Tumor suppressor gene merlin

A novel tumor suppressor protein, merlin, is described, including DNA sequences encoding merlin, and recombinant vectors and hosts capable of expressing merlin. Method for the diagnosis and treatment of merlin-associated tumors, and for the diagnosis and treatment of the disease neurofibromatosis 2 (NF2) are also provided.

FIELD OF THE INVENTION 
The invention is in the field of genetic disease diagnosis, tumor detection 
and treatment, and genetic therapy. Specifically, the invention is 
directed to the merlin gene, merlin protein, and the use of the gene 
and/or protein for (1) detecting a predisposition to develop tumors 
especially neurofibromatosis-2, (2) diagnosing certain tumors, especially 
neurofibromatosis-2, (3) treating tumors, especially neurofibromatosis-2, 
(4) monitoring the course of tumor treatment, especially 
neurofibromatosis-2 treatment, and (5) gene replacement in affected 
non-tumor tissues or cells. 
BACKGROUND OF THE INVENTION 
Neurofibromatosis (NF) describes two major human genetic disorders that 
display autosomal dominant inheritance, involve tumors of the nervous 
system, and which are distinct clinical entities (Mulvihill, J. J. et al., 
Ann. Intern. Med. 113:39-52 (1990)). NF1, or von Recklinghausen NF, is 
more common (incidence of 1/4,000) and is characterized by the highly 
variable expression of an array of features that include neurofibromas, 
cafe-au-lait macules, Lisch nodules of the iris, and a predisposition to 
certain malignant tumors (Riccardi, V. M. et al., N. Engl. J. Med. 
305:1617-1627 (1981); Riccardi, V. M. et al., "Neurofibromatosis: 
Phenotype, Natural History, and Pathogenesis," Johns Hopkins Univ. Press, 
Baltimore, Md. (1986)). It is caused by defects in a gene on chromosome 17 
that has recently been isolated and characterized (Viskochil, D. et al., 
Cell 62:187-192 (1990); Cawthon, R. M. et al., Cell 62:193-201 (1990); 
Wallace, M. R. et al., Science 249:183-186 (1990)). The NF1 gene product, 
neurofibromin, is a large protein with a GAP-related domain and is 
presumably involved in modulating a signal transduction pathway whose 
disruption can lead to tumor formation (Ballester, R. et al., Cell 
63:851-859 (1990); Buchberg, A. M. et al., Nature 347:291-294 (1990); Xu, 
G. et al., Cell 62:599-608 (1990); DeClue, J. E. et al., Cell 69:265-273 
(1992); Basu, T. N. et al., Nature 356:713-715 (1992)). 
In contrast, neurofibromatosis-2 (NF2), which occurs in about 1/40,000 
livebirths (Evans, D. G. R. et al., J. Med. Genet. 29:841-846 (1992)), is 
characterized by bilateral schwannomas that develop on the vestibular 
branch of the 8th cranial nerve. Pressure from these tumors often causes 
hearing loss and vestibular symptoms in the second and third decade. Other 
tumors of the brain, especially meningiomas and schwannomas of other 
cranial nerves and spinal nerve roots (Martuza, R. L. et al., N. Engl. J. 
Med. 318:684-688 (1988)), and posterior capsular lens opacities 
(Kaiser-Kupfer, M. I. et al., Arch. Ophthalmol. 107:541-544 (1989) are 
commonly present in the young affected adult. 
The NF2 gene is highly penetrant. Ninety-five percent of persons with the 
genotype develop bilateral vestibular schwannomas. NF2 is often more 
severe than NF1. Teenage or early adulthood onset of multiple slow growing 
tumors that can gradually cause deafness, balance disorder, paralysis or 
increasing neurologic problems necessitating repeated surgical procedures, 
characterizes NF2. 
NF2 has been shown to be genetically distinct from NF1 by linkage studies 
that assigned the NF2 gene to chromosome 22 (Rouleau, G. A. et al., Nature 
329:246-248 (1987); Wertelecki, W. et al., N. Engl. J. Med. 319:278-283 
(1988); Rouleau, G. A. et al., Am. J. Hum. Genet. 46:323-328 (1990); 
Narod, S. A. et al., Am. J. Hum. Genet. 51:486-496 (1992)). The tumor 
types that occur in NF2 can be seen in the general population as solitary, 
sporadic tumors. Since frequent loss of alleles on chromosome 22 from both 
sporadic vestibular schwannomas and meningiomas, and from their 
counterparts in NF2 had been noted previously, the localization of the 
inherited defect to the same chromosome region suggested that the NF2 
locus encodes a recessive tumor suppressor gene (Knudson, A. G. et al., 
Proc. Natl. Acad. Sci. U.S.A. 68:820-823 (1971)) whose inactivation leads 
to tumor formation (Seizinger, B. R. et al., Nature 322:644-647 (1986); 
Seizinger, B. R. et al., Science 236:317-319 (1987); Seizinger, B. R. et 
al., Proc. Natl. Acad. Sci. U.S.A. 84:5419-5423 (1987). 
A number of studies of sporadic tumors and tumors from NF2 patients have 
provided support for this hypothesis (Couturier, J. et al., Cancer Genet. 
Cytogenet. 45:55-62 (1990); Rouleau, G. A. et al., Am. J. Hum. Genet. 
46:323-328 (1990); Fiedler, W. et al., Genomics 10:786-791 (1991); 
Fontaine, B. et al., Ann. Neurol. 29:183-196 (1991); Fontaine, B. et al., 
Genomics 10:280-283 (1991); Bijlsma, E. K. et al., Genes Chromosom. Cancer 
5:201-205 (1992); Wolff, R. K. et al., Am. J. Hum. Genet. 51:478-485 
(1992)). The combined use of family studies and tumor deletion mapping has 
progressively narrowed the location of NF2 within the q12 band of 
chromosome 22, and defined a candidate region in which to search for the 
NF2 genetic defect (Rouleau, G. A. et al., Am. J. Hum. Genet. 46:323-328 
(1990); Wolff, R. K. et al., Am. J. Hum. Genet. 51:478-485 (1992)).

SUMMARY OF THE INVENTION 
The invention is directed to the protein merlin, mutants thereof, nucleic 
acid encoding this protein, nucleic acid encoding merlin regulatory 
regions and exons, mutant nucleic acid sequences, and uses thereof. 
Accordingly, in a first embodiment, the invention is directed to purified 
preparations of the protein merlin, or mutants thereof. 
In a further embodiment, the invention is directed to an isolated nucleic 
acid sequence encoding merlin, or mutants thereof. 
In a further embodiment, the invention is directed to a recombinant 
construct containing nucleic acid encoding merlin, or mutants thereof. 
In a further embodiment, the invention is directed to a vector containing 
nucleic acid encoding merlin, or mutants thereof. 
In a further embodiment, the invention is directed to a host transformed 
with the vector. 
In a further embodiment, the invention is directed to a method for 
producing merlin from the recombinant host. 
In a further embodiment, the invention is directed to a method for 
diagnosing a merlin-associated tumor, such tumor being a tumor 
characterized by a loss, alteration, or decrease of the activity of the 
merlin tumor suppressor in the cells of said tumor, and especially by a 
loss or mutation of the merlin gene, in such cells. in one specific 
embodiment, the mutation is a change from A.fwdarw.T in the first position 
(base) of the amino acid at position 220 according to FIG. 3, especially 
wherein tyrosine is substituted for asparagine at amino acid position 220. 
The nucleic acid change can be detected by RsaI. In more general 
embodiments, the mutations include the genetic sequence alterations in the 
merlin gene, described in Example 6 herein, and which contribute to tumor 
formation and especially NF2. 
In a further embodiment, the invention is directed to a method for treating 
a merlin-associated tumor in a patient, where the growth of such tumor 
reflects a functional change in merlin, a decreased level, or lack of 
merlin tumor suppressor activity in the tumor cell, the method comprising 
providing a functional merlin gene to the tumor cells of the patient, in a 
manner that permits the expression of the merlin protein provided by the 
gene, for a time and in a quantity sufficient to inhibit the growth of the 
tumor in the patient. 
In a further embodiment, the invention is directed to a method of gene 
therapy of a symptomatic or presymptomatic patient, the method comprising 
providing a functional merlin gene to the relevant cells of the patient, 
both normal or tumor, in need of the therapy, in a manner that permits the 
expression of the merlin protein provided by the gene, for a time and in a 
quantity sufficient to provide the tumor suppressor function of merlin to 
the cells of the patient. 
In a further embodiment, the invention is directed to a method for 
diagnosing NF2, the method comprising detecting a mutation in, or loss of, 
the merlin gene, or merlin protein, in a sample of non-tumor biological 
material from the subject to be diagnosed. This includes but is not 
limited to patients and single cells, such as embryonic cells or pre-natal 
cells from amniotic fluid. In a specific embodiment, the detection is of a 
mutant merlin protein encoded by a mutation of A.fwdarw.T at the first 
position (base) of amino acid 220, especially where tyrosine has been 
substituted for asparagine at amino acid position 220, or of a mutated 
nucleotide coding sequence involving an A.fwdarw.T transversion at 
position the first base of amino acid 220, detectable by RsaI digestion. 
In a more general embodiment, the detection is of a mutant merlin protein 
encoded by DNA containing mutations including those described in Example 6 
herein and which produce an altered merlin protein. 
In a further embodiment, the invention is directed to a method for 
screening an individual for future likelihood of developing 
merlin-associated tumors, or the disease NF2, the method comprising 
detecting a mutation in, or loss of, the merlin gene, or merlin protein, 
in a sample of biological material from the individual. In a specific 
embodiment, the detection is of a mutant merlin protein encoded by a 
mutation of A.fwdarw.T at the first position (base) of amino acid 220, 
especially where tyrosine has been substituted for asparagine at amino 
acid position 220, or of a mutated nucleotide coding sequence involving an 
A.fwdarw.T transversion at the first base of amino acid 220, detectable by 
RsaI digestion. In more general embodiments, the detection is of a mutant 
merlin protein or mutant merlin DNA, wherein the mutation includes but is 
not limited to any of the mutations described herein, and that are 
clinically relevant. 
In a further embodiment, the invention is directed to a method for treating 
NF2 in a patient, the method comprising providing a functional merlin gene 
to the desired cells of the patient, in a manner that permits the 
expression of the merlin protein provided by the gene, for a time and in a 
quantity sufficient to treat the patient. 
In a preferred embodiment, a method is provided for identifying DNA 
sequence differences representing potential mutations within amplified 
coding sequences using single stranded conformational polymorphism 
analysis (SSCP). In this method, individual exons from the patient's DNA 
are first amplified by PCR. The amplification products are then denatured 
to separate the complementary strands and diluted to allow each 
single-stranded DNA molecule to assume a secondary structure conformation 
by folding on itself. Gel electrophoresis under non-denaturing conditions 
allows the detection of sequence changes compared to the normal 
(non-mutant) sequence. 
In a further preferred embodiment, mutations discovered by application of 
the above method are used as standards of comparison for DNA from 
individuals suspected of being affected or known to be affected, so as to 
identify the mutation (if any) without full application of the above 
method. Knowledge of the exact mutation then allows the design of 
molecular therapeutic vehicles for gene therapy. 
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
In the following description, reference will be made to various 
methodologies known to those of skill in the art of molecular genetics and 
biology. Publications and other materials setting forth these known 
methodologies to which reference is made are incorporated herein by 
reference in their entireties as though set forth in full. 
The "merlin" gene described herein is a gene found on human chromosome 22 
that, as shown herein, contains non-overlapping and interstitial deletions 
in four independent NF2 patients and a single base change in affected 
individuals from five sibling pairs within an affected kindred; these 
results form the basis for concluding that this gene encodes a protein 
called "merlin," which possesses a tumor suppressor activity. merlin has 
previously been termed "the NF2 tumor suppressor." The merlin gene, 
therefore, includes the DNA sequences shown in FIGS. 3 and 15A-Q herein 
and all functional equivalents. The gene includes not only the coding 
sequences, but all regulatory regions specifically controlling the 
expression of the merlin coding sequence, including promoter, enhancer, 
and terminator regions. In addition, introns and other DNA sequences which 
are spliced from the final merlin RNA transcript are also considered part 
of the merlin gene as encompassed herein. For example, the merlin gene 
encompasses the exonic and intronic sequences shown in FIGS. 15A-Q. 
Additionally, it is to be understood that the merlin gene includes the 
corresponding genetic and functional sequences in non-human animal 
species. 
The merlin gene of the invention encodes a novel protein, merlin, that is 
related to the moesin-ezrin-radixin family of cytoskeleton-associated 
proteins (Gould, K. L. et al., EMBO J. 8:4133-4142 (1989); Turunen, O. et 
al., J. Biol. Chem. 264:16727-16732 (1989); Funayama, N. et al., J. Cell 
Biol. 115:1039-1048 (1991); Lankes, W. T. et al., Proc. Natl. Acad. Sci. 
U.S.A. 88:8297-8301 (1991); Sato, N. et al., J. Cell. Sci. 103:131-143 
(1992)). This protein, which is herein named "merlin" 
(moesin-ezrin-radixin-like protein), represents a new class of tumor 
suppressor whose function is mediated by interactions with the 
cytoskeleton. Merlin is found on human chromosome 22 between the known 
markers D22S1 and D22S28. 
I. cloning of Merlin DNA And Expression Of Merlin Protein 
The identification of merlin cDNA and protein as the mutated or missing 
gene in tumors from NF2 patients is exemplified below. In addition to 
utilizing the exemplified methods and results for the identification of 
additional mutations or deletions of the merlin gene in NF2 patients, and 
for the isolation of the native human merlin gene, the sequence 
information presented in FIGS. 3 and 15A-Q represents nucleic acid and 
protein sequences, that, when inserted into a linear or circular 
recombinant nucleic acid construct such as a vector, and used to transform 
a host cell, will provide copies of merlin DNA and merlin protein that are 
useful sources for the merlin DNA and merlin protein for the methods of 
the invention. Such methods are known in the art and are briefly outlined 
below. 
The process for genetically engineering the merlin coding sequence, for 
expression under a desired promoter, is facilitated through the cloning of 
genetic sequences which are capable of encoding the merlin protein. These 
cloning technologies can utilize techniques known in the art for 
construction of a DNA sequence encoding the merlin protein, such as 
polymerase chain reaction technologies utilizing the merlin sequence 
disclosed herein to isolate the merlin gene de novo, or polynucleotide 
synthesis methods to construct the nucleotide sequence using chemical 
methods. Expression of the cloned merlin DNA provides merlin protein. 
As used herein, the term "genetic sequences" is intended to refer to a 
nucleic acid molecule (preferably DNA). Genetic sequences that are capable 
of being operably linked to DNA encoding merlin protein, so as to provide 
for its expression and maintenance in a host cell, are obtained from a 
variety of sources, including commercial sources, genomic DNA, cDNA, 
synthetic DNA, and combinations thereof. Since the genetic code is 
universal, it is to be expected that any DNA encoding the merlin amino 
acid sequence of the invention will be useful to express merlin protein in 
any host, including prokaryotic (bacterial) and eukaryotic (plants, 
mammals (especially human), insects, yeast, and especially cultured cell 
populations). 
If it is desired to select a gene encoding merlin de novo from a library 
that is thought to contain a merlin gene, the library can be screened and 
the desired gene sequence identified by any means which specifically 
selects for a sequence coding for the merlin gene or expressed merlin 
protein such as, a) by hybridization (under stringent conditions for 
DNA:DNA hybridization) with an appropriate merlin DNA probe(s) containing 
a sequence specific for the DNA of this protein, the sequence being that 
provided in FIG. 3 or a functional derivative thereof (that is, a 
shortened form that is of sufficient length to identify a clone containing 
the merlin gene), or b) by hybridization-selected translational analysis 
in which native merlin mRNA which hybridizes to the clone in question is 
translated in vitro and the translation products are further characterized 
for the presence of a biological activity of merlin, or, c) by 
immunoprecipitation of a translated merlin protein product from the host 
expressing the merlin protein. 
When a human allde does not encode the identical sequence to that of FIGS. 
3 or 15A-Q, it can be isolated and identified as being merlin DNA using 
the same techniques used herein. Many polymorphic probes useful in the 
fine localization of genes on chromosome 22 are known and available (see, 
for example, "ATCC/NIH Repository Catalogue of Human and Mouse DNA Probes 
and Libraries," fifth edition, 1991, pages 23-24; "Human gene mapping 10: 
tenth international workshop on human gene mapping," Cytogenet. Cell Genet 
51:1-1148 (1989) and Rouleau, G. A. et al., "A genetic linkage map of the 
long arm of human chromosome 22," Genomics 4:1-6 (1989)). 
A useful D22S1 probe is clone designation pMS3-18, a BglII-RFLP (allele 1: 
8.2 kb; allele 2: 3.6 kb); as described in Fontaine, B. et al., Ann. 
Neurol. 29:183-186 (1991) and Barker et al., Cell 36:131-138 (1984). An 
equivalent D22S1 probe, pEDF139, is available from the ATCC (ATCC 59688 
and ATCC 59689). 
Other useful probes include: D22S28 (clone W23C: ATCC 61636 and ATCC 
61637); D22S15; D22S32 (plasmid pEZF31: ATCC 50274 and ATCC 59275); 
D22S42; D22S46; D22S56, LIF (the leukemia inhibitory factor gene); and 
NEFH (the neurofilament heavy chain gene). 
Human chromosome 22-specific libraries are known in the art and available 
from the ATCC for the isolation of probes ("ATCC/NIH Repository Catalogue 
of Human and Mouse DNA Probes and Libraries," fifth edition, 1991, pages 
72-73). Especially, LL22NS01, (ATCC 57714; Budarf et al., Genomics 
10:996-1002 (1991)) is useful for these purposes (Frazer, K. A. et al., 
Genomics 14:574-584 (1992)). 
It is not necessary to utilize the exact vector constructs exemplified in 
the invention; equivalent vectors can be constructed using techniques 
known in the art. For example, the sequence of the NEFH probe on plasmid 
pJL215 is published (see FIG. 3 in Lees, J. L. et al., EMBO J. 7:1947-1955 
(1988)). Since it is this sequence that provides the specificity for the 
NEFH gene, it is only necessary that a desired probe contain this 
sequence, or a portion thereof sufficient to provide a positive indication 
of the presence of the NEFH gene. 
Merlin genomic DNA may or may not include naturally occurring introns. 
Moreover, the genomic DNA can be obtained in association with the native 
merlin 5' promoter region of the gene sequences and/or with the native 
merlin 3' transcriptional termination region. 
Merlin genomic DNA can also be obtained in association with the genetic 
sequences which encode the 5' non-translated region of the merlin mRNA 
and/or with the genetic sequences which encode the merlin 3' 
non-translated region. To the extent that a host cell can recognize the 
transcriptional and/or translational regulatory signals associated with 
the expression of merlin mRNA and protein, then the 5' and/or 3' 
non-transcribed regions of the native merlin gene, and/or, the 5' and/or 
3' non-translated regions of the merlin mRNA can be retained and employed 
for transcriptional and translational regulation. 
