Methods and compositions useful in the recognition, binding and expression of ribonucleic acids involved in cell growth, neoplasia and immunoregulation

A peptide, Hel-N1 (SEQ ID NO: 2), which can bind to a 3'-untranslated mRNA sequence (which encompasses the "instability sequence") that is uniquely present in the messenger RNAs that encode oncoproteins and lymphokines, and mediates the specific destruction of the messenger RNAs, is described. Full-length Hel-N1 is capable of suppressing cell growth and causing cellular differentiation. Hel-N1 (SEQ ID NO: 2) possess three RNA recognition motifs. One of these forms an RNA-binding domain which, when transfected alone into cells, causes them to undergo rapid growth.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to proteins which contain amino acid sequences that 
bind to 3'-untranslated regions of mRNAs, particularly mRNA sequences 
containing "instability sequences" (Shaw et al, Cell (1986) 46: 659-667). 
2. Discussion of the Background 
General features of primary sequence that characterize RNA- and DNA-binding 
proteins have begun to become apparent. The helix-turn-helix (Pabo et al, 
Annu. Rev. Biochem., (1984) 53: 293-321) and zinc-binding finger (Evans et 
al, Cell 1988) 52: 1-3) arrangements have both been observed as structural 
features of sequence-specific DNA-binding proteins. In eukaryotes, the 
homeobox domain seems to represent a widespread primary sequence motif for 
specific DNA-binding (Levine et al, Cell (1988) 55: 537-540; Robertson, 
Nature (1988) 336: 522-524, and references therein), and the members of 
the steroid hormone receptor superfamily of DNA-binding proteins utilize a 
common motif which forms zinc-binding fingers (Evans, Science (1988) 240: 
889-895). 
Early on RNA-binding proteins were less well studied than DNA-binding 
proteins; general features of RNA-binding proteins were not evident until 
the recognition of an amino acid octamer present in four proteins 
associated with mammalian nuclear RNAs (Adam et al, Mol. Cell Biol. (1986) 
6: 2932-2943). The recognition of RNA by proteins has appeared to the 
inventors to be a key reaction in the regulation of expression of the 
genetic material of all cells. 
One of the present inventors has studied RNA binding proteins of this group 
for many years and in 1983 isolated the first eukaryotic recombinant cDNA 
member of this family of proteins that encodes the human La RNA binding 
protein (Chambers et al, Proc. Natl. Acad. Sci. (USA) (1985) 82: 
2115-2119; Chambers et al, J. Biol. Chem. (1988) 263: 18043-18051). 
Subsequently, the observation by Dreyfuss and coworkers (Adam et al, Mol. 
Cell. Biol. (1986) 6: 2932-2943; Swanson et al, Mol. Cell. Biol. (1987) 1: 
1731-1739) of an "RNP consensus" octamer in several eukaryotic proteins 
associated with RNA was an early indication that an amino acid sequence 
common among some RNA-binding proteins might exist. 
Other publications by the Dreyfuss group (Dreyfuss et al, TIBS (1988) 13: 
86-91) and from many other laboratories (Amrein et al, Cell (1988) 55, 
1025-1035; Bell et al, Cell (1988) 55, 1037-1046; Bugler et al, J. Biol. 
Chem. (1987) 262: 10922-1-925; Chambers et al (1988), ibid; Deutscher et 
al, Proc. Natl. Acad. Sci. (USA) (1988) 85: 9479-9483; Goralski et al, 
Cell (1989) 56, 1101-1108; Keene, J. D., J. Autoimmunity (1989) 2: 
329-337; Merrill et al, J. Biol. Chem. (1988) 263, 3307-3313; Sachs et al, 
Mol. Cell. Biol. (1986) 7, 3268-3276) noted the presence of related 
sequences surrounding the octamer and speculated that these regions might 
participate in RNA binding. It was not known at that time however whether 
these sequences might endow specific as opposed to nonspecific recognition 
of RNA or if discontinuous regions involving long-range interactions 
within these proteins might be required for RNA binding. 
Some authors speculated that the octamer and its surrounding residues 
constituted an RNA binding domain and Dreyfuss and coauthors (ibid) chose 
an arbitrary size of 100 amino acids. Their theory was based upon the 
occurrence of similar sequences in a set of proteins that were all thought 
to be associated with RNA. Evidence for direct binding of such regions to 
specific RNA sequences was not available and no domains of proteins with 
binding activity were defined experimentally. 
Included in this theory was the suggestion that the 70K U1 snRNP protein 
contained an RNA binding domain of 93 amino acids from positions 94 to 
186. Other investigators (Theissen et al, EMBO J. (1986) 5: 3209-3217) had 
speculated that a different region of the 70K U1 snRNP protein 
encompassing amino acid residues 241 to 437 as well as the same region 
speculated by Dreyfuss were either one or both involved in RNA binding. 
These speculations were based upon the relationship of the highly basic 
(positively charged) region at amino acids 241 to 437 of 70K protein to 
regions of other proteins (e.g., protamines and histones) known to bind 
nucleic acid. No experimental evidence was available to support these 
suggestions. 
Although the 70K protein is one of ten proteins known to be associated with 
the U1 snRNP complex (Pettersson et al, J. Biol. Chem. (1984) 259: 
5907-5914), there was no evidence of specific RNA protein contact between 
the 70K protein and any RNA species until the discovery of a specific 
binding of the 70K protein to U1 RNA. Furthermore, of the other members of 
this group of proteins studied in our laboratory, as well as, in many 
other laboratories, none was shown to directly bind to a specific RNA 
sequence until one of the present inventors discovered the 
sequence-specific interaction between 70K U1 snRNP protein and U1 RNA. 
The region of the protein involved in this specific binding involves a 
different amino acid sequence of 70K protein than that speculated by 
Theissen et al or by Dreyfuss et al. In fact, one of the sequences 
proposed by Theissen as being responsible for RNA binding actually 
interferes with the detection of specific binding activity. 
In addition, the discovery of the precise RNA binding domain of the 70K 
protein includes additional important amino acid sequences not previously 
recognized by the theory of Dreyfuss et al, by the published work of other 
workers mentioned above or by some of the inventors themselves in their 
earlier studies of La (Chambers et al, ibid) and the 60 kD Ro (Deutscher 
et al, ibid) protein members of the group. 
RNA binding proteins are now known to be involved in the control of a 
variety of cellular regulatory and developmental processes, such as RNA 
processing and compartmentalization, mRNA translation and viral gene 
expression. Some proteins that recognize and bind RNA can be classified 
into families based upon primary sequence homology, as well as higher 
order structure. 
The family of RNA binding proteins containing an RNP consensus octamer and 
an 80 amino acid motif implicated in RNA recognition (RRM) has been the 
subject of intense investigation. Query et al, Cell (1989) 57: 89-101; 
Kenan et al, Trends Biochem. Sci. (1991) 16: 214-220. Based upon 
crystallographic and NMR spectroscopic studies of the U1 RNA binding 
domain of the U1 snRNP-A protein a model of the tertiary structure has 
been derived. The tertiary structural model together with RNA binding 
studies have led to the suggestion that the RNA binding surface resides on 
a monomeric unit with four anti-parallel .beta.-strands which contains 
solvent exposed aromatic and basic residues. Kenan et al (1991) supra. 
Additional biochemical data have demonstrated that a determinant of RNA 
binding specificity resides in a loop which connects two .beta.-strands. 
Bentley et al, Mol. Cell. Biol. (1991) 11: 1829-1839. 
More than forty members of the RRM superfamily have been reported to date, 
the majority of which reside in all tissues and are ubiquitously conserved 
in phylogeny. Kenan et al (1991) supra. Tissue-specific members of the RRM 
family are less common, including X16 which is expressed in pre-B cells, 
Bj6 which is a puff-specific Drosophila protein and elav (embryonic lethal 
abnormal vision) which is neuronal-specific in Drosophila. For some RRM 
proteins the natural RNA ligands have been identified or surmised, but the 
RNA-binding sequences are not known in most cases. 
The RNA ligands for the tissue-specific RRM proteins have not been reported 
and may prove difficult to determine because of their specialized roles in 
certain developmental processes. However, in order to understand their 
functions in cellular RNA metabolism and development, it will be essential 
to identify the RNA sequences to which they bind. 
Oncogenes encode growth factors that affect the rate of cell proliferation 
by influencing cell cycle events such as mitosis, intracellular signaling 
pathways and gene expression. Some well known oncogenes are c-src, c-myc 
and c-fos. Lymphokines, which affect the growth properties of 
immunoregulatory cells, also function as growth factors similar to 
oncogene products. Although oncogene products (oncoproteins) are central 
components in the origin of the neoplastic state, they work through a 
variety of complex and largely unknown pathways. Consequently, methods to 
specifically control the functions of oncoproteins have not materialized. 
The more recent discovery of suppressor oncogenes (anti-oncogenes) has held 
promise for being able to counter the effects of oncogenes. Some examples 
of anti-oncogenes include: retinoblastoma (Rb) and p53. It is hoped that 
these factors can be used to counter the effects of oncoproteins and thus, 
provide new treatments for cancer. For example, breast tumors show a 
consistent defect in the p53 gene, thus, preventing p53 from countering 
the oncogenes that cause uncontrolled proliferation of the breast tumors. 
Unfortunately, there are likely to be dozens of anti-oncogenes, each being 
specific to a given type of cancer. 
Accordingly, there is a strongly felt need for the discovery of materials 
generally useful in the recognition, binding and/or expression of 
ribonucleic acids involved in the growth, neoplasia and immunoregulation. 
Such materials would have many uses, including regulation of cell 
proliferation in vitro and in vivo, regulation of immune cell expression, 
stimulation of cell growth, the production of transgenic animals and cell 
lines for pharmaceutical tests of cancer, immune function and neurological 
diseases, diagnostic reagents for the detection of autoantibodies 
associated with cancers, in vivo targeting systems, in diagnosing 
pathology specimens of neuronal origin, and/or as genetic or neurogenetic 
disease markers involving malformations of the central nervous system. 
SUMMARY OF THE INVENTION 
Accordingly, one object of this invention is to provide novel proteins 
which can bind to mRNAs which encode oncoproteins or lymphokines. 
It is another object of this invention to provide novel proteins which can 
bind to 3'-untranslated regions of mRNAs, particularly mRNA instability 
sequences, in eukaryotic cells. 
It is another object of this invention to provide novel proteins, and their 
corresponding DNA and mRNA sequences, which can provide in cell cultures 
or in vivo modulation of the expression of oncogenes and/or 
lymphokine-encoding genes in eukaryotic cells. 
It is another object of this invention to provide novel proteins, and their 
corresponding DNA and mRNA sequences, useful in the regulation of cell 
proliferation in cell cultures and in vivo. 
It is another object of this invention to provide novel proteins, and their 
corresponding DNA and mRNA sequences, which can be used to take cells out 
of a proliferative state and into a state of differentiation. 
It is another object of this invention to provide novel proteins, and their 
corresponding DNA and mRNA sequences, useful in the regulation of immune 
cell gene expression. 
It is another object of this invention to provide novel proteins, and their 
corresponding DNA and mRNA sequences, useful for stimulating or 
suppressing mammalian cell growth. 
It is another object of this invention to provide novel proteins, and their 
corresponding DNA and mRNA sequences, useful to produce transgenic animals 
and cell lines for pharmaceutical tests of cancer, immune function and/or 
neurological diseases. 
It is another object of this invention to provide novel proteins, and their 
corresponding DNA and mRNA sequences, useful as diagnostic and/or 
therapeutic reagents for the detection or therapy of autoantibodies 
present in the body of a cancer patient. 
It is another object of this invention to provide novel proteins, and their 
corresponding DNA and mRNA sequences, which can be used for the in vivo 
targeting of certain substances. 
It is another object of this invention to provide novel proteins, and their 
corresponding DNA and mRNA sequences, useful for diagnosing pathology 
specimens of neuronal origin. 
It is another object of this invention to provide novel proteins, and their 
corresponding DNA and mRNA sequences, useful as genetic or neurogenetic 
disease markers in the diagnosis and/or therapy of patients in need 
thereof. 
