Note: Descriptions are shown in the official language in which they were submitted.
WO g~/30693 2 1 8 8 4 5 ~ P~llu~
l~T ~ ,, ' D:~ ~ (NeuroD) Genes andProteins
This inYention was made with ~sU.~ support under grant CA42506
awarded by the National ~nstitutes of Healith. The ~5U.. has certiain rights in
the inverltion.
This apphcation is a, part of U.S. Serial No. 08/239,238, filed
May 6, 1994.
Field of the Inv~-ntinn
The invention reliates to molecular biology and in particular to genes and
proteins involved in vertebrate neurali J~
R~n~roundoftheInvention
There are currently several examples of i . regulatory proteins
sharing a basic helix-loop-helix (bHLH~ secondary structure. bHLH proteins form
and ~ t~ " comples binding DNA in the 5' regulatory regions
of genes controlling expression. Among the bHLH proteins, " M[yoD and
Drosophila AS-C are presently thought to play J~ , ' roles in muscle
d~ and in sensory organ ~ ' . t, ~ . Both proteins are
thought to ert their effects by binding 5' regulatory nucleotide sequences in genes
that seem specifically d ~_ of cellular Ji~ t~iuai and fate. However, the
specific d~,~lu~ tal roles of the genes affected by MyoD and AS-C remain largelyunknowrl, as are the moleculiar details of the d~,~lu~ pathways regulated by
these genes. The presently disclosed NeuroD represents a new sub-family of bHLH
proteins and is implicated in vertebrate neuronal d~.~lu~
Neural tissues and endocrine tissues do not regenerate. Damage is permanent.
Paralysis, loss of vision or hearing, and hormonal illa~l~h~ ~ are also permanent.
woss/306s3 218~450 P~l~u.. ~.~r~4l ~
--2--
Tumors in neural and endocrine tissues can also be very diffiGult to treat because of
the toxic side effects tbat Gu~ , '- drugs may have on nervous
tissues. The medical community and public would greatly benefit from the availability
of agents aGtive in triggering Ji:~.,., in ~ u~lvd~ stem celis. Such
5 neuronal J;~ .dfi..~ agents could be used for: of test cell lines, assays
for identifying candidate therapeutic agents capable of inducing ~ of
neuronal and endocrine tissues, gene therapy, and dilF~ .~ of tumor Gells.
of the Invention
r~ ~ and amphibian NeuroD proteins were identified, and
10 pol~,l.,l~vL.i~ molecules encoding NeuroD were isolated and sequenced. NeuroDencodes a p}otein that is a distinGtive member of the bHLH family. In addition, the
present invention provides a family of NeuroD proteins that share a higbly conserved
~H region. In the neurula stage of the mouse embryo (elO), neuroD is highly
expressed in the neurogenic derivatives of neural crest cells, the G~anial and dorsal
root ganglia, and postn~itotic cells in the central nervous system (CNS). Duringmouse d~, ' r t, neuroD is expressed transiently and ~ with neuronal
t;aliu,l m !~'~ ~ '- '- _ neurons in sensory organs such as in nasal epithelium
and retina. In Xenopus embryos ectopic expression of neuroD in I ' cells
induced formation of neurons.
A l~ ive nucleotide sequence of murine neuroD is shown in SEQ ID
NO:I. The IILH coding domain of murine neuroD resides between nucleotides 577
and 696 in SEQ ID NO:I. The deduced amino acid sequence ûf murine NeuroD is
shown in SEQ ]D NO:2. There is a highly conserved region follov~ing the helix-2
domain from amino acid 150 through amino acid 199 of SEQ ID NO:2 that is not
shared by other bHL~I proteins.
A l.~ ~live nucleotide sequence of Xenopus neuroD is shov~n in SEQ ID
NO:3. The IILH: coding domain of Xenopus neuroD resides between nucleotides 376
and 495 in SEQ ID NO:3. The deduced amino acid sequence of murine NeuroD is
shov~n m SEQ ID NO:4. There is a highly conserved region follov~ing the hehx-2
domain from amino acid 157 through amino acid 199 of SEQ ID NO:4 that is not
shared by other bHLH proteins.
IIuman neuroD sequences are also disclosed. R.,"l~ ~. nucleotide and
deduced amino acid sequences of the human NeuroD family of are shown in SEQ ID
NOS:8-11. Thedisclosedhumanclones, 9FI and 14BI,haveanidenticalHL~motif:
amino acid residues 117-156 in SEQ n) NO:9 and residues 91-130 in SEQ ]D
NO:II.
~ wos~/306s3 21 8845a P~./.i~ S0 14l
--3--
Brief Description of the Drawings
~IGURE 1 ' "~v depicts the dornain structure of the murine and
Xenopus NeuroD b~H proteins.
Detailed Descrivtion of the Preferred r ~ ~ :
Tissue-specific bHLH proteins thst regulate early ~ u~lo~
-~ ~L~u.. were discovered using expression cloning snd screerling assays
designed to identify possible blILH proteins capable of interacting with the protein
product of the Drosoph~la ~ . k~ gene. These proteins belong to a family of
proteins that share conserved residues in the ~: region.
Iû NeuroD is a member of a novel protein fsmily and is found to be trsnsiently
expressed in ~ neurons during ~lllbl~U~ . Its expression is also
detected in adult brain, in the granule layer of l -. . . and cerebellum. NeuroDcontains the bssic helix-loop-helix (bE~ domain structure that has been implicated
irl the binding of bHLH proteins to upstream recognition sequences and activation of
15 dv~ target genes. The present invention provides l~ NeuroD
proteins, which include the murine NeuroD protein of SEQ ]D NO:2 and the
amphibian NeuroD protein of ~iEQ lD NO.4. Based on homology with other bHLEI
proteins, the bHLEI dornain for the murine NeuroD protein is predicted to residebetween arnino acids 102 and 155 of SEQ ID NO:2, and between arnino acids 101
and 157 of SEQ ID NO:4 for the amphibian NeuroD protein. As detsiled below, the
present invention provides the ' ' of the human neuroD and, in ~ddition,
provides an, . I 1,.. ~1.. ~,,.. ~ gene of the ssme family based on th~ almost
identical sequence across the HLH domain shsred between the two human genes at
the amino acid level. NeuroD proteins are i . ' activators that control
. of dv.. ~ target genes that cause neuronal ~lU"_.~i.Ul~ to
t~.~; into rnature neurons. As discussed in more detail below, NeuroD
proteins are expressed in '~ . _ neurons and are capable of causing the
conversion of non-neuronal cells into neurons. The present invention ~ .
NeuroD variants that, for example, are modified in a manner that results in a NeuroD
30 protein capable of binding to its recognition site, but unable to activate 1UW11~11~11
genes. NeuroD proteins encompass proteins retrieved from naturally occurring
~naterials and closely related, " ".~, similar proteins retrieved by antisera specific
to NeuroD, and ll ' '.~, expressed proteins encoded by genetic materials
(DNA, RNA, cDNA) retrieved on the basis of their similarity to the unique regions in
35 the neuroD fsmily of genes.
wo ss/306s3 ~ ~ g g 4 5 0 r~ /41 ~
4-
The present invention discloses lc~Jlci.~ aLiv~ isolated and purified
pGl~..a~.l~LiJ~, molecules encoding proteins of the NeuroD fiunily. R~
pG~....~,l~uLi~c molecules encoding NeuroD include the sequences presented in SEQ
ID NOS:l, 3, 8, and 10. rGlJ..v.,lc.JLill. molecules encoding NeuroD include those
5 sequences resulting in minor genetic pGI~ , differences between species,
those that contain amino acid ' , additions, and/or deletions.
In some instances, one may employ such changes in the sequence of
IC ' NeuroD to ' ".~, decrease or even increase the biological activity
of NeuroD, depending on the intended use of the preparation Such changes may also
be directed towards _.. 1~c,.. ~ neuroD sequences using, for example, gene therapy
methods to alter the gene product.
The NeuroD proteins of the present invention are capable of inducing the
expression of neuronal-specific genes, such as N~AM, ~-tubulin, and Xen-l,
rl~ r- ' M (NF-M~, Xen-2, tanabin-l, shaker-l, and frog HSCL, in a frog
embryo. As described below, NeuroD activity may be detected when NeuroD is
ectopicaDy expressed in frog oocytes following, for example, injection of neuroDRNA into one of the two ceDs in a two-ceD stage Xenopus embryo, and mor~itoring
expression of neuronal-specific genes in the injected as compared fo un-injected side
of the embryo by ' y or in situ h~SIiJi~iull.
"Ov ~l~ " means an increased level of NeuroD protein or r~euroD
transcripts in a l~ ' i r I host cell relative to the level of protein or
transcripts in the parental ceD from which the host ceD is derived.
As noted above, the present invention provides isolated and purified
PrJI~ ICVLidC molecules encoding NeuroD and other members of the NeuroD family.
The disclosed sequences may be used to identify and isolate neuroD prJI~ ,Lidc
molecules from suitable host ceDs such as canine, ovine, bovine, eaprine, I gr . h,
or avian. In particular, the r~ucleotide sequences encoding the HLH region may be
used to identify pu.,' .,_lwtid~. molecules encoding oth proteins of the NeuroDfamily. Cl .' y DNA molecules encoding NeuroD family members may be
obtained by ~ ,, a eDNA hbrary rnRNA from, for example, fetal brain. DNA
molecules encoding NeurûD family membs may be isolated from such a hbrary
using the disclosed sequences in standard h.~vliJ;~d~;ull techniques (e.g., Sambrook et
al., ibid., and Bothwell, Y~ropo~ s and Alt, ibid.) or by r ~ of sequences
using polymerase chain reaetion (PCR) ~ (e.g, Loh et al. Science 243:
217-222, 1989; Frohlnan et al., Proc. NalL Acad Sci. USA 85: 8998-9002, 19~8;
and Erlich (ed.), PCR ~echnology: Principles and Al~,,lj,..l';.,.-. for DNA
~ woss/30693 ~ 8 8 ~ ~ r~ /41
~', 7i,'` '- , Stockton Press, 1989; which are IJulal_d by reference herein in
their entirety). In a similar manner, genomic DNA encoding NeuroD may be obtained
using probes designed from the sequences disclosed herein. Suitable probes for use in
identifying neuroD sequences may be obtained from neuroD-specific sequences that 5 are highly conserved regions between " and amphibian neuroD coding
sequences. Primers, for exarnple, from the region encoding the a~ 40
residues following the helix-2 domain are suitable for use in designing PCR prirners.
Altematively, ~, ' ' containing specific DNA sequences from a human
neuroD coding region may be used within the described methods to identify hurnanneuroD genomic and cDNA clones. Upstream reg_latory regions of ne~roD may be
obtained using the same methods. Suitable PCR primers are between 7-50
nucleotides in length, more preferably between 15 and 25 ,..I..l.r.,l;f~r~ in length.
Altematively, neuroD ~ol~ ' ' molecules may be isolated using standard
hJJ~ aliull techniques with probes of at least about 7 rll~rlPr~tirlPC in length and up
15 to and including the full coding sequence. Southem analysis of mouse genomic DNA
probed with the murine neuroD cDNA under stringent conditions showed the
presence of only one gene, suggesting that under stringent conditions bHL~I genes
from other protein families will not be identified. Other members of the neuroD
farnily can be identified using degenerate 'i~ ' ' based on the sequences
20 disclosed herein for PCR ~ or by l-y~ ~L;~.-- at moderate stringency.
A DNA molecule encoding NeuroD is inserted into a suitable expression
vector, which is in tum used to transfect or transfomm a suitable host cell. Suitable
expression vectors for use in carrying out the present invention comprise a promoter
capable of directing the l . .- ~ of a ~ u~idc molecule of interest in a host
25 cell. R~ aLiv~ expression vectors may include both plasrnid and/or viral vector
sequences. Suitable vectors include retroviral vectors, vaccinia viral vectors, CMV
viral vectors, Rl--Pr~rrirt~P vectors, baculovirus vectors, and the lilce. Promoters
capable of directing the ~ . of a cloned gene or cDNA may be inducible or
uu.... iLuLiv~ promoters and include viral and cellular promoters. For expression in
' host cells, suitable viral promoters include the immediate early
~ ,, ' . u~ promoter (Boshart et al., Cell 41: 521-530, 1985) and the SV40
promoter (Subramarli et al., MoL CelL BioL 1: 854-8~4, 1981). Suitable cellular
promoters for expression of proteins in marnmalian host cells include the mouse
" ' -1 promoter (Palmiter et al., U.S. Patent No. 4,579,821), a mouse Vlc
promoter (Bcrgman et al., Proc. NafL Acad. Sci. 81: 7041-7045, 1983; Grant et al.
Nucle~c ~ci~ Pes 15: 5496, 1987), and l~la_yl " -responsive promoter (Gossen
wos~/30693 ~ ~188~50 r~l~o~ 4l ~
-6-
and Bujdrd, Proc. NafL Acad Sci.USA 89: 5547-5551, 1992 and Pescini et al.,
Biochem. Biophys. Res Comm. 202: 1664-1667, 1994). Also contained in the
expression vectors, typically, is 21.. ~ tlllfil~dLiu~ signal located dU.. ~
of the coding sequence of interest. Suitable i . hlll~.d~iu.~ signals include
5 the early or late pGIJc.~..J' signals from SV40 ~Caufmarl and Sharp, Mo~. Cell.
Biol. 2:1304-1319, 1982), the ~ul~ J' signal from the Adenovirus 5
elB region, and the human growth hormone gene terminator ~DeNoto d al., Nucleic
Acid Res. 9: 3719-3730, 1981). r~ ~ cells, for example, may be trdnsfected
by a number of methods including calcium phosphate 1,l t~ iU.. (Wlgler et al., Cell
14: 725, 1978; Corsaro and Pearson, Somafic Cell Genefics 7: 603, 1981; Graham
snd Van der Eb, Virology 52: 456, 1973); lipofection, , and
~k.~.~lu~ lio~ (Neumalm et al., EMBO J. 1: 841û845, 1982). ~ " can be
transduced ~vith virus such as SV40, CMV, and the like. In the case of viral vectors,
cloned DNA molecules may be mtroduced by infection of susceptible cells with viral
15 particles. Retroviral vectors may be preferred for use m expressing NeuroD inmammalian cells ~Li.,~.l~l~ if NeuroD is used for gene therapy (for review, see,Miller et al. Mefhods in Fl, '~ 217: 581-599, 1994; which is ...~,ull '
herein by reference in its entirety). It may be preferable to use a selectable marker to
identify cells that contain the cloned DNA. Selectable markers are generally
20 introduced into the cells along with the cloned DNA molecules and include genes that
confer resistance to drugs, such as neomycin, h~b.ull.~. and
Selectable markers may also ~ u,.ul., .' in the host cell. Yet other
selectable markers provide detectable signals, such as beta-~O~ to identify
cells contairling the doned DNA molecules. Selectable markers may be amphfiable.25 Such amplifiable selectable markers may be used to amplify the number of sequences
integrated into the host genome.