Genomic DNA can be extracted and purified from any host cell, especially a 
human host cell possessing the long arm of chromosome 22, by means well 
known in the art. Genomic DNA can be shortened by means known in the art, 
such as physical shearing or restriction enzyme digestion, to isolate the 
desired merlin gene from a chromosomal region that otherwise would contain 
more information than necessary for the utilization of the merlin gene in 
the hosts of the invention. For example, restriction digestion can be 
utilized to cleave the full-length sequence at a desired location. 
Alternatively, or in addition, nucleases that cleave from the 3'-end of a 
DNA molecule can be used to digest a certain sequence to a shortened form, 
the desired length then being identified and purified by polymerase chain 
reaction technologies, gel electrophoresis, and DNA sequencing. Such 
nucleases include, but are not limited to, Exonuclease III and Bal31. 
Other nucleases are well known in the art. 
Alternatively, if it is known that a certain host cell population expresses 
merlin protein, then cDNA techniques known in the art can be utilized to 
synthesize a cDNA copy of the merlin mRNA present in such population. 
For cloning the genomic or cDNA nucleic acid that encodes the amino acid 
sequence of the merlin protein into a vector, the DNA preparation can be 
ligated into an appropriate vector. The DNA sequence encoding merlin 
protein can be inserted into a DNA vector in accordance with conventional 
techniques, including blunt-ending or staggered-ending termini for 
ligation, restriction enzyme digestion to provide appropriate termini, 
filling in of cohesive ends as appropriate, alkaline phosphatase treatment 
to avoid undesirable joining, and ligation with appropriate ligases. 
Techniques for such manipulations are well known in the art. 
When the merlin DNA coding sequence and an operably linked promoter are 
introduced into a recipient eukaryotic cell (preferably a human host cell) 
as a non-replicating, non-integrating, molecule, the expression of the 
encoded merlin protein can occur through the transient (nonstable) 
expression of the introduced sequence. 
Preferably the coding sequence is introduced on a DNA molecule, such as a 
closed circular or linear molecule that is capable of autonomous 
replication. If integration into the host chromosome is desired, it is 
preferable to use a linear molecule. If stable maintenance of the merlin 
gene is desired on an extrachromosomal element, then it is preferable to 
use a circular plasmid form, with the appropriate plasmid element for 
autonomous replication in the desired host. 
The desired gene construct, providing a gene coding for the merlin protein, 
and the necessary regulatory elements operably linked thereto, can be 
introduced into desired host cells by transformation, transfection, or any 
method capable of providing the construct to the host cell. A marker gene 
for the detection of a host cell that has accepted the merlin DNA can be 
on the same vector as the merlin DNA or on a separate construct for 
co-transformation with the merlin coding sequence construct into the host 
cell. The nature of the vector will depend on the host organism. 
Suitable selection markers will depend upon the host cell. For example, the 
marker can provide biocide resistance, e.g., resistance to antibiotics, or 
heavy metals, such as copper, or the like. 
Factors of importance in selecting a particular plasmid or viral vector 
include: the ease with which recipient cells that contain the vector can 
be recognized and selected from those recipient cells which do not contain 
the vector; the number of copies of the vector which are desired in a 
particular host; and whether it is desirable to be able to "shuttle" the 
vector between host cells of different species. 
When it is desired to use S. cerevisiae as a host for a shuttle vector, 
preferred S. cerevisiae yeast plasmids include those containing the 
2-micron circle, etc., or their derivatives. Such plasmids are well known 
in the art and are commercially available. 
Oligonucleotide probes specific for the merlin sequence can be used to 
identify clones to merlin and can be designed de novo from the knowledge 
of the amino acid sequence of the protein as provided herein in FIG. 3 or 
from the knowledge of the nucleic acid sequence of the DNA encoding such 
protein as provided herein in FIGS. 3 and 15A-15Q or of a related protein. 
Alternatively, antibodies can be raised against the merlin protein and 
used to identify the presence of unique protein determinants in 
transformants that express the desired cloned protein. 
A nucleic acid molecule, such as DNA, is said to be "capable of expressing" 
a merlin protein if that nucleic acid contains expression control 
sequences which contain transcriptional regulatory information and such 
sequences are "operably linked" to the merlin nucleotide sequence which 
encode the merlin polypeptide. 
An operable linkage is a linkage in which a sequence is connected to a 
regulatory sequence (or sequences) in such a way as to place expression of 
the sequence under the influence or control of the regulatory sequence. If 
the two DNA sequences are a coding sequence and a promoter region sequence 
linked to the 5' end of the coding sequence, they are operably linked if 
induction of promoter function results in the transcription of mRNA 
encoding the desired protein and if the nature of the linkage between the 
two DNA sequences does not (1) result in the introduction of a frame-shift 
mutation, (2) interfere with the ability of the expression regulatory 
sequences to direct the expression of the protein, antisense RNA, or (3) 
interfere with the ability of the DNA template to be transcribed. Thus, a 
promoter region is operably linked to a DNA sequence if the promoter is 
capable of effecting transcription of that DNA sequence. 
The precise nature of the regulatory regions needed for gene expression can 
vary between species or cell types, but includes, as necessary, 5' 
non-transcribing and 5' non-translating (non-coding) sequences involved 
with initiation of transcription and translation respectively, such as the 
TATA box, capping sequence, CAAT sequence, and the like, with those 
elements necessary for the promoter sequence being provided by the 
promoters of the invention. Such transcriptional control sequences can 
also include enhancer sequences or upstream activator sequences, as 
desired. 
The vectors of the invention can further comprise other operably linked 
regulatory elements sucregulatory elements such as DNA elements which 
confer antibiotic resistance, or origins of replication for maintenance of 
the vector in one or more host cells. 
In another embodiment, especially for maintenance of the vectors of the 
invention in prokaryotic cells, or in yeast S. cerevisiae cells, the 
introduced sequence is incorporated into a plasmid or viral vector capable 
of autonomous replication in the recipient host. Any of a wide variety of 
vectors can be employed for this purpose. In Bacillus hosts, integration 
of the desired DNA may be necessary. 
Expression of a protein in eukaryotic hosts such as a human cell requires 
the use of regulatory regions functional in such hosts. A wide variety of 
transcriptional and translational regulatory sequences can be employed, 
depending upon the nature of the host. Preferably, these regulatory 
signals are associated in their native state with a particular gene which 
is capable of a high level of expression in the specific host cell, such 
as a specific human tissue type. In eukaryotes, where transcription is not 
linked to translation, such control regions may or may not provide an 
initiator methionine (AUG) codon, depending on whether the cloned sequence 
contains such a methionine. Such regions will, in general, include a 
promoter region sufficient to direct the initiation of RNA synthesis in 
the host cell. 
If desired, the non-transcribed and/or non-translated regions 3' to the 
sequence coding for the merlin protein can be obtained by the 
above-described cloning methods. The 3'-non-transcribed region of the 
native human merlin gene can be retained for its transcriptional 
termination regulatory sequence elements, or for those elements which 
direct polyadenylation in eukaryotic cells. Where the native expression 
control sequences signals do not function satisfactorily in a host cell, 
sequences functional in the host cell can be substituted. 
It may be desired to construct a fusion product that contains a partial 
coding sequence (usually at the amino terminal end) of a first protein or 
small peptide and a second coding sequence (partial or complete) of the 
merlin protein at the carboxyl end. The coding sequence of the first 
protein can, for example, function as a signal sequence for secretion of 
the merlin protein from the host cell. Such first protein can also provide 
for tissue targeting or localization of the merlin protein if it is to be 
made in one cell type in a multicellular organism and delivered to another 
cell type in the same organism. Such fusion protein sequences can be 
designed with or without specific protease sites such that a desired 
peptide sequence is amenable to subsequent removal. 
The expressed merlin protein can be isolated and purified from the medium 
of the host in accordance with conventional conditions, such as 
extraction, precipitation, chromatography, affinity chromatography, 
electrophoresis, or the like. For example, affinity purification with 
anti-merlin antibody can be used. A protein having the amino acid sequence 
shown in FIG. 3 can be made, or a shortened peptide of this sequence can 
be made, and used to raised antibodies using methods well known in the 
art. These antibodies can be used to affinity purify or quantitate merlin 
protein from any desired source. 
If it is necessary to extract merlin protein from the intracellular regions 
of the host cells, the host cells can be collected by centrifugation, or 
with suitable buffers, lysed, and the protein isolated by column 
chromatography, for example, on DEAE-cellulose, phosphocellulose, 
polyribocytidylic acid-agarose, hydroxyapatite or by electrophoresis or 
immunoprecipitation. 
It is to be understood that all of the above procedures that are applicable 
to cloning and expressing merlin sequences apply equally to normal and 
mutant sequences. Mutations may be in any of the regions, i.e., coding, 
non-coding, exonic, intronic, regulatory and the like. 
II. Characterization of Mutations in An Affected Individual 
The definition of the sequence alteration predicted to change the sequence 
of the merlin protein in the extended NF2 family originally employed to 
map the genetic defect on chromosome 22 is exemplified below (Wertelecki, 
W. et al., New Engl. J. Med. 319:278-283 (1988); Narod, S. A. et al., Am. 
J. Hum. Genet. 51:486-496 (1992)). The characterization of this mutation 
allows the early detection of the disorder in "at risk" family members 
before clinical symptoms appear. Although the exemplary material is 
directed to the detection of the disorder in a specific kindred, the 
exemplary methods can be applied to any family to define the precise 
molecular lesion underlying NF2, and this information can then be used to 
accurately determine whether at-risk members of the family have inherited 
the disorder. 
Further, this information can be used to assess any given individual for 
the merlin genotype in that individual. Therefore, affected or other 
(e.g., carrier) individuals can be assayed for the presence of new 
mutations as well as for the presence of mutations that are previously 
defined. Accordingly, the exemplary material shows a preferred approach to 
isolating and characterizing the mutations associated with NF2 or other 
merlin-dependent pathologies. 
New mutations in any desired kindred or affected individual or individual 
suspected of being affected can be defined using single-strand 
conformational polymorphism (SSCP) and sequence analysis of DNA amplified 
from the NF2 gene merlin. DNA alterations in the merlin coding sequence 
cause a shift on SSCP gels that is characteristic of the disease 
chromosome being transmitted with the disorder and present in only 
affected members of the pedigree. For example, as described below, an 
A.fwdarw.T transversion causes substitution of a tyrosine for an 
asparagine at position 220 of the merlin protein, in a region highly 
conserved in closely related members of the family of 
cytoskeletal-associated proteins. This alteration caused a shift on SSCP 
gels that was characteristic of the disease chromosome in this NF2 
pedigree, being transmitted with the disorder, present only in affected 
members of the pedigree, absent in unaffected members of the family and 
absent from 158 unrelated individuals. Because of this identification, it 
is now possible to significantly alter the management of "at risk" members 
of this extended kindred, based on a precise definition of which 
individuals carry the disease gene, and which have escaped inheritance of 
the defect. A similar approach is applicable to defining the underlying 
lesion/DNA defect and thereby improving presymptomatic or prenatal 
diagnosis in any other NF2 family that does not have this specific 
mutation. As shown below in the exemplary material, an array of various 
mutations have been associated with the merlin locus. Accordingly, any of 
these relevant mutations may be used for purposes of diagnosis in specific 
kindreds or individuals. 
Accordingly, a combination of amplification, for example using the 
polymerase chain reaction (PCR) with SSCP, permits the identification of 
other mutations in a merlin gene as simple as single base changes. Any 
regions of the merlin gene can be assessed by this procedure: The sequence 
now made available by the inventors allows the design of primers and 
amplification of any desired region including coding, intronic, and 
regulatory non-coding or non-transcribed. 
The SSCP technique uses strategically placed primer sets to amplify small 
regions of the NF2 gene directly from genomic DNA using PCR. The 
double-stranded PCR product containing the amplified region can then be 
used as a template to generate single strands by priming multiple rounds 
of DNA synthesis with one of the oligonucleotides previously used in a 
double strand reaction. The amplified products can be used for SSCP 
analysis, cloned, and sequenced. 
The amplification products, denatured to DNparate the complementary DNA 
strands, are diluted to allow each single-stranded DNA molecule to assume 
a secondary structure conformation by folding on itself. The 
single-stranded molecules are then subjected to electrophoresis, for 
example by polyacrylamide, under non-denaturing conditions. The secondary 
structure, which is highly dependent on the precise DNA sequence, affects 
mobility of the strand on the gel. Even a single base change or deletion 
can produce a visible shift in the final band position on the gel. Altered 
SSCP patterns thus can be analyzed based on a comparison with the SSCP 
patterns from affected family members, unrelated individuals, sporadic 
tumors, et cetera. The altered SSCP fragment pattern can then be 
correlated with inheritance of the disorder and associated with affected 
members of the pedigree. 
Following SSCP analysis, the precise DNA alteration that causes the 
shifting mobility pattern can be ascertained by direct DNA sequencing of 
the PCR amplification product from which the altered single-stranded 
molecule was derived. 
After a specific mutation is confirmed following PCR and SSCP analysis and 
direct sequencing, the presence of this mutation can be rapidly confirmed 
for any desired member of the kindred or otherwise by performing the same 
type of analysis. 
Alternatively, an approach may be taken exploiting restriction enzyme 
digestion following PCR amplification in the area of the defined mutation. 
If the defined mutation creates a new restriction site, the presence of 
this mutation can be rapidly confirmed in other members of the kindred or 
otherwise by using primers in the area of mutation to amplify sequences 
that include the mutation and which, when subjected to restriction enzyme 
digestion, create restriction fragments that are specific to affected 
individuals and absent in non-affected individuals. Thus, following PCR, 
the fragments from affected individuals may be subjected to 
electrophoresis and merely stained by ethidium bromide without the resort 
to cumbersome radiolabeling or other labeling techniques. 
In a specific disclosed embodiment, the genomic DNA of patients and 
unaffected control individuals was amplified using primers in the intron 
flanking exon 7. SSCP analysis was then performed. The amplified products 
were also cloned and sequenced. Intron DNA sequence flanking each exon was 
determined. DNA primers that permit amplification of the exon sequences 
from genomic DNA using PCR were developed. A mobility shift was detected 
in the amplification products of exon 7 comprising base pair 819 to 894 of 
the merlin coding sequence. DNA sequencing was used to confirm the precise 
DNA alteration that caused the shifted mobility pattern in the SSCP 
analysis. 
Normal merlin DNA displays an AAC codon encoding asparagine at position 220 
of the merlin protein. The DNA with the shifted mobility reveals both an A 
and a T residue at the first position of this codon, suggesting a normal 
AAC codon on one chromosome and a mutated TAC on the other. The TAC would 
substitute a tyrosine at position 220. 
The sequence change created a GTAC stretch that can be recognized by the 
restriction endonuclease RsaI. The site created nine base pairs 3' to a 
preexisting RsaI site in this exon. Rsa digests on the amplified PCR 
product confirm the presence of the same DNA change in other affected 
members of the family, its cotransmission with NF2, and its absence in 
unaffected members of the pedigree. Unaffected members display two RsaI 
fragments of 96 base pairs and 76 base pairs, whereas the affected 
individuals produce an additional third fragment of 67 base pairs 
generated from the Rsa site created by the A.fwdarw.T change at codon 220. 
Accordingly, in one embodiment of the invention, this specific mutation 
could be identified in individuals having or suspected of having NF2 by 
simple assays, e.g., Southern blots, involving restriction enzyme 
digestion with RsaI. Similarly, if mutations in merlin create or abolish 
other restriction enzyme sites, this alteration can be exploited to 
recognize the mutation in individuals other than the one in whom the 
mutation was discovered by restriction enzyme cleavage assays. 
In specific embodiments of the invention, SSCP analysis was performed to 
scan all seventeen exons of the entire merlin gene for mutations. In 
schwannomas from NF2 patients, base changes, deletions, and insertions 
were observed at various locations which resulted in missense, frameshift, 
and possible splice donor and splice acceptor alterations. The NF2 gene 
was also examined in sporadic schwannomas. Deletions, base changes, and 
insertions were observed in various locations in both intron and exon 
sequences. These mutations created frameshift, nonsense, and missense 
mutations, as well as actual or presumed alterations in the splice 
acceptor site, splice donor site, or acceptor branch site. Accordingly, in 
specific embodiments of the invention, these mutations may be useful as 
standards of comparison for the examination of at-risk individuals. 
III. Use Of Merlin For Diagnostic And Treatment Putposes 
It is to be understood that although the following discussion is 
specifically directed to human patients, the teachings are also applicable 
to any animal that expresses merlin and in which loss or mutation of 
merlin leads to pathological manifestations as in the human patient. 
It is also to be understood that the methods referred to herein are 
applicable to any merlin-associated tumor or subject suspected of 
developing or having such a tumor, whether such tumor is sporadic or 
associated with a condition such as NF2. 
A "merlin-associated" tumor is a tumor characterized in that the growth of 
such tumor reflects a decrease, functional alteration, or lack of merlin 
activity, especially if the decrease, change, or lack of merlin activity 
reflects a mutation or loss of the merlin gene or its regulatory regions. 
An example of such a tumor would include, but is not limited to, a 
schwannoma, (such as, for example, a vestibular schwannoma, and especially 
a bilateral vestibular schwannoma, a schwannoma of a cranial nerve, 
especially on the vestibular branch of the eighth cranial nerve, or a 
schwannoma of a spinal nerve root), or a meningioma (such as, for example, 
a meningioma of a cranial nerve, a vestibular meningioma, or a meningioma 
of a spinal nerve root). 
The diagnostic and screening methods of the invention are especially useful 
for a person suspected of being at risk for developing merlin-associated 
tumor or disease and/or NF2 based on family history, or a person in which 
it is desired to diagnose or eliminate the presence of a merlin-associated 
condition or tumor or the NF2 condition as a causative agent behind a 
tumor growth. 
By "predisposition to develop a merlin-associated tumor" is intended a 
genotype wherein a subject has the tendency to develop a genotype leading 
to the expression of an aberrant merlin gene. This could involve a subject 
heterozygous for a merlin mutation who subsequently becomes homozygous for 
the mutation such that the mutation is now expressed. Such a 
predisposition can be detected by molecular assays capable of detecting 
mutations or molecular changes in DNA, RNA, or merlin protein. Thus, even 
though a subject may be heterozygous for the merlin mutation, the 
mutation, if expressed on the affected chromosome, could then be detected, 
or, alternatively, the mutation in the aberrant chromosome could be 
directly detected at the nucleic acid level. 