The present invention which satisfies all of the above objects of the 
invention, and others as can be seen from the description of the invention 
given hereinbelow, relates to a novel protein, named Hel-N1 by the 
inventors, and related proteins, discovered by the inventors as being able 
to bind to 3'-untranslated mRNAs, including a sequence (the "instability 
sequence") that is uniquely present in the messenger RNAs that encode 
oncoproteins and lymphokines. The "instability sequence", discovered by 
Shaw et al (Cell (1986) 46: 659-667), resides in the 3'-noncoding region 
of mRNAs which encode oncoproteins and lymphokines. The present invention 
also provides DNA and mRNA sequences corresponding to Hel-N1 and the 
related proteins.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
In this text, the following standard nomenclature is used. 
TABLE 1 
______________________________________ 
Amino acid symbols. 
Three-letter 
One-letter 
Amino acid symbol symbol 
______________________________________ 
Alanine Ala A 
Arginine Arg R 
Asparagine Asn N 
Aspartic acid Asp D 
Asn + Asp Asx B 
Cysteine Cys C 
Glutamine Gln Q 
Glutamic acid Glu E 
Gln + Glu Glx Z 
Glycine Gly G 
Histidine His H 
Isoleucine Ile I 
Leucine Leu L 
Lysine Lys K 
Methionine Met M 
Phenylalanine Phe F 
Proline Pro P 
Serine Ser S 
Threonine Thr T 
Tryptophan Trp W 
Tyrosine Tyr Y 
Valine Val V 
______________________________________ 
The inventors have been isolating and characterizing RNA binding proteins, 
and studying their RNA-binding specificities. More particularly, as 
described in greater detail in application Ser. Nos. 07/536,943 and 
07/436,779, filed on Jun. 12, 1990 and Nov. 15, 1989, respectively, both 
of which are hereby incorporated by reference, in studying the RNA-binding 
properties of the U1 RNA-associated 70K protein to elucidate regions of 
RNA-protein interaction, one of the inventors of the present invention, 
together with others, identified a central amino acid sequence involved in 
the specificity of gene expression at the level of pre-messenger RNA 
splicing in cells. While several structural motifs of proteins important 
in sequence-specific DNA-binding had been identified (e.g., 
helix-turn-helix and zinc-binding fingers) and two primary sequence motifs 
recently have been implicated directly in DNA-binding (homeoboxes and 
sequences within the steroid receptor family which form zinc-binding 
fingers), the structure or primary sequences of RNA-binding domains were 
not known prior to the invention of application Ser. Nos. 07/536,943 and 
07/436,779. 
Elav is known to be involved in the early development of the central 
nervous system (CNS). Homozygous mutations of this gene locus give rise to 
numerous structural defects and hypotrophy of the CNS leading to embryonic 
lethality. Its role in neuronal growth and differentiation of the 
Drosophila nervous system is also underscored by the temporal appearance 
of elav transcripts during the differentiation of neuroblasts into 
primitive neurons. 
In probing for rat and human elav counterparts, the inventors relied on a 
novel approach of using degenerate primers designed to simulate the RNP-1 
octamer sequence present in two of the three RRMs of Drosophila elav and 
thereby isolated cDNA encoding a novel neuron-specific protein, named 
Hel-N1 by them, from human brain by a combination of degenerate PCR 
probing and hybridization and found it to contain three RNA-recognition 
motifs (RRMs),. FIG. 1 provides the complete amino acid sequence of 
Hel-N1. 
FNT * The term recognition motif is used herein to designate an amino acid 
relationship; the term "RNA binding domain" designates a peptide segment 
shown to possess binding activity. 
In in vitro studies they found that, in RNA binding, Hel-N1 prefers short 
stretches of uridylate residues and can bind the 3'-untranslated regions 
of c-myc, c-fos, and GM-CSF messenger RNAs, and that although Hel-N1 has 
three RRMs, only the third one (the most C-terminal binding domain 
situated between about amino acid positions 259 and 359) is responsible 
for mRNA 3'-untranslated region (which encompasses the instability 
sequence) binding activity. The inventors further discovered that full 
length Hel-N1, when transfected into a cell, caused cellular growth to 
cease. But, by contrast, and quite surprisingly, when only the third RNA 
binding domain was transfected into cells, the opposite result was 
obtained--the cells underwent rapid growth (as illustrated in FIG. 5). 
It is not clear at this point whether transfection with the third RNA 
binding domain alone causes cellular transformation in the sense of an 
oncogene. RNA binding data obtained to date indicates that the single 
domain alone interferes with the ability of the full length Hel-N1 protein 
to bind in a multimeric fashion along the unstable oncoprotein or 
lymphokine mRNA. Thus, apparently the mRNA is rendered more stable and 
thus, more rapid proliferation results. In this sense, RBD3 may be a 
dominant negative suppressor of the instability function of Hel-N1. 
The inventors' data demonstrates that the Hel-N1 protein binds as a 
multimer along the mRNA, presumably enhancing its instability and/or 
regulating its translatability and/or deadenylating it (thus, less 
proliferation). This protein may be responsible for the growth cessation 
of neurons. 
Interestingly, recently Szabo et al (Cell (1991) 67: 325-333) reported the 
isolation of a cDNA encoding another human protein, termed HuD, based upon 
its reactivity with antisera from patients with paraneoplastic 
encephalomyelitis. But Szabo et al do not describe any binding by HuD to 
mRNA 3'-untranslated sequences, or mRNA instability sequences.HuD is also 
homologous to elav in the RRMs, but differs from elav, K3 and Hel-N1 at 
its amino terminus and other places (see FIG. 7. Thus, it appears that 
four members of this subfamily have been identified and more are likely to 
be discovered. 
Due to the high level of homology between them, the segments of elav found 
between amino acid positions about 393 and about 483, of K3 found between 
amino acid positions about 345 and about 444, and of HuD found between 
amino acid positions about 280 and about 380 can be used in accordance 
with the invention in lieu of the third domain of Hel-N1. (The amino acid 
sequences of elav and of K3 are set forth in FIG. 1, that for HuD is set 
forth in FIG. 6.) 
The present invention thus relates to the Hel-N1 protein, to its third 
domain and related elav K3 and HuD segments, and to the exploitation of 
any of these proteins and their binding reaction to the 3'-untranslated 
regions containing the instability sequence of oncoprotein and lymphokine 
mRNAs (Shaw et al, 1986) as well as to different structural fusions that 
can be produced to target these mRNAs for up or down regulation. 
The present proteins, namely either full length Hel-N1 or its third domain, 
can be used to obtain a binding reaction between two ligands in a manner 
analogous to that described in application Ser. Nos. 07/536,943 and 
07/436,779, noted supra. For example, any number of other adducts (RNA or 
protein) can be attached to either of these ligands to create novel and 
useful ribonucleoproteins, or a ribonuclease can be attached to the RNA 
binding domain 3 using known techniques to directly target any of these 
mRNAs for destruction. 
The proteins of the present invention can therefore be used as therapeutic 
reagents to provide for either growth suppression or growth stimulation. 
Full-length Hel-N1 can be used to cause growth suppression of cultured 
cells, presumably mediated through effects on the stability of messenger 
RNAs encoding growth factors. In accordance with the present invention, 
one can alter the growth properties of cells in which oncogenes and 
lymphokine genes are overexpressed. Thus, cancer cells, which may be 
targeted by any known standard means, including gene therapy, 
liposome-mediated delivery, retrovirus-mediated infection or direct 
infusion with Hel-N1 DNA, RNA or protein will consequently be retarded in 
their growth. 
Likewise, immune cells regulated by lymphokines, such as interleukins, 
interferons and others can be growth suppressed using Hel-N1. In this 
embodiment, leukemic and lymphocytic cells targeted by delivery of Hel-N1 
DNA, RNA or protein to the bone, thymus or bloodstream using known 
techniques become incapacitated. For example, immune B or T cells 
overproducing autoantibodies or other harmful antibodies can be targeted 
using antigens or antibodies imbedded in lyposomes or other known carriers 
which in turn, deliver Hel-N1 DNA, RNA or protein as a growth suppressor 
to destroy their ability to proliferate. The cells producing the harmful 
antibodies become thus incapacitated and immunosuppressive therapy can be 
enhanced in a specific manner. 
In these regiments, Hel-N1 DNA, RNA or protein can be injected directly 
into cancer patients using known techniques to affect tumor growth. 
Likewise it can be injected into patients to suppress the proliferation of 
immune cells. Thus, with many variations on these themes, it can be seen 
that delivery of Hel-N1 DNA, RNA or protein which can block cell 
proliferation by suppression of growth factor messenger RNAs is highly 
advantageous. 
As noted above, the inventors have found that the third RNA recognition 
motif of Hel-N1, found between amino acid positions 259 and about 349 of 
the Hel-N1 amino acid sequence provided in FIG. 1, constitutes the core of 
the oncoprotein and lymphokine mRNA binding domain. This approximately 100 
amino acid-long fragment is responsible for the specific instability 
sequence binding activity. 
The inventors also made the startling discovery that expression of this 
domain, by itself, results in rapid proliferation of cells. This is a 
result opposite to that obtained by using full length Hel-N1. Expression 
of RNA binding domain 3 of Hel-N1 caused an eightfold increase in the 
growth of cultured cells after 3 days, as illustrated in FIG. 5. This is a 
striking alteration in a rate of proliferation. Thus, the RNA binding 
fragment of the growth suppression protein, Hel-N1, can itself be used to 
lead to the reverse effects, rapid cell growth. 
Delivery of this fragment to tissue can be used to regenerate growth of 
cells in that tissue. One can use this embodiment to regenerate nervous 
tissue, heart tissue, skin and other tissues of limbs and organs. 
Likewise, RNA binding domain 3 can be delivered to tissues involved in 
wound healing and at other sites that are unable to be otherwise 
stimulated. Immune cells that produce autoantibodies and other factors 
needed for protection of the body can be growth stimulated using this 
invention. 
Hel-N1 is an autoimmune protein in certain patients who show central 
nervous system manifestations of cancer called paraneoplastic cerebellar 
degeneration of (PCD), paraneoplastic encephalomyelitis (PE) or 
paraneoplastic sensory neuropathy (PSN). A therapeutic regiment could 
involve injection of Hel-N1 or peptides derived from Hel-N1 in order to 
block the immune effect or cellular immune recognition for properties in 
these diseases. Large amounts of pure Hel-N1 or its third domain are 
readily available using standard DNA cloning technologies or protein 
synthesis technologies. The purified protein can be used for immuno 
depletion of harmful autoantibodies or autoantibody--producing cells using 
methods of apheresis or dialysis. 
The inventors also surprisingly discovered that full-length Hel-N1 can take 
cells out of a proliferative state and into a state of differentiation. 
Illustratively, whereas the third RNA binding domain of Hel-N1 was 
discovered to cause increased cell growth and the whole Hel-N1 protein 
discovered to cause cessation of cell growth, the inventors also observed 
that when certain neuroblastoma cells of (B104) were subjected to 
expression of whole Hel-N1 protein the cells developed an altered 
morphology. The cells became elongated like muscle cells and began to 
produce myotubules consisting of myosin and actin fibrils. 
A cell derived from brain tissue was caused to enter an apparent myogenic 
pathway of differentiation by use of a protein of the present invention. 
This effect was due to the presence of a growth factor whose mRNA 
contained an instability sequence to which Hel-N1 was able to bind. In 
this case, the growth factor appears to be the Id protein which is known 
to suppress muscle differentiation. In the case of other similar growth 
factors, Hel-N1 may affect the differentiation of any cell which depends 
upon the continued expression of a growth factor encoded by an mRNA 
containing an instability sequence. 
Thus, in another embodiment, Hel-N1 can be used in somatic or germline 
therapy to cause cells to undergo a desired pathway of differentiation. 
Hel-N1 has the further ability to control the balance between 
proliferation and differentiation that determines the developmental versus 
neoplastic consequences of gene expression. 
The proteins of the present invention are also useful in therapeutic 
testing. An important need in the field of cancer research and immunology 
is for animal models which manifest altered growth properties or immune 
disregulation. Transgenic expression of polypeptides described in this 
application, using known techniques, can provide animals in which specific 
tissues or organs have been targeted to proliferate more rapidly or more 
slowly, thus allowing animal models of cancer or immune regulation to be 
produced. These animals are useful for testing the effect of 
chemotherapeutic drugs, radiation therapies, immune irregulatory agents, 
such as immunosuppressors and immunostimulators. Furthermore, Hel-N1 is 
itself an autoantigen to which patients with certain paraneoplastic 
diseases produce an autoantibody. The expression of Hel-N1 in transgenic 
tissues can allow production of an animal model for this autoimmune-type 
of cancer. 
