Ala Val Ser Ser Thr Trp His Trp
Thr Gly Hi #s Asn Val Lys His Lys 35 # 40 # 45 Ser Ala Ile Val Thr
Leu Thr Tyr Asp Ser Gl #u Trp Gln Arg Asp Gln 50 # 55 # 60 Phe Leu
Ser Gln Val Lys Ile Pro Lys Thr Il #e Thr Val Ser Thr Gly 65 #70
#75 #80 Phe Met Ser Ile (2) INFORMATION FOR SEQ ID NO:96: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 83 amino #acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:96: Ser Gly Asn Thr Thr Pro
Ile Ile His Leu Ly #s Gly Asp Arg Asn Ser 1 5 # 10 # 15 Leu Lys Cys
Leu Arg Tyr Arg Leu Arg Lys Hi #s Ser Asp His Tyr Arg 20 # 25 # 30
Asp Ile Ser Ser Thr Trp His Trp Thr Gly Al #a Gly Asn Glu Lys Thr
35 # 40 # 45 Gly Ile Leu Thr Val Thr Tyr His Ser Glu Th #r Gln Arg
Thr Lys Phe 50 # 55 # 60 Leu Asn Thr Val Ala Ile Pro Asp Ser Val Gl
#n Ile Leu Val Gly Tyr 65 #70 #75 #80 Met Thr Met (2) INFORMATION
FOR SEQ ID NO:97: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 84
amino #acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:97: Ser Gly Asn Thr Ala Pro Ile Val His Leu Ly #s Gly Glu Ser
Asn Ser 1 5 # 10 # 15 Leu Lys Cys Leu Arg Tyr Arg Leu Lys Pro Ty #r
Lys Glu Leu Tyr Ser 20 # 25 # 30 Ser Met Ser Ser Thr Trp His Trp
Thr Ser As #p Asn Lys Asn Ser Lys 35 # 40 # 45 Asn Gly Ile Val Thr
Val Thr Phe Val Thr Gl #u Gln Gln Gln Gln Met 50 # 55 # 60 Phe Leu
Gly Thr Val Lys Ile Pro Pro Thr Va #l Gln Ile Ser Thr Gly 65 #70
#75 #80 Phe Met Thr Leu (2) INFORMATION FOR SEQ ID NO:98: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 89 amino #acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:98: Ser Gly Asn Thr Ser Cys
Phe Ala Leu Ile Se #r Gly Thr Ala Asn Gln 1 5 # 10 # 15 Val Lys Cys
Tyr Arg Phe Arg Val Lys Lys As #n His Arg His Arg Tyr 20 # 25 # 30
Glu Asn Cys Thr Thr Thr Trp Phe Thr Val Al #a Asp Asn Gly Ala Glu
35 # 40 # 45 Arg Gln Gly Gln Ala Gln Ile Leu Ile Thr Ph #e Gly Ser
Pro Ser Gln 50 # 55 # 60 Arg Gln Asp Phe Leu Lys His Val Pro Leu Pr
#o Pro Gly Met Asn Ile 65 #70 #75 #80 Ser Gly Phe Thr Ala Ser Leu
Asp Phe 85 (2) INFORMATION FOR SEQ ID NO:99: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 7 amino #acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: C-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:99: Ser Asn Lys Lys Thr Thr
Ala 1 5 (2) INFORMATION FOR SEQ ID NO:100: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 4 amino #acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:100: Asn Ser Asn Thr 1 (2)
INFORMATION FOR SEQ ID NO:101: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 4 amino #acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:
NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:101: Ser Gly Asn Thr 1 (2) INFORMATION FOR SEQ ID NO:102: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino #acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:102: Ser Ser Gly Ser Ser Gly 1
5 (2) INFORMATION FOR SEQ ID NO:103: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino #acids (B) TYPE: amino acid (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:103: Cys Tyr Pro Glu Ile Lys Asp Lys Glu Glu
Va #l Gln Arg Lys Arg 1 5 # 10 # 15 (2) INFORMATION FOR SEQ ID
NO:104: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 66 amino #acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT
TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104: Met Glu
Gln Arg Ile Thr Leu Lys Asp Tyr Al #a Met Arg Phe Gly Gln 1
5 # 10 # 15 Thr Lys Thr Ala Lys Asp Leu Gly Val Tyr Gl #n Ser Ala
Ile Asn Lys 20 # 25 # 30 Ala Ile His Ala Gly Arg Lys Ile Phe Leu Th
#r Ile Asn Ala Asp Gly 35 # 40 # 45 Ser Val Tyr Ala Glu Glu Val Lys
Pro Phe Pr #o Ser Asn Lys Lys Thr 50 # 55 # 60 Thr Ala 65 (2)
INFORMATION FOR SEQ ID NO:105: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 66 amino #acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:
NO (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:105: Met Glu Gln Glu Ile Thr Leu Lys Asp Tyr Al #a Met Arg Phe
Gly Gln 1 5 # 10 # 15 Thr Lys Thr Ala Lys Asp Leu Gly Val Tyr Gl #n
Ser Ala Ile Asn Lys 20 # 25 # 30 Ala Ile His Ala Gly Arg Lys Ile
Phe Leu Th #r Ile Asn Ala Asp Gly 35 # 40 # 45 Ser Val Tyr Ala Glu
Glu Val Lys Pro Phe Pr #o Ser Asn Lys Lys Thr 50 # 55 # 60 Thr Ala
65 (2) INFORMATION FOR SEQ ID NO:106: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 66 amino #acids (B) TYPE: amino acid (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:106: Met Arg Gln Arg Ile Thr Leu Lys Asp Tyr
Al #a Met Arg Phe Gly Gln 1 5 # 10 # 15 Thr Lys Thr Ala Lys Asp Leu
Gly Val Tyr Gl #n Ser Ala Ile Asn Lys 20 # 25 # 30 Ala Ile His Ala
Gly Arg Lys Ile Phe Leu Th #r Ile Asn Ala Asp Gly 35 # 40 # 45 Ser
Val Tyr Ala Glu Glu Val Lys Pro Phe Pr #o Ser Asn Lys Lys Thr 50 #
55 # 60 Thr Ala 65 (2) INFORMATION FOR SEQ ID NO:107: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 96 amino #acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:107: Ser Thr Lys Lys Lys Pro
Leu Thr Gln Glu Gl #n Leu Glu Asp Ala Arg 1 5 # 10 # 15 Arg Leu Lys
Ala Ile Tyr Glu Lys Lys Lys As #n Glu Leu Gly Leu Ser 20 # 25 # 30