As would be evident to one of ordinary skill in the art, the po4~ k,vLJr~
molecules of the present invention may be expressed Su~,~,hu~., cerevisiae,
r~ fungi, arld E coli. Methods for expressing doned genes in
30 S- ~ G~ cerevisiae are generally known in the art (see, "Gene Expression
Technology," Mefhods in E:r~J 'c~, Vol. 185, Goeddel (ed.), Academic Press, San
Diego, CA, 1990, and "Guide to Yeast Genetics and Molecular Biology," Methods inEir~J ' ~.v. Guthrie and Fink (eds.), Academic Press, Sam Diego, CA, 1991; whichare i~ul~ul~ltd herein by reference). r ~ fungi may also be used to express
35 the proteins of the present invention; for example, strains of the fungi Aspergillus
(McKnight et al., U.S. Patent No. 4,935,349, which is; I~ l hereirl by
woss/30693 2 ~ ~45~ r~l,.n~ ,4l
-.7-
reference). Methods for expressing genes and cDNAs in cultured ' ceDs
and in E coli is discussed in detail in Sambrook et al. (Molea(lar Clo~ing A
Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989; which is
~, ' herein by reference). As would be evident to one skiDed in the arf, one
5 could express the protein of the instant invention in other host ceDs such as avian,
insect, and plant ceDs using regulatory sequences, vectors and methods weD
established in the literature.
The term "capable of hybridizing under stringent conditions" as used herein
means that the subject nucleic acid molecules (whether DNA or RNA) anneal to an
rll;O, - Ir-~ - of 15 or more contiguous mlr~ofi~1oC of SEQ ID NO:I, SEQ lD
NO:3, SEQ ID NO:8, or SEQ lD NO:10.
NeuroD proteins produced according to the present invention may be purified
using a number of established methods such as afrlnity ~ usi~g anti-
NeuroD antibodies coupled to a solid support. Fusion proteins of antigenic tag and
NeuroD can be purified using antibodies to the tag. Additional ~ may be
achieved using ' ~ iWilUl~ means such as liquid - ~c ~
gradient ~ y~ and gel l~ll~r' . 0;O, among others. Methods of protein
are known in the art (see generaDy, Scopes, R, Protein r ~
Springer-Verlag, NY, 1982, which is UUI~I~d herein by reference) and may be
applied to the purification of ll ' NeuroD described herein.
The choice of }.~ ~.iu.. conditions wiD be evident to one skiDed in the art
and wiD generaDy be guided by the purpose of the ~,.,.idi~Liu.., the type of
~IDIir~iiul~ (DNA-DNA or DNA-RNA), and the level of desired relatedness
between the sequences. Methods for ~b~id;~Liull are weD established in the
25 literature; See, for example: Sambrook, ibid.; Hames and Higgins, eds, Nucleic Acid
h'y~r ;~ r,., A Pract~cal Approach, ~L Press, Washington DC, 1985; Berger and
Kimmel, eds, Mefhods in Fl, T~,~, Vol. 52, Guide to Molecular Cloning
lechniques, Academic Press Inc., New York, NY, 1987; and BothweD, Y
and Alt, eds, Methods for Cloning and Analysis of Eukaryotic Genes, Jones and
30 Bartlett Publishers, Boston, MA 1990; which are; ~ rl by reference herein in
f~heir entirety. One of ordinary skiD in the art realizes that the stability of nucleic acid
duplexes wiD decrease with an increased number and location of ' ' bases;
fhus, the stringency of hj~.i.li,~iu.. may be used to maximize or minimize the stability
of such duplexes. II~u~id;~hùl~ stringency can be altered by: adjusting the
35 ~ UI~ of l.yu.;d;~hu.., adjusting the percentage of helix- I ' " ,, agents,
such as formamide, in the l~ybi;d;~;ul. mix; and adjusting the . ~ and salt
woss/30693 2 1 8 8 ~ 5 0 r~ c~4l ~
--8 -
of the wash solutions. In general, the stringency of hr ' is
adjusted during the pL~st ~ ' washes by varying the salt . and/or
the tu...~,.aLule. Stringency of h~lhli~d~iu.. may be reduced by reducing the
percentage of formamide in the 5~1idU aLiu~l solution or by decreasing the
5 tu..llJ~alul~; of the wash solution. High stringency conditions may involve high
t~ ~a~ ; h~lhli~L;ull (e.g., 65-68C in aqueous solution containing 4-6 X SSC,
or 42C in 50% formamide) combined with high t~ ...a~u~ (e.g., 5-25C below the
Tm) and a low salt .. ,l, -ll~. (e.g., 0.1 X SSC). Reduced stringency
conditions may involve lower llyblhl;~d~iull i , (e.g., 35-42C in 20-50%
10 r ~ ) with ' , (e.g., 40-60C) and washes in a higher
salt ~ (e.g., 2-6 X SSC). Moderate stringency conditions, which may
involve ll~ a~iùl~ at a ~ e between 50C and 55C and washes in
0.1 X SSC, 0.1% SDS at between 50C and 55C, may be used to identify clones
encoding members ûf the NeuroD family.
The invention provides isolated and purified p~l~ ' ' molecules
encoding NeuroD capable of hybridizing under stringent conditions an li"
of 15 or more contiguous nucleotides of SEQ ID NO:l, SEQ ID NO:3, SEQ ID
NO:8, SEQ ID NO:10, and their c - ~ - y strands. The subject isolated
neuroD ~ul~ ' ' molecules preferably encode NeuroD proteins that trigger
diiT~l~ilLdLiul. in ectodermal cells, ~c~-L;uul~uly i~.llL~ stem cells, and in
more committed cells of that lineage, for example, epidermal precursor cells. Such
neuroD expression products typically form h~ ' bHL~ protein complexes
that bind in the 5'-regulatory regions of target genes and enhance or suppress
~"" '` ' ;1'1 '" of the target gene.
In some instances, cancer cells may contain non-functional NeuroD protein or
may contain no NeuroD protein due to genetic mutætion or somatic mutations such
that these cells fail to ~--rr I '- ' For cancers of this type, the cancer cells may be
treated in a manner to cause the over-eA~ of wild-type NeuroD protein to forced;~.. L.lt;dLiul~ of the cancer cells.
Antisense neuroD nucleotide sequences may be used to block expression of
mutant neuroD expression in neuronal precursor cells to generate and halvest
neuronal stem cells. The use of antisense O1;L~ and their ~ . ' have
been reviewed in the literature (see, for example, Mol and Van der Krul, eds.,
Antisense Nucletc Acids and Profeins ~ ' ' an,d ~ , New York
NY, 1992; which is i~,ul~ula~e~ by reference herein in its entirety). Suitable
antisense ~'i" ' ' are at least 11 nucleotide in length and may include
woss/30693 21 ~5~ U~ /41
g
' (upstrearn or intron) and associated coding sequences. As will be eYident
to one skilled in the art, the optimal length of antisense ~ 'i,, ' '- is its on the
strength of the interaction between the antisense ,-'i,, ' ' and its . '
on the rnRNA, the t~ Luli and ionic ~,.. UII.l.. lL translation takes place, the base
- 5 sequence ofthe antisense fll;f,.~ ;-lr, and the presence of secondary and tertiary
structure in the n~RNA and/or in the antisense ~I;L;" l~ u~ Suitable tsrget
sequences for antisense -'iv ' ' include ~ ~u.. junctions (to preYent
proper splicing), regions in which DNAIRNA hybrids will prevent transport of mRNA
from the nucleus to the cytoplasm, irlitiation factor binding sites, ribosome binding
lû sites, and sites that interfere with ribosome IJIU~ ;U... A ~olLi~,ulr~ preferred
target region for antisense -'i~ ' ' is the 5~ lul~lulu~
region of the gene of interest. Antisense r11~.. 1.. ,1;~1f; may be prepared by the
insertion of a DNA molecule containing the target DNA sequence into a suitable
expression vector such that the DNA molecule is inserted du....~.l~ll ûf a promoter
15 in a reYerse orientation as compared to the gene itself. The expression vector may
then be i ' 1, ~I .-fi... d or transfected into a suitable cell resulting in theexpression of antisense ~ ,, Ir.~ . . A' ' ~d~ antisense f. li,, ' '
may be synthesized using standard manual or automated synthesis techniques.
Synthesi_ed f ~iV ' _' ~ may be introduced into suitable cells by a variety of
2û means including fl~llu~JulaLull, calcium phosphate ~ iUII, or IlliWUl-l;~ iU.I.
The sdection of a suitable antisense -'i, ' ' ~ ' method will be
eYident to one skilled in the art. Wlth respect to ~thf Ci7~d, ~i~ ' the
stability of antisense 'i" ' '- ~NA hybrids may be increased by the addition
of stabilizing agents to the 'i_ ' ' Stabilizing agents include
agents that are covalently attached to either or both ends of the ~ 'i, ' '
n~ may be made resistant to nucleases by, for example" . ~ to
the, ' . ' - ' backbone by the udu~,liu.. of I ' . ' , ' . '
i' ."''' ~' ,1~ .' ~ ' ' ,1' .' '' ,orl' .' "''
n~i~ ' ' may also be made nuclease resistant by the synthesis of the
3û -'i~ ' ' with alpha-anomers of the d~ . il u .... Ir ..1 :.1. _
NeuroD binds to 5' regulatory regions of neurogenic genes that are involved in
L. .llU~ l~.d~ . I dilr~l. .lti~ iUll, including d~ .~lu~ of neural and endocrine
tissues. The NeuroD protein alters expression of the subject gene by, for example,
down-regulating or up-regulating 11. - ;I.l;~l or by inducing a change in
1~ to an alternative open reading frame. The subject pGl~ dw~;dc
2~ 88450
w0 95/30693 . F~~ /41 ~
-10-
molecules find a variety of uses, e.g., in preparing ~ ';v~ l ul; ~ probes, expression
vectors, and i r ~ host cells, as disclosed below in the following Examples.
DNA sequences recogr~ized by NeuroD may be deterrnined using a number of
methods known in the literature including iu~ullullu~Jlcu;~ a~iu~ (r ' ' ., et al,
5 Nature 335: 835-837, 1988, Kinzler and Vorgelstein, Nuc. AcfdsRes. 17: 3645-3653,
1989; and Sompayrac and Danna, Proc. NatL Acad Sci. USA 87: 3274-3278, 1990;
which are ill~,ullJula~cJ by reference herein), protein affinity columns (Oliphant et al.,
MoL CelL BioL 9: 2944-2949, 1989; which is i..~,c,l~Ju.a~cJ by reference herein), gel
mobility shifts (131ackwell and Weintraub, Science 250: 1104-1110, 1990; which is
10 , ' by reference herein), and Suulh~._Jh.ll blots (Keller and Maniatis, NUGAcidsRes. 17:4675-4680, 1991; which is ul~,ul~Ju~a~cJ by reference herein).
One, -I-o~ 1 of the present invention involves the UUll:>tlU~liU.. of inter-
species hybrid NeuroD proteins to facilitate structure-function analyses or to alter
NeuroD activity by increasing or decreasing the i . ' activation of
15 neurogenic genes by NeuroD relative to the wild-type NeuroD. Hybrid proteins of
the present invention may contain the l.r' of one or more contiguous amino
acids ofthe nativeNeuroD with the analogous amino acid(s) of NeuroD from anotherspecies. Such . - hybrid proteins include hybrids having whole or partial
domain l~ t~ As would be evident to one skilled in the art, such hybrid
20 proteins may be obtained using ., ' DNA techniques. Briefiy, DNA
molecules encoding the hybrid NeuroD proteins of interest are prepared using
generally available methods such as PCR . ~ d..cut~
arld/or restriction digestion and ligation. The hybrid DNA is then inserted intoexpression vectors and i ~ ' or transfected irlto suitable host cells. The
25 biological activity may be assessed essentially as described in the assays set forth in
more detail in the Examples that follow.
The invention also provides synthetic peptides, ' ~!~ derived
peptides, fusion proteins, and the like. The subject peptides have an amulo acidsequence encoded by a nucleic acid which hybridizes under stringent conditions with
30 an .,l~g,. l~l;~. of 15 or more contiguous mlrl~rti~l~e of SEQ ~) NO:1, SEQ ID
NO:3, SEQ lD NO:8, or SEQ ID NO:10. R~ ;ve amino acid sequences of
the subject peptides are disclosed in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:9,
andSEQlDNO:11. Thesubjectpeptidesfindavarietyofuses,includingpreparation
of specific antibodies.
As noted above, the invention provides antibodies which bind to NeuroD.
The production of non-human antisera or ' ' antibodies (e.g., murine,
wo ss~30693 2 1 8 8 4 5 0 P~ /41
_ .' porcine, equine) is well known and may be ~ , ' ' ' by, for
example, ~ an animal with NeuroD protein or peptides. For the production
of ' ' antibodies, antibody producing cells are obtained from imr~unized
ar,imals, " ' and screened, or screened fir$ for the production of the
- 5 antibody that binds to the NeuroD protein or peptides and then iI~IIIIUI i 1' ~ It may
be desirable to transfer the antigen binding regions (i.e., F(ab')2 or ~.J~ ~ial~
regions) of non-human antibodies into the framework of a human antibody by
1~ ' DNA techniques to produce a ' "~, human molecule. Methods
for producing such "' "' molecules are generally well known and described in,
lû for example, U.S. Patent No. 4,816,397; which is . I by reference herein in
its entirety. Alternatively, a human ' ' antibody or portions thereof may be
identified by first screening a human B-cell cDNA library for DNA molecules thatencode antibodies that specifically bind to NeuroD according to the method generally
set forth by E~use et al. (Science 246: 1275-1281, 1989, which is illwl~ul~t~ d by
reference herein in its entirety). The DNA molecule may then be cloned and amplified
to obtain sequences that encode the antibody (or binding domain) of the desired
specificity.