Proteins of the present invention are also useful in diagnostic 
applications. As a histological probe, Hel-N1 can be used to identify 
certain neuron types, such as granule cells or basket cells of the 
cerebellum. For example, in the pathology laboratory it is useful to stain 
cells with antibodies specific for Hel-N1 to determine the tissue origin 
of the specimen in question. Because Hel-N1 is present in certain neurons 
and not others, its presence in a tissue sample is an indicator of the 
type of tissue being examined. 
Hel-N1 DNA constitutes a novel genetic marker for potential malformations 
of the central nervous system. For example, in the testing for genetic 
defects during prenatal examinations, many normal as well as abnormal 
markers are needed. For example, Hel-N1, in keeping with known oncogenes 
and antioncogenes, may be defective in patients suffering frmo natural 
cancers and leukemias. Full-length Hel-N1 DNA, RNA or protein may be used 
in the diagnosis and/or therapy of such individuals. Such therapy includes 
gene therapy, or targeted DNA, RNA or protein delivery. Hel-N1 is a 
useful, neuronal-specific probe. In testing for cystic fibrosis, Down's 
syndrome and similar genetic defects, one can get additional information 
on the status of CNS gene by monitoring Hel-N1 levels. 
Thus in one embodiment, the present invention provides a polypeptide having 
the amino sequence of at least from the amino acid position 259 to 349 of 
Hel-N1 set forth in FIG. 1, and up to the whole amino acid sequence of 
Hel-N1. In another embodiment, the present invention provides a 
polypeptide which can be used to promote cell growth, where the 
polypeptide has the amino acid sequence of from amino acid position about 
259 to about 349 of Hel-N1, or about position 393 to about position 483 of 
elav or about position 345 to about position 444 of K3, or about position 
280 to about position 380 of HuD. In another embodiment, the present 
invention provides a polypeptide which can be used to suppress cell 
growth, and in particular expression of oncogenes and/or lymphokine 
encoding genes, by using a polypeptide having the whole amino acid 
sequence of Hel-N1. 
In other embodiments, the present invention provides the corresponding DNA 
sequences and RNA sequences, optionally present in a liposome formulation, 
which may be either targeted or not targeted, or in a retroviral 
formulation, or in another formulation suitable for in vitro or in vivo 
delivery to cells or tissue. In other embodiments, these DNA and RNA 
sequences may be used in conjunction with gene therapy technology or to 
produce transgenic animals. 
Another embodiment of the present invention relates to method for 
regenerating a mammalian tissue, including neuronal tissue, by 
administering to the tissue a polypeptide having the amino acid sequence 
of from about position 259 to about position 349 of Hel-N1 or the 
corresponding elav, K3 or HuD segments. The polypeptide may be 
administered to the tissue using any known means to deliver a polypeptide 
to a cell culture or in vivo to the cells of certain tissue, including 
gene therapy, liposome-mediated delivery, retrovirus-mediated infection, 
or direct infusion with the corresponding DNA, RNA or protein. 
In another embodiment, the present invention is used to suppress the 
expression of an oncogene in a cell and/or of a lymphokine encoding gene 
in a cell, by causing the cell to express a polypeptide having about the 
whole amino acid sequence of Hel-N1. As with tissue regeneration, this may 
be achieved by using any standard means to cause the cell to express the 
desired polypeptide, including gene therapy, liposome-mediated delivery, 
retrovirus-mediated infection, or direct infusion with Hel-N1 DNA, RNA or 
protein. Particular oncogenes which may be targeted, include c-myc, c-fos 
or c-src, and others. Specific lymphokines which may be targeted in 
accordance with the present invention include GM-CSF, any interferon, or 
any interleukin, or others. 
Hel-N1 and its associated DNAs and RNAs can also be used to produce 
transgenic animals and cell lines, using standard and known technologies, 
for pharmaceutical tests of cancer, immune functions and/or neurological 
diseases. 
Having generally described this invention, a further understanding can be 
obtained by reference to certain specific examples which are provided 
herein for purposed of illustration only and are not intended to be 
limiting unless otherwise specified. 
Hel-N1 and a rat cDNA, Rel-N1, appear to be homologous to Drosophila elav 
within the RNA recognition motifs; however, these proteins differ markedly 
in other regions. Analysis of mRNA expression in rat tissues demonstrated 
that Rel-N1, like elav, was specific to brain tissue. In situ 
hybridization localized Rel-N1 mRNA to neurons of the hippocampus and 
neocortex, but not to Purkinje cells, glial cells, or white matter. 
The mRNA of the rat counterpart of elav was found to reside in a subset of 
neurons in the brain. It was not detected in glial cells or white matter 
and was found within the hippocampus and cerebral cortex of the rat. Using 
in vitro RNA binding methods, it was found that the human counterpart, 
Hel-N1 (Human elav-like Neuronal protein-1) could bind in vitro to the 
3'-untranslated regions (3'-UTR) of certain mRNAs, including the mRNA 
"instability regions" of c-myc, c-fos and GM-CSF mRNAs. 
These growth regulatory proteins are known to play important roles in cell 
proliferation, differentiation and immunoregulation. Thus, these 
observations show that Hel-N1, and perhaps other members of the elav 
sub-family, represent tissue-specific transacting factors involved in 
post-transcriptional mRNA metabolism. 
Rat and human cDNA counterparts of the Drosophila neuronal protein, elav, 
were isolated using degenerate oligonucleotides, PCR, and library 
screening. RNAs capable of binding the human neuronal protein, Hel-N1, 
include 3'-UTRs of mRNAs encoding the oncoproteins, c-myc and c-fos and 
the lymphokine, GM-CSF. These RNA sequences encompass the "instability 
region" that is known to correlate with lability of these mRNAs (Meijlink 
et al, Proc. Nat. Acad. Sci. (USA) (1985) 82: 4987-4991; Shaw et al, Cell 
(1986) 46: 659-667; Jones et al, Mol. Cell Biol., (1987) 7: 4513-4521). 
RNA binding results were obtained using recombinant Hel-N1 followed by: (1) 
selection of uridylate stretches from a degenerate pool of RNAs, (2) 
immunoprecipitation of c-myc, c-fos and GM-CSF mRNAs using two types of 
Hel-N1-specific antibodies, and (3) crosslinking to c-myc and GM-CSF 
3'-UTR with uv light. The 3-UTR of these mRNAs are U-rich, but also 
contain other identifiable features of primary sequence. For example, the 
pentameric sequence, AUUUA defined by Malter Science (1989) 246: 664-666 
and the octameric sequence, UUAUUUAU proposed by Caput et al, (1986), are 
common among the 3'-UTR of these mRNAs. These findings indicate that 
Hel-N1 and related proteins participate in the post-transcriptional 
regulation of unstable messenger RNAs. 
Shaw et al Cell (1986) 46: 659-667, demonstrated a role for the A/U-rich 
3'-UTR of protooncogene and lymphokine mRNAs in the instability of the 
RNA. In addition, they demonstrated that instability could be conferred to 
otherwise stable mRNAs by placement of the instability region in the 
3'-UTR. 
However, it should be noted that other regions of certain mRNAs, including 
c-myc, c-fos, histone and transferin receptor, have also been implicated 
in destabilizing yhr mRNA (reviewed by Cleveland and Yen, 1989; Atwater et 
al, 1990). Verma and coworkers (Meilink et al, 1985) demonstrated that 
removal of the 3'-UTR from c-fos mRNA resulted in increased levels of 
c-fos protein and cell transformation. 
These studies show that regulatory events at the 3'-UTR are important for 
growth control. However, the A/U-rich 3'-UTR sequences span hundreds of 
nucleotides and the precise sequences involved in instability have not 
been identified. Recent work suggests that the AUUUA sequences are not 
required for instability, but that an upstream secondary structure in the 
3'-UTR is more important. Thus, the role of the sequence elements within 
the 3'-UTR of these proto-oncogene and lymphokine mRNAs are not clearly 
defined at this time. 
Proteins that interact with the 3'-UTR of oncoprotein and lymphokine mRNAs 
are poorly understood. Cross-linking with UV light and label transfer 
experiments by Vakalopoulou et al, Mol. Cell. Biol. (1991) 11: 3355-3364, 
noted a 32 kD protein that binds this region. Malter, Science (1989) 246: 
664-666, observed a factor composed of three subunits, termed AUBF, in 
Jurkat cells that crosslinked to four repeats of the pentameric AUUUA 
sequence. More recently, Myer et al, Proc. Nat. Acad. Sci. (USA) (1992) 
found that small RNA transcripts from herpes simplex virus contain the 
AUUUA sequence and are capable of being UV cross-linked to the 32 kD 
protein from HeLa cell extracts. These findings suggest that there may be 
many proteins capable of recognizing sequences in the 3'-UTR. The binding 
specificity of Hel-N1 to the 3'-UTR of c-myc, c-fos, GM-CSF represents the 
only defined RNA-protein interaction in this region. 
Using an in vitro RNA degradation assay Brewer (1991) identified and 
partially purified an activity termed, Auf, from human erythroleukemia 
cells that appears to be involved in instability of c-myc mRNA. Based upon 
a mobility shift assay, he postulated that proteins of 37 kD and a 40 kD 
present in these fractions were involved in binding to c-myc RNA. Although 
these factors were implicated in instability, they have not been 
characterized as to sequence or binding specificity. 
Hel-N1 represents an amino acid sequence containing an RNA-binding domain 
that can recognize and bind to 3'-UTR of mRNAs containing the instability 
sequence. It is possible that Hel-N1 represents a neuron-specific 
counterpart of one of several proteins shown to bind A/U-rich 3'-UTR 
sequences in UV crosslinking studies. Given that it contains three 
different RRMS, it appears that Hel-N1 functions as a structural component 
of an RNP which interacts in the 3'-UTR through one RNA binding domain and 
carries another small RNA to that site. Alternatively, the RNA binding 
domains could perform a structural role in RNA bridging interactions as 
proposed for the U1 snRNP-A protein (Lutz-Freyermuth et al, Proc. Nat. 
Acad. Sci. (USA) (1990) 87: 6393-6397). 
As an RNP or a bridging protein, Hel-N1 (or elav) may play a role in other 
post-transcriptional processes such as mRNA compartmentalization or 
translation. By this analogy, Hel-N1 may be involved in neuron-specific 
localization of mRNAs in the central nervous system. 
Thus, members of the elav subfamily might recognize similar RNAs, but be 
functionally distinct based upon differences in their amino-terminal 
sequences. Expansion of the subfamily and determination of the tissue 
specificity and developmental regulation of each member will be required 
to address these possibilities. 
Hel-N1, like HuD, was observed by the inventors to be reactive with an 
autoantibody present in the sera of patients with paraneoplastic disease, 
putting it in the category of other human autoantigens that are members of 
the RRM superfamily (Query et al, Mol. Cell. Biol. (1989), 9: 4872-4881). 
The potential to bind to oncoprotein mRNAs adds an element of intrigue 
because these patients are a subset of those inflicted with small lung 
cell carcinoma in which levels of c-myc protein are elevated. However, the 
mechanism of initiation of the autoimmune response to these self antigens 
remains as elusive as that of the systemic snRNP autoantigens. In 
addition, there is no evidence that Hel-N1 or HuD play a role in the 
derivation of the paraneoplastic syndrome or of small cell carcinoma. 
Additional information concerning the influence of Hel-N1 and related 
proteins on the production of cellular growth factors will be required to 
argue for such a link. 
cDNAs encoding a variety of putative RNA-binding proteins were isolated by 
probing with degenerate oligonucleotides derived from conserved portions 
of the RRM. For members of the RRM family that contain multiple RRMs, 
oligonucleotides derived from the sequence of the RNP1 octamers were used. 