Gln Glu Ser Val Ala Asp Lys Met Gly Met Gl #y Gln Ser Gly Val Gly
35 # 40 # 45 Ala Leu Phe Asn Gly Ile Asn Ala Leu Asn Al #a Tyr Asn
Ala Ala Leu 50 # 55 # 60 Leu Ala Lys Ile Leu Lys Val Ser Val Glu Gl
#u Phe Ser Pro Ser Ile 65 #70 #75 #80 Ala Arg Glu Ile Tyr Glu Met
Tyr Glu Ala Va #l Ser Met Glu Pro Ser 85 # 90 # 95 (2) INFORMATION
FOR SEQ ID NO:108: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 96
amino #acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:108: Ser Thr Lys Lys Lys Pro Leu Thr Gln Glu Gl #n Leu Glu Asp
Ala Arg 1 5 # 10 # 15 Arg Leu Lys Ala Ile Tyr Glu Lys Lys Lys As #n
Glu Leu Gly Leu Ser 20 # 25 # 30 Gln Glu Ser Val Ala Asp Lys Met
Gly Met Gl #y Gln Ser Gly Val Gly 35 # 40 # 45 Ala Leu Phe Asn Gly
Ile Asn Ala Leu Asn Al #a Tyr Asn Ala Ala Leu 50 # 55 # 60 Leu Ala
Lys Ile Leu Lys Val Ser Val Glu Gl #u Phe Ser Pro Ser Ile 65 #70
#75 #80 Ala Arg Glu Ile Tyr Glu Met Cys Glu Ala Va #l Ser Met Glu
Pro Ser 85 # 90 # 95 (2) INFORMATION FOR SEQ ID NO:109: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 180 amino #acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:109: Gly Ile Val Glu Gln Cys
Cys Thr Ser Ile Cy #s Ser Leu Tyr Gln Leu 1 5 # 10 # 15 Glu Asn Tyr
Cys Asn Met Ser Met Glu Gln Ar #g Ile Thr Leu Lys Asp 20 # 25 # 30
Tyr Ala Met Arg Phe Gly Gln Thr Lys Thr Al #a Lys Asp Leu Gly Val
35 # 40 # 45 Tyr Gln Ser Ala Ile Asn Lys Ala Ile His Al #a Gly Arg
Lys Ile Phe 50 # 55 # 60 Leu Thr Ile Asn Ala Asp Gly Ser Val Tyr Al
#a Glu Glu Val Lys Pro 65 #70 #75 #80 Phe Pro Ser Asn Lys Lys Thr
Thr Ala Ser As #n Lys Lys Thr Thr Ala 85 # 90 # 95 Asn Ser Asn Thr
Thr Pro Ile Val His Leu Ly #s Gly Asp Ala Asn Thr 100 # 105 # 110
Leu Lys Cys Leu Arg Tyr Arg Phe Lys Lys Hi #s Cys Thr Leu Tyr Thr
115 # 120 # 125 Ala Val Ser Ser Thr Trp His Trp Thr Gly Hi #s Asn
Val Lys His Lys 130 # 135 # 140 Ser Ala Ile Val Thr Leu Thr Tyr Asp
Ser Gl #u Trp Gln Arg Asp Gln 145 1 #50 1 #55 1 #60 Phe Leu Ser Gln
Val Lys Ile Pro Lys Thr Il #e Thr Val Ser Thr Gly 165 # 170 # 175
Phe Met Ser Ile 180 (2) INFORMATION FOR SEQ ID NO:110: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 113 amino #acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:110: Gly Ile Val Glu Gln Cys
Cys Thr Ser Ile Cy #s Ser Leu Tyr Gln Leu 1 5 # 10 # 15 Glu Asn Tyr
Cys Asn Met Ser Met Glu Gln Ar #g Ile Thr Leu Lys Asp 20 # 25 # 30
Tyr Ala Met Arg Phe Gly Gln Thr Lys Thr Al #a Lys Asp Leu Gly Val
35 # 40 # 45 Tyr Gln Ser Ala Ile Asn Lys Ala Ile His Al #a Gly Arg
Lys Ile Phe 50 # 55 # 60 Leu Thr Ile Asn Ala Asp Gly Ser Val Tyr Al