The invention also provides methods for inducing the expression of genes
associated with neuronal phenotype in a cell that does not normally express those
genes. Examples of neuronal phenotypes that may be modulated by NeuroD
expression include expression of ~ or ~ ' ' y factors. Cells
that can be used for the purpose of ~ n of gene expression by NeuroD include
cells of the l.~,~llU-- I.,A~ .,,,.1 Iineage, glial cells, neural crest cells, and epidermal
epithelial basal stem cells, and all types of both ' ' and . I ' ' lineage
cells.
As illustrated in Example 10, the expression ûf NeuroD protein in stem cells
causes redirection of epidermal cell ' ~ and induces terminal !"~ ~ '' "
into neurons, i.e., instead of epidermal cells. Epithelial basal stem cells (i.e., in skin
and mucosal tissues) are one of the few . '~ Iry.. ~ cell types in an
adult mammal. Lltlu~,~iùl~ of the subject nucleotide sequences into an epithelial
basal stem cell may be ~- r-"~ )n vi~ro or in vivo using a suitable gene therapyvector delivery system (e.g., a retroviral vector), a Illl~lUlllj~LiUII technique (see, for
example, Tam, Basic Life Sciences 37: 187-194, 1986, which is . ' by
reference herein in its entirety), or a l~ ~fi~ method (e.g., naked or liposome
. . ' ' DNA or RNA) (see, for example, Trends in Gene~ics 5: 138, 1989;
Chen and Okayama, ~~'~ ' ., 6: 632-638, 1988; Mannino and Gould-Fogerite,
WO 95/30693 2 1 8 ~ ~ 5 ~ PCTIUS95105741
-12-
r. -~. , 6: 682-690, 1988; l~ojima et al., Biochem. Biophys. Res. Comm. 207:
8-12, 199'i; which are illl,ul~Julat~,l by reference herein in their entirety). The
illLIuJu~,Liun method may be chosen to achieve a transient expression of NeuroD in
the host cell, or it may be preferable to achieve .,u.... iLuLiv~ or regulated expression in
'i a tissue specific manner.
TrPn~fnrn~ host celis of the present invention find a variety of in v~tro uses;
for =ple: i) as convenient sources of neuronal growth factors, u) in transient and
continuous cultures for screening anti-cancer drugs capable of driving terminal
~' ~ ~.ti...io,l in neural tumors, and ui) as sources of ' ~y expressed
10 NeuroD protein for use as an antigen in preparing .~ and polyclonal
antibodies usefui in diagnostic assays.
TrPn~fnrrn.orl host cells of the present invention also find a variety of in viwuses, for example, for i , ' at sites of traumatic neural injury where motor
or sensory neural activity has been lost. R~ patient ~ that may
1'i benefit from i . ' include: patients with hearing or vision loss due to optical
or auditory nene damage, patients with peripheral nerve damage and loss or motor or
sensory neural activity, and patients with brain or spinal cord damage from traumatic
injury. For example, donor celis from a patient such as epithelial basai stem celis are
cultured in vitro and then i " ' or transduced with a neuroD nucleotide
20 sequence. The i ~ ' celis are then returned to the patient by , ; at
the site of neurai ~ ~
R~ ;ve uses of the nucleotide sequences of the invention include the
foliowing:
1. Construction of cDNA and ~ li, ' ' probes usefui in Northern,
25 Southern, and dot-blot assays for identifying and quantifying the level of expression of
neuroD in a cell. High level expression of neuroD in ~ lu...lu~ tumors and in
rapidly ~lu~ g regions of embryonic neurai Ju~.lu~ (see below) indicates
that measuring the level of neuroD expression may provide prognostic markers forassessing the growth rate and ~ of a neurai turnor. In addition,; ' ~
30 the important role of NeuroD in embryonic d~ r ' it is thought highiy likely
that birth defects and abortions may result from expression of an abnorrnai NeuroD
protein. In this cæe, NeuroD may prove highiy useful in prenatai screening of
mothers and/or for ~n utero testing of fetuses.
2. Cnn~n~ nn of ~ - -- cell lines, ova, and transgenic embryos
35 and animais including ' .._~.Liv~ and "knock-out" Ir~ celi lines in
which the l r ~ - reguiatory activity of NeuroD protein is down-reguiated or
.
21 8~45~
wossl306s3 E~l/~
-13-
eiiminated. Such ceiis may contain aitered neuroD coding sequences that result in the
expression of a NeuroD protein that is not capable of enhancing, ~u~ or
activating ~ of the target gene. The subject ceii iines amd animais find uses
in screening for candidate therapeutic agents capable of either ~ for a
5 function performed by NeuroD or correcting the celiular defect caused by a defective
NeuroD. C~ ' _ the important reguiatory role of NeuroD in embryonic
d~ . birth defects may occur from expression of mutant NeuroD proteins,
and tnese defects may be correctable in ufero or in early post-natai i:ife tilrough the
use of c- ~ identified in screening assays using NeuroD. In addition, neuroD
10 pul~ ,ut;J~ moleculesmaybejoinedtoreportergenes,suchas ~B-grl~ 1~ - or
luciferase, and inserted into the genome of a suitable embryonic host ceii such as an
mouse embryonic stem ceii by, for exampie, l ~' _ ~ ' (for review,
see Capecchi, Trends in Genefics 5: 70-76, 1989; wilich is; ~,u ... ~ -1 by reference).
Cells expressing NeuroD may then be obtained by subjecting the ~i;lf~
15 embryonic cells to ceii sorting, leading to the l r '- of a population of
neuroblasts. ~l~,ulubi~a may be useful for studying neuroblast sensitivity to growth
factors or ~' ' agents. The neurobiasts may aiso be used as a source
from which to purify specific protein products or gene transcripts. These products
maybeusedfortheisolationofgrowthfactors,orforthe;~ ~;r 1;----ofceiisurface0 markers tilat can be used to purify stem ceii popuiation from a donor for
. . ..
3. C~ of gene transfer vectors (e.g., retroviral vectors, and the
iike) wherein neuroD is inserted into the coding region of the vector under the control
of a promoter. NeuroD gene therapy may be used to correct traumatic neurai injury
25 that has resulted in loss of motor or sensory neurai function. For these therapies,
gene transfer vectors may either be injected directly at the site of the traumatic injury,
or the vectors may be used to construct i '` ' host celis that are then injected at
the site of the traumatic injury. The resuits disclosed in Example 10 indica~e that
Jll~lU~i'U~liU.. of neuroD induces a non-neuronai cell to become a neuron. This
30 discovery raises for the fust time the possibiiity of using i , ' and/or genetherapy to repair neurai defects resulting from traumatic injury. In addition, the
discovery of neuroD provid the possibiiity of providmg specific gene therapy for the
treatment of certain l~,~ulu~ai disorders such as Alzheimer's disease, ~ _ 's
disease, and Parkinson's disease, in which a population of neurons have been
35 damaged. Two basic methods of neuroD utiiization can be envisioned in this regard.
In one method, neuroD is expressed in existing P--~ of neurons to modulate
_ = = _ _ . _ . . . ... .. . . . _ . . .. ... . _
wo gs/30693 . ~ 1 8 8 ~ 5 a r~ /41
-14-
aspects of their neuronal phenotype (e.g., I..,.llu~ . expression or synapse
targeting) to make the neurons express a factor or phenotype to overcome the
deficiency that contributes to the disease. In this method, ll ' neuroD
sequences are introduced into existing neurons or r-~ neuroD expression is
5 induced. In another method, neuroD is expressed in r,u~ Ullal cells (e.g., glial
cells in the brain or another non-neuronal cell type such as basal epithelial cells) to
irlduce expression of genes that confer a complete or partial neuronal phenotype that
ameliorates aspects of the disease. As an example, Parkinson's disease is caused, at
least in part, by the death of neurons that supply the l.~.~IIUI "" '- ' i' dopamine to the
10 basal ganglia. Increasing the levels of I ,.llUi ' ~m~ c the symptoms of
Parkinson's disease. F~xpression of neuroD in basal ganglia neurons or glial cells rnay
induce aspects of a neuronal phenotype such that the l..,"l~ dopamine is
produced directly in these cells. It may also be possible to express neuroD in donor
cells for i . ' into the affected region, either as syngeneic or allogeneic
4. rl~. of . ' ' ' ., ' neuronal precursor cell
~('1'"~";" from embryonic ectodermal cells, non-neural basal stem cells, and the]ike. F ' '' '- _ cultures of l~o.. Ii,, neuronal cells for use in therapeutic
screening assays has proven to be a difficult task. The isolated ~CI~ G
20 molecules encoding NeuroD of the present invention permit the . '' ' of
primary (or ) cultures of ~Jl uLC~Lill~ embryonic neuronal stem cells under
conditions mimicking those that are ac~ive in ~.'ovi ' . and cancer. The resultant
cell lines flnd uses: i) as sources of novel neural growth factors, ii) in screening assays
for anti-cancer ~ l ' and iii) in assays for identifying novel neuronal growth
25 factors. ~igh level expression of neuroD in the embryorlic optic tectum (see below)
indicates that NeuroD protein may regulate expression of factors trophic for growing
retinal cells. Such cells may be useful sources of growth factors, and may be useful in
screening assays for candidate therapeutic, ~ '
The cell lines and i ,: regulatory factors disclosed herein offer the
30 unique advantage that since they are active very early in embryonic J;ff~ t~Liull
they represent potential switches, e.g., ON~OFF or OFP~ON, controDing
subsequent cell fate. If the switch can be shown to be reversible ~I.e., ON~OFF), the
NeuroD . regulatory factor and neuroD nucleic acids disclosed hereir
provide exciting O,Ul~Ul' '- for restoring lost neural andlor endocrine fiunctions in a
35 subject.
w095/30693 21 8845~ r~ m ,~ /41
-15-
The foliowing amples are offered by way of iilustration and not by way of
limitation.
- EXAMPLE 1
Cnnctn.rtifm of the embryonic stem ceil " 179" cDNA library.
S A continuous murine embryonic stem ceii iine (i.e., the ES ceil Iine) having
mutant E2A (the putative binding partner of myoD) was used as a celi source to
develop a panel of embryonic stem celi tumors. R~ '' ' ES stem ceiis were
(i.e., usmg I ' .~ ' ) wherein both aileles of the
putative myoD binding partner E2A were replaced with i-uc l~,ld-.~l-, marker genes.
ES celis do not make functional E12 or E47 proteins, both of which are E2A gene
products. ES ceiis form ~ tumors in congenic mice (i.e., 129J) that
appear to contain Ic~ c lia~ of many different embryonal ceii types as judged
' ,, ".~ and through the use of RT-PCR gene expression assays. Individual
embryonic stem ceii tumors were induced in male 129J strain mice by
injection of 1 x 107 celis/site. Three weeks later each tumor was harvested and used
to prepare an individual sample of RNAs. Foliowing random priming and second
strand synthesis the ds-cDNAs were selected based on their size on 0.7% agarose gels
and those cDNAs in the range of 400-800 bp were ligated to either Bam HI or Bgi II
linkers. (Lir.tkers were used to minimize the possibiiity that an intemai Bam ~I site in
a cDNA might ill~ tlllly be cut during cloning, leading to an abnomlaily sized or
out-of-frame expression product.) The resultant individual stem cell tumor DNAs
were ". ' 't.~ iigated into the Bam Hl cloning site in the "fl-VP16" 2~ yeast
expression vector. This expression vector, fl-VP16, contains the VP16 activationdomain of Herpes sintpiex vi~us (EISV) located between Xnd m ~ and Eco Rl
25 ~U) sites and under the control of the Su,hu, cereviseae aicohol
d~ nc~. promoter, with ~FU2 and Ampiciiiin-resistance selectable markers.
Insertion of a DNA molecuie of interest into the Xnd m site of the fl-VP16 vector
(i.e., 5' to the VP16 nucleotide sequence), or into a Bam Hl site (i.e., 3' to tile VP16
sequence but 5' to the Eco Rl site), resuits in expression of a VP16 fusion protein
having the protein of interest joined in-frame with VP16. The resultant cDNA iibrary
was termed the "179-library".
EXAI~iPLE 2
T~l. . ,1; 1~. ~' ;. . and cDNA cloning of neYroD.
A two-hybrid yeast screening assay was used essentialiy as described by Fields
and Song (Nature 340:245, 1989) and modified as described herein was used to
screen the 179-iibrary described in Exaînple 1. Yeast two-hybrid screens are
wogs/3~693 ~1 8~8~50 r~ vCv/4l ~
-16-
reviewed as disclosed in Fields and Sternglanz (Trends in Genetics 10: 286-292,
1994). The library was screened for cDNAs that interacted with LexA-Da, a fusionprotein between the Drosophila Da (r _' ' ) bHLH domain and the ~u~ul;~,
LexA-D~A binding domain. 1~,' ' ' LexA binding sites were cloned upstream
5 of two reporter genes, the ~IS3 gene and the ~-y~ gene. The S. cereviseae
str4in L40 containing a plasmid encoding the LexA-Da fusion protein was j
with CsCI gradient-purified fl-VP16-179-cDNA library. T- r ' were
maintained on me&um selecting both plasmids (the LexA-Da plasmid and the cDNA
library plasmid) for 16 hours before being subjected to histidine selection on plates
10 lacking histidine, leucine, ~'Yl r~ , uracil, and Iysine. Clones that were HIS+ were
r~ , 'y assayed for the e~pression of LacZ. To eliminate possible non-
specific cloning artifacts, plasmids from HlS+/LacZ+ were isolated and
r " . ~ into S. cereviseae strain L40 containing a plasmid encoding a LexA-
Lamin fusiûn. Clones that scored positive in the interaction with lamin were
discarded. AL~,U~ , 400 cDNA clones, which ~ t-l 60 different
transcripts, were identified as positive in these assays. Twenty-five percent of the
original clones were ' , ~ shown to be known bHLH genes on the basis of
their reactivity with specific cDNA probes. One cDNA clone encoding a VP16-
fusion protein that interacted with Da but not ]amin was identified as unique bysequence analysis. This clone, initially termed tango, is now referred to as
neuroD.