Primers representing sense and antisense strands of the RNP 1 of RRM 1 and 
the RNP 1 of RRM 2 of elav DNA (Robinow et al, Science (1986) 242: 
1570-1572) were used to probe mRNA from rat pup brain following reverse 
transcription with random primers. A PCR product was isolated and found to 
contain an ORF with an amino acid sequence termed, Rel-N1, which was, in 
turn, used to screen a human fetal brain library under high stringency 
conditions. A 2.2 kb DNA insert containing an open reading frame (ORF) of 
359 amino acids was obtained. In vitro transcription and translation of 
the human cDNA produced a protein, termed Hel-N1, of the predicted size. 
Hel-N1 and Rel-N1 were identical in amino acid sequence and greater than 
92% homologous in nucleic acid sequence. 
As shown in FIGS. 1 and 2, Hel-N1 contains three RNA binding domains as 
evidenced by RRMs 1, 2 and 3, which matched the structural criteria of 
Kenan et al (1991), supra, and each contained an RNP1 octamer (boxed and 
shaded) and an RNP2 hexamer (boxed) sequence. Sequence comparison of elav 
and a related Drosophila protein, K3, with Hel-N1, revealed strong 
similarities in the RRMs (FIGS. 1 and 2). On the other hand, Hel-N1 was 
only 76% the length of elav because the region amino terminal to the first 
RRM of the proteins demonstrated striking sequence differences (FIG. 1). 
The amino terminus of Hel-N1 lacks the homopolymeric stretches of alanine, 
asparagine and glutamine seen in the amino termini of elav and K3, leaving 
it considerably shorter in length. This divergence is of unclear 
significance, especially in light of rescue studies done in Drosophila 
bearing the lethal mutation elavE5. These studies demonstrated that 
deletion of a 40 amino acid portion in the amino terminal does not prevent 
rescue from lethality. Thus, elav, Hel-N1 and K3 represent members of a 
subfamily of the RRM superfamily of RNA-associated proteins (Kenan et al, 
(1991), supra. This shows the existence of an elav-like subfamily of RNA 
binding proteins and, except for authentic elav, they can be designated by 
species as human (H) or rat (R) and tissue as neuronal (N) of origin. 
Kenan et al, Trends. Biochem. Sci. (1991) 16: 214-220, have proposed that 
pPTB and hnRNP-L represent a distinct subset of the RRM superfamily of RNA 
binding proteins in that they lack the characteristic RNP 1 and RNP 2 
sequences. Also evident in FIG. 2 are the sequence differences in loop 3 
that connects .beta.-strand 2 to .beta.-stand 3 (RNP 1). Loop 3 has been 
described as highly variable among RRM family members (Bentley et al, Mol. 
Cell. Biol. (1991) 11: 1829-1839. In the case of the U1 snRNP-A protein, 
sequences residing in loop 3 were shown to affect the specificity of RNA 
recognition (Bentley et al, 1991; reviewed in Kenan et al, 1991); thus, 
representing one determinant of specificity. It is apparent that Hel-N1 
differs from elav most strikingly in RRM 1, while RRMs 2 and 3 are highly 
similar (FIG. 2). This may indicate that the potential RNA-binding domains 
at RRM 1 of elav and Hel-N1 recognize very different RNA ligands. 
Rel-N1 is neuron-specific 
RNAs extracted from various rat tissues were analyzed by ribonuclease 
protection assays using Rel-N1 as probe. Protected bands were found only 
in RNA from rat brain; however, longer exposures revealed a small amount 
of RNA detectable in rat testes. To identify the specific neuroanatomic 
loci expressing Rel-N1 mRNA, 4% paraformaldehyde-fixed rat brain sections 
were hybridized with [35S]-labeled antisense RNA derived from the PCR 
fragment of Rel-N1 using the method of Fremeau et al, EMBO J (1990) 9: 
3533-3538. 
Data revealed that Rel-N1 mRNA was heterogeneously distributed in adult rat 
brain. Prominent hybridization signals were observed throughout all layers 
of the cerebral cortex and within the hippocampus. High levels of 
expression were observed in the CA3-CA4 fields of Ammon's Horn. In 
contrast, only low levels of expression were observed in the CA1 field of 
Ammon's horn and the granule calls of the dentate gyrus. Prominent 
hybridization signals were also observed throughout the thalamus and 
brainstem. Particularly intense hybridization signals were observed in the 
parafascicular and midline thalamic nuclei. In the cerebellum, only a 
small percentage of labeled cells were observed in the granule cell layer 
while only background labeling was observed over the molecular layer, the 
Purkinje cell layer, and the white reafter tracts. Grains were not 
observed over the choroid plexus, ependymal cells of the cerebral 
ventricles, and control sections hybridized with a sense-strand probe. 
In sum, these data indicate that Rel-N1 mRNA is expressed most highly in 
the hippocampus and cerebral cortex, as well as in certain neurons in the 
granule cell layer of the cerebellum, but not in Purkinje cells of the 
cerebellum. 
Our initial approach, given that the RNA binding ligands are not known for 
any of the four known elav sub-family members, was to use several standard 
RNA binding assays (Lerner et al, Proc. Nat. Acad. Sci. (USA), (1979) 76: 
5495-5499) using total 32P labeled RNA isolated from HeLa, glioblastoma 
and neuroblastoma cells. In addition, in vitro RNA binding procedures 
which have been used effectively for other members of the RRM family of 
proteins (Query et al, Cell (1989) 57: 89-101; Lutz-Freyermuth et al, 
Proc. Nat. Acad. Sci. (USA), (1990) 87: 6393-6397; Bentley et al, 1991) 
did not reveal a cognate RNA species for Hel-N1. 
As an alternative approach, we used a random RNA selection procedure to 
define the RNA ligand site for Hel-N1. A synthetic oligodeoxynucleotide 
containing a stretch of 25 degenerate nucleotides was used to create a 
large heterogeneous pool of RNA sequences for selection of binding ligands 
(Tsai et al, Nucl. Acids Res. (1991) 19: 4931-4936). Binding of the 
degenerate RNA pool to recombinant Hel-N1, followed by immunoprecipitation 
of the complex using the epitope tag, glO, was carried out as described 
previously (Lutz-Freyermuth et al, 1990; Bentley et al, 1991). 
After three complete cycles of binding and selection, 30 independent 
clones, representing individual coimmunoprecipitated RNA species were 
evaluated by sequence analysis. The sequences of the bound RNAs showed a 
preponderance of uridylate residues in short stretches interrupted by 
other nucleotides. However, two of the 30 sequences (B-17 and B-5) did not 
contain this U-rich pattern. These variants were rare in the population 
and thus, may represent ligands of lower binding affinity. Alternatively, 
because Hel-N1 contains three potential RNA binding domains, these other 
sequences may represent ligands which were bound by one of the domains not 
involved in recognition of the U-rich regions. This possibility is 
compatible with the proposal that Hel-N1 may exist as an RNP that bridges 
between two or more RNAs via its multiple RRMs as proposed for the U1 
snRNP-A protein (Lutz-Freyermuth et al, 1990). 
This random RNA selection procedure has proved useful in our laboratory 
with other members of the RRM family of proteins to derive RNA ligand 
consensus sequences (Tsai et al, 1991), but in no other case has a U-rich 
sequence been selected. In the experiments using Hel-N1, RNA sequences 
with a Urich character were derived using the selection procedure, but a 
single consensus sequence was not evident. 
The sequences selected from the in vitro RNA selection protocol were 
suggestive of biologically relevant sites known to exist in mammalian RNAs 
such as 3' UTRs in labile RNAs, the polypyrimidine tract near 3' splice 
junctions, sequence 5' of the polyadenylation signal, and in mitochondrial 
telomeres. The most striking feature was that short uridylate stretches 
flanked by either A, G or C could be located within the 3' UTRs listed by 
Shaw et al, Cell (1986) 46: 659-667 in their study of the instability 
sequences of proto-oncogene and lymphokine messenger RNAs. Thus, we 
conducted a series of direct RNA binding experiments to examine this 
possibility. 
DNA constructs encoding portions of the 3' UTR of c-myc, GM-CSF, and c-fos 
mRNAs were used to synthesize radiolabeled transcripts for binding to 
recombinant Hel-N1 protein using our standard methods (Bentley et al, 
1991). We utilized .sup.32 p labeled transcripts corresponding to the 3' 
UTR sequences, as well as to a variety of unrelated RNAs. As with the RNA 
selection procedure used above, Hel-N1 was fused to the glO epitope for 
precipitation. c-fos, GM-CSF and c-myc transcripts were precipitable, 
while other transcripts were not precipitable. 
The specificity of Hel-N1 binding to 3'-UTR of c-myc, GM-CSF, and c-fos 3' 
UTR was substantiated by the use of many control RNAs including total HeLa 
cell RNA, transcripts of various small RNAs, precursor mRNAs, various 
vector RNA transcripts and other RNAs. In addition, RNA binding was always 
in the presence of carrier transfer RNA and poly A (Query et al, 1989; 
Bentley et al, 1991). 
Control transcripts for RNA binding specificity also included hY3 antisense 
RNA that contained a single AUUUA pentamer. This sequence has been 
suggested to represent the most conserved element present in the 3' UTR of 
the unstable protooncogene and lymphokine RNAs (Shaw et al, Cell (1986) 
46: 659-667; Caput et al, (1986); Malter, Science (1989) 246: 664-666. 
Vakalopoulou et al, Mol. Cell. Biol. (1990) 11: 3355-3364, showed 
previously that the specificity for binding of these 3' UTRs to a 32 Kd 
protein present in Hela nuclear cell extracts resided in multiple copies 
of an AUUUA motif contained within a uridylate-rich region. 
It should be noted that the hY3 RNA did not contain a uridylate-rich region 
surrounding the AUUUA. N-myc was also used as a control transcript because 
it contained a stretch of thirteen uridylates, but no AUUUA pentamer. None 
of these various control RNAs were significantly immunoprecipitated 
indicating that binding to Hel-N1 did not occur. 
Among the control transcripts, we employed precursor mRNA-in-pieces (PIP 
vectors) which encode uridylate-rich stretches of RNA that are active in 
in vitro splicing and can be cross-linked with uv light to pPTB 
(Garcia-Blanco et al, 1990), supra. PIP transcripts also failed to bind 
Hel-N1. Several other RNA transcripts failed to bind Hel-N1 including 
coding regions of N-myc mRNA, U1RNA, a transcript encoding neomycin 
resistance, noncoding regions of U1 snRNP-70K mRNA, and coding regions of 
the dopamine 1 receptor. 
In these studies, RNAs in the supernatants of the binding reactions were 
analyzed for the presence of intact non-bound RNA to rule out degradation. 
Although Hel-N1 binding to other untested U-rich sequences remains a 
possibility, its preference for the instability sequences at the 3' UTR of 
c-myc, GM-CSF, and c-fos mRNAs was compelling. 
As an alternative confirmation of the RNA-binding specificity of Hel-N1 
with the 3'-UTRs of these rapidly degraded mRNAs, label transfer 
experiments involving uv crosslinking with 32P labeled RNA were performed 
using standard procedures. HeLa cell nuclear extract and recombinant 
Hel-N1 in an E. coli extract were incubated with 32P labeled c-myc or 
GM-CSF mRNAs and exposed to UV light to mediate covalent cross-linking 
between the RNA and associated proteins. After cross-linking, excess RNA 
was digested with RNase A and analyzed on an SDS-acrylamide gel. 
The label transfer to Hel-N1 revealed two predominant bands of 70 kD and 28 
kD; similar results were obtained with GM-CSF (data not shown). The higher 
molecular weight band was found to be an artifact of IPTG induction, since 
control E. coli extracts lacking Hel-N1 also showed the 70 kD cross-linked 
band. The 28 Kd band (termed Hel-Ni) was 10 Kd smaller than the expected 
size of Hel-N1. While it is possible that the bound RNA or the 
cross-linking protocol caused Hel-N1 to migrate aberrantly, we observed 
that the 28 Kd band contained Hel-N1 epitopes (see below). 
Direct label transfer experiments using HeLa cell extracts and radiolabeled 
c-myc mRNA demonstrated the ability to uv crosslink several proteins 
similar to that reported by Vakaloupoulu et al (1991). To determine 
whether Hel-N1 can compete with cross-linked proteins in the HeLa cell 
nuclear extract for binding to c-myc, increasing amounts of Hel-N1 were 
added prior to UV exposure. Neither the 32 kD protein identified by 
Valakopoulou et al (1991) nor hnRNP C protein (45 kD) diminished 
significantly upon addition of Hel-N1. 