#a Glu Glu Val Lys Pro 65 #70 #75 #80 Phe Pro Ser Asn Lys Lys Thr
Thr Ala Ser As #n Lys Lys Thr Thr Ala 85 # 90 # 95 Cys Asp Thr Asp
Asp Arg His Arg Ile Glu Gl #u Lys Arg Lys Arg Lys 100 # 105 # 110
Thr (2) INFORMATION FOR SEQ ID NO:111: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 292 amino #acids (B) TYPE: amino acid
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:111: Phe Val Asn Gln His Leu
Cys Gly Ser His Le #u Val Glu Ala Leu Tyr 1 5 # 10 # 15 Leu Val Cys
Gly Glu Arg Gly Phe Phe Tyr Th #r Pro Lys Thr Met Ser 20 # 25 # 30
Met Glu Gln Glu Ile Thr Leu Lys Asp Tyr Al #a Met Arg Phe Gly Gln
35 # 40 # 45 Thr Lys Thr Ala Lys Asp Leu Gly Val Tyr Gl #n Ser Ala
Ile Asn Lys 50 # 55 # 60 Ala Ile His Ala Gly Arg Lys Ile Phe Leu Th
#r Ile Asn Ala Asp Gly 65 #70 #75 #80 Ser Val Tyr Ala Glu Glu Val
Lys Pro Phe Pr #o Ser Asn Lys Lys Thr 85 # 90 # 95 Thr Ala Ser Asn
Lys Lys Thr Thr Ala Ser Se #r Gly Ser Ser Gly Ser 100 # 105 # 110
Gly Ile Val Pro Gln Leu Gln Asn Ile Val Se #r Thr Val Asn Leu Gly
115 # 120 # 125 Cys Lys Leu Asp Leu Lys Thr Ile Ala Leu Ar #g Ala
Arg Asn Ala Glu 130 # 135 # 140 Tyr Asn Pro Lys Arg Phe Ala Ala Val
Ile Me #t Arg Ile Arg Glu Pro 145 1 #50 1 #55 1 #60 Arg Thr Thr Ala
Leu Ile Phe Ser Ser Gly Ly #s Met Val Cys Thr Gly 165 # 170 # 175
Ala Lys Ser Glu Glu Gln Ser Arg Leu Ala Al #a Arg Lys Tyr Ala Arg
180 # 185 # 190 Val Val Gln Lys Leu Gly Phe Pro Ala Lys Ph #e Leu
Asp Phe Lys Ile 195 # 200 # 205 Gln Asn Met Val Gly Ser Cys Asp Val
Lys Ph #e Pro Ile Arg Leu Glu 210 # 215 # 220 Gly Leu Val Leu Thr
His Gln Gln Phe Ser Se #r Tyr Glu Pro Glu Leu 225 2 #30 2 #35 2 #40
Phe Pro Gly Leu Ile Tyr Arg Met Ile Lys Pr #o Arg Ile Val Leu Leu
245 # 250 # 255 Ile Phe Val Ser Gly Lys Val Val Leu Thr Gl #y Ala
Lys Val Arg Ala 260 # 265 # 270 Glu Ile Tyr Glu Ala Phe Glu Asn Ile
Tyr Pr #o Ile Leu Lys Gly Phe 275 # 280 # 285 Arg Lys Thr Thr 290
(2) INFORMATION FOR SEQ ID NO:112: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 273 amino #acids (B) TYPE: amino acid (D) TOPOLOGY:
linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:112: Phe Val Asn Gln His Leu Cys Gly Ser His
Le #u Val Glu Ala Leu Tyr 1 5 # 10 # 15 Leu Val Cys Gly Glu Arg Gly
Phe Phe Tyr Th #r Pro Lys Thr Met Ser 20 # 25 # 30 Met Arg Gln Arg
Ile Thr Leu Lys Asp Tyr Al #a Met Arg Phe Gly Gln 35 # 40 # 45 Thr
Lys Thr Ala Lys Asp Leu Gly Val Tyr Gl #n Ser Ala Ile Asn Lys 50 #
55 # 60 Ala Ile His Ala Gly Arg Lys Ile Phe Leu Th #r Ile Asn Ala
Asp Gly 65 #70 #75 #80 Ser Val Tyr Ala Glu Glu Val Lys Pro Phe Pr