The unique cDNA identified above, VP16-neuroD, contained an
~UAU1~ I 450 bp insert that spanned the bHLH region. Sequence analysis
showed that the clone contained an insert encoding a complete bHLH amino acid
sequence mûtif that was unique and previously ~, ' Further analysis
suggested that while the cDNA contained conserved residues common to all
members of the bHLH protein family, several residues were unique and made it
distinct from previously identified bHLH proteins. The neuroD cDNA insert was
subcloned as a Bam HI-Not I insert into Bam HI-Not I linear,7ed r~ .: SK+.
The resulting plasmid was designated pSK+ 1-83.
The neuroD insert contained in the VP16-neuroD plasmid was used to re-
probe a mouse cDNA library prepared from mouse embryos at d~ lu~ stage
elO.5. Candidate clones were isolated and sequenced essentially as described above.
Several clones were isolated. One clone, designated pKS+ m7a RX, was deposited at
the American Type Culture Collection, 12301 Parkiawn Drive, Roch ille, MD 20852
USA, on May 6, 1994, under accession number 75768. Plasmid pKS m7a RX
wossl306s3 21 ~45~) r~ /41
-17-
contains 1646 bp of murine neuroD cDNA as an EcoRI-XhoI insert. The amino acid
sequace encoded by the insert begins at amino acid residue +73 and extends to the
carboxy-taminus of the NeuroD protein. The plasmid contains about 855 bp of
NeuroD coding sequence. (ancoding amino acids 73-536).
- 5 None of the mouse cDNAs contained the complete 5' coding sequence. Toobtain the 5' neuroD coding sequence, a mouse strain 129/Sv genomic DNA Gbrary
was screened with the VP16-neuroD plasmid insert (450 bp). Genomic clones were
isolated and sequenced and the sequences were aGgned with the cDNA sequences.
AGgnment of the sequence and ~ . of the genomic 5' coding sequances with
the Xenopus neuroD clone (Example g) conflrmed the 5' neuroD coding sequence.
The complete neuroD coding sequence and deduced amino acid sequence are shown
in SEQ ID NOS:1 and 2.
EXAMPLE 3
NeuroD/neuroD
bHLH proteins share common structural similarities that include a basic region
that binds DNA and an HL~I region involved in protein-protein required
for the formation of I - - ' and l~t~,.. ' complexes. A; . of the
amino acid sequence of the basic region of murine NeuroD (amino acids 102 to 113
of SEQ lD NO:2) with basic regions of otha bHLH proteins revealed that murine
20 NeuroD contained all of the conserved residues ~ ; among this family of
proteins. However, in addition, NeuroD contained several unique residues. These
unique amino acid residues were not found in any other known HLH, making NeuroD
a distinctive new member of the bHLH family. The NARERNR basic region motif in
NeuroD (amino acids 107-113 of SEQ ID NO:2) is also found in the Drosophila AS-
25 C protein, a protein thought to be involved in i~,,.ua~ ,. Similar, but not identical,NARERRR and NERERNR motifs (SEQ ID NOS:5 and 6, ~ ) have been
found in the Drosophila Atonal and MASH ( ' achaete-scute homolog)
proteins, l~ .t;~ , which are also thought to be involved in n~ Jb_~oh~ The
NARER motif (SEQ ID NO:7) of neuroD is shared by other liHLH proteins, and the
30 Drosophila r! .~ a) and r ~ E proteins. The basic region of bHLH
proteins is important for DNA binding site ll, and there is homology
between NeuroD and other neuro-proteins in this functional region. Wlthin the
important dimer-' g HLH region of NeuroD, a low level of homology was
recorded with mouse twist protein (i.e., 51% homology) and with MASH (i.e., 46%
35 homology). NeuroD contains several regions of unique peptide sequence within the
bHLH domain including the junction sequence (MHG).
w095/30693 2t ~8450 r~ 4l ~
-18-
EXAMPLE 4
NeuroD is expressed in ~ neurons
during embryonic d~
~euroD expression was analyzed during embryonic .1~ , of mouse
5 embryos using in situ ~G,;.Ii~liu.~ with an antisense neuroD single-stranded
riboprobe labeled with ~ 130ehringer r ~ ' ) Brie'dy, a riboprobe was
prepared f+om plasmid pSK+1-83 using T7 pc,l~ . and .I:~s"~ ll-UTP for
labeling. The hybridized probe was detected using anti-~1i,, ,, antibody
conjugated with alkaline ~ Color d~ ~,lu~ was carried out according
10 to the r ' ~ S instruction. Stages of d.,~. lu~ are cornmonly expressed as
days following copulation and where formation of the vaginal plug is eO.5. The
results recorded in the in situ ~.rl,li.li~i.,.. studies were as follows:
In the e9.5 mouse embryo, neuroD expression was observed in the developing
trigerminal ganglia.
In the elO.5 mouse embryo, a distinctive pattern of neuroD expression was
observed in all the cranial ganglia (i.e., V-XI) and in dorsal root ganglia (DRG) in the
trunk region of the embryo. At this time neuroD expression was also observed in the
central nervous system in p ~st ~ cells in the brain and spinal cord t~hat were
,, g neuronal !'-rr In the spinal cord, the ventral portion of the cord
from which the motor neurons arise and I IT~l ~ was observed to express neuroD
at high levels; and expression in the posterior-ventral spinal cord was higher when
compared to more mature anterior-ventral spinal cord.
In the ell.5 mouse embryo, the ganglionic expression pattem of neuroD
observed in elO.5 persisted. Expression in the spinal cord was increased over the
level of expression observed in elO.5 embryos, which is consistent with the presence
of more ~ t~hl~ neurons at this stage. At this stage neuroD expression is also
observed in other sensory organs in which neuronal d~ occurs, for
example, in the nasal epithelium, otic vesicle, and retina of the eye. ln both of these
organs neuroD expression was observed in the region containirlg -rr ~ .- .-
neurons.
In the el4.5 mouse embryo, expression of neuroD was observed in cranial
ganglia and DRG, but expression of neuroD persisted in the neuronal regions of
developing sensory organs and the central nervous system (CNS). Thus, neuroD
expression was observed to be transient during neuronal d~
In surnmary, expression of neuroD in the neurula stage of the embryo (elO), in
the neurogenic derivatives of neural crest cells, the cranial and dorsal root ganglia,
WO 95/30693 2 18 8 ~ 5 ~ r~, ll~J' ~ )/'~J/4l
-19-
and post mitotic cells in the CNS suggests an important possible Gnk between
expression and generation of sensory and motor nerves. Expression occurring later in
embryonic d.,~,lu~ .. in d~t; ~ neurons in the CNS and in sensory organs
(i.e., nasal epithelium and retina) also supports a role in d~ . of the CNS and
5 sensory nervous tissue. Since neuroD expression is transient, the results suggest that
neuroD expression is operative as a switch controlling formation of sensory nervous
tissue. It is ~ , that in these studies neuroD expression was not observed in
embryonic ~ and enteric ganglia (also derived from migrating neural crest
cells). OYerall, the results indicate that neuroD plays an important role in neuronal
EXAMPLE 5
NeuroD is expressed in neural and brain tumor cells:
murine probes identify human neuroD.
Given the expression pattem in mouse embryo (Example 4), Northem blots of
15 tumor cell line rnRNAs were examined using murine neuroD cDNA (Example 2) as a
molecular probe. As a first step, cell lines that have the potential for developing into
neurons were screened. The D283 human - ' " ' ' cell Gne, which expressed
many neuronal markers, expressed high levels of neuroD by Northem blot analysis.NeuroD was also transcribed at various levels by different human .~..l~ ' ' cell20 lines and in certain ~ wulll.~u~ ulll~ lines that are capable of converting to neurons.
Murine PC12 ~ ' ' Ull~ ; cells and P19 ...~.~u.,~, ce~s ~rr . .
into neurons in tissue culture in the presence of ~..u~ t~, inducers, i.e., nenegrowth factor and retinoic acid, .~ . When induced, murine Pl9 but not
PC12 cells expressed neuroD transcripts. However, ~u.. ' ' murine PC12 cells,
25 Pl9 cells, and control 3T3 fibroblasts did not produce detectable levels of neuroD
transcripts. Thus, PC12 and Pl9 cells represent cell types that are potentially useful
m screening assays for identifying inducers of neuroD expression that rnay ~timulate
nerve l~ . and ~ of neural tumor cells.
EXAMPLE 6
3û Rl ' cellsexpressingNeuroD.
R~ ' murine 3T3 fibroblast cells expressing either a myc-tagged
murine NeuroD protein or myc-tagged Xenopus NeuroD protein were made. The
' cells were used as a test system for identifying antibody to NeuroD
described below.
Xenopus NeuroD protein was tagged with the antigenic marker Myc to aUow
the ~ of the specificity of anti-NeuroD antibodies to be d~ '
_ _
WO95/30693 ~i ~84 5~ r~ J ./41 ~
-20-
Plasmid CS2+MT was used to produce the Myc fusion protein. The CS2+MT vector
(Turner and Weintraub, ibid.) contains the simian iyi , ' ~..u~ lE94
(and an SP6 promoter in the 5' ~ ' ' region of the lE94- -
driven transcript to aOow in vitro RNA synthesis) operatively linked to a DNA
5 sequence encoding six copies of the Myc epitope tag (Roth et al, ~ Cen B~ol. 115:
587-596, 1991; which is . ' herein in its entirety), a polylinker for insertion
of coding sequences, and an SV40 late p~ .,' site. CS2-MT was digested
with Xho I to lineanze the plasmid nt the polylinker site du.. ~.. of the DNA
sequence encoding the myc tag. The linearized plasrnid was blunt-ended using
10 Klenow and dNTPs. A full length Xenopus cDNA clone was digested with Xho I and
Eae I and ' ' ~ d using Klenow and dNTPs, and the 1.245 kb fragment of the
Xenopus neuroD cDNA was isolated. The neuroD fraglnent and the linearized vectorwere ligated to form plasrnid CS2+MT x1-83.
CS2+MT was digested with Eco RI to linearize the plasmid at the polylinker
site du ........ , ~ of the DNA sequence encoding the myc tag. The linearized plasmid
was h~ c..~c~ using Klenow and dNTPs and digested with X_o I to obh~in a
linearized plasmid having an Xho I adhesive end and a blunt end. Plasmid pKS+m7aconh~ining a partial murine NeuroD cDNA was digested with Xho I, and the NeuroD
conhlining fragment was ' ' e..~c~ and digested with Xba I to obtain the
~ 1.6 kb fragment of the murine neuroD cDNA. The neuroD fragment
and the linearized vector were ligated to form plasmid CS2+MT Ml-83(m7a).
Plasmids CS2+MT x1-83 and CS2+MT Ml-83(m7a) were each
into murine 3T3 fibroblast ceOs and used as a test system for identifying amhbody
against NeuroD (Example 7).
EXAMPLE 7
Anhbodies to NeuroD.
A .~ ' fiusion protein of maltose binding protein (MBP) and amino
acid residues 70-355 of murine NeuroD was used as an anhgen to evoke anhbodies in
rabbits. Specificity of hhe resultant anhsera was confirmed by _ of hhe
3û ,~ ' 3T3 ceOs described above. Du~l~ _ of the .~ '
ceOs was observed wihh ' ' antibodies to Myc (i.e., hhe conhrol antigenic hag
on the hransfected DNA) and with rabbit anh-murine NeuroD in ' with
anti-rabbit IgG. The specificity of hhe resultant ...~..hle NeuroD sera was
~_~L;~:~t~ filrther by preparing mouse 3T3 fibroblasts cells h~nsfected with differe~t
35 porhons of NeuroD DNA Specificity seemed to map to hhe glutamic acid-rich
dûmain (i.e., amino acid~ 66-73 of SEQ ID NO:2). The anh-murine antisera did not
~ wossl306s3 2 ~ 8~45~ r~ '/41
-21-
react with cells transfected with the myc-tagged Xenopus neuroD. In a similar
marmer, Xenopus NeuroD was used to generate rabbit anti-NeuroD antisera. The
- antisera was ~; ~ L~ , and did not cross react with cells transfected with myc-
tagged murine neuroD.
EXA~LE 8
NeuroD is a highly ~v~llul;u.~il~ conserved prûtein:
sequence of XenopusNeuroD.
A"l.. u~..d~ one miDion clones from a stage 17 Xenopus head library made
by Kintner and Melton (D~ r ' 99: 311, 1987) were screened with the mouse
10 cDNA insert as a probe at low stringency. The h~ GiiUII was performed with
50% r ' ~ Iq X Ssc at 33C and washed with 2 X SSC/0. 1% SDS at 40C.
Positive clones were identified and sequenced. Analysis of the Xenopus
neuroD cDNA sequence (SEQ ID NO.3) revealed that NeuroD is a highly conserved
protein between frog and mouse. The deduced amino acid sequences of frog and
mouse (SEQ ID NOS:2 and 4) show 96% identity in the bHLH domain (50 of 52
amino acids are identical) and 80% identity in the region that is carboxy-terminal to
the bHLH domain (159 of 198 amino acids are identical). The domairl structures of
mu~ine and Xenopus NeuroD are highly I ' ., with an "acidic" N-terrninal
domain (i.e., glutarnic or aspartic acid rich); a basic region; helix 1, loop, helix 2; and
a proline rich C-temlinal region. Although the amino temlinal regions of murine and
Xenopus NeuroD differ in amino acid sequence, both retain a glutamic or aspartic acid
rich "acidic domain" (amino acids 102 to 113 of SEQ ID NO:2 and amino acids 56 to
79 of SEQ ID NO:4). It is highly likely that the acidic domain constitutes an
iYdtiUII" domain for the NeuroD protein, in a manner analogous to the activation' currently l ' - d for other known ~ re~ulatory factors.
EXAMPLE 9
Neuronal expression of Xenopus neuroD.