Patients suspected of having NF2 will generally first present with a 
diagnosis of NF2 made according to the criteria set forward by the 
National Institutes of Health Consensus Development Conference on 
Neurofibromatosis (Arch. Neurol. 45:575-578 (1988); Mulvihill, J. J. et 
al., Ann. Intern. Med. 113:39-52 (1990). Specifically, for NF2, the 
NIHCDCN diagnostic criteria are met if a person has either of the 
following: 
1) Bilateral eighth nerve masses seen with appropriate imaging techniques 
(for example, computerized tomographic or magnetic resonance imaging); 
2) A first-degree relative with NF2 and either unilateral eighth nerve mass 
or two of the following: neurofibroma, meningioma, glioma, schwannoma, and 
juvenile posterior subcapsular lenticular opacity. 
According to the invention, presymptomatic screening of an individual in 
need of such screening is now possible using DNA encoding the merlin 
protein of the invention, and specifically, DNA having the sequence of the 
native human merlin gene. The screening method of the invention allows a 
presymptomatic diagnosis, including prenatal diagnosis, of the presence of 
a missing or aberrant merlin gene in individuals, and thus an opinion 
concerning the likelihood that such individual would develop or has 
developed merlin-associated tumors and/or NF2. This is especially valuable 
for the identification of carriers of altered or missing merlin genes, for 
example, from individuals with a family history of merlin-associated 
tumors and/or NF2. This is also especially valuable for those patients 
where the chances of hearing preservation are optimal with early 
microsurgical removal of a vestibular schwannoma. Early diagnosis is also 
desired to maximize appropriate timely intervention as to any expected 
sequelae of the patient's tumor growth and lens opacities. 
For example, in the method of screening, a tissue sample would be taken 
from such individual, and screened for (1) the presence of the `normal` 
human merlin gene; (2) the presence of merlin mRNA and/or (3) the presence 
of merlin protein. The normal human gene can be characterized based upon, 
for example, detection of restriction digestion patterns in `normal` 
versus the patient's DNA, including RFLP analysis, using DNA probes 
prepared against the merlin sequence (or a functional fragment thereof) 
taught in the invention. Similarly, merlin mRNA can be characterized and 
compared to normal merlin mRNA (a) levels and/or (b) size as found in a 
human population not at risk of developing merlin-associated tumors and/or 
NF2 using similar probes. Lastly, merlin protein can be (a) detected 
and/or (b) quantitated using a biological assay for merlin activity (its 
ability to suppress tumor growth) or using an immunological assay and 
anti-merlin antibodies. When assaying merlin protein, the immunological 
assay is preferred for its speed. 
An (1) aberrant merlin DNA size pattern or sequence, and/or (2) aberrant 
merlin mRNA size, level, or sequence, and/or (3) aberrant merlin protein 
or level thereof would indicate that the patient is at risk for developing 
a merlin-associated tumor and/or NF2 and is likely to develop a 
merlin-associated tumor and/or NF2. 
Similarly, if the tissue sample was derived from a tumor taken from a 
patient suspected of having a merlin-associated tumor and/or NF2, then (1) 
aberrant merlin DNA size, pattern, or sequence, and/or (2) aberrant merlin 
mRNA size, sequence, or level and/or (3) aberrant merlin protein or levels 
thereof would indicate that the patient has developed a merlin-associated 
tumor and/or NF2. These tumors can be treated with the methods of the 
invention as described below. 
In accordance with the inventors' characterization of a specific merlin 
mutation in a particular kindred, and the guidance provided to extend the 
knowledge and approaches disclosed herein to the identification of other 
mutations and the identification of the mutation disclosed herein in other 
kindreds, preferred methods of screening tissue samples from 
presymptomatic, asymptomatic, or symptomatic individuals involve, rather 
than comparison with normal merlin protein, DNA, or RNA, direct detection 
of abnormal merlin genes and gene products. 
Accordingly, a preferred strategy for identifying DNA sequences 
representing potential mutations within amplified coding sequences is the 
use of SSCP analysis combined with amplification and DNA sequencing. 
Accordingly, screening individual exons from a subject's DNA that have 
been amplified by PCR is a first approach. SSCP followed by direct DNA 
sequencing is then performed. In more preferred embodiments, mutations 
previously identified using this protocol provide a standard for 
comparison of the tissue sample to be assayed. The sample is thus 
amplified using primers known to be adjacent to the mutation and the 
amplification product either subjected to SSCP or subjected to restriction 
enzyme analysis in the case wherein the mutation creates or abolishes 
restriction sites found in the normal merlin gene. 
Similarly, mutant mRNAs may also be the basis for assay ofa subject's RNA 
in Northern blots where the abnormal RNA has a characteristic pattern as 
in electrophoresis. Alternatively, cDNA transcripts from the RNA of a 
subject can be analyzed by comparing such transcripts to the transcripts 
from known mutant merlin genes. 
Alternatively, aberrant merlin proteins with characterized mutations may 
serve as the basis for comparison with proteins derived from the 
individual undergoing the diagnostic treatment. Thus, recognition by 
monoclonal or polyclonal antibodies using standard immunological assays 
may reveal the presence of mutated proteins that can be identified by 
comparison with previous mutations. Alternatively, methods of identifying 
mutant proteins using known mutants as comparisons include, but are not 
limited to, tryptic peptide digests. 
Accordingly, a repository of mutant DNA, RNA/cDNA and protein patterns 
gathered from an analysis of the NF2 mutations from various kindreds may 
serve as standards for rapid and accurate identification of affected cells 
or individuals. 
The screening and diagnostic methods of the invention do not require that 
the entire merlin DNA coding sequence be used as a probe. Rather, it is 
only necessary to use a fragment or length of nucleic acid that is 
sufficient to detect the presence of the merlin gene in a DNA preparation 
from a normal or affected individual, the absence of such gene, or an 
altered physical property of such gene (such as a change in 
electrophoretic migration pattern). 
Prenatal diagnosis can be performed when desired, using any known method to 
obtain fetal cells, including amniocentesis, chorionic villous sampling, 
and fetoscopy. Prenatal chromosome analysis can be used to determine if 
the portion of chromosome 22 possessing the normal merlin gene is present 
in a heterozygous state. 
The merlin DNA can be synthesized, and, if desired, labeled with a 
radioactive or nonradioactive reporter group, using techniques known in 
the art (for example, see Eckstein, F., ed., Oligonucleotides and 
Analogues: A Practical Approach, IRS Press at Oxford University Press, New 
York, 1992); and Kricka, L. J., ed., Nonisotopic DNA Probe Techniques, 
Academic Press, San Diego, (1992)). 
Although the method is specifically described for DNA-DNA probes, it is to 
be understood that RNA possessing the same sequence information as the DNA 
of the invention can be used when desired. 
In the method of treating NF2 in a patient in need of such treatment, 
functional merlin DNA is provided to the cells of such patient, especially 
the tumor cells, in a manner and amount that permits the expression of the 
merlin protein provided by such gene, for a time and in a quantity 
sufficient to treat such patient. Many vector systems are known in the art 
to provide such delivery to human patients in need of a gene or protein 
missing from the cell. For example, retrovirus systems can be used, 
especially modified retrovirus systems and especially herpes simplex virus 
systems, such as those described in U.S. application Ser. No. 07/913,977 
(filed Jul. 16, 1992); U.S. application Ser. No. 07/956,949 (filed Oct. 6, 
1992), U.S. application Ser. No. 07/895,364 (filed Jun. 9, 1992); each 
incorporated herein fully by reference. In addition, such methods are 
provided for, in, for example, the teachings of Breakefield, X. A. et al., 
The New Biologist 3:203-218 (1991); Huang, Q. et al., Experimental 
Neurology 115:303-316 (1992), WO93/03743 and WO90/09441 each incorporated 
herein fully by reference. 
Delivery of a DNA sequence encoding a functional merlin protein, such as 
the amino acid encoding sequence of FIGS. 3 and 15A-15Q, will effectively 
replace the missing or mutated merlin gene of the invention, and inhibit, 
and/or stop and/or regress tumor growth that arose due to the loss of the 
merlin tumor suppressor. 
This method is especially effective in the tumor types such as those 
classically associated with NF2, and especially with a schwannoma, (such 
as, for example, a bilateral vestibular schwannoma, a schwannoma of a 
cranial nerve, especially on the vestibular branch of the eighth cranial 
nerve, or a schwannoma of a spinal nerve root), meningiomas (such as, for 
example, a meningioma of a cranial nerve, a vestibular meningioma, or a 
meningioma of a spinal nerve root). 
The method of the invention is also useful to treat conditions such as 
posterior capsular lens opacities, deafness, balance disorder, paralysis 
or other neurological problem when such problem is due to the presence of 
a merlin-associated tumor or NF2 condition. 
The manner and method of carrying out the present invention can be more 
fully understood by those of skill by reference to the following examples, 
which examples are not intended in any manner to limit the scope of the 
present invention or of the claims directed thereto. 
EXAMPLES 
Experimental Procedure for Examples 1-4 
NF2 Cell Lines 
Lymphoblast cell lines were established (Anderson, M. A. et al., In Vitro 
20:856-858 (1984)) from affected members of NF2 pedigrees and from their 
unaffected relatives. Diagnosis of NF2 conformed to the criteria set 
forward by the National Institutes of Health Consensus Development 
Conference on Neurofibromatosis (Mulvihill, J. J. et al., Ann. Intern. 
Med. 113:39-52 (1990)), except for the patient whose meningioma displayed 
a 4 bp deletion. This patient had a right vestibular schwannoma, and 
multiple meningiomas. Although she did not have a history of NF2, she 
probably represents a new mutation. Primary meningioma cells were cultured 
as described (Logan, J. A. et al., Cancer Genet. Cytogenet. 45:41-47 
(1990)) and analyzed after less than five passages. 
Somatic cell hybrids were prepared by fusing GUS5069 lymphoblasts with a 
Chinese hamster cell line deficient in HPRT activity (CHTG49); Athwall, R. 
S. et al., Proc. Natl. Acad. Sci. U.S.A. 74:2943-2947 (1977)) using GIBCO 
PEG 4,000. Fused cell lines were selected by their ability to grow in 
media containing hypoxanthine, aminopterin and thymidine (HAT). Hybrids 
were screened for the chromosome 22 homologues using the polymorphic SSR 
marker, TOPIP2 (Trofatter, J. A. et al., Hum. Mol. Genet. 1:455 (1992)). 
Control hybrids GM10888 and Eye3FA6 (NA10027) are described in the listing 
the NIGMS Human Genetic Mutant Cell Repository collection (Coriell 
Institute, Camden, N.J.). 
DNA/RNA Blotting 
DNA was prepared from cultured cells and DNA blots prepared and hybridized 
as described (Gusella, J. F. et al., Proc. Natl. Acad. Sci. U.S.A. 
76:5239-5243 (1979); Gusella, J. F. et al., Nature 306:234-238 (1983)). 
For pulsed-field gel analysis, agarose DNA plug preparation, and 
electrophoresis were carried out as described (Bucan, M. et al., Genomics 
6:1-15 (1990)). RNA was prepared and Northern blotting performed as 
described in Buckler et al. Buckler, A. J. et al., Proc. Natl. Acad. Sci. 
U.S.A. 88:4005-4009 (1991). 
Cosmid Walking 
The NEFH probe used for blot analysis and to initiate cosmid walking was 
pJL215, representing a 4.4 kb KpnI/XbaI genomic fragment containing exon 4 
and 3'UTR (Lees, J. F. et al., EMBO J. 7:1947-1955 (1988)). The NEFH probe 
pJL215 was obtained from Dr. Greg Elder and Dr. Robert Lazzarini, The 
Laboratory of Molecular Genetics, National Institute of Neurological and 
Communicative Disorders and Stroke, National Institutes of Health, 
Bethesda, Md., 29892. Cosmid walking was performed in an arrayed cosmid 
library prepared from DNA of flow-sorted human chromosome 22 (LL22NC03); 
Dr. Pieter DeJong, Lawrence Livermore National Laboratory). Cosmid 
overlaps were identified by either hybridization of whole cosmid DNA or 
isolated fragments to filter replicas of the gridded arrays, or by PCR 
screening of row and column DNA pools. STSs were developed by direct 
cosmid sequencing using the T3 or T7 end-primers (McClatchey, A. I. et 
al., Hum. Mol. Genet. 1:521-527 (1992)). 
cDNA Isolation and Characterization 
Human frontal cortex and hippocampus cDNA libraries in lambdaZAPll 
(Stratagene) were screened using exon probes isolated and prepared as 
described by Buckler et al. (Buckler, A. J. et al., Proc. Natl. Acad. Sci. 
U.S.A. 88:4005-4009 (1991)). cDNA clones and trapped exon were sequenced 
as described (Sanger, T. et al., Proc. Natl. Acad. Sci. U.S.A. 
74:5463-5467 (1977)). Direct PCR sequencing was performed as described 
(McClatchey, A. I. et al., Cell 68:769-774 (1992)). Screening for 
variations by SSCP analysis followed the procedure described in Ambrose et 
al. (Ambrose, G. et al., Hum. Mol. Genet. 1:697-703 (1992)). RNA was 
reverse transcribed using an oligo(dT) primer (BRL reverse transcriptase) 
to prepare first strand cDNA. Portions of the cDNA were amplified using 
the following primer sets: 
5'CCAGCCAGCTCCCTATGGATG3' SEQ ID No: 11! and 
5'AGCTGAAATGGAATATCTGAAG3' SEQ ID No: 12! 
to amplify bp 824-2100 and 
5'GCCTTCTCCTCCCTGGCCTG3' SEQ ID No: 13! and 
5'GATGGAGTTCAATTGCGAGATG3' SEQ ID No: 14! 
to amplify bp 3 14-1207. These cold PCR products were then reamplified with 
specific regional primers for SSCP as described in the legend to FIGS. 6, 
6A and 6B. 
Example 1 
Scanning the NF2 Candidate Region for Rearrangement 
The region on chromosome 22 that was examined for the presence of the NF2 
gene was between D22S1 and D22S28; this region was estimated to encompass 
6 Mb of band q12 (Frazer, K. A. et al., Genomics 14:574-584 (1992)). 
By scanning the human chromosome 22 region between D22S1 and D22S28 for 
loss of DNA it was determined whether some germline NF2 mutations might 
involve a deletion of the tumor suppressor gene as has been found in 
Wilms' tumor and retinoblastoma (Riccardi, V. M. et al., Pediatrics 
61:604-610 (1978); Francke, U. et al., Cytogenet. Cell Genet. 24:185-192 
(1979); Dryja, T. P. et al., Proc. Natl. Acad. Sci. U.S.A. 83:7391-7394 
(1986)). Pulsed-field gel blots containing lymphoblast DNA from various 
NF2 patients were probed for several loci in the candidate region, 
including D22S1, D22S15, D22S28, D22S32, D22S42, D22S46, D22S56, LIF (the 
leukemia inhibitory factor gene) and NEFH (the neurofilament heavy chain 
gene). This analysis revealed that in a lymphoblast cell line (GUS5069) 
derived from a female NF2 patient, a probe for the NEFH locus hybridized 
to apparently altered fragments of reduced size with both NotI and NruI. 
In NotI-digested DNA (FIG. 1), the NEFH probe detected fragments of 
approximately 600 kb, 400 kb and 230 kb in most lymphoblast cell lines. It 
was not possible to confirm that the 600 kb fragment originated from 
chromosome 22. Thus, it was possible that hybridization had occurred with 
a related locus (Menon et al., unpublished results). Variable intensity of 
the 230 kb fragment in many samples suggested that it resulted from 
partial digestion of the 400 kb fragment. In GUS5069, additional fragments 
of approximately 370 kb and 200 kb were observed. These results are 
consistent with the possibility of a deletion within the region common to 
the 400 kb and 230 kb fragments. The alteration was transmitted along with 
NF2 from the patient to her affected daughter (represented by GUS5068) 
(FIG. 1). 
Example 2 
Chromosome Walking Toward the Merlin Gene 
To isolate DNA corresponding to the region of chromosome 22 apparently 
deleted in GUS5069, a bi-directional cosmid walk was initiated from NEFH. 
At each step, single restriction fragments of the cosraids were used as 
probes on pulsed-field gels to establish the location of the putalive 
deletion relative to NEFH. On the 5' side of NEFH, a NotI site that was 
rarely cleaved in lymphoblast DNA was identified. Probes beyond the NotI 
site detected the same approximately 400 kb NotI fragment along with a 170 
kb fragment of variable intensity. Thus, infrequent cleavage of this Notl 
site divides the 400 kb fragment into fragments of 230 kb and 170 kb. 
Since the putative deletion in GUS5069 affects the 230 kb fragment but not 
the 170 kb fragment, further experiments continued to walk only 3' of 
NEFH. Pulsed-field gel blots containing DNA from GUS5069 were again 
probed. 
The Notl pulsed-field gel map and a minimal set of clones representing the 
cosmid walk and the extent of the genomic deletion (see FIG. 5A) are shown 
in FIG. 2. The deletion was reached when a probe (FIG. 2, Probe A) was 
tested from cosmid 96C10 which failed to detect the altered Notl fragment 
in GUS5069. However, various probes from cosmid 28H6 and 121G10 did detect 
the altered fragment. 
To estimate the extent of deletion, probes B and C (FIG. 2) were tested. 
Probe B is an 8 kb HindIII fragment from 28H6 which overlaps with the T3 
end of 96C10. Probe C is a 9 kb HindllI fragment from the T7 end of 96C10. 
Probe B detected both the normal and the altered Notl PFG fragment, but 
probe C detected only the normal fragment. 
For more precise analysis of the deletion, the altered chromosome 22 from 
GUS5069 was segregated from its normal counterpart in human X hamster 
somatic cell hybrids. STS assays for the T3 and T7 ends of 96C10 were 
created and hybrids containing the separated chromosomes 22 were tested. 
In contrast to the above hybridization results, the T3 end of 96C10 was 
absent in hybrid GUSH134A3, containing the deleted chromosome but present 
in GUSH134A10 containing the normal chromosome. Moreover, the T7 assay was 
positive in both hybrids. The locations of probes B and C, and of both STS 
assays were confirmed on the cosmid walk. Thus, the failure of probe C to 
detect the altered fragment suggests that the deletion spans most but not 
all of this sequence. Similarly, the other deletion breakpoint must occur 
within the region spanned by probe B. Therefore, the results of 
hybridization and PCR indicate that the deletion must encompass almost all 
of 96C10 and up to an additional 5 kb of 28H6. This 35-45 kb region is 
expanded below the cosmid walk in FIG. 2. 
Example 3 
Identification and Characterization of the merlin cDNA 
Exon amplification (Buckler, A. J. et al., Proc. Natl. Acad. Sci. U.S.A. 
88:4005-4009 (1991)), which produces cloned "trapped exons," was applied 
to cosmids 28H6, 96C10, 121G10, 123F5, 10H11, and 7C4 surrounding the site 
of the NF2 deletion as a rapid method of obtaining exonic probes for cDNA 
cloning. Each exon clone can represent a single exon, or multiple exons 
spliced together in the trapping procedure. Twenty-four exon clones were 
obtained and sequenced, 6 of which displayed sequence similarity with the 
cytoskeleton-associated proteins moesin, ezrin and radixin (see below). 