#o Ser Asn Lys Lys Thr 85 # 90 # 95 Thr Ala Ser Asn Lys Lys Thr Thr
Ala Gly As #p Pro Gly Lys Lys Lys 100 # 105 # 110 Gln His Ile Cys
His Ile Gln Gly Cys Gly Ly #s Val Tyr Gly Lys Thr 115 # 120 # 125
Ser His Leu Arg Ala His Leu Arg Trp His Th #r Gly Glu Arg Pro Phe
130 # 135 # 140 Met Cys Thr Trp Ser Tyr Cys Gly Lys Arg Ph #e Thr
Arg Ser Asp Glu 145 1 #50 1 #55 1 #60 Leu Gln Arg His Lys Arg Thr
His Thr Gly Gl #u Lys Lys Phe Ala Cys 165 # 170 # 175 Pro Glu Cys
Pro Lys Arg Phe Met Arg Ser As #p His Leu Ser Lys His 180 # 185 #
190 Ile Lys Thr His Gln Asn Lys Lys Gly Gly Pr #o Gly Val Ala Leu
Ser 195 # 200 # 205 Val Gly Thr Leu Pro Leu Asp Ser Gly Ala Gl #y
Ser Glu Gly Ser Gly 210 # 215 # 220 Thr Ala Thr Pro Ser Ala Leu Ile
Thr Thr As #n Met Val Ala Met Glu 225 2 #30 2 #35 2 #40 Ala Ile Cys
Pro Glu Gly Ile Ala Arg Leu Al #a Asn Ser Gly Ile Asn 245 # 250 #
255 Val Met Gln Val Ala Asp Leu Gln Ser Ile As #n Ile Ser Gly Asn
Gly 260 # 265 # 270 Phe (2) INFORMATION FOR SEQ ID NO:113: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 421 amino #acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:113: Gln Leu Tyr Ser Ala Leu
Ala Asn Lys Cys Cy #s His Val Gly Cys Ile 1 5 # 10 # 15 Lys Arg Ser
Leu Ala Arg Phe Cys Met Ser Me #t Arg Gln Arg Ile Thr 20 # 25 # 30
Leu Lys Asp Tyr Ala
Met Arg Phe Gly Gln Th #r Lys Thr Ala Lys Asp 35 # 40 # 45 Leu Gly
Val Tyr Gln Ser Ala Ile Asn Lys Al #a Ile His Ala Gly Arg 50 # 55 #
60 Lys Ile Phe Leu Thr Ile Asn Ala Asp Gly Se #r Val Tyr Ala Glu
Glu 65 #70 #75 #80 Val Lys Pro Phe Pro Ser Asn Lys Lys Thr Th #r
Ala Ser Asn Lys Lys 85 # 90 # 95 Thr Thr Ala Met Ala Asp Asp Asp
Pro Tyr Gl #y Thr Gly Gln Met Phe 100 # 105 # 110 His Leu Asn Thr
Ala Leu Thr His Ser Ile Ph #e Asn Ala Glu Leu Tyr 115 # 120 # 125
Ser Pro Glu Ile Pro Leu Ser Thr Asp Gly Pr #o Tyr Leu Gln Ile Leu
130 # 135 # 140 Glu Gln Pro Lys Gln Arg Gly Phe Arg Phe Ar #g Tyr
Val Cys Glu Gly 145 1 #50 1 #55 1 #60 Pro Ser His Gly Gly Leu Pro
Gly Ala Ser Se #r Glu Lys Asn Lys Lys 165 # 170 # 175 Ser Tyr Pro
Gln Val Lys Ile Cys Asn Tyr Va #l Gly Pro Ala Lys Val 180 # 185 #
190 Ile Val Gln Leu Val Thr Asn Gly Lys Asn Il #e His Leu His Ala
His 195 # 200 # 205 Ser Leu Val Gly Lys His Cys Glu Asp Gly Va #l
Cys Thr Val Thr Ala 210 # 215 # 220 Gly Pro Lys Asp Met Val Val Gly
Phe Ala As #n Leu Gly Ile Leu His 225 2 #30 2 #35 2 #40 Val Thr Lys
Lys Lys Val Phe Glu Thr Leu Gl #u Ala Arg Met Thr Glu 245 # 250 #
255 Ala Cys Ile Arg Gly Tyr Asn Pro Gly Leu Le #u Val His Ser Asp