The expression pattem of neu~oD in whole mount Xenopus embryos was
determined using in situ hj~Dli~i;u~l with a sirlgle stranded ~
Xenopus neuroD antisense cDNA riboprobe. Embryos were examined at several
different stages.
Consistent with the mouse expression pattem, by late stage, aD crarlial ganglia
showed very strong staining patterns. ~ Xenopus, as in other vertebrate organisms,
neural crest cells give rise to skeletal ~ of the head, aD ganglia of the
peripheral nervous system, and pigment ceDs. Among these derivatives, the cranial
sensory ganglia, which are of mixed crest and placode origin, represent the only group
wo 9~130693 -22-
of ceDs that express neuroD. High levels of neuroD expression in the eye were also
obsened, correlating with active neuronal i;L[c-t~L~liu~l in the retina at this stage.
Expression is obsened in the developing olfactory placodes and otic vesicles, as was
seen in mice. The pineai gland also expressed neuroD. AD of this expression in
5 transient, suggesting that neuroD functions during the d;lrcl~ t;d~iu.. process but is
not required for of these 1;~ t;~cd ceD types.
As early as stage 14 (i.e., the ' .._~llUir. stage) neuroD expression was
obsened in the cranial neural crest region where trigerninal gangiia d;~c~
Primary ' y neurons in the spinal cord, also referred to as Rohon-Beard
10 ceDs and primary motor neurons, showed neuroD expression at this stage.
By stage 24, aD of the developing cranial ganglia, i ~ l, facio-scoustic,
glosso-pharyngeal, and vagal nenous tissues showed a high level of neuroD
expression. High levels of expression of neuroD was also obsened in the eye at this
stage. (Note that in Xenopus neuronal "~ c in the retina occurs at a much
15 earlier stage than in mice, and neuroD expression was .,ull~-r " ~ eariier and
stronger in this animai modei.)
In summary, in Xenopus as in mouse, neuroD expression was correiated Witi
sites of neuronai ~' ^` clliid iull. The remarkable ~. ' y ~,u...._.~,~i;u.. of the
pattem of neuroD expression in iiLFc~-t;~lk.o neurons supports the notion that
20 NeuroD has been cvulutiu~ consened botil structuraDy and r ' ~ in these
distant ciasses, which L ~ ~D the criticai role performed by this protein in
~rnbryOlliC sle~ ~ r
EXAMPLE 10
Ectopic expression of neuroD converts
~., , ' ceDsinto neurons.
To further anaiyze the biologicai functions of NeuroD, a gain-of-function
assay was conducted. In this assay, RNA was . _.u...;~ i into one of the two ceDs
in a 2-ceD stage Xenopus ernbryo, and the effects on iater d~,~. ' . of neuronalphenotype was evaluated. For these ~ myc-tagged neuroD transcripts were
30 synthesized in vi~ro using SP6 RNA pGI) The myc tagged-neuroD transcripts
were .. _., , ' into one of the two cells in a Xenopus 2-ceD embryo, and the other
ceD of the embryo sened as an intemai control. Antibodies to Xenopus N-CAM, a
neural adhesion molecule, anti-Myc (to ddect the exogenous protein), and
. techniques were used to evaiuate phenotypic expression of the
35 neuronai marker (and control) gene during the subsequent d~,~lu~ ~i stages ofthe uu~;~dt~i embryos. Remarkably, an evaiuation of over 130 embryos that were
. .
~ WO gs/30693 ~ 1 ~ 8 4 5 0 PCT/US95/057~11
-23-
injected with neuroD RNA showed a strildng increase in ectopic expression of N-
CAM on the llu~,luu~;~.c~d side of the embryo (i.e., Myc~), as judged by increased
~ The increased staining was observed in the region from which neural
crest ceDs normally migrate. It is considered likely that ectopic expression (or over-
- 5 expression) of neuroD caused neural crest stem cells to follow a neurogenic cell fate.
Outside the neural tube, the ectopic ,, was observed in the facio-cranial
region and epidermal layer, and in some cases the stained cells were in the verltral
region of the embryo far from the neural tube. The ' ceDs not only
expressed N-CAM ectopically, but displayed a l..vl. ' ' " ' phenotype of neuronal
10 cells. At high ~ the N-CAM expressing cells exhibited typical neuronal
processes l~ of axonal processes.
To confrm that the ectopic N-CAM expression resulted from a direct effect
on the ~ ..,Ulll~Liv~ epidermal cells and not from aberrant neural cell migration into
the lateral and ventral epidermis, neuroD RNA was injected into the top tier of 32-cell
15 stage embryos, in order to target the injection into cells destined to becomeepidermis. N-CAM staining was observed in the lateral and ventral epidemlis vvithout
any noticeable effect on the .. I~,~" .. ~ nervous system, indicating that the staining
of N-CAM in the epidem~is represents the conversion of epidemmal cell fate into
neuronal cell fate.
Ectopic generation of neurons by neuroD was conflrmed with other neural
specific markers, such as neural-specific class II ~-tubulin ~Richter et al., Proc. NatL
Aca~L Sci. ~A 85: 8066, 1988), acetylated alpha-tubulin ~Pipemo and Fuller, 1 CelL
BioL 101: 2085, 1985), tanabin ~EIemmati-Brinvanlou et al., Neuron 9: 417, 1992),
F)-M (Szaro et al., ~ Comp. NeuroL 273: 344, 1988), and Xen-1,2
Q~iz i Altaba, Dl, lr ' 115: 67, 1992). The embryos were subjected to
' y as described by Tumer and Weintraub (Genes Dev. 8: 1434, 1994,
which is ~~q ' by reference herein) using primary antibodies detected with
aUcaline ~ conjugated goat anti-mouse or anti-rabbit antibodies diluted to
1:2000 (Ro~in~er ~ ' ) Anti ~' ' alpha-tubulin was diluted 1:2000.
Anti-Xen-l was diluted 1:1. Anti-NF-M was diluted 1:2000. Embryos stained for
NF-M were fixed in Dent's fixative (20% " 'Iyls~lL, i~L/8~/O methanol) and
cleared in 2:1 benzyl ~ 1,".~jl alcohol as described by Dent et al.
(Dc~, . 105:61, 1989, which is i~.~ulpulal~d by reference herein). In situ
h~bli~ iul~ of embryos was carried out essentiaUy as described by Harland (in
Methods in Cell Bio~ogJ~, BX. Kay, ~J. Pend, Eds, Academic Press, New York NY,
Vol 36, pp. 675-685, 1991, which is i,,~ullJulal~d by reference herein) as modified by
... ...... . . _ _ _ _ _ ... . .
woss/30693 ~8&45C r~ /41
-24-
Turner and Weintraub (ibid.). In si~u .hJUI; iiL~iUII with ~-tubulin without RNase
treatment can also detect tubulin expression in the ciiiated epidermal ceils. All of
these markers displayed ectopic staining on the neuroD RNA injected side. Injection
of neuroD mRNA into vegetal celis led to no ectopic expression of neural markers5 except in one embryo that showed internal N-CAM staining in the trunk region,
suggesting the absence of cofactors or the presence of inhibitors in vegetal ceils.
However, the one embryo that showed ectopic neurons in the internal organ tissuesuggests that it may be possible to convert non-ectodermal lineage ceiis into neurons
under certain conditions.
The ernbryos were also stained with markers that detect Rohon-Beard ceiis
(cells in which neuroD is normally expressed). T ' ' ~ using the method
described above for Rohon-Beard cell-specif c markers such as HNK-1 (Nu l" '
Dev. Brain Res. 50: 147, 1989, which is il~Cul~ul_:~,;i by reference herein) at a
dilution of 1:1, Islet-l ~Ericson et al., Science 256: 1555, 1992 and Korzh et al.,
15 Dc,. '~ 118: 417, 1993) at a dilution of 1:500, and in sifu hyblidi~liul~ as
described above with shaker-l (Ribera et al., J~ Neurosci. 13: 4988, 1993) showed
more ceiis staining on the injected side of the embryûs.
Tile combined results support the notion that ectopic expression of NeuroD
induced i;~ t~iu.. of neuronai ceils from ceiis that, without neuroD
20 ,; wouid have given rise to l._u.u.. i cells. In summary, these
"r support the notion that ectopic neuroD expression can be used to convert
a non-neuronal cell (i.e., I ' neurai crest cells and epidermal epitheiiai basaistem ceDs) into a neuron. These findings offer for the first time the potentiai for gene
therapy to induce neuron formation in injured neurai tissues.
Interesting .' ', ' ~' " were observed in the ~; '
embryos. In many cases the eye on the .~ ; ' side of the embryo faiied to
deveiop. In other embryos, the spinai cord on the ~ ; ' side of ti~ e embryo
failed to develop properly, and the tissues were strongiy r ~ when stained
with anti-N-CAM. In addition, at the mid-neurula stage many ....~,.u...;~,t~,~i embryos
30 exhibited an increase in ceil mass in the craniai region of the embryo from which (in a
normai embryo) the neurai crest cells and tbeir derivatives (i.e., craniai gangiionic
cells) would migrate. The observed craniai bulge exi~ibited strong
with antibodies specific for N-CAM. These results were interpreted to mean that
."u.~ l~i changes in the eye, neural crest, and spinai cord resulted from
35 premature neurai "~ ~ which aitered the migration of neurai and neurai crest
precursor cells.
~ WO 9S/30693 2 1 8 8 4 5 0 ~ r /41
-25-
NeuroD-injected embryos were also assayed for alteration in the expression of
Xtwist, the Xenopus homolog of Drosophila twist, to determine whether neuroD
- converted non-neuronal ~ r ' of neural crest cells into the neural lineage. Tn
wild-type embryos, Xtwist is strongly expressed in the non-neuronal population
cephalic neural crest cells that give rise to the connective tissue and skeleton of the
head. NeuroD-injected embryos were completely missing Xtwist expression in the
migrating cranial neural crest cells on the injected side. The failure to generate
sufficient cranial ', ' neural crest precursors in neuroD-injected embryos
was also observed ,' ~ , since many of the injected embryos exhibited
poor branchial arch d~,~,' . in the head. r. c, the increased mass of
cells in the cephalic region stained very strongly for N-CAM, ~-tubulin, and Xen-l,
indicating that these cells were neural in character.
The converse periment in which frog embryos were injected with Xtwist
mRNA showed that ectopic expression of Xtwist _ ~ , decreased neuroD
expression on the injected side. Thus, two members of the bHLH family, neuroD and
Xtwist, may compete for defining the identity of different cell types derived from the
neural crest. In the neuroD-injected embryos, exogenous neuroD may induce
~UI~ _ ' y neural crest to ~ into neurons in siiu, and ~ ly they
fail to migrate to their normal positions.
The effect of ud~ ;ul~ of exogenous neuroD on the fate of cells that
normally express neuroD, such as cranial ganglia, eye, otic vesicle, olfactory organs,
and primary neurons, and on other CNS cells that normally do not express neuroD,was determined by staining for d;~ ;a~;ull markers. When the cranial region of the
embryo is severely affected by ectopic neuroD, the injected side of the embryos
displayed either small or no eyes in addition to poorly organized brains, otic vesicles,
and olfactory organs. Moreover, as the embryos grew, the spinal cord showed
retarded growth, remaining thinner and shorter on the neuroD-injected side.
N-CAM staining in the normal embryo at early stages was not uniform
throughout the entire neural plate, but rather was more prominent in the medial region
of the neural plate. Injected embryos analyzed for N-CAM expression show that the
neural plate on the injected side of the early stage embryos was stained more il~tensely
and more laterally. The increase in N-CAM staining was not associated with any
lateral expansion of the neural plate as assayed by visual inspection and staining with
the epidermal marker EpA This was in contrast to what has been observed with
35 XAS~-3 injection that causes neural plate expansion. These ul~ L;u.~ suggest
wo g~l30693 2 1 8 8 ~ ~ 0 r~ 4l ~
-26-
that the first effects of neuroD are to cause neuronal precursors rn the neural plate to
el.~.
To determine whether neuroD caused neuronal precursors to ~
l~u~l~Lu~el~, rnjected embrvos were stained using two neuronal markers that are
5 expressed in '^` Cll~;dt~ neurons, neural specific ~-tubulin and tanabin. In situ
h~hL.d~;ull for ~-tubulin and tanabin was carried out as described above. Over-
expression of neuroD ' "~ increased the ~-tubulin signals in the region of the
neural plate contarn~ng both motor neurons and Rohon-Beard cells at stage 14. The
earliest ectopic ~-tubulin positive cells on the injected side were observed at the end
10 of guaLlul~lL;oll when the control side did not yet show any p-tubulin positive cells.
Tanabin was also expressed in more cells in the spinal cord in the neuroD injected side
of the embryos at stage 14. These results suggest that neuroD can cause premature
!" ~ '' ' ' of the neural precursors rnto d;~cl cllL;dtcl neurons. This is a powerful
i;ndication that, when ectopically expressed or over-expressed, NeuroD can5 d;~.tu..c mitotic cells into non-dividrng mature neurons.
EXAMPLE 1 1
Human genomic NeYroD.
Genoniic clones encodrng human NeuroD were obtained by probing a h=
fibroblast genomic library with the mouse neuroD cDNA Host E. coli strain LE392
2û (New England Biolabs) were grown in LB + 10 rnM MgSO4 0.2% maltose overnight
at 37C. The cells were harvested and Ic...-~w.d~l in 10 rnM MgSO4 to a final
OD600 of 2. The ,~,.~.~ cells were used as hosts for phage infection. The
optrmal volume of phage stock for use in this screer~ing was determined by usingserial dilutions of the phage stock of a human fibroblast genomic library rn lambda
25 ~ ~ t~g~n~) to infect LE392 cells (New Englamd Biolabs). To obtain
SU,OUO plaques per plate, a 2.5 l11 aliquot of the phage stock was used
to infect 600 111 of the .c..~.~...d.,.l LE392 cells. The cells were rncubated with the
phage for 15 minutes at 37C, af~er which the cells were mixed with 6.5 ml of top
~gar warmed to 5ûC. The top agar was plated on solid LB, and incubated overnight
3û at 37C. A total of 22 15-cm plates were prepared in this manner.