The latter exons were used to screen human frontal cortex and hippocampus 
cDNA libraries. 
FIG. 3 shows the complete DNA sequence of JJR-1, the longest clone obtained 
in cDNA screening. JJR-1 has been deposited with the American Type Culture 
Collection and assigned ATCC75509. This sequence contains eight of the 
cloned exon segments as shown in FIG. 5. The cDNA is 2,257 bp long and 
shows no evidence of a poly(A) tail. However, two shorter cDNA clones, 
JJR-6 and JJR-9, which overlapped the restriction map of JJR-1, had 
apparent poly(A) tails beginning at base 2231. JJR-1 contains an open 
reading frame of 1785 bp, encoding a predicted protein of 69 kD. There is 
one in-frame stop codon within 90 bp upstream of the putative initiator 
methionine. The JJR-1 cDNA spans at least 50 kb of genomic DNA, and is 
transcribed in the same orientation as NEFH as shown by the arrows in FIG. 
2. 
Both the JJR-1 DNA sequence and the predicted protein product were used to 
search for similarity in nucleic acid and protein databases using the 
BLAST network service of the National Center for Biotechnology Information 
(Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990)). The DNA 
sequence displayed significant similarity to moesin and ezrin genes from 
several species, including man (P=9.0e.sup.-125 and 9.0e.sup.-122), to 
mouse radixin (1.1e.sup.-102) and to Echinococcus multilocularis tegument 
protein (2.4e.sup.-21). Striking similarity was also detected at the amino 
acid level with these same proteins (2.5e.sup.-146, 5.0e.sup.-146, 
2.7e.sup.-145 and 7.6.sup.-73, respectively) and to a potential product of 
a sequence tag from Caenorhabditis elegans (3.7e.sup.-43). Weaker 
similarities were detected to the sequences of two protel n tyrosine 
phosphatases, PTP-MEG and PTP-H1 (1.3e.sup.-17 and 9.6.sup.-16, 
respectively), to erythrocyte protein 4.1 (9.9e.sup.-14) and to a wide 
range of myosin, tropomyosin and paramyosin proteins. Because this novel 
gene is most closely related to moesin, ezrin and radixin (45-47% 
identity), it is called "merlin." 
Northern blot analysis using total RNA from various cultured human tumor 
cell lines (FIG. 4) revealed two major hybridizing species of 2.6 kb and 7 
kb, and a less intensely hybridizing RNA of 4.4 kb. A similar pattern was 
detected in poly(A)+RNA from various human tissues, including heart, 
brain, lung, skeletal muscle, kidney, pancreas and weakly in liver 
indicating that the merlin gene is expressed widely. The apparent poly(A) 
tails detected in JJR-6 and JJR-9 suggest that these clones may have 
derived from the approximately 2.6 kb RNA. The JJR-1 clone likely derived 
from one of the larger RNAs which apparently has a much longer 3' UTR. 
However, it cannot be excluded that the larger RNAs arise by alternative 
splicing that alters the length and composition of the coding sequence or 
by hybridization to related family members. 
Example 4 
Non-Overlapping Deletions Interrupt the Candidate NF2 Gene 
To determine whether the deletion detected in GUS5069 interrupts the merlin 
gene, exon probes were prepared from across the coding sequence (FIG. 5) 
and Southern blots containing DNA from GUSH134A3 and GUSH 134B1 (two 
independent hybrid lines containing the deleted chromosome 22) were 
analyzed. The results for probes I and II, shown in FIGS. 5A and 5B, 
demonstrate that the probe I sequence was absent from both hybrids, while 
the probe II sequence was present in both. Thus, the genomic deletion 
truncates the merlin gene within the coding sequence between probes I and 
II, removing the 5' end. 
In a search for additional alterations in the merlin gene, blots of 
restriction-digested DNA from 33 unrelated NF2 patients were scanned using 
the cDNA as probe. One patient, represented by cell line GUS5722, 
displayed altered fragments with several restriction enzymes suggestive of 
a small .about.3.4 kb genomic deletion. This patient was analyzed using 
Southern blotting as shown in FIGS. 5C, 5D, and 5E. Probes III, IV and V 
all reside on the same 21 kb EcoRI fragment. In GUS5722, probes III and V 
detected both the normal EcoRI fragment and second fragment reduced in 
size by the deletion. Probe IV failed to detect the altered fragment in 
GUS5722 because it lies within the region deleted. PCR amplification of 
first strand cDNA from GUS5722 was performed and confirmed the presence of 
two types of PCR product (FIG. 6). Direct sequencing revealed that the 
novel PCR product was missing bases 1559 to 1792 of the cDNA, representing 
deletion of at least two exons. The absence of this segment would remove 
78 amino acids from the protein, while leaving the reading frame intact. 
The GUS5722 cell line was generated from a member of a large NF2 kindred 
(Family 3 in Narod, S. A. et al., Am. J. Hum. Genet. 51:486-496 (1992)), 
and the deletion was present in five affected members and absent in eleven 
unaffected members of this pedigree. 
The presence of non-overlapping deletions affecting the merlin gene in two 
independent families supported the conclusion that this gene represents 
the NF2 tumor suppressor. The presence of additional alterations were 
determined by single-strand conformational polymorphism (SSCP) analysis of 
PCR amplified first strand cDNA from tumor and lymphoblast samples. mRNA 
from four primary cultures of meningiomas (3 from NF2 patients with a 
family history of the disorder, 1 from a probable new mutation to NF2) was 
used and only selective regions of the mRNA were analyzed. Two of the 
tumors yielded aberrant patterns. 
A meningioma from a female patient likely to have NF2 (see Experimental 
Procedures) displayed a reduced size for the expected non-denatured PCR 
product on SSCP gels (FIG. 6A). The vastly reduced level of the 
normal-sized PCR product suggests that this tumor had lost alleles in this 
region of chromosome 22. However, lymphocyte DNA was not available from 
this patient to confirm this. Direct sequence analysis of the PCR product 
confirmed the presence of a 4 bp deletion which removes bases 1781 to 
1784. This deletion alters the reading frame and generates a shorter 
protein. 
A meningioma from a male patient with NF2 (see Experimental Procedures) 
displayed an altered pattern on SSCP analysis (FIG. 6B). This meningioma 
was known to have lost heterozygosity on chromosome 22 based on comparison 
of polymorphic markers in blood and tumor DNA. Thus, the tumor suppressor 
model would suggest that the normal homologue had been lost and that the 
remaining copy of the gene represented the altered NF2 allele. Direct 
sequence analysis revealed a single base pair deletion at position 488 
(FIG. 3) which introduces a frameshift which dramatically alters the 
predicted protein by introducing a stop codon within 100 bases. 
Discussion 
The delineation of non-overlapping deletions affecting different portions 
of the same chromosome 22 gene in two independent NF2 families is strong 
evidence that "merlin" is the NF2 tumor suppressor. Although it is 
possible that one or both of these deletions may affect a second gene in 
the area, should this gene in fact be the NF2 tumor suppressor, it would 
have to be affected by both deletions and must therefore be composed of 
exons interspersed with those of the merlin gene. 
The larger of the deletions truncates the 5' end of the merlin gene, 
removing at least 120 amino acids. In addition, the extent of this 
deletion suggests that the 5' regulatory elements may also be missing. The 
smaller germline deletion removes 78 amino acids from the C-terminal 
portion of the protein. It is likely that such alterations would have 
drastic consequences for the function of the merlin protein. 
The four base pairs and single base pair deletions in meningiomas from 
unrelated NF2 patients could possibly be of somatic origin and unrelated 
to the inherited predisposition. However, the almost exclusive expression 
of the altered copy of the merlin gene suggests that the normal sequence 
has been lost as a somatic event in tumor formation. This is consistent 
with the tumor suppressor model, and would suggest that the frameshift 
alterations actually represent germline mutations in these patients. 
The merlin protein encoded at the candidate NF2 locus is a novel member of 
a growing family of proteins that have been proposed to act as links 
between the cell membrane and the cytoskeleton (Luna, E. J. et al., 
Science 258:955-964 (1992); Sato, N. et al., J. Cell. Sci. 103:131-143 
(1992)). All members of the family (which includes moesin, ezrin, radixin, 
erythrocyte protein 4.1 and talin) contain a homologous domain of 
approximately 200 amino acids near the N-terminus followed by a segment 
that is predicted to be rich in a .alpha.-helix structure, and a highly 
charged C-terminal domain. Where they have been characterized from more 
than one mammalian species, members of this family are remarkably 
conserved. Moreover, highly related genes have been detected in the 
nematode, Caenorhabditis elegans (Waterson, R. etal., Nature Genet. 
1:114-123 (1992)), and in the parasitic cestode, Echinococcus 
multilocularis (Frosch, P. M. et al., Mol. Biochem. Parasitol. 
48:121-130). 
Although most distantly related to merlin, protein 4.1 and talin are the 
best studied members of this family of proteins and have contributed the 
most towards understanding the function of the gene family. Protein 4.1 
plays a critical role in maintaining membrane stability and cell shape in 
the erythrocyte by connecting the integral membrane proteins glycophorin 
and protein 3 (the anion channel) to the spectrin-actin lattice of the 
cytoskeleton (Leto, T. L. et al., J. Biol. Chem. 259:4603-4608 (1984); 
Conboy, J. et al., Proc. Natl. Acad. Sci. U.S.A. 83:9512-9516 (1986)). 
Genetic defects in protein 4.1 lead to one form of hereditary 
elliptocytosis (Tchernia, G. et al., J. Clin. Invest. 68:454-460 (1981); 
Delaunay, J. et al., Nucleic Acids Res. 12:387-395 (1984)). The binding 
site for glycophorin in protein 4.1 has been mapped to the N-terminal 
domain, suggesting that the homologous region in other family members 
might also bind to proteins in the membrane (Leto, T. L. et al. in 
Membrane Skeletons and Cytoskeletal Membrane Associations, Bennett et al., 
eds. Liss, New York (1986), pp. 201-209). Interestingly, a related domain 
is also found in two protein tyrosine phosphatases, PTP-MEG and PTP-H 1, 
perhaps allowing these enzymes to associate with the membrane or the 
cytoskeleton (Gu, M. et al., Proc. Natl. Acad. Sci. U.S.A. 88:5867-5871 
(1991); Yang, Q. et al., Proc. Natl. Acad. U.S.A. 88:5949-5953 (1991)). 
Binding of protein 4.1 to spectrin is mediated by the .alpha.-helical 
region of the protein, suggesting that the analogous segments of the other 
family members might also bind to cytoskeletal components (Correas, I. et 
al., J. Biol. Chem. 261:3310-3315 (1986)). Talin, a large protein found in 
regions of focal adhesions at cell-cell or cell-substrate contacts, 
appears to behave similarly, binding to the integrins in the cell membrane 
and to vinculin, thereby connecting the extracellular adhesion matrix to 
the cytoskeleton (Rees, D. J. G. et al., Nature 347:685-689 (1990); Luna, 
E. J. et al., Science 258:955-964 (1992)). 
Moesin, ezrin and radixin are highly related proteins (.about.70-75% amino 
acid identity) that have each been postulated to provide a link between 
the cytoskeleton and the cell membrane. Each of these proteins shares 
45-47% amino acid identity with merlin. Moesin (membrane-organizing 
extension spike protein), originally proposed as a receptor for heparin 
sulfate, has been found at or near the membrane in filopodia and other 
cell surface protrusions (Lankes, W. T. et al., Proc. Natl. Acad. Sci. 
U.S.A. 88:8297-8301 (1991); Furthmayr, H. et al., Kidney lnt. 41:665-670 
(1992)). Ezrin (cytovillin) has been seen in association with microviili 
and cellular protrusions in many cell types (Pakkanen, R. et al., J. Cell. 
Biochem. 38:65-75 (1988); Gould, K. L. et al., EMBO J. 8:4133-4142 (1989); 
Turunen, O. et al., J. Biol. Chem. 264:16727-16732 (1989); Hanzel, D. et 
al., EMBO J. 10:2363-2373 (1991); Birbauer, E. et al., J. Neurosci. Res. 
30:232-241 (1989)). Rapid redistribution of ezrin to regions of membrane 
remodeling, such as microvillar formation and membrane ruffling in 
response to growth factor stimulation, may be regulated by phosphorylation 
of the protein on both tyrosine and serine residues (Bretscher, A., J. 
Cell Biol. 108:921-930 (1989); Krieg, J. et al., J. Biol. Chem. 
267:19258-19265 (1992)). Radixin was isolated from the cell-cell adherents 
junction, where it is proposed to cap actin filaments and provide for 
their attachment to the cell membrane (Tchernia, G. et al., J. Clin. 
Invest. 68:454-460 (1981); Funayama, N. et al., J. Cell Biol. 
115:1039-1048 (1991)). Interestingly, in milotic cells, radixin is 
concentrated at the cleavage furrow (Sato, N. et al., J. Cell. Biol. 
113:321-320 (1991)). 
Merlin possesses an N-terminal domain that is similar to protein 4.1 (28% 
identity), and to talin (21% identity). It is much more closely related, 
however, to moesin, ezrin and radixin (FIG. 7). Amino acid identity 
between merlin and the three latter proteins is concentrated in the first 
342 residues (.about.63% identity). Like these other family members, the 
merlin protein is predicted to have a very long .alpha.-helical domain 
spanning 160-170 amino acids, beginning around residue 300. The first 
third of this domain overlaps with the region of strongest hornology to 
moesin, ezrin and radixin. However, the remaining stretch shows limited 
similarity with these proteins and with a wide variety of myosins and 
tropomyosins. The C-terminal region of merlin contains a hydrophilic 
domain analogous to those of other family members. The similarity in 
structure of merlin to the other members of this family suggests that it 
too may normally act as a link between the cytoskeleton and the cell 
membrane and may thus represent a new class of tumor suppressor gene. 
The cytoskeleton of mammalian cells is a complicated lattice-work of many 
different kinds of interconnected filaments (Luna, E.J. et al., Science 
258:955-964 (1992)). It participates in a wide range of crucial cellular 
activities, including determining and altering shape, movement, cell 
division, cell-cell communication, cell anchorage, and organization of the 
intracellular milieu (Bernal, S. D. et al., Crit. Rev. Oncol. Hematol 
3:191-204 (1985)). A defect in a protein which connects some component of 
this network to the plasma membrane could affect any of these processes, 
and have a consequent effect on growth control. For example, inactivation 
of the merlin protein may disregulate growth by disrupting a signal 
transduction pathway, by altering anchorage dependence, by upsetting the 
cell cycle regulation, or by some other mechanism remains to be 
determined. However, the characteristic structure of the merlin protein 
suggests that a search for its membrane and cytoskeletal binding targets 
might provide a logical route to exploring this question. 
Example 5 
Altered Coding Sequence of the Merlin Tumor Suppressor Permitting DNA 
Diagnosis in an Extended Pedigree with NF2 
The objective of this example was to define the DNA mutation causing NF2 in 
a large, well-studied NF2 pedigree previously used to chromosomally map 
and to isolate the disease gene. The design was to use SSCP and sequence 
analysis of DNA amplified from the NF2 gene of affected and unaffected 
persons. The participants in the study set forth in this example were 
affected, unaffected, and at-risk members of a large pedigree segregating 
NF2. The results of the study showed a DNA alteration in the merlin coding 
sequence causing a shift on SSCP gels that was characteristic of the 
disease chromosome in this NF2 pedigree, being transmitted with the 
disorder, present only in affected members of the pedigree, absent in 
unaffected members of the family, and absent from 158 unrelated 
individuals. The alteration caused substitution of a tyrosine for an 
asparagine at position 220 of the merlin protein, in a region highly 
conserved in closely-related members of the family of 
cytoskeletal-associated proteins. The DNA change could also be detected by 
restriction enzyme digestion with RsaI. 
A. Materials and Methods 
1. Patients 
The family studied in this Example has been extensively characterized 
clinically and described in detail previously (Wertelecki, W., et al., New 
Engl. J. Med. 319:278-283 (1988)). Medical records, histologic slides, 
death certificates and autopsy reports were sought for all symptomatic 
family members. Clinical assessments were performed including a search for 
signs of neurofibromatosis type 1. The results of computed tomography, 
MRI, and ophthalmologic and audiologic examinations were also sought. 
Diagnostic criteria used were those of the NIH consensus statement on 
neurofibromatosis (National Institutes of Health Consensus Development 
Conference Statement: Neurofibromatosis Arch Neurol. 45:575-578 (1988)). 
Lymphoblast lines were established from peripheral blood samples for all 
patients and relatives as previously described (Anderson et al., In Vitro 
20:856-858 (1984)). DNA was isolated from peripheral or cultured 
ieukocytes as described herein. 
2. Polymerase Chain Reaction (PCR) Amplification of Exon 7 
The genomic DNA of the patients and the unaffected control individuals were 
amplified using primers in the intron flanking exon 7. Approximately 30 ng 
of genomic DNA was amplified using the primer pair: 
5'-CCATCTCACTTAGCTCCAATG-'3 SEQ ID NO: 17! and 5'-CTCACTCAGTCTCTGTCTAC-'3 
SEQ ID NO: 18!. Amplification conditions include 20 .mu.M each of dATP, 
dGTP, dCTP and dTTP, 4 pmoles of each primer, 0.5 units Taq polymerase, 10 
mM Tris pH 8.3, 1.5 mM MgCl.sub.2, 50 mM KCl, and 0.1 mg/ml gelatin in a 
total volume of 10 .mu.l. Each reaction was cycled 35 times using the 
following steps: denaturation (at 95.degree. C. for 1.5 minutes), primer 
annealing (at 60.degree. C. for 1.5 minutes), and elongation (at 
72.degree. C. for 30 seconds). 
3. Single Strand Conformational Polymorphism Analysis (SSCP) 
SSCP analysis was performed according to the procedure of Orita et al. with 
minor modifications (Orita, M., et al., Genomics 5:874-879 (1989)). PCR 
amplification was carried out as described above except each reaction 
included 0.1 .mu.l (10 mCi/ml) of .alpha.-.sup.32 P dATP (Amersham Life 
Sciences). The amplified products were diluted 1:20 in 0.05% SDS, 6 mM 
EDTA, 40% formamide, 0.5 mg/ml xylene cyanol and 0.5 mg/ml bromphenol blue 
and heated to 90.degree. C. for 3 minutes to denature the DNA. Samples 
were immediately cooled on ice and loaded on an 8% polyacrylamide gel 
containing 8% glycerol. Electrophoresis was carried out at room 
temperature for 12 to 16 hours at a constant power of 6-10 W. Gels were 
dried and exposed overnight to Kodak X-Omat AR film. 