Leu 260 # 265 # 270 Ala Tyr Leu Gln Ala Glu Gly Gly Gly Asp Ar #g
Gln Leu Thr Asp Arg 275 # 280 # 285 Glu Lys Glu Ile Ile Arg Gln Ala
Ala Val Gl #n Gln Thr Lys Glu Met 290 # 295 # 300 Asp Leu Ser Val
Val Arg Leu Met Phe Thr Al #a Phe Leu Pro Asp Ser 305 3 #10 3 #15 3
#20 Thr Gly Ser Phe Thr Arg Arg Leu Glu Pro Va #l Val Ser Asp Ala
Ile 325 # 330 # 335 Tyr Asp Ser Lys Ala Pro Asn Ala Ser Asn Le #u
Lys Ile Val Arg Met 340 # 345 # 350 Asp Arg Thr Ala Gly Cys Val Thr
Gly Gly Gl #u Glu Ile Tyr Leu Leu 355 # 360 # 365 Cys Asp Lys Val
Gln Lys Asp Asp Ile Gln Il #e Arg Phe Tyr Glu Glu 370 # 375 # 380
Glu Glu Asn Gly Gly Val Trp Glu Gly Phe Gl #y Asp Phe Ser Pro Thr
385 3 #90 3 #95 4 #00 Asp Val His Arg Gln Phe Ala Ile Val Phe Ly #s
Thr Pro Lys Tyr Lys 405 # 410 # 415 Asp Val Asn Ile Thr 420 (2)
INFORMATION FOR SEQ ID NO:114: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 391 amino #acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:
NO (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:114: Met Arg Gln Arg Ile Thr Leu Lys Asp Tyr Al #a Met Arg Phe
Gly Gln 1 5 # 10 # 15 Thr Lys Thr Ala Lys Asp Leu Gly Val Tyr Gl #n
Ser Ala Ile Asn Lys 20 # 25 # 30 Ala Ile His Ala Gly Arg Lys Ile
Phe Leu Th #r Ile Asn Ala Asp Gly 35 # 40 # 45 Ser Val Tyr Ala Glu
Glu Val Lys Pro Phe Pr #o Ser Asn Lys Lys Thr 50 # 55 # 60 Thr Ala
Met Ala Glu Asp Asp Pro Tyr Leu Gl #y Arg Pro Glu Gln Met 65 #70
#75 #80 Phe His Leu Asp Pro Ser Leu Thr His Thr Il #e Phe Asn Pro
Glu Val 85 # 90 # 95 Phe Gln Pro Gln Met Ala Leu Pro Thr Ala As #p
Gly Pro Tyr Leu Gln 100 # 105 # 110 Ile Leu Glu Gln Pro Lys Gln Arg
Gly Phe Ar #g Phe Arg Tyr Val Cys 115 # 120 # 125 Glu Gly Pro Ser
His Gly Gly Leu Pro Gly Al #a Ser Ser Glu Lys Asn 130 # 135 # 140
Lys Lys Ser Tyr Pro Gln Val Lys Ile Cys As #n Tyr Val Gly Pro Ala
145 1 #50 1 #55 1 #60 Lys Val Ile Val Gln Leu Val Thr Asn Gly Ly #s
Asn Ile His Leu His 165 # 170 # 175 Ala His Ser Leu Val Gly Lys His
Cys Glu As #p Gly Ile Cys Thr Val 180 # 185 # 190 Thr Ala Gly Pro
Glu Asp Cys Val His Gly Ph #e Ala Asn Leu Gly Ile 195 # 200 # 205
Leu His Val Thr Lys Lys Lys Val Phe Glu Th #r Leu Glu Ala Arg Met
210 # 215 # 220 Thr Glu Ala Cys Ile Arg Gly Tyr Asn Pro Gl #y Leu
Leu Val His Pro 225 2 #30 2 #35 2 #40 Asp Leu Ala Tyr Leu Gln Ala
Glu Gly Gly Gl #y Asp Arg Gln Leu Gly 245 # 250 # 255 Asp Arg Glu
Lys Glu Leu Ile Arg Gln Ala Al #a Leu Gln Gln Thr Lys 260 # 265 #
270 Glu Met Asp Leu Ser Val Val Arg Leu Met Ph #e Thr Ala Phe Leu
Pro 275 # 280 # 285 Asp Ser Thr Gly Ser Phe Thr Arg Arg Leu Gl #u