In addition, the 28 kD Hei-N1 band (Hel-Ni) appeared during the crosslink 
competition. 
These results indicate that Hel-N1, the 32 kD protein, and hnRNP-C protein 
can bind simultaneously to the 3'-UTR of c-myc MRNA. On the other hand, a 
band of 65 kD was competed by Hel-N1, while E. coli extracts lacking 
Hel-N1 had no effect. The identity of the competed 65 kD protein remains 
unknown. These data suggest that while the HeLa 32 Kd protein and hnRNP C 
may share similar RNA binding characteristics with Hel-N1, their binding 
sites as defined by uv crosslinking are not identical. 
Recent studies into several paraneoplastic neurologic disorders including 
paraneoplastic sensory neuropathy (PSN), paraneoplastic cerebellar 
degeneration (PCD), and paraneoplastic encephalomyelitis (PEM) have 
reported the identification of several antigens recognized by the sera of 
patients with these disorders (Dropcho et al, Proc. Nat. Acad. Sci. (USA) 
(1987) 84: 4552-4556; Anderson et a, Neurology (1988) 38: 1018-1026; 
Dalmau et al, Ann.. Neurol. (1990) 27: 544-557; Szabo et al, Cell (1991) 
67: 325-333). 
One such antigen, HuD, displays strong similarity to recombinant Hel-N1, 
but possesses important differences. Both HuD and Hel-N1 contain three 
RRMs which share approximately 70% overall homology. The major differences 
exist in the amino termini and in a stretch of thirteen amino acids 
between the second and third RRMs. 
Using anti-HuD sera, we demonstrated cross reactivity with Hel-N1 by 
Western blotting. When used in the RNA binding protocol in place of the gl 
0 serum, an anti-Hu serum was found to immunoprecipitate c-myc transcripts 
that bound to Hel-N1 in vitro. Control RNAs did not bind Hel-N1. 
Furthermore, four normal human sera lacked the ability to 
immunoprecipitate these mRNPs. These experiments demonstrate that the 
complex formed between HuD antibodies and Hel-N1 does not interfere with 
the ability of the protein to recognize its RNA ligand. 
To confirm the HuD RNA binding assay, the label transfer experiments using 
cmyc 3'-UTR and g 10-Hel-N1 as described above were followed by 
immunoprecipitation of the 28 Kd Hel-N1'band with HuD sera. Normal human 
sera were always negative. In addition, the 70Kd E. coli band was not 
immunoprecipitated by any of these sera, as expected of the nonspecific E. 
coli protein. These data show that HuD sera can also immunoprecipitate a 
preformed complex of RNA bound to Hel-N1. Thus, Hel-N1, and presumably 
HuD, appear to possess autoantigenic epitopes that are distinct from the 
RNA-binding domain(s) that recognize the uridylates. 
It is interesting to note that the 28 Kd band (Hel-N1') was 
immunoprecipitated with the HuD sera, but not with the glO serum or normal 
sera. Thus, it was assumed that the amino terminus was lost by cleavage. 
Estimation of the resultant size of Hel-N1' suggests that cleavage 
occurred at a site C-terminal to the first RRM, leaving a fragment 
containing RRMs 2 and 3. The source of this unexpected cleavage event is 
currently under investigation. These results suggest that the interaction 
between c-myc mRNA and Hel-N1 is specific to the second or third RRM; one 
of which may constitute the RNA binding domain. 
Experimental Procedures 
Cloning Rel-N1 and Hel-N1 by PCR and hybridization 
Degenerate PCR primers were synthesized based on the first seven amino 
acids of the RNP1 consensus sequence in the first (sense) and second 
(antisense) RRMs of elav. Inosine residues were placed in positions 
degenerate for all 4 nucleotides and Eco R1 restriction sites were placed 
at the 5' end of each oligonucleotide. cDNA was prepared by reverse 
transcribing total cytoplasmic RNA from a Sprague-Dawley rat pup brain 
according to the manufacturer's specifications (Cetus.RTM.): 6 mg total 
RNA, 1 mM dNTPs, 100 picomoles of random hexamers (Pharmacia.RTM.), 
GeneAmp buffer, 20 U RNASIN (Promega.RTM.), 200U BRL reverse 
transcriptase. 40 cycles of PCR amplification were carried out using an 
annealing temperature of 37 and an extension temperature of 55 C. (cycles 
1-4) and 72 (cycles 540). A PCR product of 281 bp was purified on a 1% 
agarose gel using Geneclean.RTM. (Bio 101) and subcloned into a TA vector 
(in Vitrogen.RTM.). The clone Rel-N1, was sequenced and found to have a 
high degree of homology with elav, including a 100% homologous RNP2 
consensus sequence within the second RRM. 
A random primed cDNA probe was generated using Rel-N1 and used to screen a 
.lambda.ZAPII human fetal brain library (Stratagene.RTM.). Seven positive 
plaques were isolated from an initial population of 500,000 phage screened 
using the following hybridization conditions: 50% formamide, 6.times. SSC, 
0.1% SDS and 0.01% Blotto. Filters were hybridized for 18 hours at 42 C. 
and then washed two times at room temperature (10 minutes each) in 
2.times.SSC/0.1% SDS followed by a final wash at 65 C. in 0.2xSSC0.1% SDS 
for 45 minutes. The Bluescript.RTM. plasmids of the positive phage were 
then isolated according to the manufacturer's specifications 
(Stratagene.RTM.). 
Sequencing Hel-N1 cDNA 
EcoR1 inserts within the Bluescript.RTM. plasmids were sequenced by 
exonuclease digestion and primer extension using the dideoxynucleotide 
chain termination with a modified T7 DNA polymerase from the Sequenase 
system (USB). Oligonucleotides were synthesized on an Applied 
Biosystems.RTM. 391 DNA synthesizer. 
Expression of Hel-N1 in E. coli 
An inducible T7 RNA polymerase expression system (Rosenberg et al, (1987) 
Gene, 56, 125-135 was used for production of Hel-N1 protein. By using PCR 
mutagenesis, a conservative point mutation was introduced into the carboxy 
portion of the ORF to delete an Ncol site, such that the only Ncol site 
remaining was at the translation-initiation methionine. An Ncol-EcoR1 
insert from this construct was then subcloned in frame into pET-3c 
containing the T7 12-amino acid (g10) sequence at the 5cloning site. After 
transfection of this construct into BL21(DE3)pLysS, the bacteria were 
induced with IPTG. The cells were washed twice in SM buffer and then 
resuspended in a small volume of E. coli lysis buffer (1 XTBS, 10 mM EDTA, 
0.05% Tween, 3mM DTT and PMSF). Lysis was completed by freeze-thawing the 
cells. The extract was centrifuged at 10,000.times.g to remove insoluble 
debris. The amount of induction was evaluated by sodium 
dodecylsulfate-polyacrylamide gel electrophoresis and Western blotting as 
well as Coomassie staining. 
In situ Hybridization 
In situ hybridization was conducted on 4% paraformaldehyde-postfixed adult 
rat brain sections as previously described (Fremeau et al, EMBO J (1990) 
9: 3533-3538). Briefly, adult Sprague-Dawley rats were anesthetized with 
300 mg of sodium pentobarbital, and killed by decapitation. Brains were 
removed and frozen on an aluminum block cooled with liquid nitrogen. 
Frozen sections (10 u) were prepared in a cryostat, mounted onto room 
temperature slides (Onasco Biotech.RTM.; Houston, Tex.) and stored at 
-70.degree. C. until processed for in situ hybridization. 
Tissue sections were thawed and fixed for 10 min in 4% paraformaldehyde in 
phosphate-buffered saline at 4.degree. C. The sections were then rinsed in 
2.times.SSC, covered with a minimal volume of 2.times.SSC, and illuminated 
with a germicidal UV-lamp (30W, wide spectrum UV light) for 5 min at a 
distance of 30 cm. The sections were then rinsed in 2.times.SSC, and 
covered with prehybridization buffer (50% formamide, 0.6M NaC1, 10mM 
Tris-HCl (pH 7.5), 0.02% Ficoll, 0.02% polyvinyl pyrollidine, 0.1% bovine 
serum albumin, 1 mM EDTA (pH 8.0), 50 ug/ml salmon sperm DNA, 500 ug/ml 
yeast total RNA, 50 ugml yeast tRNA and stored at 50.degree. C. for 1 hr. 
Prehybridization buffer was removed, and the slides were covered with 
hybridization buffer (50% formamide, 0.6M NaCl, 10 mM Tris-HCl (pH 7.5) 
0.02% Ficoll, 0.02% polyvinyl pyrollidone, 0.1% bovine serum albumin, 1 mM 
EDTA (pH 8.0), 10 ug/ml salmon sperm DNA, 50 ug/ml yeast total RNA, 50 
ug/ml yeast tRNA, 10 mM dithiothreitol, 10% dextran sulphate containing 
35S-labeled probes (2.5-5.O.times.10.sup.6 cpm/ml; heat-denatured for 15 
min at 65.degree. C.). 
Hybridization was performed for 16-18 hrs at 50.degree. C. Following 
hybridization, the sections were washed for 60 min in 2.times.SSC at 
50.degree. C. and then treated with RNase A (50 ug/ml) for 60 min at 
37.degree. C. The sections were then washed in 2.times.SSC for 60 min at 
50.degree. C. followed by a final high stringency wash in 0.1.times.SSC, 
14 mM b-mercaptoethanol, 0.15% sodium pyrophosphate for 3 hr at 50.degree. 
C., the heat was then turned off and the slides were allowed to gradually 
cool to room temperature overnight. The hybridized sections were 
dehydrated through graded ethanols containing 0.3M ammonium acetate, 
vacuum dried, and dipped in Kodak.RTM. NTB2 emulsion diluted 1:1 with 
H.sub.2 O. After 4-6 week exposure times, the slides were developed as 
previously described (Fremeau et al, 1990) and photographed under 
dark-field illumination with kodachrome 160 tungsten slide film 
(Kodak.RTM.). 
RNA Probes 
Rel-N1 cDNA was excised from the TA vector and subcloned into pGEM-3Zf(+) 
and linearized. .sup.35 S (for in situ hybridization) or 32P (for RNAse 
protection assay) labeled single stranded antisense RNA probes were 
synthesized using T7 RNA polymerase in the presence of [35S]UTP (New 
England Nuclear.RTM.) or [32P]UTP (ICN). Sense RNA probe, made in a 
similar way, was used as a control for the in situ hybridization 
experiments. Unincorporated nucleotides were removed by G50 Sephadex 
(Pharmacia.RTM.) columns. 
Ribonuclease Protection Assays 
Total cellular RNA was prepared from various tissues of an adult male 
Sprague-Dawley rat according to standard methods. Assays were carried out 
using 15 ug of total RNA from each tissue source essentially as described 
by Zinn et al (1983) Cell, 34, 865-879. Protected fragments were 
electrophoresed on a denaturing 5% polyacrylamide gel. The integrity of 
the RNA was ascertained by protection assay using 32P labeled antisense 
RNA transcribed from mouse .beta.-microglobulin cDNA. 
RNA selection procedure 
The RNA selection process was done according to the method described by 
Tsai et al (1991). Briefly, an oligodeoxynucleotide containing a T7 
promoter sequence (T7Univ) at one end, followed by 25 degenerate 
nucleotides and then a reverse universal primer sequence (RevUniv) at the 
other end was used in a PCR reaction (1 min. 94, 1 min. 50, 2 min. 72 in 
10 mM Tris-HCl (pH 8.3), 50 mM KC1, 1.5 mM MgCl.sub.2, 0.01% gelatin, 0.1 
mg of T7Univ and RevUniv primers, 200 mm dNTPs and 2.5 U of Taq DNA 
polymerase) to create double stranded template for transcription. RNA was 
synthesized using T7 polymerase using standard methods (Maniatis, 1990). 
In binding conditions described previously (Query et al, 1989), the 
degenerate pool of RNA was then incubated with g10-Hel-N1 fusion protein 
which had been prebound to protein-A beads (Sigma) using the g10 antibody. 