Duplicate plaque li~s were prepared. A first set of Hybond membranes
(Amersham) were placed onto the plates and allowed to sit for 2 minutes. The initial
membranes were removed and the duplicate membranes were laid on the plates for 4mir~utes. The membranes were allowed to air dry; then the phage were denatured r~
35 û.5 M NaOX 1.5 M NaCI for 7 minutes. The membranes were neutralized with two
washes in ' buffer (1.5M NaCI, û.5 M Tris, pH 7.2). After
wo gs/30693 -27- r~ J,.. . /41
' the membranes were crossGnked by exposure to W. A I kb Eco RI-
IIind m fragment contau ing murine neuroD coding sequences was random primed
- using the Random Priming Rit ~3oehringer Mannheim) according to the
~.~s r~ ~ were prepared for h.~: ' by placing
S six membr~mes in 10 ml of FBI h~' ' buffer [100 g p~ i glycol 800,
350 ml 20% SDS, 75 ml 20X SSPE; add water to a final volume of one Gter.] and
irlcubating the membranes at 65C for 10 minutes. Af~er 10 minutes, denatured
salmon sperm DNA was added to a final ~ of 10 llglml and denatured
probe was added to a final . of 0.25-0.5 x 107 cpm/ml. The membranes
were hybridized at 65C for a period of 8 hours to overnight. Afler incubation, the
excess probe was removed, and the ' were washed first irl 2 X SSC, 0.1%
SDS for 30 minutes at 50C. The first wash was followed by a final wash in 0.1 XSSC, 0.1% SDS for 30 minutes at 55C. A..l-..;..li~ of the ' were
prepared. The first screen identified 55 putative positive plaques. Thirty-one of the
plaques were subjected to a secondary screen using the method essentially set forth
above. Ten positive clones were identified and subjected to a tertiary screen asdescribed above. Eight positive clones were identified after the tertiary screen.
Phage DNA was prepared from dones 14B1, 9F1, and 20A1. The 14Bl and
20AI phage DNA were digested with Pst I to isolate the 1.2 kb and 1.6 kb fragments,
,. ~.,Li~. I~, that hybridized to the mouse neuroD probe. The 9FI phage DNA was
dGgested with Eco RI and SacI to obtain an a~ V 2.2 kb fragment that
hybridizes with the mouse neuroD probe. The fragments were each subcloned into
plasmid Bluescript SR (Stratagene) that had been Gnearized with the a~
restriction enzyme(s). The fragments were sequenced using Sequenase Version 2.0
from USB (US F- - ' ') and the following primers: the universal primer M13-21,the T',7 primer, and the T3 primer. Sequence analysis of clones 9FI, (SEQ ID NO:8)
and 14BI (SEQ lD NO:I0) showed a high similarity be~ween the mouse and human
coding sequences at both the amino acid and nucleotide level. In addition, whileclones 9FI and 14BI shared 100% identity in the HLH region at the amino acid level
(i.e.,residuesll7-156inSEQlDNO:9andresidues91-130mSEQlDNO:ll),they
diverged in the : ' of the b~I. This finding strongly suggests that
14BI is a member of the NeuroD family of genes. Sequence analysis d ....~ '".t. _
that clone 9FI has a high degree of homology throughout the sequence region thatspans the translation start site to the end of the bHLEI region. The 9FI clone has
100% identity to mouse NeuroD in the HLH region (i.e., residues 117-156 in
SEQIDNO:9 and residues 117-156 in SEQ lD NO:2), and an overall identity of
WOg5/30693 2 ~ 88 4 5~ P~ /4I ~
-28-
94%. The 14Bl clone also has 100% identity to the HLH region ~I.e., residues 91-130 irl SEQ lD NO: l l and residues 117-156 in SEQ lD NO:2), but only 40% identity
to 9Fl and 39% identity to mouse NeuroD in the amino-terminal region. This
~1.. ,~1~.1. - that 9Fl is the human homolog of mouse neuroD, whereas the strong5 Cu~ dtiu.. of the neuroD HLH identifies 14B 1 as another member of the neuroD
HLH subfamily.
EXAMPLE 12
Ch.ullluDull._ mapping of human neuroD clones.
FISH k~uyuL~u~g was performed on fixed metaphase spreads of the microcell
hybrids essentially as described (Trask et al., Am. J. Hum. Genet. 48: 1-15, 1991; arld
Brandriff et al., Genomics 10: 75-82, 1991, which are l l by reference
herein in their entirety). NeuroD sequences were detected using the 9Fl or 20Al
phage DNA as probes labeled using ~ ,r ~, dUTP (R~ Mannheim)
according to the r ' ~I~S ;11~1l~ Phage DNA was 1 ,' ' by
15 random prin~ing (Gibco/BRL BioNlck Kit) and hybridized in situ to derlatured
metaphase .,Iu, spreads for 24-48 hours. Probes were detected with
' ' ~conjugated antibodies to ~l;., .- c,. - and ~', were
' DAPI (Sigma). Signals were viewed through a ~lhu~
UD.,UIJ., and i ' ., . ' were taken with color slide film. FISH analysis
20 indicated done 9Fl maps to human ~,IUU~UDUIII~ 2q, and clone 20Al maps to human
~,lu, 5.
(~ UIIIUDUII~ mapping was also carried out on a Ilull~Jl~ ' somatic cell
hybrid panel (National Institute of General Medical Sciences; Camden NJ). This
panel consists of DNA isolated from 24 1 l~u~ somatic cell hybrids retaining
25 one human clu~ For one set of ~.. the panel of DNA's were
digested with Eco RI and elC_LI~ d on an agarose gel. The DNA was
transferred to Hybond-N membranes (Amersham). A random primed (Boehringer
M~nnheim) 4 kb Eco RI-Sac I fragment of clone 9Fl was prepared. The filter was
~u}~ lid~d in 10 rnl of FBI h~.id;~Liu.. buffer (see above) at 65C for 10 minutes.
After ~ denatured salmon sperm DNA was added to a final
of 10 llg/ml; denatured probe was added to a final ~ of one
million cpm/rnl. The filter was hybridized at 65C for a period of 8 hours to
overrlight. After incubatio4 excess probe was removed, and the filter was washedf~rst in 2 X SSC, 0.1% SDS for 30 minutes at 65C. The first wash was followed by a
final wash in 0.1 X SSC, 0.1% SDS for 30 minutes at 65C. An ~ y,.~ of
the filter was prepared. A..l ... ,.~lif.~ ~IIID confirmed the FISH mapping results.
~ WO 95130693 21 8 8 4 5 0 PCT/IJS95/05741
-29-
In the second . r t, the panel was digested with Pst I, ~Ic_llu~ v.~ d
and transferred essentially as described above. A random-primed (Boehringer
Mannheim) 1.6 kb Pst I fragment of clone 20Al was prepared. The membrane was
, hybridized with the 20Al probe and washed as described above.
5 .~ .. A.~ .h - of the Southern showed that 20Al mapped to human ' UIIIUDUIIlC 5
and confirmed the FISH mapping results. After A~ h~, the 20Al-probed
membrane was stripped by a wash in 0.5 M NaOH, 1.5 M NaCI. The membrane was
neutralized in 0.5 M Tris-HCI (pH 7.4), 1.5 M NaCI. The filter was washed in 0.1 X
SSC before ylellyl,lil~tiull. A random-primed (Boehringer r r ' ) 1.2 kb Pst I
10 fragment of clone 14BI was prepared. The washed membrane was ~ }~ and
hybridized with the 14BI probe as described above. After washing under the
previously described conditions, the membrane was ~ ' " .' '
A.,~ d ~et' that clone 14Bl mapped to eh,~ 17.
EXAMPLE 13
HumanneuroD .' yDNA
To obtain a human neuroD cDNA, one million plaque forming units (pfu)
were plated onto twenty LB + 10 mM MgSO" (150 mm) plates using the bacterial
Dtrain XL-I Blue (Stratagene). Plating and membrane lifts were performed using
standard methods, as described in Example 11. After IJV cross-linl~mg, the
' were prc hj~ in an aqueous }.~. " solutiûn (1% bovine
serum albumin, I mM EDTA, 0.5 M Na2HPO" (pH 7.4), 7% SDS) at 50C for twû
hûurs.
The neuroD cDNA insert was prepared by digesting the pKS+ m7a RX
plasmid with Eco RI and Xho I, and isolating the fragment containing the cDNA by25 ~,l~i~u~,h~liul~. A probe was made with the cDNA containing fragment by random
primed synthesis with random ' ' ' , dGTP,dATP, dTTP, alpha-32P-labeled
dCTP, and Klenow in a buffered solution (25 mM Tris (pH6.9), 50mM KCL 5mM
MgCI2, lmM DTT). The probe was purified from the I , . ' nucleotides on
a G-50 sepharose column. The purified prûbe was heat denatured at 90C for 3
30 minutes.
After ~ h~hli~iù~l~ the denatured probe was added to the membranes in
~Jbli~ iull solution. The membranes were hybridized for 24 hours at 50C. Excessprobe was removed from the mPn.h~rp,.Pc and the membranes were washed in 0.1 X
SSC, 0.1% SDS for 20 minutes at 50C. The wash solution was changed five times.
35 The membrsnes were blotted dry and covered with plastic film before being subjected
w09s/30~93 21 88450 r~l,u~,~c~,4l ~
-30-
to u~ s~ ..1 h~ y of the filters identified 68 positive dones. The
clones are plaque-purified and rescreened to obtain pure, positive clones.
The plasmid vector containing cDNA insert was excised in vivo from the
lambda phage clone according to the Strategene . ~ hlhvJ. Briefiy, eluted phage
arld XL-I Blue cells (200 microliters of OD 600=1) were mixed with R408 helper
phage provided by Strategene for 15 rninutes at 37C. Five milliliters of rich bacterial
growth media (2 X YT, see Sambrook et al., ibid.) was added, and the cultures were
incubated for 3 hours at 37C. The tubes were heated at 70C for 20 minutes and
spun for 5 minutes at 4,000 X g. After c~ u~ 200 microliters of supemant
was added to the s~me volume of XL-I Blue cells (OD=1), and the mixture was
irlcubated for 15 rninutes at 37C, after which the bacterial cells were plated onto LB
plates containing 50 llg/ml ampicillin. Each colony was picked and grown for
sequencing template preparation. The clones are sequenced and compared to the
hurnan genornic sequence.
From the foregoing it will be ~ that, although specific ~
of the invention have been described herein for purposes of illustration, various
~'~ may be rnade without deviating from the spirit and scope of the
invention.
21 88450
WO95/30693 r~.l,o~ _ I /41
-31-
SEQUENCE LISTING
(1) GENER~L INFORMATION:
(i) APPLICANT: Weintraub, Harold
Lee, J?-T'''l In~ E.
Tapscott, StepheD J.
!lollenberg, Stanley M.
(ii) TITLE OF INVENTION: Neurog~nic n; ff~r~.nt; ~ti ~n (NeuroD)~ne
and Protein
(iii) NUMBER OF SEQUENCES: 11
(iv) ~.U~ NlJ~ ADDRESS:
(A) ADDRESSEE: Christensen O'Connor Johnson Kindness
(B) STREET: 1420 Fifth Avenue, Suite 2800
(C) CITY: Seattle
(D) STATE: WA
( E ) COUNTRY: USA
(F) ZIP: 98101-2347
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy digk
(B) COMPUTER: IBM PC t;hl~
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOE~WARE: P~tentIn Rele~e #1.0, Versi~n #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY~AGENT INFORMATION:
(A) NAME: Rro~ r; rl~, Thomas F.
(B) ~ ~AllON NUMBER: 31,332
(C) REFERENCE/DOCKET NUM3ER: FHCR-1-8504
(iX) TFT,-~ INFORMATION:
(A) TELEPHONE: 206-682-8100
(B) TELEFAX: 206-225-0709
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE r~ rTT:~t~TICS:
(A) LENGT~: 2089 base pairs
(B) TYPE: nucleic ~cid
(C) STRhNDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
_ _ _ _ _ _ .
WO95/30693 21 88450 r~u~ 4l ~
-32-
( ix ) FEATURE:
~A) NAME/REY: CDS
~S) LOCATION: 229..1302
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
ACTACGCAGC ACCGAGGTAC AGACACGCCA GCATGAAGCA CTGCGTTTAA ~ .G
AGGCATCCAT TTTGCAGTGG ACTCCTGTGT ATTTCTATTT bl~.,~I~l CTGTAGGATT
120
ACCCACI~CCC AGCTGAAGGC TTATCCAGCT TTTA~ATATA GCGGGTGGAT ~ u~,e~.