4. DNA Sequencing 
The double-stranded PCR product containing the amplified exon was used as a 
template to generate single-strands by priming multiple rounds of DNA 
synthesis with one of the oligonucleotides previously used in the 
double-strand reaction (Gibbs, A., et al., Proc. Natl. Acad. Sci. USA 
86:1919-1923 (1989)). Conditions for the single strand-producing reactions 
were identical to the PCR amplification of individual exons as described 
above. After 30 cycles, the product was ethanol precipitated in the 
presence of ammonium acetate and resuspended in 7 .mu.l water for 
subsequent sequence analysis. The sequencing reactions were performed as 
described in Park et al. (Hum. Mut. 1:293-297 (1992)) utilizing Sequenase 
(US Biochemicals). The amplified products were also cloned in T vector 
(Novagen) and the DNA obtained from the individual clones were sequenced 
using the Sequenase kit (US Biochemicals). 
B. Results 
1. The Extended NF2 Pedigree 
The NF2 gene shown in previous Examples was mapped precisely using genetic 
linkage analysis in an extremely large disease pedigree from which 137 
blood samples were tested for polymorphic DNA markers on chromosome 22 
(Wertelecki, W., et al., New Engl. J. Med. 319:278-283 (1988)). For this 
Example, a subset of the members of this kindred was selected to use for 
identification of the underlying NF2 mutation. The relationships of the 
family members used is shown in FIG. 8. 
2. Scanning for NF2 Mutation Affecting Expression of Merlin 
The NF2 gene has been identified herein based on non-overlapping genomic 
DNA deletions that altered its coding sequence in three independent NF2 
families and in a meningioma from an unrelated NF2 patient. The merlin 
mRNA sequence consists of more than 2250 bases. The translated portion of 
the mRNA, an open reading frame of 1785 bases encoding the predicted 595 
amino acid merlin protein is bracketed by 5' and 3' untranslated regions 
whose extent and variability remain to be defined completely. The merlin 
mRNA is spliced together from at least 17 exons that are distributed 
across about 100 kb of chromosome 22. In order to scan for mutations in 
the NF2 gene, the intron DNA sequence flanking each exon was determined, 
and DNA primers that permit amplification of the exon sequences from 
genomic DNA using the PCR have been developed. 
The strategy for identifying DNA sequence differences representing 
potential mutations within the amplified coding sequences is the use of 
SSCP analysis (Orita, M., et al., Genomics 5:874-879 (1989)). In this 
method, individual exons from the patient's DNA are first amplified by 
PCR. The amplification products are then denatured to separate the 
complementary DNA strands and diluted to allow each single stranded DNA 
molecule to assume a secondary structure conformation by folding on 
itself. The single stranded DNA molecules are then subjected to 
polyacrylamide gel electrophoresis under nondenaturing conditions. The 
secondary structure, which is highly dependent on the precise DNA 
sequence, affects mobility of the strand on the gel. Even a single base 
change or deletion can produce a visible shift in the final band position 
on the gel. 
The SSCP technique was applied to the DNA samples from the family in FIG. 
8. A mobility shift was detected when comparing amplified PCR products 
from the affected family member, designated by an arrow in FIG. 8, 
relative to those from several normal unrelated individuals and from 
members of other unrelated NF2 families. This shift was detected by 
amplification of exon 7, comprising bp 819 to 894 of the merlin coding 
sequence. To confirm that this alteration is characteristic of the disease 
allele and transmitted with the disorder, additional affected and 
unaffected members of the NF2 pedigree were typed. FIG. 8 shows the 
deduced genotype of each family member tested in this and other analyses. 
FIG. 9 displays the results of one SSCP analysis in which five sibling 
pairs, each consisting of one affected and one unaffected individual, were 
tested. The altered SSCP pattern shows a clear correlation with 
inheritance of the disorder, and is present only in affected members of 
the pedigree. This SSCP pattern was not observed in a search of more than 
300 independent chromosomes, derived from normal controls, other NF2 
families, and sporadic tumors of various types (colon, astrocytoma, 
schwannoma etc.). 
3. Identification of the DNA Sequence Alteration Underlying the SSCP 
Variation 
To determine the precise DNA alteration that caused the shifted mobility 
pattern in the SSCP analysis, direct DNA sequencing of the PCR 
amplification product from an affected member of the pedigree and an 
unrelated normal control was performed. The change in DNA sequence in the 
affected individual and its absence in an unaffected member of the 
pedigree was reconfirmed using cloned PCR product. The results of sequence 
analysis are shown in FIG. 10. 
The control sequence displays an AAC codon encoding asparagine at position 
220 of the merlin protein. The DNA from the patient reveals both an A and 
a T residue at the first position of this codon, suggesting a normal AAC 
codon on one chromosome, and a mutated TAC on the other. The TAC codon 
would substitute a tyrosine at position 220. Since this SSCP variation was 
absent from more than 300 independent chromosomes, it is unlikely that 
this change is simply a polymorphism. Rather, the change from the 
aliphatic side chain of asparagine to the bulky aromatic side chain of the 
tyrosine is likely to have significant consequences for the structure of 
the protein. 
4. Confirmation of the Sequence Alteration by RsaI Digestion 
The sequence change outlined above creates a GTAC stretch that is 
recognized and cleaved by the restriction endonuclease RsaI. This site is 
created 9 bp 3' to a pre-existing RsaI site in this exon. Thus, to confirm 
the presence of the same DNA change in other affected members of the 
family, its cotransmission with NF2, and its absence in unaffected members 
of the pedigree, we performed RsaI digests on the amplified PCR product 
from several family members. Typical results are shown in FIG. 11. The 
unaffected members of the family (U) and other control individuals display 
two RsaI fragments of 96 bp and 76 bp, whereas the affected individuals 
(A) produce an additional third fragment of 67 bp generated from the RsaI 
site created by the A.fwdarw.T change at codon 220. 
Discussion 
The isolation and characterization of the NF2 gene, encoding the merlin 
protein, creates new possibilities for accurate predictive testing in NF2. 
Until the NF2 gene was mapped to chromosome 22, no genetic testing was 
possible. Once the gene was mapped, it became possible to predict NF2 
carrier status using linked DNA markers (Narod, S. A. et al., Am. J. Hum. 
Genet. 51:486-496 (1992)). Unfortunately, testing by linkage is limited by 
the availability of multiple family members for comparison, by lack of 
complete informativeness of linked markers, and by the diminished accuracy 
inherent in using a marker not located precisely within the disease gene. 
The precise identity of the NF2 gene discovered by the inventors now makes 
it possible to overcome these diagnostic hurdles by defining the exact 
molecular lesion associated with the disorder in any given pedigree. 
Because mutations in NF2 were expected in general to inactivate the gene, 
they could have fallen into several different categories. DNA 
rearrangements, such as deletions, inversions, insertions, or duplications 
could be expected. Mutations that eliminate expression of the entire 
transcript, interfere with exon splicing, or disrupt its stability could 
effectively inactivate the gene. Finally, DNA sequence alterations within 
the coding sequence that cause premature termination of translation, 
yielding a truncated protein, or that result in a change in an amino acid 
residue critical for normal function of the merlin protein were also 
likely. 
In this application, as shown in the exemplary material, the SSCP technique 
was applied as a rapid means to locate and confirm the probable molecular 
basis of NF2 in a large and well studied kindred (Wertelecki, W. et al., 
New Engl. J. Med. 319:278-283 (1988)). The technique used strategically 
placed primer sets to amplify small regions of the NF2 gene directly from 
genomic DNA using PCR. The findings were highly specific; none of the 
unaffected 158 control samples produced an altered SSCP pattern. As would 
be predicted, all samples from affected members of the same kindred 
produced the same altered SSCP pattern, reflecting the identical nature of 
the underlying molecular defect. 
The sequence alteration in this family resulted in the substitution of a 
tyrosine residue for the asparagine at position 220 of the merlin protein. 
This change affects one of the most conserved stretches in the merlin 
protein as shown in FIG. 12. This particular asparagine residue, and all 
surrounding residues are present in all three closely related members of 
the family of cytoskeletal associated proteins, moesin, ezrin and radixin. 
The sequence conservation in this protein domain suggests that this region 
plays a crucial role in the function of the protein. A change from 
asparagine to tyrosine would be expected to significantly disrupt the 
structure of this domain. The drastic nature of this amino acid change, 
and the absence of the same DNA change from more than 300 independent 
chromosomes argue that this alteration is the cause of NF2 in this 
pedigree. 
Example 6 
Analysis of merlin in Sporadic and Inherited Tumors 
To facilitate the search for mutations in NF2 and related tumors, the 
exon-intron junctions of the NF2 locus were sequenced, including the E16 
additional exon that is alternatively spliced (Bianchi, A. B., et al., 
Nature Genet., in press, 1994; Haase, V. H., et al., Human Mol. Genet., in 
press, 1994). PCR assays were developed for all seventeen exons, and were 
used to scan the entire gene for mutations in sporadic and inherited 
vestibular schwannomas. The high proportion of tumors in which 
inactivating mutations were found indicates that the NF2 gene plays a 
fundamental role in schwannoma tumorigenesis. 
Materials and Methods 
Tissue Samples 
Tumor specimens were obtained at the time of surgery and frozen for DNA 
analysis. Blood samples were also obtained at the time of surgery to serve 
as normal tissue controls. High molecular weight DNA was extracted from 
peripheral blood leukocytes and from frozen pulverized tumor tissue by 
SDS-proteinase K digestion followed by phenol and chloroform extraction 
(Seizinger, B. R., et al., Nature 322:644-647 (1986)). 
Design of Primer Pairs 
Exonic primers were designed within the NF2 coding sequence near the 
intron-exon borders as determined by the results of exon trapping 
(Trofatter, J. A., et al., Cell 72:791-800 (1993)). For those regions not 
isolated by trapping, primers were synthesized at approximate 100 base 
pair intervals. Using these primers, an intronic sequence was obtained by 
directly sequencing cosraids containing the gene using a cycle sequencing 
kit (US Biochemical). Intronic primer pairs were then designed to amplify 
the splice donor and acceptor sites as well as the exon itself. In the 
case of E12, it was necessary to construct two overlapping primer sets to 
maintain a product length of less than 300 base pairs. 
SSCP Analysis 
SSCP analysis was performed according to the procedure of Orita et al. 
(Orita, M., et al., Genomics 5:874-879 (1989)) with minor modifications. 
Approximately 50 ng of genomic DNA was amplified using appropriate 5 
intronic primer pairs (Table 2). Each 10 .mu.l reaction contained 70 .mu.M 
each of dATP, dCTP, dGTP and dTTP, 4 pmoles of each primer, 0.5 units Taq 
polymerase, 10 mM Tris pH 8.3, 1.5 mM MgCl.sub.2, 50 mM KCl, 0.01% 
gelatin, and 0.1 .mu.l .alpha..sup.33 P!dATP (Amersham, 10 mCi/ml). For 
E1, a MgCl.sub.2 concentration of 0.5 mM was used. Amplification was 
carried out for 30 cycles as follows: 94.degree. C. for 1 min., 
55.degree.-60.degree. C. for 1 min., and 72.degree. C. for 1 min., after 
an initial denaturation step at 94.degree. C. for 4 min. One .mu.l of 
labeled amplified DNA was diluted into 9 .mu.l of 0.1% SDS and 10 mM EDTA, 
and an equal volume of loading dye (95% formamide, 0.5M EDTA, 0.05% 
bromphenol blue and 0.05% xylene cyanol) was added. The samples were 
denatured for 2 min. at 90.degree. C. and separated on 6-8% polyacrylamide 
gels containing 8% glycerol for 16 hrs. at 6-8 W. 6els were dried and 
exposed to Kodak X-OMAT film. 
DNA Sequencing 
For DNA sequencing, PCR amplifications were performed in 50 .mu.l volumes 
as described above for SSCP analysis except that 200 .mu.M dNTPs were 
added and a radioactive nucleotide was omitted. The product was sequenced 
by one of two methods. In the first, the double-stranded product was used 
as a template to generate single-strands by priming multiple rounds of DNA 
synthesis with one of the oligonucleotides previously used in the 
double-strand reaction (Gibbs, A., et al., Proc. Natl. Acad. Sci. USA 
86:1919-1923 (1989)). Conditions for asymmetric PCR amplification were 
identical except that only one primer was added. The product was ethanol 
precipitated in the presence of ammonium acetate and resuspended in 10 
.mu.l H.sub.2 O for subsequent sequence analysis. The sequencing reactions 
were performed by the dideoxy chain-termination method using Sequenase (T7 
DNA polymerase, US Biochemical) under conditions recommended by the 
supplier. Alternatively, the PCR products were purified with BioSpin 
columns (BioRad) and ethanol precipitated. DNA sequencing was performed 
according to a standard cycle-sequencing protocol using VentR (exo-) DNA 
polymerase and the CircumVent Thermal Cycle kit (New England BioLabs). 
Both strands of exons with SSCP mobility shifts were analyzed in all 
cases. 
Loss of Heterozygosity Analysis 
Genomic DNA was amplified using primer pairs for the polymorphic 
dinucleotide repeats at markers D22S193 (Trofatter et al., in preparation) 
or D22S268 (Marineau, C., et al., Hum. Mol. Genet. 2:336 (1993)). The CA 
strand primer was 5' end-labeled with polynucleotide kinase and 
gamma-.sup.32 P ATP, and PCR was performed as described previously (Louis, 
D. N., et al., Am. J. Pathol. 141:777-782 (1992)). In some cases, loss of 
chromosome 22 alleles was determined by Southern blot analysis using 
probes at the following loci: D22S22, D22S29, D22S28, D22S15, D22S1, 
CRYB2, D22S10 and D22S9 (Rouleau, G. A., et al., Genomics 4:1-6 (1989)). 
Results 
Exon Structure of the NF2 Gene 
The internal exons range in size from 45 base pairs to 218 base pairs, with 
an average of 111 base pairs. Of the fifteen internal exons, ten (E3, 
E5-7, E9-11, E14-16) were isolated using exon amplification, the technique 
described herein, which led to the isolation of the NF2 locus from cloned 
genomic DNA (see also Trofatter, J. A., et al., Cell 72:791-800 (1993)). 
Table 1 shows the DNA sequences immediately surrounding the intron-exon 
junctions, which all match the consensus for splice acceptor and donor 
sites. Additional intron sequences on both 5' and 3' sides that were used 
to design primers for PCR amplification are described herein in the 
section "Description of the Preferred Embodiments" (FIGS. 15A-Q herein). 
PCR Assays for the Seventeen NF2 Gene Exons 
For each internal exon, primers were chosen in flanking intron sequences to 
develop an assay for PCR amplification of the exon directly from genomic 
DNA. For E12, two overlapping primer sets were chosen to yield products in 
a size range amenable to SSCP analysis. Because E12 is 218 base pairs, two 
sets of primers were employed, one set spanning the 5' intron-exon 
junction and the 5' region of the exon and the other overlapping set 
spanning the remainder of the exon and the 3' exon-intron junction. In 
this way amplified fragments of a suitable length for SSCP analysis (140 
and 284 base pairs, respectively) were generated. All of the coding region 
from E1 was amplified using one primer in the 5' untranslated region (UTR) 
and one in intron 1. For E17, a primer from the final intron was paired 
with a primer in the 3'UTR to amplify all of the E17 coding sequence, 
(along with the first 100 base pairs of 3'UTR). All assays (except that 
for E1) were performed according to standardized amplification conditions. 
As discussed in the exemplary material, E1 was amplified using a 
MgCl.sub.2 concentration of 0.5 mM rather than 1.5 mM (to optimize the 
yield of PCR product). The annealing temperature was varied for optimum 
results. Table 2 lists the primers, annealing temperature and product size 
for each exon assay. In some cases (E7 and E8), an initial set of primers 
was used, but subsequently was replaced with a second primer pair. In some 
cases, the original primer was too far removed from the exon for 
convenient DNA sequencing or too close to the exon to detect potential 
intronic mutations. In such cases, primer pairs were used. In these 
instances both sets of primer pairs are listed. 
Scanning for Mutations by SSCP Analysis of Blood-Tumor Pairs 
The exon PCR assays listed in Table 2 were used to scan the entire merlin 
coding sequence for mutations in schwannomas. SSCP analysis was applied to 
DNA extracted from thirty eight primary tumor specimens, including eight 
vestibular schwannomas from NF2 patients (Table 3), twenty-seven sporadic 
vestibular schwannomas (Table 4) and three sporadic spinal schwannomas 
(Table 4, S9, S25 and S27). DNA extracted from a blood sample of the 
corresponding individual was used for comparison in each case. All 
tumor-blood pairs were assayed for all exons. 
Representative results of the SSCP analyses for three of the exons, (E2, 
E10 and E14) are shown in FIG. 13. For each assay, a lane of PCR product 
was run without denaturation (ND) to identify fully reannealed, 
double-stranded DNA in the test lanes. The other bands in the test lanes 
represent various conformations of the single-stranded DNAs in the 
product. For E2, the normal pattern is seen only in the blood DNA (B) of 
S29. S11 and S33 display mobility shifts that are detected in both blood 
and tumor DNA. This result indicates germline alterations. S29 displays a 
mobility shift only in the tumor DNA. This result indicates a somatic 
mutation. For E10 and E14, all blood DNAs display the normal pattern and 
all tumor DNAs display different mobility shifts. Both normal and altered 
PCR products were compared by direct sequence analysis to identify the 
precise base change(s) involved. 
NF2 Mutations in Schwannomas 
The blood and tumor DNAs were genotyped using polymorphic DNA markers to 
detect a loss of heterozygosity that could indicate deletion of one NF2 
allele (Tables 3 and 4). General testing had predicted that at least 
sixteen of the thirty-eight tumors had lost one NF2 allele. Thus, the 
entire NF2 coding sequence for 58-60 independent alleles was examined by 
SSCP. No obvious polymorphism affecting coding or non-coding sequences was 
observed. This result indicates a remarkable degree of homogeneity for 
this gene sequence in the population. In contrast, mutations in both NF2 
and sporadic tumors were readily detected. DNA differences from normal 
have been confirmed in twenty-seven of these tumors. Seven germline and 
twenty-five somatic alterations have been identified by sequence analysis. 
The results are summarized in Tables 3 and 4. 
Germline mutations, present in both blood and tumor DNA, were delineated in 
five of the eight patients with a confirmed diagnosis of NF2 (Table 3). 
These changes occurred in disparate locations and included 1) point 
mutations creating stop codons at residues 57 and 60 of E2 (S11 and S33) 
and 1396 of E13 (S4); 2) a 28 base pair deletion creating a frame shift 
and premature stop codon in E10 (S1) and an insertion of one base into a 
splice donor site in E12 (S32). Two other germline alterations were found 
in blood and tumor DNA from individuals without a confirmed NF2 diagnosis 
(Table 4). These included 1) a single base change in the intron upstream 
of E7 in a sporadic spinal schwannoma (S9) and 2) a substitution of Cys 
for Arg at residue 418 in a sporadic vestibular schwannoma (S44). Although 
the two intron changes and the apparent missense mutation could 
conceivably represent polymorphisms, they were not found by SSCP analysis 
of 150 independent DNA samples from normal individuals or individuals with 
other types of tumors. 