The beads were subsequently washed 5 times with NT2 buffer, and the 
immunoprecipitated RNA was then phenol extracted and ethanol precipitated 
in the presence of 10 ug of carrier tRNA (Sigma.RTM.). The RNA was 
resuspended in 10 ul of doubly distilled water, and 3 ul was used for PCR 
amplification under conditions described above. The T7 and RevUniv primers 
had Bam-H1 restriction sites incorporated in the 5' ends such that any 
multimer products were reduced to monomers with Bam-H1 digestion. The same 
process was then carried out two more times. After the final PCR 
amplification and Bam-H1 digestion, the product was subcloned into 
pGEM-3Zf(+) and sequenced. 
Plasmids and mRNA transcripts 
The 3' end of the GM-CSF gene (240 bp fragment between Nco I and Eco RI 
cleavage sites) inserted into the polylinker, pGem3 containing the 3' end 
of the human c-fos gene (250 bp Rsal-Tth111l) inserted into the Hinc II 
site, and pGem3 containing the NsiI-AflII fragment of the 3' end of the 
human c-myc gene were used. The plasmids were linearized as follows: 
GM-CSF in PSP64 was cut at BgIII and transcribed with Sp6 RNA polymerase; 
pGEM 3 containing c-fos was linearized with Kpn 1 and transcribed with T7 
RNA polymerase; pGEM3 containing c-myc DNA was linearized with BamHI and 
transcribed with Sp6 RNA polymerase. 
Linearized plasmid DNA was transcribed with SP6 RNA polymerase for c-myc 
and GM-CSF, or T7 RNA polymerase for c-fos. These reactions were carried 
out in the presence of 1.25 mm ATP, CTP, GTP; 0.75 mm UTP, and 5 ul of 1 u 
Ci/ul 32P UTP. 
RNA Binding to Hel-N1 
For each binding reaction 4 mg of Protein A beads were washed three times 
in NT2 Buffer (150 mm NaCl, 50 mm Tris-HCl pH 7.4, and 0.05% NP40). 5 ul 
of rabbit anti-g10 antibody, or 20 ul of human serum, was incubated with 
Protein A for 10 minutes on ice and washed three times with NT2 buffer. 35 
ul of Hel-N1 E. coli extract was then added and incubated for ten minutes 
on ice and washed three times with NT2 buffer. After the final wash, the 
protein complex was resuspended in 0.1 ml of RNA Binding Buffer and 
equimolar amounts of labeled transcripts were added. 
After a 5 min. incubation at room temperature, the binding reaction was 
washed five times with NT2 buffer and resuspended in 0.1 ml of NT2 buffer. 
0.1 ml of the supernatant from the first wash was saved and treated 
identically as the bound pellet. 0.1 ml of diethyl pyrocarbonate treated 
water was added as well as 13 ul of 5M NaCl and 1 ul of 10 mg/ml of tRNA. 
The reactions were PCI extracted and EtOH precipitated. The pelleted RNA 
was run on a 6% urea polyacrylamide gel. 
UV Cross Linking 
Hela cell nuclear extract was prepared as described by Dignam (1983) and 
label transfer from RNA to protein was carried out as described by Wilusz 
et al, Cell (1988) 52: 221-228. 500,000 cpm of labeled transcripts were 
incubated with 5 ug of nuclear extract in a total reaction volume of 10 
ul. The reaction was performed in a microtiter plate and irradiated for 10 
minutes on ice. RNase A was added for a final concentration of 1 mg/ml and 
incubated for 15 minutes at 37.degree. C. The reactions were mixed with 
Laemmli buffer and run on a 10% SDS polyacrylamide gel. 
Hel-N1 crosslinking was carried out as above, except that the protein was 
dissolved in a uv cross-linking buffer (20 mm Hepes, 1 mm MgCl.sub.2, 60 
mm KCl 10% glycerol). Competition experiments included 5 ug of Hela cell 
nuclear extract in the presence of increasing amounts of Hel-N1 
maintaining a total reaction volume of 10 ul. 
Cellular Growth 
NIH 3T3 cells where transfected with a pBC vector derived from the CMV 
promoter containing DNA expression RNA binding domain 3 (RBD3) of Hel-N1 
using the calcium phosphate method. Cells were co-transfected with a 
plasmid encoding resistance to neomycin and colonies were selected with 
neomycin in the growth medium. After approximately three weeks of 
selection cells were examined by immunoflorescents and found to express 
RBD3(=), while control cells transfected with neomycin resistance alone 
(+) did not express RBD3. Cells were counted at passage and planted on 
culture plates for determination of growth rate. At days 1, 2 and 3 a 
plate of each was sacrificed and the cell numbers determined. It was 
readily evident that those cells expressing RBD3 entered into rapid 
proliferation, while the control cells grew at the same rate as normal 3T3 
cells. 
Obviously, numerous modifications and variations of the present invention 
are possible in light of the above teachings. It is therefore to be 
understood that within the scope of the appended claims, the invention may 
be practiced otherwise than as specifically described herein. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 51 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 485 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
AspCysMetAspPhe IleMetAlaAsnThrGlyAlaGlyGlyGlyVal 
151015 
AspThrGlnAlaGlnLeuMetGlnSerAlaAlaAlaAlaAlaAlaVal 
20 2530 
AlaAlaThrAsnAlaAlaAlaAlaProValGlnAsnAlaAlaAlaVal 
354045 
AlaAlaAlaAlaGlnLeu GlnGlnGlnGlnValGlnGlnAlaIleLeu 
505560 
GlnValGlnGlnGlnGlnThrGlnGlnAlaValAlaAlaAlaAlaAla 
6570 7580 
AlaValThrGlnGlnLeuGlnGlnGlnGlnGlnAlaValValAlaGln 
859095 
GlnAlaValValGlnGln GlnGlnGlnGlnAlaAlaAlaValValGln 
100105110 
GlnAlaAlaValGlnGlnAlaValValProGlnProGlnGlnAlaGln 
115 120125 
ProAsnThrAsnGlyAsnAlaGlySerGlySerGlnAsnGlySerAsn 
130135140 
GlySerThrGluThrArgThrAsnLeuI leValAsnTyrLeuProGln 
145150155160 
ThrMetThrGluAspGluIleArgSerLeuPheSerSerValGlyGlu 
165 170175 
IleGluSerValLysLeuIleArgAspLysSerGlnValTyrIleAsp 
180185190 
ProLeuAsnProGlnAlaPr oSerLysGlyGlnSerLeuGlyXaaGly 
195200205 
PheValXaaTyrValArgProGlnAspAlaGluGlnAlaValAsnVal 
210215 220 
LeuAsnGlyLeuArgLeuGlnAsnLysThrIleLysValSerPheAla 
225230235240 
ArgProSerSerAspAlaIleLys GlyAlaAsnLeuTyrValSerGly 
245250255 
LeuProLysThrMetThrGlnGlnGluLeuGluAlaIlePheAlaPro 
260 265270 
PheGlyAlaIleIleThrSerArgIleLeuGlnAsnAlaGlyAsnAsp 
275280285 
ThrGlnThrLysGlyValGlyPhe IleArgPheAspLysArgGluGlu 
290295300 
AlaThrArgAlaIleIleAlaLeuAsnGlyThrThrProSerSerCys 
305310 315320 
ThrAspProIleValValLysPheSerAsnThrProGlySerThrSer 
325330335 
LysIleIleGlnProGlnLeuP roAlaPheLeuAsnProGlnLeuVal 
340345350 
ArgArgIleGlyGlyAlaMetHisThrProValAsnLysGlyLeuAla 
355 360365 
ArgPheSerProMetAlaGlyAspMetLeuAspValMetLeuProAsn 
370375380 
GlyLeuGlyAlaAlaAlaAlaAlaAlaThrTh rLeuAlaSerGlyPro 
385390395400 
GlyGlyAlaTyrProIlePheIleTyrAsnLeuAlaProGluThrGlu 
405 410415 
GluAlaAlaLeuTrpGlnLeuPheGlyProPheGlyAlaValGlnSer 
420425430 
ValLysIleValLysAspProThr ThrAsnGlnCysLysGlyTyrGly 
435440445 
PheValSerMetThrAsnTyrAspGluAlaAlaMetAlaIleArgAla 
450455 460 
LeuAsnGlyTyrThrMetGlyAsnArgValLeuGlnValSerPheLys 
465470475480 
ThrAsnLysAlaLys 
485 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 359 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetGluThrGlnLeuSerAsnGlyProThrCysAsnAsnThrAlaAsn 
1 51015 
GlyProThrThrIleAsnAsnAsnCysSerSerProValAspSerGly 
202530 
AsnTh rGluAspSerLysThrAsnLeuIleValAsnTyrLeuProGln 
354045 
AsnMetThrGlnGluGluLeuLysSerLeuPheGlySerIleGlyGlu 
50 5560 
IleGluSerCysLysLeuValArgAspLysIleThrGlyGlnSerLeu 
65707580 
GlyTyrGlyPh eValXaaTyrIleAspProLysAspAlaGluLysAla 
859095 
IleAsnThrLeuAsnGlyLeuArgLeuGlnThrLysThrIleLysVal 
100105110 
SerTyrAlaArgProSerSerAlaSerIleArgAspAlaAsnLeuTyr 
115120125 
ValSerGlyLeu ProLysThrMetThrGlnLysGluLeuGluGlnLeu 
130135140 
PheSerGlnTyrGlyArgIleIleThrSerArgIleLeuValAspGln 
1451 50155160 
ValThrGlyIleSerArgGlyValGlyPheIleArgPheAspLysArg 
165170175 
IleGluAlaG luGluAlaIleLysGlyLeuAsnGlyGlnLysProPro 
180185190 
GlyAlaThrGluProIleThrValLysPheAlaAsnAsnProSerGln 
19 5200205 
LysThrAsnGlnAlaIleLeuSerGlnLeuTyrGlnSerProAsnArg 
210215220 
ArgTyrProGlyProLeuAl aGlnGlnAlaGlnArgPheArgLeuAsp 
225230235240 
AsnLeuLeuAsnMetAlaTyrGlyValLysArgPheSerProMetThr 
245250255 
IleAspGlyMetThrSerLeuAlaGlyIleAsnIleProGlyHisPro 
260265270 
GlyThrGlyTrp CysIlePheValTyrAsnLeuAlaProAspAlaAsp 
275280285 
GluSerIleLeuTrpGlnMetPheGlyProPheGlyAlaValThrAsn 
290 295300 
ValLysValIleArgAspPheAsnThrAsnLysCysLysGlyPheGly 
305310315320 
PheValThrMetThr AsnTyrAspGluAlaAlaMetAlaIleArgSer 
325330335 
LeuAsnGlyTyrArgLeuGlyAspArgValLeuGlnValSerPheLys 
3 40345350 
ThrAsnLysThrHisLysAla 
355 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 444 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
MetValGluGlyGlnThrAlaValGlnGlnGlnGlnGlnGlnProSer 
151015 
GlyAlaGlyGlyAlaSerGlyValGlySerThrThrGly SerAlaGly 
202530 
GlyProAlaThrAlaAsnAsnValThrAsnSerGlnAlaGlnThrAsn 
354045 
GlyGlyThrThrAlaThrThrThrAlaAlaAlaGlyAlaGlySerThr 
505560 
ThrAsnAlaAlaValGlyGlnAlaThrAlaAsnAsnAlaAlaSerAsn 
65707580 
AsnAsnAsnAsnAsnAsnAsnThrAsnAsnAsnAsnAsnAsnAsnAla 
859095 
ThrAlaAsnAsnAsnAsnAsnAsnGluProAspProLysThrAsnLeu 