180
,l b~ , A~ACAGGA~ GTGGAaAC ATG ACC AAA
237
Met Thr Lys
TCA TAC AGC GAG AGC GGG CTG ATG GGC GAG CCT CAG CCC CAA GGT CCC
285
Ser Tyr Ser Glu Ser Gly Leu Met Gly Glu Pro Gln Pro Gln Gly Pro
5 10 lS
CCA AGC TGG ACA GAT GAG TGT CTC AGT TCT CAG GAC GAG GAA CAC GAG
333
Pro Ser Trp Thr Asp Glu Cya Leu Ser Ser Gln Aap Glu Glu h~is Glu
20 25 30 35
GCA GAC AAG A~A GAG GAC GAG CTT GAA GCC ATG aAT GCA GAG GAG GAC
391la A~p Ly~ Lys Glu Asp Glu Leu Glu Ala Met Asn Ala Glu Glu Asp
40 45 50
TCT CTG AGA AAC GGG GGA GAG GAG GAG GAG GAA GAT GAG GAT CTA GAG
429er L~u Arg Aln Gly Gly Glu Glu Glu Glu Glu Aap Glu Asp Leu Glu
55 60 65
GAA GAG GAG GAA GAA GAA GAG GAG GAG GAG GAT CAA A~G CCC AAG AGA
477lu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Gln Lys Pro Lys Arg
70 75 80
CGG GGT CCC A~A AAG AAA AAG ATG ACC AAG ~-r~ f~ GAA CGT TTT
525
Arg Gly Pro Lys Lys Ly~ Lya Met Thr Lys Ala Arg Leu Glu Arg Phe
WO 95/30693 _33 r~ t~,/41
A~A TTA AGG CGC ATG A~G GCC APC GCC CGC GAG CGG AAC CGC ATG CAC
573
Lys Leu Arg Arg Met Lys Ala Asn Ala Arg Glu Arg Asn Arg Met His
- 100 105 110 115
GGG CTG AAC GCG GCG CTG GAC AAC CTG CGC A~G GTG GTA CCT TGC TAC
621
Gly Leu Asn Ala Ala Leu Asp Asn Leu Arg Lys Vai Val Pro Cys Tyr
120 125 130
TCC AAG ACC CAG A~A CTG TCT A~A ATA GAG ACA CTG CGC TTG GCC A~G
669
Ser Lys Thr Gln Lys Leu Ser Lys Ile Glu Thr Leu Arg Leu Ala Lys
135 140 145
AAC TAC ATC TGG GCT CTG TQ GAG ATC CTG CGC TCA GGC A~A AGC CCT
717
Asn Tyr Ile Trp Ala Leu Ser Glu Ile Leu Arg Ser Gly Lys Ser Pro
150 155 160
GAT CTG GTC TCC TTC GTA CAG ACG CTC TGC A~A GGT TTG TCC CAG CCC
765
Asp Leu Val S Phe Val Gln Thr Leu Cys Lys Gly Leu Ser Gln Pro
165 170 175
ACT ACC A~T TTG GTC GCC GGC TGC CTG CAG CTC A~C CCT CGG ACT TTC
813
Thr Thr Asn Leu Val Ala Gly Cys Leu Gln L~u Asn Pro Arg Thr Phe
180 185 190 195
TTG CCT GAG CAG A~C CCG GAC ATG CCC CCG CAT CTG CCA ACC GCC AGC
861
Leu Pro Glu Gln Asn Pro Asp Met Pro Pro His Leu Pro Thr Ala Ser
200 205 210
GCT TCC TTC CCG GTG CAT CCC TAC TCC TAC CAG TCC CCT GGA CTG CCC
909
Ala Ser Phe Pro Val Hi~ Pro Tyr Ser Tyr Gln Ser Pro Gly Leu Pro
215 220 225
AGC CCG CCC TAC GGC ACC ATG GAC AGC TCC CAC GTC TTC C~C GTC A~G
957
Ser Pro Pro Tyr Gly Thr Met Asp Ser Ser Hi~ Val Phe His Val Ly~
230 235 240
CCG CCG CCA CAC GCC TAC AGC GCA GCT CTG GAG CCC TTC TTT GAA AGC
1005
Pro Pro Pro His Ald Tyr Ser Ala Ala Leu Glu Pro Phe Phe Glu Ser
245 250 255
CCC CTA ACT GAC TGC ACC AGC CCT TCC TTT GAC GGA CCC CTC AGC CCG
1053
Pro Leu Thr Asp Cys Thr Ser Pro Ser Phe ~sp Gly Pro Leu Ser Pro
260 265 270 275
WO 9S/30693 21 ~ 8 4 5 ~ : PCT/~JS95/05741
-34-
CCG CTC AGC ATC AAT GGC AAC TTC TCT TTC AAA CAC GAA CCA TCC GCC
1101ro Leu Ser Ile Asn Gly Asn Phe Ser Phe Lys His Glu Pro Ser Ala
280 285 290
GAG TTT GAA A~A AAT TAT GCC TTT ACC ATG CAC TAC CCT GCA GCG ACG
1149lu Phe Glu Lys A~n Tyr Ala Phe Thr Met His Tyr Pro Ala Ala Thr
295 300 305
CTG GCA GGG CCC CAA AGC CAC GGA TCA ATC TTC TCT TCC GGT GCC GCT
1197
L~u Al~ Gly Pro Gln Ser Hia Gly Ser Ile Phe Ser Ser Gly Ala Ala
310 315 320
GCC CCT CGC TGC GAG ATC CCC ATA GAC AAC ATT ATG TCT T,TC GAT AGC
1245
Ala Pro Arg Cys Glu Ile Pro Ile ASp Asn Ile Met Ser Phe A~p Ser
325 330 335
CAT TCG CAT CAT GAG CGA GTC ATG AGT GCC CAG CTT AAT GCC ATC TTT
1293
Hi~ Ser Hi~ Hi~ Glu Arg Val Met Ser Ala Gln Leu Asn Ala Ile Phe
340 345 350 355
CAC GAT TAGAGGGCAC GTCAGTTTCA ~,lA~ , GA~ACGAATC CACTGTGCGT
1349
Hi~ Asp
ACAGTGACTG TCCTGTTTAC AGAAGGCAGC C~ ,blA AGATTGCTGC A~AGTGCAhA
1409
TACTCA~AGC TTCAAGTGAT ATATGTATTT All~ C A~;AAACAGG
14 69
GGATCA~AGT TCCTGTTCAC CTTATGTATT bl 1 1 1 ~ lA GCTCTTCTAT TTTAMAATA
1529
ATAATACAGT A~AGTAMA~ AGAMATGTG TACCACGAAT TTCGTGTAGC TGTATTCAGA
1589
TCGTATTAAT TATCTGATCG GGATAMAAA AATQCAAGC AATAATTAGG ATCTATGCAA
1649
TTTTTA~ACT AGTAATGGGC CAATTAPIAAT ATATATAAAT ATATATTTTT CAACCAGCAT
1709
TTTACTACCT GTGACCTTTC CCATGCTGAA TTATTTTGTT ~.lW llll~.l ACAGAATTTT
1769
TAATGACTTT TTATAACGTG GATTTCCTAT TTTAMACQ TGCAGCTTCA TCAATTTTTA
1829
WO 95130693 _35_ P_llu~,,~ /41
TACATATCAG A~AAGTAGAA TTATATCTAA TTTATACAAA ATAATTTAAC TAATTTA~AC
1889
CAGCAGA~AA GTGCTTAGAA AGTTATTGCG TTGCCTTAGC ACTTCTTTCT TCTCTAATTG
1949
TAAAAAAGAA AZ~A7~ A~AAAACTCG ~ rCir~C CGGTACCCAG ~ ~U
2009
CTTTAGTGAG GGTTAATTGC 6-.b~ b-.b TAATCATGGT CATAGCTGTT l ~ l .. A
2069
ATTGTTATCC GCTCACAATT
2089
(2) INFORMAT}ON FOR SEQ ID NO:2:
(i) SEQUENCE CEARACTERISTICS:
(A) LENGT~i: 357 amino acid~
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE L~ U~llUN: SEQ ID NO:2:
et Thr Lys Ser Tyr Ser Glu Ser Gly Leu Met Gly Glu Pro Gln Pro
5 10 15
ln Gly Pro Pro Ser Trp Thr Asp Glu Cy- Leu Ser Ser Gln Asp Glu
20 25 30
lu h'i~ Glu Ala Asp Ly~ Ly~ Glu Asp Glu Leu Glu Ala Met Asn Ala
35 40 45
Glu Glu Asp Ser Leu Arg Asn Gly Gly Glu Glu Glu Glu Glu Asp Glu
50 55 60
Asp Leu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Gln Ly3
65 70 75 80
ro Ly~ Arg Arg Gly Pro Lys Lys Ly~ Lys Met Thr Lys Ala Arg Leu
85 90 95
lu Arg Phe Lys Leu Arg Arg Met Lys Ala Asn Ala Arg Glu Arg Asn
100 105 110
Arg Met his Gly Leu Asn Ala Ala Leu Asp Asn Leu Arg Lys Val Val
115 120 125
Pro Cys Tyr Ser Lys Thr Gln Lys Leu Ser Lys Ile Glu Thr Leu Arg
130 135 140
_
W095/30693 2~1 8845~ r~ /41
-36-
, . 1,
Leu Ala Lys Asn Tyr Ile Trp Ala Leu Ser Glu Ile Leu Arg Ser Gly
145 150 155 160
y~ Ser Pro Asp Leu Val Ser Phe Val Gln Thr Leu Cys Lys Gly Leu
165 170 175
er Gln Pro Thr Thr Asn Leu Val Ala Gly Cys Leu Gln Leu Asn Pro
180 185 190
Arg Thr Phe Leu Pro Glu Gln Asn Pro Asp Met Pro Pro His Leu Pro
195 200 205
Thr Ala Ser Ala Ser Phe Pro Val His Pro Tyr Ser Tyr Gln Ser Pro
210 215 Z20
Gly Leu Pro Ser Pro Pro Tyr Gly Thr Met Asp Ser Ser His Val Phe
225 230 235 240
is Val Lys Pro Pro Pro His Ala Tyr Ser Ala Ala Leu Glu Pro Phe
245 250 255
he Glu Ser Pro Leu Thr Asp Cys Thr Ser Pro Ser Phe Asp Gly Pro
260 265 270
Leu Ser Pro Pro Leu Ser Ile Asn Gly A5n Phe Ser Phe Lys His Glu
275 280 285
Pro Ser Ala Glu Phe Glu Lys Asn Tyr Ala Phe Thr Met His T Pro
290 295 300 yr
Ala Ala Thr Leu Ala Gly Pro Gln Ser His Gly Ser Ile Phe Ser Ser
305 310 315 320
ly Ala Ala Ala Pro Arg Cys Glu Ile Pro Ile Asp Asn Ile Met Ser
325 330 335
he Asp Ser His Ser His His Glu Arg Val Met Ser Ala Gln Leu A~n
340 345 350
Ala Ile Phe His Asp
355
2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHA~ACTERISTICS:
(A) LENGTH: 1275 base pairs
(B) TYPE: nucleic ~cid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
~ W095130693 21 8845~ r~ 5~c~
-37-
~vi) ORIGINAL SOURCE:
(A) ORGANISM: Xenopus laeviJ
- (ix) FEATURE:
(A) NAME/KEY: CDS
(~3) LOCATION: 25. .1083
(xi) SEQUENCE L~ ,nl~lUN: SEQ ID NO:3:
,u~ . TCCAGATCTA AAAA ATG ACC A~A TCG TAT GGA GAG AAT GGG
51
Met Thr Lys Ser Tyr Gly Glu Asn Gly
CTG ATC CTG GCC GAG ACT CCG GGC TGC AGA GGA TGG GTG GAC GAA TGC
99
Leu Ile Leu Ala Glu Thr Pro Gly Cys Arg Gly Trp Val Asp Glu Cys
10 15 20 25
CTG AGT TCT CAG GAT GAA AAC GAT CTG GAG AF~A AAG GAG GGA GAG TTG
147
Leu Ser Ser Gln Asp Glu Asn Asp Leu Glu LYJ Ly3 Glu Gly Glu Leu
30 35 40
ATG A~A GAA GAC GAT GAA GAC TCA CTG AAT C~T CAC AAT GGA GAG GAG
195
Met Lys Glu Asp Asp Glu Asp Ser Leu Asn Lis HiJ Asn Gly Glu Glu
45 50 55
AAC GAG GAA GAG GAT GAA GGG GAT GAG GAG GAG GAG GAC GAT GAA GAT
243
Asn Glu Glu Glu Asp Glu Gly Asp Glu Glu Glu Glu Asp Asp Glu Asp
60 65 70
GAT GAT GAG GAT GAC GAC CAG A~A CCC A~A AGG CGA GGA CCG A~A AAG
291
A~p Asp Glu Asp Asp Asp Gln Lys Pro Lys Arg Arg Gly Pro Lys LyJ
75 80 85
A~A A~A ATG ACG A~A GCC CGG GTG GAG CGA TTT AAA GTG AGA CGC ATG
339
Ly~ Lys Met Thr Lys Ala Arg Val Glu Arg Phe Lys Val Arg Arg Met
go 95 100 105
AAG GCA AAC GCC AGG GAG AGG AAT CGC ATG CAC GGA CTC AAC GAT GCC
- 387
Lys Ala Asn Ala Arg Glu Arg Asn Arg Met Elis Gly Leu Asn Asp Ala
110 115 120
CTG GAC AGT CTG CGC AAA GTT GTG CCC TGC TAC TCC AAA ACA CAA AAG
435
Leu Asp Ser Leu Arg Lys Val Val Pro CyJ Tyr Ser Lys Thr Gln Ly~
125 130 135
W0 95130693 ~18 ~ 4 5 ~ r~l,u~ ~J~4l ~
-38-
TTG TCT A~G ATT GAA ACT CTG CGC CTG GCT AAG AAC TAC ATC TGG GCT
483
L~u Ser Lys Ile Glu Thr Leu Arg Leu Ala Lys Asn Tyr Ile Trp Ala
140 145 lS0
CTT TCT GAG ATT TTA AGG TCC GGC A~A AGC CCA GAC CTG GTG TCC TTT
531
Leu Ser Glu Ile Leu Arg Ser Gly Lys Ser Pro Asp Leu Val Ser Phe
lSS 160 165
GTA CAA ACT CTC TGC A~A GGT TTG TCG l ~G CCC ACC ACC AAT CTA GTA
579
Val Gln Thr Leu Cys Lys Gly Leu Ser Gln Pro Thr Thr Asn Leu Val
170 175 180 185
GCG GGG TGT CTG CAG CTG AAC CCC AGA ACT TTC CTT CCT GAG CAG AGT
627l~ Gly Cys Leu Gln Leu Asn Pro Arg Thr Phe Leu Pro Glu Gln Ser
190 195 200
CAG GAC ATC CAG TCG CAC ATG CAA ACA GCG AGC TCT TCC TTC CCT CTG
6~5ln Aap Ile Gln Ser His Met Gln Thr Ala Ser Ser Ser Phe Pro Leu
205 210 215
C~G GGC TAT CCC TAT CAG TCC CCT GGT CTT CCC AGT CCC CCC TAT GGT
723
Gln Gly Tyr Pro Tyr Gln Ser Pro Gly Leu Pro Ser Pro Pro Tyr Gly
220 225 230
ACC ATG GAC AGC TCC CAT GTA TTC CAC GTC AAG CCT CAC TCC TAT GGG
771
Thr Met Asp Ser Ser His Val Phe His Val Lys Pro His Ser Tyr Gly
235 240 245