Somatic mutations were observed in tumors from five of the eight patients 
with NF2. These included two patients (S1 and S4) in whom a germline 
mutation was also detected (Table 3). In thirty sporadic tumors, a total 
of twenty somatic mutations were found (Table 4). In three cases in which 
chromosome 22 heterozygosity was maintained (S18, S24, and S29), two 
distinct somatic alterations were found in each tumor. For example, tumor 
S29 displayed small deletions of one base pair and four base pairs in E2 
and E8, respectively (FIG. 14). In one tumor (S35), a complex of two 
adjacent deletions was detected in a single allele. One deletion removed 
five codons in-frame. The other deletion (beginning three base pairs 
downstream) caused a frameshift. 
The twenty-five somatic mutations from NF2 and sporadic tumors were found 
throughout the gene and were associated with E1, E2, E3, E4, E7, E8, E9, 
E10, E12, E14 and E15. By far the most frequent lesions detected (19/25) 
were small deletions of one to sixty-one base pairs, that had either an 
obvious or presumed effect on splicing or that produced frameshifts that 
led to truncated proteins of altered sequence. A single mutation involved 
a frameshift resulting from a single base insertion (S42). The remaining 
five somatic changes were point mutations that either altered splice donor 
(S22) or acceptor (S12, S37) sites, produced a stop codon at residue 212 
(S24), or generated a Met for Val substitution at residue 219 (S1). 
Discussion 
The results of the studies herein defining specific mutations immediately 
allow improved prenatal, presymptomatic and unaffected diagnosis for 
family members at risk. Highly accurate prenatal testing is now possible 
by direct examination of fetal DNA for the presence of characteristic SSCP 
shifts, altered DNA sequences, or gain or loss of restriction sites. 
Similarly, presymptomatic testing using these approaches could eliminate 
the need for up to half of at risk family members to undergo expensive and 
time consuming clinical monitoring, reducing the considerable financial 
and psychological burdens on these individuals. For those that test 
positive for the specific changes, medical care should be improved by the 
clarification of their status earlier in the course of their disease, 
which in turn might allow earlier consideration of surgical intervention. 
With the delineation of a larger number of mutations, it now becomes 
possible to discover whether the NF2 gene contains mutational "hot spots" 
which would simplify scanning. 
NF2 is a disorder consistent with a "two-hit" model of tumorigenesis, in 
which homozygous inactivation of a gene that normally suppresses tumor 
growth is the critical event in tumor formation (Knudson, A. G., Proc. 
Natl. Acad. Sci. USA 68:820-823 (1971)). The same types of tumors that are 
present as multiple independent growths in NF2 patients occur as sporadic, 
solitary cases in the general population (Martuza and Eldridge, New Eng. 
J. Med. 318:684-688 (1988); Mulvihill, J., et al., Ann. Intern. Med. 
113:39-52 (1990)). The familial and sporadic tumors both display frequent 
loss of genetic material from chromosome 22, in a region to which the NF2 
gene defect has been mapped by linkage analysis (Kaiser-Kupfer, M. I., et 
al., Arch. Ophthalmol. 107:541-544 (1989); Rouleau, G. A., et al., Nature 
329:246-248 (1987); Wertelecki, W., et al., New Engl. J. Med. 319:278-283 
(1988); Rouleau, G. A., et al., Am. J. Hum. Genet. 46:323-328 (1990); 
Narod, S. A., et al., Am. J. Hum. Genet. 51:486-496 (1992); Seizinger, B. 
R., et al., Nature 322:644-647 (1986); Seizinger, B. R., et al., Science 
236:317-319 (1987); Seizinger, B. R., et al., Proc. Natl. Acad. Sci. USA 
84:5419-5423 (1987); Couturier, J., et al., Cancer Genet. Cytogenet. 
45:55-62 (1990); Bijlsma, E. K., et al., Genes Chromosom. Cancer 5:201-205 
(1992); Fontaine, B., et al., Ann. Neurol. 29:183-196 (1991); Fontaine, 
B., et al., Genomics 10:280-283 (1991); Fiedler, W., et al., Genomics 
10:786-791 (1991); Wolff, R. K., et al., Am. J. Hum. Genet. 51:478-485 
(1992)). Thus, it is presumed that the NF2 locus encodes a tumor 
suppressor and that inactivation of both alleles by loss or mutation in 
specific cells results in unregulated proliferation. However, only 
specific cell types are affected in this way as the vast majority of 
tumors seen in NF2 are schwannomas, and particularly vestibular 
schwannomas and meningiomas. 
The results herein implicate merlin as a tumor suppressor. Whereas germline 
mutations are present in both blood and tumor DNA from NF2 patients, 
somatic mutation of merlin is a frequent event in schwannomas. In several 
cases, two inactivating mutations were detected in the same tumor. In many 
others, a single mutant allele remained following the loss of the second 
copy of the locus. The alterations occured throughout the NF2 gene. Most 
exons displayed at least one mutation. 
One alteration, conversion of the Arg codon at position 57 to a stop codon, 
has been seen twice before in independent NF2 patients (Rouleau, G. A., et 
al., Nature 363:515-521 (1993)), suggesting that this site containing a 
cCpG dinucleotide may be particularly prone to C.fwdarw.T transitions. The 
presence of this change in the blood DNA of S11 combined with this absence 
from either parent demonstrate that this is a case of a new mutation to 
NF2. 
Two new missense mutations were identified, Val219Met and Arg418Cys, which 
may target these residues as particularly important in merlin's tumor 
suppressor function. The Val at position 219, one residue away from the 
previously reported Asn220Tyr mutation, is located within the protein 4.1 
domain that is characteristic of this family and is conserved in human 
moesin, radixin, and protein 4.1, but changed to Ile in human ezrin. The 
Arg at position 418 is located in the long .alpha.-helical domain that 
comprises most of the C-terminal half of merlin and its relatives but is 
not strictly conserved in the other human members of this protein family. 
A surprising number of somatic mutations involve changes in intron 
sequences. These may occur at a distance from the exon-intron junction. 
The absence of these alterations in blood-derived DNA of the same 
individuals, and the failure to detect the same change in any other 
individuals, indicate that these mutations are de novo events associated 
with tumor formation. 
In addition to expanding the number and variety of germline NF2 mutations 
described, the examples herein suggest that germline alterations occur in 
patients not yet diagnosed clinically with NF2. One patient, with a single 
spinal schwannoma, displayed a single base alteration in an intron 
sequence. Another with a single vestibular schwannoma, displayed the 
Arg418Cys change described above. Careful clinical follow-up is indicated 
for these patients. It is possible that they represent a class of 
individuals with mutations that only mildly affect normal merlin function, 
and consequently do not produce the full NF2 phenotype. 
The development of reliable PCR assays for each exon of the NF2 gene should 
facilitate greatly the cataloging of mutations in NF2 patients and their 
tumors by genomic scanning. It can be expected that a detailed mutational 
analysis of the NF2 gene, identifying sites particularly prone to 
alteration, pinpointing amino acid residues crucial for normal function, 
and providing a basis for relating specific alterations with variations in 
phenotype will result. Perhaps most important, however, the ability to 
rapidly scan the NF2 gene for mutations will accelerate the assessment of 
a role for merlin in other tumor types. 
TABLE 1 
__________________________________________________________________________ 
Intron-Exon Boundaries of the NF2 Gene 
__________________________________________________________________________ 
Splice Start 
Exon 
Exon Exon 
End Splice 
Exon 
Acceptor 
(bp).sup.1 
Start 
Length 
End 
(bp) 
Donor 
__________________________________________________________________________ 
1 GAG 
114 GTAACCGGCC 
2 GTTATTGCAG 
115 ATG 
126 AAG 
240 GTTGGGCTAG 
3 AATTCTGCAG 
241 GTA 
123 CAG 
363 GTACATCAGT 
4 CTCCTTTCAG 
364 GTA 
84 AAG 
447 GTAGGCTCAA 
5 TTCTTTCCAG 
448 TAT 
69 AGG 
516 GTAAGAGATT 
6 TTTTTGGTAG 
517 GTA 
83 CAG 
599 GTGAGGCCCA 
7 CTCCCCACAG 
600 GGA 
76 CGG 
675 GTGTGTTGAA 
8 GGATCCACAG 
676 AAT 
135 GAG 
810 GTAGGACATG 
9 ATTCTTCCAG 
811 TTT 
75 CTG 
885 GTAAGTTGAG 
10 GTGGCCACAG 
886 ATT 
114 CAG 
999 GTGAGCACAA 
11 CCCCTCGCAG 
1000 
ATG 
123 CTG 
1122 
GTGATTTCTG 
12 TGCCCTCCAG 
1123 
ATG 
218 GAG 
1340 
GTGAGGGGGC 
13 TTCCTTGCAG 
1341 
GGC 
106 CCG 
1446 
GTGAGCCTGG 
14 TCATTAACAG 
1447 
CCC 
128 AAA 
1574 
GTATGTAGCC 
15 TTGCCGGCAG 
1575 
AGT 
163 AAG 
1737 
GTACCCAGGG 
16 GCTGGTTTAG 
1738 
CCT 
45 AAA 
1782 
GTAGGTTGTT 
17 TTTCTTACAG 
1783 
CTC 
__________________________________________________________________________ 
Exon Splice Acceptor 
Splice Donor 
__________________________________________________________________________ 
1 SEQ ID NO. 19 
2 SEQ ID NO. 20 
SEQ ID NO. 21 
3 SEQ ID NO. 22 
SEQ ID NO. 23 
4 SEQ ID NO. 24 
SEQ ID NO. 25 
5 SEQ ID NO. 26 
SEQ ID NO. 27 
6 SEQ ID NO. 28 
SEQ ID NO. 29 
7 SEQ ID NO. 30 
SEQ ID NO. 31 
8 SEQ ID NO. 32 
SEQ ID NO. 33 
9 SEQ ID NO. 34 
SEQ ID NO. 35 
10 SEQ ID NO. 36 
SEQ ID NO. 37 
11 SEQ ID NO. 38 
SEQ ID NO. 39 
12 SEQ ID NO. 40 
SEQ ID NO. 41 
13 SEQ ID NO. 42 
SEQ ID NO. 43 
14 SEQ ID NO. 44 
SEQ ID NO. 45 
15 SEQ ID NO. 46 
SEQ ID NO. 47 
16 SEQ ID NO. 48 
SEQ ID NO. 49 
17 SEQ ID NO. 50 
__________________________________________________________________________ 
.sup.1 Base pair numbering is based on #1 being the A of the initiator AT 
TABLE 2 
__________________________________________________________________________ 
Primers for Exon PCR Assays 
__________________________________________________________________________ 
Exon.sup.1 
Product 
Temp.sup.2 (.degree.C.) 
Primer #1 (5'-3') Primer #2 (5'-3') 
__________________________________________________________________________ 
1 235 58 GCTAAAGGGCTCAGAGTGCAG 
GAGAACCTCTCGAGCTTCCAC 
2 182 60 TGTCCTTCCCCATTGGTTTG 
CAGTTTCATCGAGTTCTAGCC 
244 58 AGTGCAGAGAAAAGGTTTTATTAATGAT 
TGGAAAGCTCACGTCAGCC 
3 272 60 GCTTCTTTGAGGGTAGCACA 
GGTCAACTCTGAGGCCAACT 
4 188 59 CCTCACTTCCCCTCACAGAG 
CCCATGACCCAAATTAACGC 
5 148 60 GCTCTCCCTTTCTTCTTTCC 
TCCTTCAAGTCCTTTGGTTAGC 
171 58 TGGCAGTTATCTTTAGAATCTC 
TTAGACCACATATCTGCTATG 
6 161 60 CATGTGTAGGTTTTTTATTTTGC 
GCCCATAAAGGAATGTAAACC 
7 173 60 CCATCTCACTTAGCTCCAATG 
CTCACTCAGTCTCTGTCTAC 
170 60 GAATGCTTGATTTGGTGCCC 
GAGGTTTCAACACACCCGGA 
8 247 60 GAAGGTTGAATAAAATTTTGAGCCTC 
GACAGGGAAAGATCTGCTGGACC 
232 60 CTGTTCTTATTGGATCCACAG 
AACAACCACACCCTCAAAGC 
9 300 58 GACTTGGTGCTCCTAATTCCC 
CCATTATCAGTAATGAAAACCAGG 
10 260 59 TGCTACCTGCAAGAGCTCAA 
CTGACCACACAGTGACATC 
11 268 60 TCTTTGGCCCTTGTGGCAC CAGGAGACCAAGCTCCAGAA 
12A 140 60 TTCAGCTAAGAGCACTGTGC 
CGCTGCATTTCCTGCTCAG 
12B 284 58 GCTGAAAAGGCCCAGATCA CTTGAGGACAACTGCTGTAG 
13 228 60 GGTGTCTTTTCCTGCTACCT 
GGGAGGAAAGAGAACATCAC 
14 254 60 TGTGCCATTGCCTCTGTG AGGGCACAGGGGGCTACA 
15 317 58 TGGCCAAGTAGAGACGTGA TACAAGAAAGAGACCCTGGG 
248 58 TCTGCCCAAGCCCTGATGC TGGTCCTGATCAGCAAAATAC 
16 148 60 GGCATTGTTGATATCACAGGG 
GGCAGCACCATCACCACATA 
17 177 60 CTCTCAGCTTCTTCTCTGCT 
CCAGCCAGCTCCTATGGATG 
__________________________________________________________________________ 
Exon Product Primer#1 
Primer #2 
__________________________________________________________________________ 
1 235 SEQ ID NO: 51 
SEQ ID NO: 52 
2 182 SEQ ID NO: 53 
SEQ ID NO: 54 
244 SEQ ID NO: 55 
SEQ ID NO: 56 
3 272 SEQ ID NO: 57 
SEQ ID NO: 58 
4 188 SEQ ID NO: 59 
SEQ ID NO: 60 
5 148 SEQ ID NO: 61 
SEQ ID NO: 62 
171 SEQ ID NO: 63 
SEQ ID NO: 64 
6 161 SEQ ID NO: 65 
SEQ ID NO: 66 
7 173 SEQ ID NO: 67 
SEQ ID NO: 68 
170 SEQ ID NO: 69 
SEQ ID NO: 70 
8 247 SEQ ID NO: 71 
SEQ ID NO: 72 
232 SEQ ID NO: 73 
SEQ ID NO: 74 
9 300 SEQ ID NO: 75 
SEQ ID NO: 76 
10 260 SEQ ID NO: 77 
SEQ ID NO: 78 
11 268 SEQ ID NO: 79 
SEQ ID NO: 80 
12A 140 SEQ ID NO: 81 
SEQ ID NO: 82 
12B 284 SEQ ID NO: 83 
SEQ ID NO: 84 
13 228 SEQ ID NO: 85 
SEQ ID NO: 86 
14 254 SEQ ID NO: 87 
SEQ ID NO: 88 
15 317 SEQ ID NO: 89 
SEQ ID NO: 90 
248 SEQ ID NO: 91 
SEQ ID NO: 92 
16 148 SEQ ID NO: 93 
SEQ ID NO: 94 
17 177 SEQ ID NO: 95 
SEQ ID NO: 96 
__________________________________________________________________________ 
.sup.1 All exons were scanned by single PCR assays except exon 12, for 
which overlapping assays (12A and 12B) were required. For exons 2, 5, 7, 
and 15, two different PCR assays were developed. 
.sup.2 Annealing temperature for PCR reaction 
TABLE 3 
__________________________________________________________________________ 
NF2 Gene Mutations in Schwannomas from NF2 Patients 
DNA Sequence 
Codon 
Tumor 
Exon 
Alteration.sup.1 
Change.sup.2 
Consequence 
Origin.sup.3 
Alleles.sup.4 
__________________________________________________________________________ 
S1 E7 655 G .fwdarw. A 
Val219Met 
Missense 
S 2 
S1 E10 
904/6 to 931/3 
Gly302fs &gt; 322X 
Frameshift 
G 
del 28 bp 
S4 E3 353 to 363 + 19 Splice donor 
S 2 
del 30 bp site 
S4 E13 
1396 C .fwdarw. T 
Arg466X Nonsense 
G 
S10 E9 844 del 1 bp (G) 
Val282fs &gt; 296X 
Frameshift 
S 2 
S11 E2 169 C .fwdarw. T 
Arg57X Nonsense 
G 2 
S32 E12 
1340 + 2 inst 1 bp 
Splice donor? 
G 2 
(t) 
S33 E2 179 G .fwdarw. A 
Trp60X Nonsense 
G 1 
S34 E14 
1451 to 1452 
Met484fs &gt; 494X 
Frameshift 
S NI 
del 2 bp (TG) 
S36 E7 600 - 28 to -5 Splice S 2 
del 24 bp acceptor? 
__________________________________________________________________________ 
.sup.1 Numbering of bases showing alteration is given relative to the cDN 
sequence with the initiator ATG beginning at base 1. All coding sequence 
bases are given in upper case. When the alteration affects intronic 
sequence, it is presented in lower case and numbered as "-" (5' intron) o 
"+" (3' intron) the requisite number of bases from the first or last base 
of the exon, respectively. For deletions, the span of deleted bases 
(numbered as above) is given, followed by the deletion size ("del"). For 
deletions of less than 5 bp, the deleted base are also named. Where the 
start position of the deletion is uncertain, the alternative ranges of 
bases deleted are shown. Insertion is indicated by "ins" followed by the 
number of bases inserted, and their identity. 
.sup.2 Original amino acid and position of the residues in the protein 
(numbered from the initiator Met as 1) is followed by new amino acid for 
missense mutation, X for nonsense mutation, or fs for frameshift, followe 
by the position of the next inframe stop codon. 
.sup.3 S = somatic mutation; G = germline mutation. 
.sup.4 Number of NF2 alleles in tumor predicted by heterozygosity testing 
with Chr 22 DNA markers. NI = not informative. 
TABLE 4 
__________________________________________________________________________ 
NF2 Gene Mutations in Sporadic Schwannomas.sup.1 
DNA Sequence 
Codon 
Tumor 
Exon 
Alteration Change Consequence 
Origin 
Alleles 
__________________________________________________________________________ 
S2 E3 241 - 22 to -13 Splice acceptor? 
S 1 
del 10 bp 
S3 NI 
S5 1 
S6 2 
S9 E7 600 - 32 t -&gt; a Acceptor-branch site? 