100105110 
IleValAsnTyrLeuProGlnThrMetSerGlnAspGluIleArg Ser 
115120125 
LeuPheValSerPheGlyGluValGluSerCysLysLeuIleArgAsp 
130135140 
LysV alThrGlyGlnSerLeuGlyTyrGlyPheValXaaTyrValLys 
145150155160 
GlnGluAspAlaGluLysAlaIleAsnAlaLeuAsnGlyLeuArgLeu 
165170175 
GlnAsnLysThrIleLysValSerIleAlaArgProSerSerGluSer 
180185190 
IleLysGlyAlaAsnLeuTyrValSerGlyLeuProLysAsnMetThr 
195200205 
GlnSerAspLeuGluSerLeuPheSerProTyrGlyLysIleIleThr 
210215220 
SerArgIleLeuCysAspAsnIleThrAspGluHisAlaAlaGlyLeu 
225230235240 
SerLysGlyValGlyPheIleArgPheAspGlnArgPheGluAlaAsp 
245250255 
ArgAlaIleLysGluLeuAsnGlyThrThrProLysAsnSerThrGl u 
260265270 
ProIleThrValLysPheAlaAsnAsnProSerSerAsnLysAsnSer 
275280285 
MetGlnProLeuAlaAlaTyrIleAlaProGlnAsnThrArgGlyGly 
290295300 
ArgAlaPheProAlaAsnAlaAlaAlaGlyAlaAlaAlaAlaAlaAla 
305 310315320 
AlaAlaAlaIleHisProAsnAlaGlyArgTyrSerSerValIleSer 
325330335 
ArgTyrSerProLeuThrSerAspLeuIleThrAsnGlyMetIleGln 
340345350 
GlyAsnThrIleAlaSerSerGlyTrpCysIlePheValTyrAsnLeu 
355360365 
AlaProGluThrGluGluAsnValLeuTrpGlnLeuPheGlyProPhe 
370375380 
GlyAlaVa lGlnSerValLysValIleArgAspLeuGlnSerAsnLys 
385390395400 
CysLysGlyPheGlyPheValThrMetThrAsnTyrGluGluAlaVal 
405410415 
LeuAlaIleGlnSerLeuAsnGlyTyrThrLeuGlyAsnArgValLeu 
420425430 
GlnValSerPheLysThrAsnLysAsnLysGlnThr 
435440 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 76 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
ValIleHisIleArgLysLeuProIleAspValThrGluGlyGluVal 
151015 
IleSerLeuGlyLeuProPheGlyLysValThrAsnLeuLeuMetL eu 
202530 
LysGlyLysAsnGlnAlaPheIleGluMetAsnThrGluGluAlaAla 
354045 
A snThrMetValAsnTyrTyrThrSerValThrProValLeuArgGly 
505560 
GlnProIleTyrIleGlnPheSerAsnHisLysGlu 
6570 75 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 78 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
ArgIleIleValGluAsnLeuPheTyrProValThrLeuAspValLeu 
151015 
MetGlnIlePheSerLysPheGlyThrValLeuLysIleIleThrPhe 
202530 
ThrLysAsnAsnGlnPheGlnAlaLeuLeuGlnTyrAlaAspProVal 
354045 
SerAlaGlnHisAlaLysLeuSerLeuAspGlyGlnAsnIleTyrAsn 
505560 
AlaCysCysThrLeuArgIleAspPheSerLysLeuThrSer 
657075 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 76 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
ValLeuLeuValSerAsnLeuAsnProGluArgValThrProGlnSer 
1510 15 
LeuPheIleLeuPheGlyValTyrGlyAspValGlnArgValLysIle 
202530 
LeuPheAsnLysLysGluAsnAlaLeuValGlnMetAlaAsp GlyAsn 
354045 
GlnAlaGlnLeuAlaMetSerHisLeuAsnGlyHisLysLeuHisGly 
505560 
Lys ProIleArgIleThrLeuSerLysHisGlnAsn 
657075 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 76 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
ValValHisIleArgGlyLeuIleAspGlyValValGluAlaAspLeu 
151015 
ValGluAlaLeuGlnGluPheGlyProIleSerTyrValVa lValMet 
202530 
ProLysLysArgGlnAlaLeuValGluPheGluAspValLeuGlyAla 
354045 
CysAsnAlaValAsnTyrAlaAlaAspAsnGlnIleTyrIleAlaGly 
505560 
HisProAlaPheValAsnTyrSerThrSerGlnLys 
65 7075 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 77 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
LeuPheThrIleLeuAsnProIleTyrSerIleThrThrAspV alLeu 
151015 
TyrThrIleCysAsnProCysGlyProValGlnArgIleValIlePhe 
2025 30 
ArgLysAsnGlyValGlnAlaMetValGluPheAspSerValGlnSer 
354045 
AlaGlnArgAlaLysAlaSerLeuAsnGlyAlaAspIleTyrSerG ly 
505560 
CysCysThrLeuLysIleGluTyrAlaLysProThrArg 
657075 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 76 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
ValLeuMetValTyrGlyLeuAspGlnSerLysMetAsnGlyAspArg 
1510 15 
ValPheAsnValPheCysLeuTyrGlyAsnValGluLysValLysPhe 
202530 
MetLysSerLysProGlyAlaAlaMetValGluMetAla AspGlyTyr 
354045 
AlaValAspArgAlaIleThrHisLeuAsnAsnAsnPheMetPheGly 
505560 
GlnLysLeuAsnValCysValSerLysGlnProAla 
657075 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 80 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
AsnLeuIleValAsnXaaLeuProGlnAspMetThrAspArgGluLeu 
151015 
TyrAlaLeuPheArgAlaIleGlyProIleAsnThrCys ArgIleMet 
202530 
ArgAspTyrLysThrGlyTyrSerPheGlyTyrAlaPheValAspPhe 
35404 5 
ThrSerGluMetAspSerGlnArgAlaIleLysValLeuAsnGlyIle 
505560 
ThrValArgAsnLysArgLeuLysValSerTyrAlaArgProGlyGly 
65707580 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 82 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
AsnLeuTyrValTh rAsnLeuProArgThrIleThrAspAspGlnLeu 
151015 
AspThrIlePheGlyLysTyrGlySerIleValGlnLysAsnIleLeu 
202530 
ArgAspLysLeuThrGlyArgProArgGlyValAlaPheValArgTyr 
354045 
AsnLysArgGluGluAl aGlnGluAlaIleSerAlaLeuAsnAsnVal 
505560 
IleProGluGlyGlySerGlnProLeuSerValArgLeuAlaGluGlu 
6570 7580 
HisGly 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 75 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
LysValTyrValGlyAsnLeuGlySe rSerAlaSerLysHisGluIle 
151015 
GluGlyAlaPheAlaLysTyrGlyProLeuArgAsnValTrpValAla 
20 2530 
ArgAsnProProGlyPheAlaPheValGluPheGluAspArgArgAsp 
354045 
AlaGluAspAlaThrArgAlaLeuAspGl yThrArgCysCysGlyThr 
505560 
ArgIleArgValGluMetSerSerGlyArgSer 
657075 
(2) INFORMATION FOR SEQ ID NO:13: 
(i ) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 81 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
IleAlaPheValGlyAsnLeuProGlnGlyLeuValGlnGlyAspVal 
15 1015 
IleLysIlePheGlnAspPheGluValLysTyrValArgLeuValLys 
202530 
AspArgGluThrAspGlnPheLysGlyP heCysTyrValGluPheGlu 
354045 
ThrLeuAspAsnLeuGluArgAlaLeuGluCysAspGlyArgIleLys 
5055 60 
LeuAspAspLeuSerAlaProLeuArgIleAspIleAlaAspArgArg 
65707580 
Lys 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 93 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
AsnLeuIleValAsnTyrLeuProGlnThrMetThrGluAspGluIle 
1510 15 
ArgSerLeuPheSerSerValGlyGluIleGluSerValLysLeuIle 
202530 
ArgAspLysSerGlnValTyrIleAspProLeuAsnProGlnA laPro 
354045 
SerLysGlyGlnSerLeuGlyTyrGlyPheValAsnTyrValArgPro 
505560 
GlnA spAlaGluGlnAlaValAsnValLeuAsnGlyLeuArgLeuGln 
65707580 
AsnLysThrIleLysValSerPheAlaArgProSerSer 
8590 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 80 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
AsnLeuIleValAsnTyrLeuProGlnThrMetSerGln AspGluIle 
151015 
ArgSerLeuPheValSerPheGlyGluValGluSerCysLysLeuIle 
2025 30 
ArgAspLysValThrGlyGlnSerLeuGlyTyrGlyPheValAsnTyr 
354045 
ValLysGlnGluAspAlaGluLysAlaIleAsnAlaLeuAsn GlyLeu 
505560 
ArgLeuGlnAsnLysThrIleLysValSerIleAlaArgProSerSer 
65707580 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 80 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
AsnLeuIleValAsnTyrLeuProGlnAsnMetThrGlnGluGluLeu 
1 51015 
LysSerLeuPheGlySerIleGlyGluIleGluSerCysLysLeuVal 
202530 
ArgAspLysIle ThrGlyGlnSerLeuGlyTyrGlyPheValAsnTyr 
354045 
IleAspProLysAspAlaGluLysAlaIleAsnThrLeuAsnGlyLeu 
50 5560 
ArgLeuGlnThrLysThrIleLysValSerTyrAlaArgProSerSer 
65707580 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 83 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
AsnLeuTyrValSerGlyLeuProLysThrMetThrGlnGlnGluLeu 
1510 15 
GluAlaIlePheAlaProPheGlyAlaIleIleThrSerArgIleLeu 
202530 
GlnAsnAlaGlyAsnAspThrGlnThrLysGlyVa lGlyPheIleArg 
354045 
PheAspLysArgGluGluAlaThrArgAlaIleIleAlaLeuAsnGly 
505560 
ThrThrProSerSerCysThrAspProIleValValLysPheSerAsn 
65707580 
ThrProGly 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 87 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
AsnLeuTyrValSerGlyLeuProLysAsnMetThrGlnSerAspLeu 
1510 15 
GluSerLeuPheSerProTyrGlyLysIleIleThrSerArgIleLeu 
202530 
CysAspAsnIleThrAspGluAsnAlaAlaGlyLeuSerLysGl yVal 
354045 
GlyPheIleArgPheAspGlnArgPheGluAlaAspArgAlaIleLys 
505560 
GluLe uAsnGlyThrThrProLysAsnSerThrGluProIleThrVal 
65707580 
LysPheAlaAsnAsnProSer 
85 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 82 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
AsnLeuTyrValSerGlyLeuProLysThrMetThrGlnLysGluLeu 
15 1015 
GluGlnLeuPheSerGlnTyrGlyArgIleIleThrSerArgIleLeu 
202530 
ValAspGlnValThrGlyIleS erArgGlyValGlyPheIleArgPhe 
354045 
AspLysArgIleGluAlaGluGluAlaIleLysGlyLeuAsnGlyGln 
5055 60 
LysProProGlyAlaThrGluProIleThrValLysPheAlaAsnAsn 
65707580 
ProSer 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 80 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
ProIlePheIleTyrAsnLeuAlaProGluThrGluGluAlaAlaLeu 
1510 15 
TrpGlnLeuPheGlyProPheGlyAlaValGlnSerValLysIleVal 
202530 
LysAspProThrThrAsnGlnCysLysGlyTyrG lyPheValSerMet 
354045 
ThrAsnTyrAspGluAlaAlaMetAlaIleArgAlaLeuAsnGlyTyr 
505560 
ThrMetGlyAsnArgValLeuGlnValSerPheLysThrAsnLysAla 
65707580 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 80 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
CysIlePheValTyrAsnLeuAlaProGluThrGluGluAsnValLeu 
151015 
TrpGln LeuPheGlyProPheGlyAlaValGlnSerValLysValIle 
202530 
ArgAspLeuGlnSerAsnLysCysLysGlyPheGlyPheValThrMet 
354045 
ThrAsnTyrGluGluAlaValLeuAlaIleGlnSerLeuAsnGlyTyr 
505560 
ThrLeuGlyAsnArgVal LeuGlnValSerPheLysThrAsnLysAsn 
65707580 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 80 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
CysIlePheValTyrAsnLeuAlaProAspAlaAspGluSerIleLeu 