Pro Val Val Ser Asp 290 # 295 # 300 Ala Ile Tyr Asp Ser Lys Ala Pro
Asn Ala Se #r Asn Leu Lys Ile Val 305 3 #10 3 #15 3 #20 Arg Met Asp
Arg Thr Ala Gly Cys Val Thr Gl #y Gly Glu Glu Ile Tyr 325 # 330 #
335 Leu Leu Cys Asp Lys Val Gln Lys Asp Asp Il #e Gln Ile Arg Phe
Tyr 340 # 345 # 350 Glu Glu Glu Glu Asn Gly Gly Val Trp Glu Gl #y
Phe Gly Asp Phe Ser 355 # 360 # 365 Pro Thr Asp Val His Arg Gln Phe
Ala Ile Va #l Phe Lys Thr Pro Lys 370 # 375 # 380 Tyr Lys Asp Ile
Asn Ile Thr 385 3 #90 (2) INFORMATION FOR SEQ ID NO:115: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 391 amino #acids (B) TYPE:
amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:115: Met Glu Gln Glu Ile Thr
Leu Lys Asp Tyr Al #a Met Arg Phe Gly Gln 1 5 # 10 # 15 Thr Lys Thr
Ala Lys Asp Leu Gly Val Tyr Gl #n Ser Ala Ile Asn Lys 20 # 25 # 30
Ala Ile His Ala Gly Arg Lys Ile Phe Leu Th #r Ile Asn Ala Asp Gly
35 # 40 # 45 Ser Val Tyr Ala Glu Glu Val Lys Pro Phe Pr #o Ser Asn
Lys Lys Thr 50 # 55 # 60 Thr Ala Met Ala Glu Asp Asp Pro Tyr Leu Gl
#y Arg Pro Glu Gln Met 65 #70 #75 #80 Phe His Leu Asp Pro Ser Leu
Thr His Thr Il #e Phe Asn Pro Glu Val 85 # 90 # 95 Phe Gln Pro Gln
Met Ala Leu Pro Thr Ala As #p Gly Pro Tyr Leu Gln 100 # 105 # 110
Ile Leu Glu Gln Pro Lys Gln Arg Gly Phe Ar #g Phe Arg Tyr Val Cys
115 # 120 # 125 Glu Gly Pro Ser His Gly Gly Leu Pro Gly Al #a Ser
Ser Glu Lys Asn 130 # 135 # 140 Lys Lys Ser Tyr Pro Gln Val Lys Ile
Cys As #n Tyr Val Gly Pro Ala 145 1 #50 1 #55 1 #60 Lys Val Ile Val
Gln Leu Val Thr Asn Gly Ly #s Asn Ile His Leu His 165 # 170 # 175
Ala His Ser Leu Val Gly Lys His Cys Glu As #p Gly Ile Cys Thr Val
180 # 185 # 190 Thr Ala Gly Pro Glu Asp Cys Val His Gly Ph #e Ala
Asn Leu Gly Ile 195 # 200 # 205 Leu His Val Thr Lys Lys Lys Val Phe
Glu Th #r Leu Glu Ala Arg Met 210 # 215 # 220 Thr Glu Ala Cys Ile
Arg Gly Tyr Asn Pro Gl #y Leu Leu Val His Pro 225 2 #30 2 #35 2 #40
Asp Leu Ala Tyr Leu Gln Ala Glu Gly Gly Gl #y Asp Arg Gln Leu Gly
245 # 250 # 255 Asp Arg Glu Lys Glu Leu Ile Arg Gln Ala Al #a Leu
Gln Gln Thr Lys 260 # 265 # 270 Glu Met Asp Leu Ser Val Val Arg Leu
Met Ph #e Thr Ala Phe Leu Pro 275 # 280 # 285 Asp Ser Thr Gly Ser
Phe Thr Arg Arg Leu Gl #u Pro Val Val Ser Asp 290 # 295 # 300 Ala
Ile Tyr Asp Ser Lys Ala Pro Asn Ala Se #r Asn Leu Lys Ile Val 305 3
#10 3 #15 3 #20 Arg Met Asp Arg Thr Ala Gly Cys Val Thr Gl #y Gly
Glu Glu Ile Tyr 325 # 330 # 335 Leu Leu Cys Asp Lys Val Gln Lys Asp
Asp Il #e Gln Ile Arg Phe Tyr 340 # 345 # 350 Glu Glu Glu Glu Asn