G 2 
S12 E7 600 - 1 g -&gt; a Splice acceptor site 
S 1 
S13 2 
S14 1 
S15 E15 
1634/6 to 1694/6 
Ile546fs &gt; 
Frameshift 
S 1 
del 61 bp 550X 
S16 E4 439 del 1 (C) 
Gln147fs &gt; 
Frameshift 
S 2 
174X 
S17 2 
S18 E8 676 - 10 to 726 Splice acceptor site 
S 2 
del 61 bp 
E12 
1266 to 1267 
Glu422fs &gt; 
Frameshift 
S 
del 2 (GA) 442X 
S19 E15 
1575 - 26/-27 to 1581/2 
Splice acceptor site 
S 1 
del 34 bp 
S22 E7 675 + 1 g -&gt; t Splice donor site 
S 1 
S23 1 
S24 E7 634 C -&gt; T Gln212X 
Nonsence S 2 
E10 
905 to 912 Gly302fs &gt; 
Frameshift 
S 
del 8 bp 331X 
S25 E10 
992 to 999 +1 del 9 bp 
Splice donor site 
S 1 
S26 1 
S27 2 
S29 E2 134 del 1 (A) 
Asp45fs &gt; 
Frameshift 
S 2 
123X 
E8 729 to 732 Ile243fs &gt; 
Frameshift 
S 
del 4 (TTAT) 
251X 
S30 E4 447 or 447 + 1 del 1 
Lys149fs &gt; 
Frameshift 
S 1 
(G or g) 174X or splice donor site 
S31 1 
535 E1 65/70 to 79/84 
del 5 aa 
Frameshift 
S 1 
del 15 bp Asp30fs &gt; 40X 
88 to 109 del 22 bp 
S37 E4 364 - 2 a -&gt; g Splice acceptor site 
S 2 
S38 E10 
933 del 1 (G) 
Arg311fs &gt; 
Frameshift 
S 2 
322X 
S39 E12 
1223 to 1227 
Glu408fs &gt; 
Frameshift 
S 1 
del 5 bp 442X 
S40 1 
S42 E14 
1517/20 ins 1 (T) 
Phe507fs &gt; 
Frameshift 
S 2 
513X 
S43 E14 
1571/4 del 1 (A) 
Lys525fs &gt; 
Frameshift 
S 2 
550X 
S44 E12 
1252 C - &gt;T 
Arg418Cys 
Missense G 2 
__________________________________________________________________________ 
.sup.1 Explanation of all symbols can be found in Table 3. 
Having now fully described the invention, it will be understood by those 
with skill in the art that the scope may be performed within a wide and 
equivalent range of conditions, parameters, and the like, without 
affecting the spirit or scope of the invention or of any embodiment 
thereof. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 120 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CAGATTGTTCATTCCAAGTGG21 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
ACCCTGAGGAATCCACTACC20 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
TGCACACACATCCTTTTCAC20 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GAGAGAGACTGCTGTCTCAAAAA23 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
AGGAGGCTGAACGCACGAG19 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
TGGTATTGTGCTTGCTGCTG20 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
CTTCAACCTGATTGGTGACAG21 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
TGGTATTGTGCTTGCTGGTG20 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
AGGTACTGGATCATGATGTTTC22 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
TTTGGAAGCAATTCCTCTTGG21 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
CCAGCCAGCTCCCTATGGATG21 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
AGCTGAAATGGAATATCTGAAG22 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
GCCTTCTCCTCCCTGGCCTG20 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
GATGGAGTTCAATTGCGAGATG22 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2257 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 220..2004 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
ACGGCAGCCGTCAGGGACCGTCCCCCAACTCCCCTTTCCGCTCAGGCAGGGTCCTCGCGG60 
CCCATGCTGGCCGCTGGGGACCCGCGCAGCCCAGACCGTTCCCGGGCCGGCCAGCCGGCA120 
CCATGGTGGCCCTGAGGCCTGTGCAGCAACTCCAGGGGGGCTAAAGGGCTCAGAGTGCAG180 
GCCGTGGGGCGCGAGGGTCCCGGGCCTGAGCCCCGCGCCATGGCCGGGGCCATC234 
MetAlaGlyAlaIle 
15 
GCTTCCCGCATGAGCTTCAGCTCTCTCAAGAGGAAGCAACCCAAGACG282 
AlaSerArgMetSerPheSerSerLeuLysArgLysGlnProLysThr 
101520 
TTCACCGTGAGGATCGTCACCATGGACGCCGAGATGGAGTTCAATTGC330 
PheThrValArgIleValThrMetAspAlaGluMetGluPheAsnCys 
253035 
GAGATGAAGTGGAAAGGGAAGGACCTCTTTGATTTGGTGTGCCGGACT378 
GluMetLysTrpLysGlyLysAspLeuPheAspLeuValCysArgThr 
404550 
CTGGGGCTCCGAGAAACCTGGTTCTTTGGACTGCAGTACACAATCAAG426 
LeuGlyLeuArgGluThrTrpPhePheGlyLeuGlnTyrThrIleLys 
556065 
GACACAGTGGCCTGGCTCAAAATGGACAAGAAGGTACTGGATCATGAT474 
AspThrValAlaTrpLeuLysMetAspLysLysValLeuAspHisAsp 
70758085 
GTTTCAAAGGAAGAACCAGTCACCTTTCACTTCTTGGCCAAATTTTAT522 
ValSerLysGluGluProValThrPheHisPheLeuAlaLysPheTyr 
9095100 
CCTGAGAATGCTGAAGAGGAGCTGGTTCAGGAGATCACACAACATTTA570 
ProGluAsnAlaGluGluGluLeuValGlnGluIleThrGlnHisLeu 
105110115 
TTCTTCTTACAGGTAAAGAAGCAGATTTTAGATGAAAAGATCTACTGC618 
PhePheLeuGlnValLysLysGlnIleLeuAspGluLysIleTyrCys 
120125130 
CCTCCTGAGGCTTCTGTGCTCCTGGCTTCTTACGCCGTCCAGGCCAAG666 
ProProGluAlaSerValLeuLeuAlaSerTyrAlaValGlnAlaLys 
135140145 
TATGGTGACTACGACCCCAGTGTTCACAAGCGGGGATTTTTGGCCCAA714 
TyrGlyAspTyrAspProSerValHisLysArgGlyPheLeuAlaGln 
150155160165 
GAGGAATTGCTTCCAAAAAGGGTAATAAATCTGTATCAGATGACTCCG762 
GluGluLeuLeuProLysArgValIleAsnLeuTyrGlnMetThrPro 
170175180 
GAAATGTGGGAGGAGAGAATTACTGCTTGGTACGCAGAGCACCGAGGC810 
GluMetTrpGluGluArgIleThrAlaTrpTyrAlaGluHisArgGly 
185190195 
CGAGCCAGGGATGAAGCTGAAATGGAATATCTGAAGATAGCTCAGGAC858 
ArgAlaArgAspGluAlaGluMetGluTyrLeuLysIleAlaGlnAsp 
200205210 
CTGGAGATGTACGGTGTGAACTACTTTGCAATCCGGAATAAAAAGGGC906 
LeuGluMetTyrGlyValAsnTyrPheAlaIleArgAsnLysLysGly 
215220225 
ACAGAGCTGCTGCTTGGAGTGGATGCCCTGGGGCTTCACATTTATGAC954 
ThrGluLeuLeuLeuGlyValAspAlaLeuGlyLeuHisIleTyrAsp 
230235240245 
CCTGAGAACAGACTGACCCCCAAGATCTCCTTCCCGTGGAATGAAATC1002 
ProGluAsnArgLeuThrProLysIleSerPheProTrpAsnGluIle 
250255260 
CGAAACATCTCGTACAGTGACAAGGAGTTTACTATTAAACCACTGGAT1050 
ArgAsnIleSerTyrSerAspLysGluPheThrIleLysProLeuAsp 
265270275 
AAGAAAATTGATGTCTTCAAGTTTAACTCCTCAAAGCTTCGTGTTAAT1098 
LysLysIleAspValPheLysPheAsnSerSerLysLeuArgValAsn 
280285290 
AAGCTGATTCTCCAGCTATGTATCGGGAACCATGATCTATTTATGAGG1146 
LysLeuIleLeuGlnLeuCysIleGlyAsnHisAspLeuPheMetArg 
295300305 
AGAAGGAAAGCCGATTCTTTGGAAGTTCAGCAGATGAAAGCCCAGGCC1194 
ArgArgLysAlaAspSerLeuGluValGlnGlnMetLysAlaGlnAla 
310315320325 
AGGGAGGAGAAGGCTAGAAAGCAGATGGAGCGGCAGCGCCTCGCTCGA1242 
ArgGluGluLysAlaArgLysGlnMetGluArgGlnArgLeuAlaArg 
330335340 
GAGAAGCAGATGAGGGAGGAGGCTGAACGCACGAGGGATGAGTTGGAG1290 
GluLysGlnMetArgGluGluAlaGluArgThrArgAspGluLeuGlu 
345350355 
AGGAGGCTGCTGCAGATGAAAGAAGAAGCAACAATGGCCAACGAAGCA1338 
ArgArgLeuLeuGlnMetLysGluGluAlaThrMetAlaAsnGluAla 
360365370 
CTGATGCGGTCTGAGGAGACAGCTGACCTGTTGGCTGAAAAGGCCCAG1386 
LeuMetArgSerGluGluThrAlaAspLeuLeuAlaGluLysAlaGln 
375380385 
ATCACCGAGGAGGAGGCAAAACTTCTGGCCCAGAAGGCCGCAGAGGCT1434 
IleThrGluGluGluAlaLysLeuLeuAlaGlnLysAlaAlaGluAla 
390395400405 
GAGCAGGAAATGCAGCGCATCAAGGCCACAGCGATTCGCACGGAGGAG1482 
GluGlnGluMetGlnArgIleLysAlaThrAlaIleArgThrGluGlu 
410415420 
GAGAAGCGCCTGATGGAGCAGAAGGTGCTGGAAGCCGAGGTGCTGGCA1530 
GluLysArgLeuMetGluGlnLysValLeuGluAlaGluValLeuAla 
425430435 
CTGAAGATGGCTGAGGAGTCAGAGAGGAGGGCCAAAGAGGCAGATCAG1578 
LeuLysMetAlaGluGluSerGluArgArgAlaLysGluAlaAspGln 
440445450 
CTGAAGCAGGACCTGCAGGAAGCACGCGAGGCGGAGCGAAGAGCCAAG1626 
LeuLysGlnAspLeuGlnGluAlaArgGluAlaGluArgArgAlaLys 
455460465 
CAGAAGCTCCTGGAGATTGCCACCAAGCCCACGTACCCGCCCATGAAC1674 
GlnLysLeuLeuGluIleAlaThrLysProThrTyrProProMetAsn 
470475480485 
CCAATTCCAGCACCGTTGCCTCCTGACATACCAAGCTTCAACCTCATT1722 
ProIleProAlaProLeuProProAspIleProSerPheAsnLeuIle 
490495500 
GGTGACAGCCTGTCTTTCGACTTCAAAGATACTGACATGAAGCGGCTT1770 
GlyAspSerLeuSerPheAspPheLysAspThrAspMetLysArgLeu 
505510515 
TCCATGGAGATAGAGAAAGAAAAAGTGGAATACATGGAAAAGAGCAAG1818 
SerMetGluIleGluLysGluLysValGluTyrMetGluLysSerLys 
520525530 
CATCTGCAGGAGCAGCTCAATGAACTCAAGACAGAAATCGAGGCCTTG1866 
HisLeuGlnGluGlnLeuAsnGluLeuLysThrGluIleGluAlaLeu 
535540545 
AAACTGAAAGAGAGGGAGACAGCTCTGGATATTCTGCACAATGAGAAC1914 
LysLeuLysGluArgGluThrAlaLeuAspIleLeuHisAsnGluAsn 
550555560565 
TCCGACAGGGGTGGCAGCAGCAAGCACAATACCATTAAAAAGCTCACC1962 
SerAspArgGlyGlySerSerLysHisAsnThrIleLysLysLeuThr 
570575580 
TTGCAGAGCGCCAAGTCCCGAGTGGCCTTCTTTGAAGAGCTC2004 
LeuGlnSerAlaLysSerArgValAlaPhePheGluGluLeu 
585590595 
TAGCAGGTGACCCAGCCACCCCAGGACCTGCCACTTCTCCTGCTACCGGGACCGCGGGAT2064 
GGACCAGATATCAAGAGAGCCATCCATAGGGAGCTGGCTGGGGGTTTCCGTGGGAGCTCC2124 
AGAACTTTCCCCAGCTGAGTGAAGAGCCCAGCCCCTCTTATGTGCAATTGCCTTGAACTA2184 
CGACCCTGTAGAGATTTCTCTCATGGCGTTCTAGTTCTCTGACCTGAGTCTTTGTTTTAA2244 
GAAGTATTTGTCT2257 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 595 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
MetAlaGlyAlaIleAlaSerArgMetSerPheSerSerLeuLysArg 
151015 
LysGlnProLysThrPheThrValArgIleValThrMetAspAlaGlu 
202530 
MetGluPheAsnCysGluMetLysTrpLysGlyLysAspLeuPheAsp 
354045 
LeuValCysArgThrLeuGlyLeuArgGluThrTrpPhePheGlyLeu 
505560 
GlnTyrThrIleLysAspThrValAlaTrpLeuLysMetAspLysLys 
65707580 
ValLeuAspHisAspValSerLysGluGluProValThrPheHisPhe 
859095 
LeuAlaLysPheTyrProGluAsnAlaGluGluGluLeuValGlnGlu 
100105110 
IleThrGlnHisLeuPhePheLeuGlnValLysLysGlnIleLeuAsp 
115120125 
GluLysIleTyrCysProProGluAlaSerValLeuLeuAlaSerTyr 
130135140 
AlaValGlnAlaLysTyrGlyAspTyrAspProSerValHisLysArg 
145150155160 
GlyPheLeuAlaGlnGluGluLeuLeuProLysArgValIleAsnLeu 
165170175 
TyrGlnMetThrProGluMetTrpGluGluArgIleThrAlaTrpTyr 
180185190 
AlaGluHisArgGlyArgAlaArgAspGluAlaGluMetGluTyrLeu 
195200205 
LysIleAlaGlnAspLeuGluMetTyrGlyValAsnTyrPheAlaIle 
210215220 
ArgAsnLysLysGlyThrGluLeuLeuLeuGlyValAspAlaLeuGly 
225230235240 
LeuHisIleTyrAspProGluAsnArgLeuThrProLysIleSerPhe 
245250255 
ProTrpAsnGluIleArgAsnIleSerTyrSerAspLysGluPheThr 
260265270 
IleLysProLeuAspLysLysIleAspValPheLysPheAsnSerSer 
275280285 
LysLeuArgValAsnLysLeuIleLeuGlnLeuCysIleGlyAsnHis 
290295300 
AspLeuPheMetArgArgArgLysAlaAspSerLeuGluValGlnGln 
305310315320 
MetLysAlaGlnAlaArgGluGluLysAlaArgLysGlnMetGluArg 
325330335 
GlnArgLeuAlaArgGluLysGlnMetArgGluGluAlaGluArgThr 
340345350 
ArgAspGluLeuGluArgArgLeuLeuGlnMetLysGluGluAlaThr 
355360365 
MetAlaAsnGluAlaLeuMetArgSerGluGluThrAlaAspLeuLeu 
370375380 
AlaGluLysAlaGlnIleThrGluGluGluAlaLysLeuLeuAlaGln 
385390395400 
LysAlaAlaGluAlaGluGlnGluMetGlnArgIleLysAlaThrAla 
405410415 
IleArgThrGluGluGluLysArgLeuMetGluGlnLysValLeuGlu 
420425430 
AlaGluValLeuAlaLeuLysMetAlaGluGluSerGluArgArgAla 
435440445 
LysGluAlaAspGlnLeuLysGlnAspLeuGlnGluAlaArgGluAla 
450455460 
GluArgArgAlaLysGlnLysLeuLeuGluIleAlaThrLysProThr 
465470475480 
TyrProProMetAsnProIleProAlaProLeuProProAspIlePro 
485490495 
SerPheAsnLeuIleGlyAspSerLeuSerPheAspPheLysAspThr 
500505510 
AspMetLysArgLeuSerMetGluIleGluLysGluLysValGluTyr 
515520525 
MetGluLysSerLysHisLeuGlnGluGlnLeuAsnGluLeuLysThr 
530535540 
GluIleGluAlaLeuLysLeuLysGluArgGluThrAlaLeuAspIle 
545550555560 
LeuHisAsnGluAsnSerAspArgGlyGlySerSerLysHisAsnThr 
565570575 
IleLysLysLeuThrLeuGlnSerAlaLysSerArgValAlaPhePhe 
580585590 
GluGluLeu 
595 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
CCATCTCACTTAGCTCCAATG21 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
CTCACTCAGTCTCTGTCTAC20 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
GTAACCGGCC10 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
GTTATTGCAG10 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
GTTGGGCTAG10 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
AATTCTGCAG10 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
GTACATCAGT10 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
CTCCTTTCAG10 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
GTAGGCTCAA10 
(2) INFORMATION FOR SEQ ID NO:26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
TTCTTTCCAG10 
(2) INFORMATION FOR SEQ ID NO:27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
GTAAGAGATT10 
(2) INFORMATION FOR SEQ ID NO:28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
TTTTTGGTAG10 
(2) INFORMATION FOR SEQ ID NO:29: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
GTGAGGCCCA10 
(2) INFORMATION FOR SEQ ID NO:30: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
CTCCCCACAG10 
(2) INFORMATION FOR SEQ ID NO:31: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
GTGTGTTGAA10 
(2) INFORMATION FOR SEQ ID NO:32: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
GGATCCACAG10 
(2) INFORMATION FOR SEQ ID NO:33: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
GTAGGACATG10 
(2) INFORMATION FOR SEQ ID NO:34: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
ATTCTTCCAG10 
(2) INFORMATION FOR SEQ ID NO:35: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: 
GTAAGTTGAG10 
(2) INFORMATION FOR SEQ ID NO:36: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: 
GTGGCCACAG10 
(2) INFORMATION FOR SEQ ID NO:37: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: 
GTGAGCACAA10 
(2) INFORMATION FOR SEQ ID NO:38: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: 
CCCCTCGCAG10 
(2) INFORMATION FOR SEQ ID NO:39: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: 
GTGATTTCTG10 
(2) INFORMATION FOR SEQ ID NO:40: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: 
TGCCCTCCAG10 
(2) INFORMATION FOR SEQ ID NO:41: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: 
GTGAGGGGGC10 
(2) INFORMATION FOR SEQ ID NO:42: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: 
TTCCTTGCAG10 
(2) INFORMATION FOR SEQ ID NO:43: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: 
GTGAGCCTGG10 
(2) INFORMATION FOR SEQ ID NO:44: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: 
TCATTAACAG10 
(2) INFORMATION FOR SEQ ID NO:45: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: 
GTATGTAGCC10 
(2) INFORMATION FOR SEQ ID NO:46: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: 
TTGCCGGCAG10 
(2) INFORMATION FOR SEQ ID NO:47: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: 
GTACCCAGGG10 
(2) INFORMATION FOR SEQ ID NO:48: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: 
GCTGGTTTAG10 
(2) INFORMATION FOR SEQ ID NO:49: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: 
GTAGGTTGTT10 
(2) INFORMATION FOR SEQ ID NO:50: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: 
TTTCTTACAG10 
(2) INFORMATION FOR SEQ ID NO:51: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: 
GCTAAAGGGCTCAGAGTGCAG21 
(2) INFORMATION FOR SEQ ID NO:52: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: 
GAGAACCTCTCGAGCTTCCAC21 
(2) INFORMATION FOR SEQ ID NO:53: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: 
TGTCCTTCCCCATTGGTTTG20 
(2) INFORMATION FOR SEQ ID NO:54: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: 
CAGTTTCATCGAGTTCTAGCC21 
(2) INFORMATION FOR SEQ ID NO:55: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55: 
AGTGCAGAGAAAAGGTTTTATTAATGAT28 
(2) INFORMATION FOR SEQ ID NO:56: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: 
TGGAAAGCTCACGTCAGCC19 
(2) INFORMATION FOR SEQ ID NO:57: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: 
GCTTCTTTGAGGGTAGCACA20 
(2) INFORMATION FOR SEQ ID NO:58: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: 
GGTCAACTCTGAGGCCAACT20 