151015 
TrpGlnMetPheGlyProPheGlyAlaVal ThrAsnValLysValIle 
202530 
ArgAspPheAsnThrAsnLysCysLysGlyPheGlyPheValThrMet 
3540 45 
ThrAsnTyrAspGluAlaAlaMetAlaIleArgSerLeuAsnGlyTyr 
505560 
ArgLeuGlyAspArgValLeuGlnValSerPheLysThrAsn LysThr 
65707580 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
UCCAGUAACCCCACCUCCUCUUUUU25 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
(xi ) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
UCAGUUAAACGUGUAAACCUUUUAA25 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
( xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
UCAUAGCACCACCUCACCCUUUUUA25 
(2) INFORMATION FOR SEQ ID NO:26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
UCAUAGCACCACCUCACCCUUUUUA25 
(2) INFORMATION FOR SEQ ID NO:27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
GGGCUAGGCUUAUCCUCCUUUCC23 
(2) INFORMATION FOR SEQ ID NO:28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
AUCAUAAAUUCAGUGUCAUUUUUCU25 
(2) INFORMATION FOR SEQ ID NO:29: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
UUAUUUAUUUGCGUCUCCUUUAUUA25 
(2) INFORMATION FOR SEQ ID NO:30: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
AACUACCGGAGUACAGAUUUUUUUA25 
(2) INFORMATION FOR SEQ ID NO:31: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
UCAGUGGCAUCUCUUUCUUUACUUU25 
(2) INFORMATION FOR SEQ ID NO:32: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(i i) MOLECULE TYPE: RNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
CACAACCCUAACUUUCAUUUGCUUU25 
(2) INFORMATION FOR SEQ ID NO:33: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
UGACCGAUACACAUUCUUUUAUUUA25 
(2) INFORMATION FOR SEQ ID NO:34: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
AUUGACUUCGUUAUUGUUUUUAUUG25 
(2) INFORMATION FOR SEQ ID NO:35: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: 
AGACGCAAUUAAUGAUUUGUUUUUA25 
(2) INFORMATION FOR SEQ ID NO:36: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: 
UAGCUCGGACAUUUAUUUUUAUUU24 
(2) INFORMATION FOR SEQ ID NO:37: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: 
UUAGGUUUCUUUUUAUUUGAGCAUA25 
(2) INFORMATION FOR SEQ ID NO:38: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D ) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: 
AUUUCUCAUUUAACGUCUCUCCUUU25 
(2) INFORMATION FOR SEQ ID NO:39: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: 
ACACCCUUUUUAGUUCCUGUAUUU24 
(2) INFORMATION FOR SEQ ID NO:40: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: 
CUAAUUUCCGAUAUUAAAGCUUAUUA26 
(2) INFORMATION FOR SEQ ID NO:41: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: 
AUGAUUUAGAUUUUCGCACAUUUCA25 
(2) INFORMATION FOR SEQ ID NO:42: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: 
UACUUUCGGUACUAAAAUCGAUCAG25 
(2) INFORMATION FOR SEQ ID NO:43: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: 
UCCUUUUUGUACCACUCUCAGUUGU25 
(2) INFORMATION FOR SEQ ID NO:44: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: 
UUAUUUAUUUGCGUCUCCUUUAUUA25 
(2) INFORMATION FOR SEQ ID NO:45: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: 
UUAUUUAUUUGCGUCUCCUUUAUUA25 
(2) INFORMATION FOR SEQ ID NO:46: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: 
UUUGUUUUCGUGUAACGCAUAUACU25 
(2) INFORMATION FOR SEQ ID NO:47: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
( C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: 
UUUAGUUUAAUAGGGAUAAUACUUA25 
(2) INFORMATION FOR SEQ ID NO:48: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: 
UUUGUUUUCGUGUAACGCAUAUACU25 
(2) INFORMATION FOR SEQ ID NO:49: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 25 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: RNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: 
UUGAUUUUCGCGCCCGCCGCCUUAG25 
(2) INFORMATION FOR SEQ ID NO:50: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1467 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: unknown 
(D) TOPOLOGY: unknown 
(ii) MOLECULE TYPE: DNA (genomic) 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 95..1234 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: 
CCAATAGTAGTCATTTTAAATATATATTCTGAAATCTTTGCAAATTTTAACAGAAGAGTC60 
GAAGCTCTGCGAGACCC AATATTTGCCAATAAGAATGGTTATGATAATTAGC112 
MetValMetIleIleSer 
15 
ACCATGGAGCCTCAGG TGTCAAATGGTCCGACATCCAATACAAGCAAT160 
ThrMetGluProGlnValSerAsnGlyProThrSerAsnThrSerAsn 
101520 
GGACCCTCCAGCAACAAC AGAAACTGTCCTTCTCCCATGCAAACAGGG208 
GlyProSerSerAsnAsnArgAsnCysProSerProMetGlnThrGly 
253035 
GCAACCACAGATGACAGCAAAACC AACCTCATCGTCAACTATTTACCC256 
AlaThrThrAspAspSerLysThrAsnLeuIleValAsnTyrLeuPro 
404550 
CAGAATATGACCCAAGAAGAATTCAGGAGTCT CTTCGGGAGCATTGGT304 
GlnAsnMetThrGlnGluGluPheArgSerLeuPheGlySerIleGly 
55606570 
GAAATAGAATCCTGCAAACTTGTGAGAG ACAAAATTACAGGACAGAGT352 
GluIleGluSerCysLysLeuValArgAspLysIleThrGlyGlnSer 
758085 
TTAGGGTATGGATTTGTTAACTATATT GATCCAAAGGATGCAGAGAAA400 
LeuGlyTyrGlyPheValAsnTyrIleAspProLysAspAlaGluLys 
9095100 
GCCATCAACACTTTAAATGGACTCAGACTC CAGACCAAAACCATAAAG448 
AlaIleAsnThrLeuAsnGlyLeuArgLeuGlnThrLysThrIleLys 
105110115 
GTCTCATATGCCCGTCCGAGCTCTGCCTCAATCAG GGATGCTAACCTC496 
ValSerTyrAlaArgProSerSerAlaSerIleArgAspAlaAsnLeu 
120125130 
TATGTTAGCGGCCTTCCCAAAACCATGACCCAGAAGGAACTGG AGCAA544 
TyrValSerGlyLeuProLysThrMetThrGlnLysGluLeuGluGln 
135140145150 
CTTTTCTCGCAATACGGCCGTATCATCACCTCACGAATC CTGGTTGAT592 
LeuPheSerGlnTyrGlyArgIleIleThrSerArgIleLeuValAsp 
155160165 
CAAGTCACAGGAGTGTCCAGAGGGGTGGGATTCATCCGC TTTGATAAG640 
GlnValThrGlyValSerArgGlyValGlyPheIleArgPheAspLys 
170175180 
AGGATTGAGGCAGAAGAAGCCATCAAAGGGCTGAATGGCCA GAAGCCC688 
ArgIleGluAlaGluGluAlaIleLysGlyLeuAsnGlyGlnLysPro 
185190195 
AGCGGTGCTACGGAACCGATTACTGTGAAGTTTGCCAACAACCCCA GC736 
SerGlyAlaThrGluProIleThrValLysPheAlaAsnAsnProSer 
200205210 
CAGAAGTCCAGCCAGGCCCTGCTCTCCCAGCTCTACCAGTCCCCTAAC784 
GlnLysSerSerGlnAlaLeuLeuSerGlnLeuTyrGlnSerProAsn 
215220225230 
CGGCGCTACCCAGGTCCACTTCACCACCAGGCTCAGAGGTTCAGGCTG 832 
ArgArgTyrProGlyProLeuHisHisGlnAlaGlnArgPheArgLeu 
235240245 
GACAATTTGCTTAATATGGCCTATGGCGTAAAGAGACTGATGTCTGGA 880 
AspAsnLeuLeuAsnMetAlaTyrGlyValLysArgLeuMetSerGly 
250255260 
CCAGTCCCCCCTTCTGCTTGTTCCCCCAGGTTCTCCCCAATTACCATT 928 
ProValProProSerAlaCysSerProArgPheSerProIleThrIle 
265270275 
GATGGAATGACAAGCCTTGTGGGAATGAACATCCCTGGTCACACAGGA976 
As pGlyMetThrSerLeuValGlyMetAsnIleProGlyHisThrGly 
280285290 
ACTGGGTGGTGCATCTTTGTCTACAACCTGTCCCCCGATTCCGATGAG1024 
ThrGlyTrpC ysIlePheValTyrAsnLeuSerProAspSerAspGlu 
295300305310 
AGTGTCCTCTGGCAGCTCTTTGGCCCCTTTGGAGCAGTGAACAACGTA1072 
SerVal LeuTrpGlnLeuPheGlyProPheGlyAlaValAsnAsnVal 
315320325 
AAGGTGATTCGTGACTTCAACACCAACAAGTGCAAGGGATTCGGCTTT1120 
LysVal IleArgAspPheAsnThrAsnLysCysLysGlyPheGlyPhe 
330335340 
GTCACCATGACCAACTATGATGAGGCGGCCATGGCCATCGCCAGCCTC1168 
ValThrMe tThrAsnTyrAspGluAlaAlaMetAlaIleAlaSerLeu 
345350355 
AACGGGTACCGCCTGGGAGACAGAGTGTTGCAAGTTTCCTTTAAAACC1216 
AsnGlyTyrArgL euGlyAspArgValLeuGlnValSerPheLysThr 
360365370 
AACAAAGCCCACAAGTCCTGAATTTCCCATTCTTACTTACTAAAATAT1264 
AsnLysAlaHisLysSer 
375 380 
ATATAGAAATATATACGAACAAAACACACGCGCGCACACACACATACACGAAAGAGAGAG1324 
AAACAAACTTTTCAAGGCTTATATTCAACCATGGACTTTATAAGCCAGTGTTGCCTAGTA1384 
TTAAAACATTGGGTTATCCTGAGGT GTACCAGGAAAGGATTATAATGCTTAGAAAAAAAA1444 
AAAGAAAAAAAAAAAACAAAAAA1467 
(2) INFORMATION FOR SEQ ID NO:51: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 380 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: 
MetValMetIleIleSerThrMetGluProGlnValSerAsnGlyPro 
151015 
ThrSerAsnThrSerAsnGlyProSerSerA snAsnArgAsnCysPro 
202530 
SerProMetGlnThrGlyAlaThrThrAspAspSerLysThrAsnLeu 
354045 
IleValAsnTyrLeuProGlnAsnMetThrGlnGluGluPheArgSer 
505560 
LeuPheGlySerIleGlyGluIleGluSerCysLysLeuValArgAsp 
65 707580 
LysIleThrGlyGlnSerLeuGlyTyrGlyPheValAsnTyrIleAsp 
859095 
ProLysAspAlaGluLysAl aIleAsnThrLeuAsnGlyLeuArgLeu 
100105110 
GlnThrLysThrIleLysValSerTyrAlaArgProSerSerAlaSer 
115120 125 
IleArgAspAlaAsnLeuTyrValSerGlyLeuProLysThrMetThr 
130135140 
GlnLysGluLeuGluGlnLeuPheSerGlnTyrGlyArgIleIleThr 
145 150155160 
SerArgIleLeuValAspGlnValThrGlyValSerArgGlyValGly 
165170175 
PheIleArg PheAspLysArgIleGluAlaGluGluAlaIleLysGly 
180185190 
LeuAsnGlyGlnLysProSerGlyAlaThrGluProIleThrValLys 
195 200205 
PheAlaAsnAsnProSerGlnLysSerSerGlnAlaLeuLeuSerGln 
210215220 
LeuTyrGlnSerProAsnArgArgTyrProGlyProLeuHi sHisGln 
225230235240 
AlaGlnArgPheArgLeuAspAsnLeuLeuAsnMetAlaTyrGlyVal 
24525025 5 
LysArgLeuMetSerGlyProValProProSerAlaCysSerProArg 
260265270 
PheSerProIleThrIleAspGlyMetThrSerLeuValGlyMetAsn 
275280285 
IleProGlyHisThrGlyThrGlyTrpCysIlePheValTyrAsnLeu 
290295300 
SerProAspSerAspGluSerValLeuTrp GlnLeuPheGlyProPhe 
305310315320 
GlyAlaValAsnAsnValLysValIleArgAspPheAsnThrAsnLys 
325330 335 
CysLysGlyPheGlyPheValThrMetThrAsnTyrAspGluAlaAla 
340345350 
MetAlaIleAlaSerLeuAsnGlyTyrArgLeuGlyAspArgVa lLeu 
355360365 
GlnValSerPheLysThrAsnLysAlaHisLysSer 
370375380