Gly Gly Val Trp Glu Gl #y Phe Gly Asp Phe Ser 355 # 360 # 365 Pro
Thr Asp Val His Arg Gln Phe Ala Ile Va #l Phe Lys Thr Pro Lys 370 #
375 # 380 Tyr Lys Asp Ile Asn Ile Thr 385 3 #90 (2) INFORMATION FOR
SEQ ID NO:116: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 241 amino
#acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE
TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (v)
FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:116:
Met Arg Gln Arg Ile Thr Leu Lys Asp Tyr Al #a Met Arg Phe Gly Gln 1
5 # 10 # 15 Thr Lys Thr Ala Lys Asp Leu Gly Val Tyr Gl #n Ser Ala
Ile Asn Lys 20 # 25 # 30 Ala Ile His Ala Gly Arg Lys Ile Phe Leu Th
#r Ile Asn Ala Asp Gly 35 # 40 # 45 Ser Val Tyr Ala Glu Glu Val Lys
Pro Phe Pr #o Ser Asn Lys Lys Thr 50 # 55 # 60 Thr Ala Ser Asn Lys
Lys Thr Thr Ala Gly As #p Pro Gly Lys Lys Lys 65 #70 #75 #80 Gln
His Ile Cys His Ile Gln Gly Cys Gly Ly #s Val Tyr Gly Lys Thr 85 #
90 # 95 Ser His Leu Arg Ala His Leu Arg Trp His Th #r Gly Glu Arg
Pro Phe 100 # 105 # 110 Met Cys Thr Trp Ser Tyr Cys Gly Lys Arg Ph
#e Thr Arg Ser Asp Glu 115 # 120 # 125 Leu Gln Arg His Lys Arg Thr
His Thr Gly Gl #u Lys Lys Phe Ala Cys 130 # 135 # 140 Pro Glu Cys
Pro Lys Arg Phe Met Arg Ser As #p His Leu Ser Lys His 145 1 #50 1
#55 1 #60 Ile Lys Thr His Gln Asn Lys Lys Gly Gly Pr #o Gly Val Ala
Leu Ser 165 # 170 # 175 Val Gly Thr Leu Pro Leu Asp Ser Gly Ala Gl
#y Ser Glu Gly Ser Gly 180 # 185 # 190 Thr Ala Thr Pro Ser Ala Leu
Ile Thr Thr As #n Met Val Ala Met Glu 195 # 200 # 205 Ala Ile Cys
Pro Glu Gly Ile Ala Arg Leu Al #a Asn Ser Gly Ile Asn 210 # 215 #
220 Val Met Gln Val Ala Asp Leu Gln Ser Ile As #n Ile Ser Gly Asn
Gly 225 2 #30 2 #35 2 #40 Phe (2) INFORMATION FOR SEQ ID NO:117:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 base #pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: both (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:117: GGGAMTNYCC # # # 10 (2)
INFORMATION FOR SEQ ID NO:118: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 72 amino #acids (B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:
NO (v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:118: Met Glu Pro Val Asp Pro Arg Leu Glu Pro Tr #p Lys His Pro
Gly Ser 1 5 # 10 # 15 Gln Pro Lys Thr Ala Cys Thr Asn Cys Tyr Cy #s
Lys Lys Cys Cys Phe 20 # 25 # 30 His Cys Gln Val Cys
Phe Ile Thr Lys Ala Le #u Gly Ile Ser Tyr Gly 35 # 40 # 45 Arg Lys
Lys Arg Arg Gln Arg Arg Arg Ala Hi #s Gln Asn Ser Gln Thr 50 # 55 #
60 His Gln Ala Ser Leu Ser Lys Gln 65 #70
* * * * *