(2) INFORMATION FOR SEQ ID NO:59: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: 
CCTCACTTCCCCTCACAGAG20 
(2) INFORMATION FOR SEQ ID NO:60: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60: 
CCCATGACCCAAATTAACGC20 
(2) INFORMATION FOR SEQ ID NO:61: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: 
GCTCTCCCTTTCTTCTTTCC20 
(2) INFORMATION FOR SEQ ID NO:62: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: 
TCCTTCAAGTCCTTTGGTTAGC22 
(2) INFORMATION FOR SEQ ID NO:63: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: 
TGGCAGTTATCTTTAGAATCTC22 
(2) INFORMATION FOR SEQ ID NO:64: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: 
TTAGACCACATATCTGCTATG21 
(2) INFORMATION FOR SEQ ID NO:65: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65: 
CATGTGTAGGTTTTTTATTTTGC23 
(2) INFORMATION FOR SEQ ID NO:66: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: 
GCCCATAAAGGAATGTAAACC21 
(2) INFORMATION FOR SEQ ID NO:67: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: 
CCATCTCACTTAGCTCCAATG21 
(2) INFORMATION FOR SEQ ID NO:68: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: 
CTCACTCAGTCTCTGTCTAC20 
(2) INFORMATION FOR SEQ ID NO:69: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: 
GAATGCTTGATTTGGTGCCC20 
(2) INFORMATION FOR SEQ ID NO:70: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70: 
GAGGTTTCAACACACCCGGA20 
(2) INFORMATION FOR SEQ ID NO:71: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: 
GAAGGTTGAATAAAATTTTGAGCCTC26 
(2) INFORMATION FOR SEQ ID NO:72: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: 
GACAGGGAAAGATCTGCTGGACC23 
(2) INFORMATION FOR SEQ ID NO:73: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: 
CTGTTCTTATTGGATCCACAG21 
(2) INFORMATION FOR SEQ ID NO:74: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: 
AACAACCACACCCTCAAAGC20 
(2) INFORMATION FOR SEQ ID NO:75: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75: 
GACTTGGTGCTCCTAATTCCC21 
(2) INFORMATION FOR SEQ ID NO:76: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: 
CCATTATCAGTAATGAAAACCAGG24 
(2) INFORMATION FOR SEQ ID NO:77: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: 
TGCTACCTGCAAGAGCTCAA20 
(2) INFORMATION FOR SEQ ID NO:78: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: 
CTGACCACACAGTGACATC19 
(2) INFORMATION FOR SEQ ID NO:79: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: 
TCTTTGGCCCTTGTGGCAC19 
(2) INFORMATION FOR SEQ ID NO:80: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:80: 
CAGGAGACCAAGCTCCAGAA20 
(2) INFORMATION FOR SEQ ID NO:81: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: 
TTCAGCTAAGAGCACTGTGC20 
(2) INFORMATION FOR SEQ ID NO:82: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: 
CGCTGCATTTCCTGCTCAG19 
(2) INFORMATION FOR SEQ ID NO:83: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: 
GCTGAAAAGGCCCAGATCA19 
(2) INFORMATION FOR SEQ ID NO:84: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:84: 
CTTGAGGACAACTGCTGTAG20 
(2) INFORMATION FOR SEQ ID NO:85: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:85: 
GGTGTCTTTTCCTGCTACCT20 
(2) INFORMATION FOR SEQ ID NO:86: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:86: 
GGGAGGAAAGAGAACATCAC20 
(2) INFORMATION FOR SEQ ID NO:87: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:87: 
TGTGCCATTGCCTCTGTG18 
(2) INFORMATION FOR SEQ ID NO:88: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:88: 
AGGGCACAGGGGGCTACA18 
(2) INFORMATION FOR SEQ ID NO:89: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:89: 
TGGCCAAGTAGAGACGTGA19 
(2) INFORMATION FOR SEQ ID NO:90: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:90: 
TACAAGAAAGAGACCCTGGG20 
(2) INFORMATION FOR SEQ ID NO:91: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:91: 
TCTGCCCAAGCCCTGATGC19 
(2) INFORMATION FOR SEQ ID NO:92: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:92: 
TGGTCCTGATCAGCAAAATAC21 
(2) INFORMATION FOR SEQ ID NO:93: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:93: 
GGCATTGTTGATATCACAGGG21 
(2) INFORMATION FOR SEQ ID NO:94: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:94: 
GGCAGCACCATCACCACATA20 
(2) INFORMATION FOR SEQ ID NO:95: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:95: 
CTCTCAGCTTCTTCTCTGCT20 
(2) INFORMATION FOR SEQ ID NO:96: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:96: 
CCAGCCAGCTCCTATGGATG20 
(2) INFORMATION FOR SEQ ID NO:97: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: both 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:97: 
TACGGTGTGWACTACTTTGCA21 
(2) INFORMATION FOR SEQ ID NO:98: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: both 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..21 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:98: 
TACGGTGTGAACTACTTTGCA21 
TyrGlyValAsnTyrPheAla 
15 
(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: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:99: 
TyrGlyValAsnTyrPheAla 
15 
(2) INFORMATION FOR SEQ ID NO:100: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: both 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:100: 
GGGAAGGACCTCTTT15 
(2) INFORMATION FOR SEQ ID NO:101: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: both 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:101: 
CACATTTATGACCC14 
(2) INFORMATION FOR SEQ ID NO:102: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 243 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: both 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:102: 
AGTGCAGAGAAAAGGTTTTATTAATGATTTTTGCTCACAGTGTCCTTCCCCATTGGTTTG60 
TTATTGCAGATGAAGTGGAAAGGGAAGGACCTCTTTGATTTGGTGTGCCGGACTCTGGGG120 
CTCCGAGAAACCTGGTTCTTTGGACTGCAGTACACAATCAAGGACACAGTGGCCTGGCTC180 
AAAATGGACAAGAAGGTTGGGCTAGAACTCGATGAAACTGGTGGGGCTGACGTGAGCTTT240 
CCA243 
(2) INFORMATION FOR SEQ ID NO:103: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 275 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: both 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:103: 
GCTTCTTTGAGGGTAGCACAGGAGGAAGTGCCAATATANNGTGTGTTTGTCTTTTGCTCT60 
GCAATTCTGCAGGTACTGGATCATGATGTTTCAAAGGAAGAACCAGTCACCTTTCACTTC120 
TTGGCCAAATTTTATCCTGAGAATGCTGAAGAGGAGCTGGTTCAGGAGATCACACAACAT180 
TTATTCTTCTTACAGGTACATCAGTCAAGGCTACCCCCCAGTTCTGAGAGAGAACTTGCC240 
CAGGAGTGGTTGCAGAGTTGGCCTCAGAGTTGACC275 
(2) INFORMATION FOR SEQ ID NO:104: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 236 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: both 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:104: 
GCTAAAGGGCTCAGAGTGCAGGCCGTGGGGCGCGAGGGTCCCGGGCCTGAGCCCCGCGCC60 
ATGGCCGGGGCCATCGCTTCCCGCATGAGCTTCAGCTCTCTCAAGAGGAAGCAACCCAAG120 
ACGTTCACCGTGAGGATCGTCACCATGGACGCCGAGATGGAGTTCAATTGCGAGGTAACC180 
GGCCGGCAGCCCCGACTGCTGCGGTGACAGTCGAGGTGGAAGCTCGAGAGGTTCTC236 
(2) INFORMATION FOR SEQ ID NO:105: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 188 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: both 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:105: 
CCTCACTTCCCCTCACAGAGTATCATGTCTCCCTTGTTGCTCCTTTCAGGTAAAGAAGCA60 
GATTTTAGATGAAAAGATCTACTGCCCTCCTGAGGCTTCTGTGCTCCTGGCTTCTTACGC120 
CGTCCAGGCCAAGGTAGGCTCAAAGAAGAAAAATGTATTTTTNNCTGGGCGTTAATTTGG180 
GTCATGGG188 
(2) INFORMATION FOR SEQ ID NO:106: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 200 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: both 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:106: 
TGGCAGTTATCTTTAGAATCTCAATCGCCTGCTCTCCCTTTCTTCTTTCCAGTATGGTGA60 
CTACGACCCCAGTGTTCACAAGCGGGGATTTTTGGCCCAAGAGGAATTGCTTCCAAAAAG120 
GGTAAGAGATTAAATTCCCTTTTCAGGAAGACATAGCAGATATGTGGTCTAAAAGAAAGC180 
TAACCAAAGGACTTGAAGGA200 
(2) INFORMATION FOR SEQ ID NO:107: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 256 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: both 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:107: 
TCTGTGTGACTACTCCTGGTGTAGCTTTAAAATAGCTTTACTGTTTGTAAAATGATGCAT60 
AATTATAAAAGTGGCAAACAATACCAAATTTACTTCATGTGTAGGTTTTTTATTTTGCTC120 
TATTTTTTGGTAGGTAATAAATCTGTATCAGATGACTCCGGAAATGTGGGAGGAGAGAAT180 
TACTGCTTGGTACGCAGAGCACCGAGGCCGAGCCAGGTGAGGCCCATTCATTGTTGGTTT240 
ACATTCCTTTATGGGC256 
(2) INFORMATION FOR SEQ ID NO:108: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 240 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: both 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:108: 
GAATGCTTGATTTGGTGGCCCACCCGCTCTCCACCCATCTCACTTAGCTCCAATGACAGT60 
GTCTTCCGTTCTCCCCACAGGGATGAAGCTGAAATGGAATATCTGAAGATAGCTCAGGAC120 
CTGGAGATGTACGGTGTGAACTACTTTGCAATCCGGGTGTGTTGAAACCTCTCTGAGCTC180 
CTTGTGTAGTAGACAGAGACTGAGTGAGGGCCAGGACTGCTAAAATGGTTACTTCTTCAT240 
(2) INFORMATION FOR SEQ ID NO:109: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 387 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: both 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:109: 
TCTGTGGACCTGCTGAACTGCACATGTGACAGTGTGTGCCAGATTCTTTGGAAGGTTGAA60 
TAAAATTTTGAGCCTCAGCTGGCGCTTACAGTAGCTGTTCTTATTGGATCCACAGAATAA120 
AAAGGGCACAGAGCTGCTGCTTGGAGTGGATGCCCTGGGGCTTCACATTTATGACCCTGA180 
GAACAGACTGACCCCCAAGATCTCCTTCCCGTGGAATGAAATCCGAAACATCTCGTACAG240 
TGACAAGGAGGTAGGACATGTGTGTACTGCAGATGGGTCCAGCAGATCTTTCCCTGTCTG300 
CCCCCCTCACTGGAGCCTCCCCAGCCAGGGCATCTCCTTGTTATTCATAGAGTCCTTTAA360 
TTCCCAGGCTTTGAGGGTGTGGTTGTT387 
(2) INFORMATION FOR SEQ ID NO:110: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 300 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: both 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:110: 
GACTTGGTGCTCCTAATTCCCTGAGGTTTAGTGCCTGGATACTGGGAAGCCAGNACAAGG60 
GCATAACNTCATGCTGGTCTGTGGCCAGTGTGGTTGCGCATTTGTGGAATTNCCAATTGC120 
TGGTAACATTCCAGGCTGTCGGACTGAAACTGTGTTCTGCTTCATTCTTCCAGTTTACTA180 
TTAAACCACTGGATAAGAAAATTGATGTCTTCAAGTTTAACTCCTCAAAGCTTCGTGTTA240 
ATAAGCTGGTAAGTTGAGATCCTGGTAAGTTGAGATCCTGGTTTTCATTACTGATAATGG300 
(2) INFORMATION FOR SEQ ID NO:111: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 260 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: both 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:111: 
TGCTACCTGCAAGAGCTCAAACTGCTATGGCACTAGTGGGCCAGTAGGCAGTGAAGTAAA60 
TTTGTGGATATTAACCTTTTTGTCTGCTTCTGTGGCCACAGATTCTCCAGCTATGTATCG120 
GGAACCATGATCTATTTATGAGGAGAAGGAAAGCCGATTCTTTGGAAGTTCAGCAGATGA180 
AAGCCCAGGCCAGGGAGGAGAAGGCTAGAAAGCAGGTGAGCACAACCTTGTTTTAACTGA240 
TGATGTCACTGTGTGGTCAG260 
(2) INFORMATION FOR SEQ ID NO:112: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 292 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: both 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:112: 
TCTTTGGCCCTTGTGGCACCCTAGGTCTCGAGCCCTGTGATTCAATGACTGTTTTTCTTC60 
ACCCCTCGCAGATGGAGCGGCAGCGCCTCGCTCGAGAGAAGCAGATGAGGGAGGAGGCTG120 
AACGCACGAGGGATGAGTTGGAGAGGAGGCTGCTGCAGATGAAAGAAGAAGCAACAATGG180 
CCAACGAAGCACTGGTGATTTCTGAGGGGCTGGGGTTCCAGGAGGCTACTTGGGGACTTC240 
CTTGGCTTTTCTGGAGCTTGGTCTCCTGAAAACATGAGTTAGCAGCGTTTGC292 
(2) INFORMATION FOR SEQ ID NO:113: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 365 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: both 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:113: 
CGGGAGAACAGCACATGATCCCACTTCAGCTAAGAGCACTGTGCCCTCCAGATGCGGTCT60 
GAGGAGACAGCTGACCTGTTGGCTGAAAAGGCCCAGATCACCGAGGAGGAGGCAAAACTT120 
CTGGCCCAGAAGGCCGCAGAGGCTGAGCAGGAAATGCAGCGCATCAAGGCCACAGCGATT180 
CGCACGGAGGAGGAGAAGCGCCTGATGGAGCAGAAGGTGCTGGAAGCCGAGGTGCTGGCA240 
CTGAAGATGGCTGAGGAGTCAGAGAGGAGGTGAGGGGGCACCGGGCACCAGACTGGCGAG300 
GAGGCTGGCGAAGGGCCGCAGACCAGCCTGCCCTGAGGCTGAGCTCTACAGCAGTTGTCC360 
TCAAG365 
(2) INFORMATION FOR SEQ ID NO:114: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 227 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: both 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:114: 
GGTGTCTTTTCCTGCTACCTGCCCTCTTCTGTGAAGCTGACATCTCATCCTTTCCTTGCA60 
GGGCCAAAGAGGCAGATCAGCTGAAGCAGGACCTGCAGGAAGCACGCGAGGCGGAGCGAA120 
GAGCCAAGCAGAAGCTCCTGGAGATTGCCACCAAGCCCACGTACCCGGTGAGCCTGGGGG180 
CCACCAGCTGGGGCTGCCTTAGTCCTGGTGATGTTCTCTTTCCTCCC227 
(2) INFORMATION FOR SEQ ID NO:115: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 281 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: both 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:115: 
TGTGCCATTGCCTCTGTGGCTGCTGGAGGATCGGTTGTCAACACAGTAGTGTCCTTCTGT60 
GCTTGTATGACCCAAGCTCCTAATCCGAAATTTCTCATTAACAGCCCATGAACCCAATTC120 
CAGCACCGTTGCCTCCTGACATACCAAGCTTCAACCTCATTGGTGACAGCCTGTCTTTCG180 
ACTTCAAAGATACTGACATGAAGCGGCTTTCCATGGAGATAGAGAAAGAAAAGTATGTAG240 
CCCCCTGTGCCCTGCTGTGGGCTTGCTGTGAACTAGACTGA281 
(2) INFORMATION FOR SEQ ID NO:116: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 335 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: both 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:116: 
TGGCCAAGTAGAGACGTGANNCCAGCNTNAAACCCTAGATCGCACACCAAGCAGCTTGTG60 
GGCCACAGAGCACCTGAGCCGTGTCTCACTGTCTGCCCAAGCCCTGATGCATGATACCCT120 
CTTGCCGGCAGAGTGGAATACATGGAAAAGAGCAAGCATCTGCAGGAGCAGCTCAATGAA180 
CTCAAGACAGAAATCGAGGCCTTGAAACTGAAAGAGAGGGAGACAGCTCTGGATATTCTG240 
CACAATGAGAACTCCGACAGGGGTGGCAGCAGCAAGCACAATACCATTAAAAAGGTACCC300 
AGGGTCTCTTTCTTGTATTTTGCTGATCAGGACCA335 
(2) INFORMATION FOR SEQ ID NO:117: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 254 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: both 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:117: 
CAAACAAAATCACTCATCACGATNTCAGGCCTATCCAAGCATTTTGCANATGGCACTTAT60 
GGCATTGTTGATATCACAGGGTATGTTTTTGTTTTTCTTCATTTTATTTTGCTGGTTTAG120 
CCTCAAGCCCAAGGCAGAAGACCTATCTGCATTTGAGCCCTCAAAGTAGCTTGTTCCCAG180 
GTACTCTCTATGTGGTGATGGTGCTGCCCTCTGTGATACTAACCCGTGCATGAGNTTGCC240 
TGTCTCTGTCTCGG254 
(2) INFORMATION FOR SEQ ID NO:118: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 339 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: both 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:118: 
AGGACCCTGTGAGACAGAGCGGAGGTCNNGTGCCCTCTCAGCTTCTTCTCTGCTTTCTTA60 
CAGCTCACCTTGCAGAGCGCCAAGTCCCGAGTGGCCTTCTTTGAAGAGCTCTAGCAGGTG120 
ACCCAGCCACCCCAGGACCTGCCACTTCTCCTGCTACCGGGACCGCGGGATGGACCAGAT180 
ATCAAGAGAGCCATCCATAGGGAGCTGGCTGGGGGTTTCCGTGGGAGCTCCAGAACTTTC240 
CCCAGCTGAGTGAAGAGCCCAGCCCCTCTTATGTGCAATTGCCTTGAACTACGACCCTGT300 
AGAGATTTCTCTCATGGCGTTCTAGTTCTCTGACCTGAG339 
(2) INFORMATION FOR SEQ ID NO:119: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: both 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:119: 
GGGAAGGCCTCTTT14 
(2) INFORMATION FOR SEQ ID NO:120: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: both 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:120: 
CACATGACCC10 
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