GCG GCC CTG GAG CCT TTC TTT GAC AGC AGC ACC GTC ACT GAG TGT ACC
819
Al~ Al~ Leu Glu Pro Phe Phe Asp Ser Ser Thr Val Thr Glu Cys Thr
250 255 260 265
AGC CCG TCA TTC GAT GGT CCC CTG AGC CCA CCC CTT AGT GTT AAT GGG
867er Pro Ser Phe Asp Gly Pro Leu Ser Pro Pro Leu Ser Val Asn Gly
270 275 280
AAC TTT ACT TI~T APA CAC GAG CAT TCG GAG TAT GAT A~A AAT TAC ACG
915~n Phe Thr Phe Ly~ Glu His Ser Glu Tyr Asp Lys A~n Tyr Thr
285 290 295
TTC ACT ATG CAC TAT CCT GCA GCC ACT ATA TCC CAG GGC CAC GGA CCA
963
Phe Thr Met His Tyr Pro Ala Ala Thr Ile Ser Gln Gly His Gly Pro
WO 95/30693 2 1 ~ ~ 4 5 0 PCI/US9~/05741
-39-
300 305 . 310
TTG TTC TCC ACG GGG GGA CCA CGC TGT GA~ ATC CCA ATA GAC ACC ATC
1011
L~u Phe Ser Thr Gly Gly Pro Arg Cys Glu Ile Pro Ile Asp Thr Ile
315 320 325
ATG TCC TAT GAC GGT CAC TCC CAC CAT GAA AGA GTC ATG AGT GCC CAG
1059
Met Ser Tyr Aap Gly His Ser His His Glu Arg Val Met Ser Ala Gln
330 335 340 345
CTA AAT GCC ATC TTT CAT GAT TAACCCTTGG AAGATCAAAA CAaCTGACTG
1110
Leu Asn Ala Ile Phe His Asp
350
TGCATTGCCA GGACTGTCTT GTTTACCAAG GGCAGACACG TGGGTAGTAA AAGTGCAAAT
117 0
GCCCCACTCT GGGGCTGTAA CAAACTTGAT ~ .Ll..~. CTTTAGATAT cCcc7~ rr~
123 0
AATGTATTAA TTCCCACCTC CTTCCAATCG ACACTCCTTT AAATT
1275
(2) INFOR~ATION FOR SEQ ID NO:4:
(i) SEQUENCE r~ ,5:
(A) LENGTH: 352 amino a~:lds
(B~ TYPE: amino arid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(Yi) SEQUENCE l1~ 110N: SEQ I0 NO:4:
et Thr Lys Ser Tyr Gly Glu Asn Gly Leu Ile Leu Ala Glu Thr Pro
5 10 15
ly Cya Arg Gly Trp Val Asp Glu Cys Leu Ser Ser Gln Asp Glu A~n
20 25 30
Asp Leu Glu Lys Lys Glu Gly Glu Leu Met Lys Glu Asp Asp Glu As
35 40 45
Ser Leu Asn His His Asn Gly Glu Glu Asn Glu Glu Glu Asp Glu Gly
50 55 60
Asp Glu Glu Glu Glu Asp Asp Glu Asp Asp Asp Glu Asp Asp Asp Gln
65 70 75 80
Ly~ Pro Lys Arg Arg Gly Pro Lys Lys Lys Lys Met Thr Lys Ala Arg
. _ _ . . _ _
WO 95M0693 2 1 ~ ~ 4 5 ~ r~ l, ~P~ /41
-40-
85 90 95
~l Glu Arg Phe Lys Val Arg Arg Met Lys Ala Asn Ala Arg Glu Ar
100 105 110
Asn Arg Met His Gly Leu Asn Asp Ala Leu Asp Ser Leu Arg Lys Val
115 120 125
Val Pro Cys Tyr Ser Lys Thr Gln Lya Leu Ser Lys Ile Glu Thr Leu
130 135 140
Arg Leu Ala Lys Asn Tyr Ile Trp Ala Leu Ser Glu Ile Leu Arg Ser
145 150 155 160
ly Lys Ser Pro Asp Leu Val Ser Phe Val Gln Thr Leu Cys Lys Gly
165 170 175
~u Ser Gln Pro Thr Thr Asn Leu Val Ala Gly Cys Leu Gln Leu Asn
180 185 190
Pro Arq Thr Phe Leu Pro Glu Gln Ser Gln Asp Ile Gln Ser His Met
195 20C 205
Gln Thr Ala Ser Ser 3er Phe Pro Leu Gln Gly Tyr Pro Tyr Gln S~r
210 215 . 220
Pro Gly Leu Pro Ser Pro Pro Tyr Gly Thr Met Asp Ser Ser His Val
225 230 235 240
he Hi~ Val Lys Pro Hi~ Ser Tyr Gly Ala Ala Leu Glu Pro Phe Phe
245 250 255
sp Ser Ser Thr Val Thr Glu Cys Thr Ser Pro Ser Phe Asp Gly Pro
260 265 270
L~u Ser Pro Pro Leu Ser Val Asn Gly Asn Phe Thr Phe Lys His Glu
275 280 285
His Ser Glu Tyr Asp Lys Asn Tyr Thr Phe Thr Met His Tyr Pro Ala
290 295 300
Ala Thr Ile Ser Gln Gly HiJ Gly Pro Leu Phe Ser Thr Gly Gly Pro
305 310 315 320
rg Cys Glu Ile Pro Ile Asp Thr Ile Met Ser Tyr Asp Gly His S~r
325 330 335
is his Glu Arg Val Met Ser Ala Gln Leu Asn Ala Ile Phe His Asp
340 345 350
~ WO 95/30693 21 8 8 ~ ~ ~ PCT/US95/05741
~1_
(2) INFORMATION FOR SEQ ID NO:5:
~i) SEQUENCE CHMACTERISTICS:
(A) LENGTH: 7 amino acida
(B) TYPE: amino acid
(D) TOPOLOGY: linear
( ii ) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
A~n Ala Arg Glu Arg Arg Arg
(2) INFORMATION FOR SEQ ID No:6:
~i) SEQUENCE CHMACTERISTICS:
~A) LENGTH: 7 amino acids
~B) TYPE: amino acid
~ D ) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FElAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Asn Glu Arg Glu Arg Asn Arg
(2) INFORMATION FOR SEQ ID NO:7:
~i) SEQUENCE CHMACTERISTICS:
(A~ LENGTH: 5 amino acidJ
(B) TYPE: amino ~cid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
r
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
A~n Ala Arg Glu Arg
W095~30693 21 ~45~ ` P~ '/41 ~
-12-
(2) INFORMATION FOR SEQ ID NO:8:
(i) 9EQUENCE CHP~ACTERISTICS:
(A) LENGT~i: 524 ~ase pairs
(B) TYPIS: nucleic acid
(C) ~l~ANLI~;L)Nh~S: double
(D) TOPOLOGY: linear
( ii ) MOLECULE TYPE: DNA ( genomi c )
(vi) ORIGINAL SOURCE:
(A) ORGANIS~: Homo sapiens
(vil) IMMEDIATE SOURCE:
( B ) CLONE: 9 Fl
( ix ) FEATURE:
(A) NAME/REY: CDS
(B) LOCATION: 57..524
~xl) SEQUENCE L~ ,n~ m: SEQ ID NO:8:
C~ C~ CCTTGTTGAA TGTAGGAAAT CGAAAC
56
ATG ACC A~A TCG TAC AGC GAG AGT GGG CTG ATG GGC GAG CCT CAG CCC
104et Thr Lys Ser Tyr Ser Glu Ser Gly Leu Met Gly Glu Pro Gln Pro
5 10 15
CAA GGT CCT CCA AGC TGG ACA GAC GAG TGT CTC AGT TCT CAG GAC GAG
lS2ln Gly Pro Pro Ser Trp Thr Asp Glu Cys Leu Ser Ser Gln Asp Glu
20 25 30
GAG CAC GAG GCA GAC AAG AZ~G GAG GAC GAC CTC GAA GCC ATG A~C GCA
200lu llis Glu Ala Asp Lys Lys Glu Asp Asp Leu Glu Ala Met Asn Ala
35 40 45
GAG GAG GAC TCA CTG AGG A~C GGG GGA GAG GAG GAG GAC GAA GAT GAG
248
Glu Glu Asp Ser Leu Arg Asn Gly Gly Glu Glu Glu Asp Glu Asp Glu
50 55 60
GAC CTG GAA GAG GAG GAA GAA GAG GAA GAG GAG GAT GAC GAT CAA AAG
296
Asp Leu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Asp Asp Gln Lys
65 70 75 80
CCC A~G AGA CGC GGC CCC A~A AAG A~G A~G ATG ACT A~G GCT CGC CTG
344
Pro Lys Arg Arg Gly Pro Lys Lys Lys Lys Met Thr Lys Ala Arg Leu
~ WO9S/30693 21 884~ P ~ rs,4l
13-
85 90 95
GAG CGT TTT A~A TTG AGA CGC ATG AAG GCT AAC GCC CGG GAG CGG AAC
392lu Arg Phe Lys Leu Arg Arg Met Lys Ala Asn Ala Arg Glu Arg Asn
100 105 110
CGC ATG QC GGA CTG AAC GCG GCG CTA GAC APC CTG CGC AAG GTG GTG
440
Arg Met Elis Gly Leu Asn Ala Ala Leu Asp A~n Leu Arg Lys Val Val
115 120 125
CCT TGC TAT TCT A~G ACG QG AAG CTG TCC AP,A ATC GAG ACT CTG CGC
488
Pro Cys Tyr Ser Lys Thr Gln Lys Leu Ser Lys Ile Glu Thr Leu Arg
130 135 140
TTG GCC A~G AAC TAC ATC TGG GCT CTG TCG GAG ATC
524
Leu Ala Ly- Asn Tyr Ile Trp Ala Leu ser Glu Ile
145 150 155
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CH~PACTEEIISTICS:
(A) LENGTH: 156 amino acid~
(B) TYPE: amino ~cid
(D) TOPOLOGY: line~r
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE L)l:.a~,~L~lL~JN: SEQ ID NO:9:
et Thr Lys Ser Tyr Ser Glu S~r Gly Leu Met Gly Glu Pro Gln Pro
5 10 15
ln Gly Pro Pro Ser Trp Thr Asp Glu Cys Leu Ser Ser Gln Asp Glu
20 25 - 30
lu l~l~ Glu Ala Asp Lys Lys Glu Asp Asp Leu Glu Ala Met Asn Ala
35 40 45
Glu Glu Asp Ser Leu Arg Asn Gly Gly Glu Glu Glu Asp Glu Asp Glu
50 55 60
A~p Leu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Aap Asp Gln Lys
65 70 75 80
ro Ly~ Arg Arg Gly Pro Lys Lys Lys Ly~ Met Thr Lys Ala Arg Leu
85 90 95
lu Arg Phe Lys Leu Arg Arg Met Ly~ Ala Asn Ala Arg Glu Arg A~n
100 105 110
.. .. , . _ . .
WO 95/3~693 21 8 g 4 5 ~ PCTlUS9~/o574l
Arg Met Nis Gly Leu Asn Al~ Ala Leu Asp Asn Leu Arg Lys Val Val
115 120 125
Pro Cys Tyr Ser Lys Thr Gln Lys Leu Ser Ly~ Ile Glu Thr Leu Ar
130 135 140
Leu Ala Lys Asn Tyr Ile Trp Ala Leu Ser Glu Ile
145 150 155
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 485 base pairs
(B) TYPE: nucleic acid
(C) STR~NDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo ~apiens
(vii) IMMEDIATE SOURCE:
(B) CLONE: 14Bl
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 3..485
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GG GCC AGG GGC TCC GGG GCC AGC CCG GGC GGC CAA GCC AGT CCC TCT
47
Ala Arg Gly Ser Gly Al~ Ser Pro Gly Gly Gln Ala Ser Pro Ser
5 10 15
CCG TGG AGA AGA GGG GAC GGA GGC CAC GTT GGC CGA GGT CAA GGA GGA
ro Trp Arg ~rg Gly Asp Gly Gly His Val Gly Arg Gly Gln Gly Gly
20 25 30
AGG CGG CTG GGG GGA GAG GAG GAG 5~aG GAA GAG GAG GAG GA~ GAA GGA
143
Arg Arg Leu Gly Gly Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Gly
35 40 45
CTG GAC GAG GCG GAG GGC GAG CGG CCC APG AAG CGC GGG CCC AAG APG
191
Leu Asp Glu Ala Glu Gly Glu Arg Pro Lys Lys Arg Gly Pro Lys Ly~
WO 9S/30693 21 8 8 ~ 5 0 r~ . .3/41
CGC AAG ATG ACC AAG GCG CGC TTG GAG CGC TCC AAG CTT CGG CGG CAG
239
Arg Lys Met Thr Lys Ala Arg Leu Glu Arg Ser Lys Leu Arg Arg Gln
65 70 75
AAG GCG AAC GCG CGG GAG AAS CGC ATG CAC GAC CTG AAC GCA GCC CTG
287
Lys Ala Asn Ala Arg Glu Asn Arg Met His Asp Leu Asn Ala Ala Leu
80 85 90 9S
GAC AAC CTG CGC AAG GTG GTG CCC TGC TAC TCC AaG ACG CAG AAG CTG
335sp Asn Leu Arg Lys Val Val Pro Cys Tyr Ser Lys Thr Gln Lys Leu
100 105 110
TCC AAG ATC GAG ACG CTG CGC CTA GCC AAG AAC TAT ATC TGG GCG CTC
383er Lys Ile Glu Thr Leu Arg L~u Ala Lys Asn Tyr Ile Trp Ala Leu
llS 120 125
TCG GAG ATC CTG SGC TCC GGC AAG CGG CCA GAC CTA GTG TCC TAC GTG
431
Ser Glu Ile L~u Arg Ser Gly Lys Arg Pro Asp Leu Val Ser Tyr Val
130 135 140
CAG ACT CTG TGC AAG GGT CTG TCG CAG CCC ASC ACC AAT CTG GTG GCC
479
Gln Thr Leu Cys Lys Gly L~u Ser Gln Pro Thr Thr Asn Leu Val Ala
145 150 155
GGC TGT
485
Gly Cy~
160
(2) lN~Ul~llUN FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 161 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE Lll:;:>U~lr~lUN: SEQ ID NO:ll:
l~ Arg Gly Ser Gly Ala Ser Yro Gly Gly Gln Ala Ser Pro Ser Pro
5 10 15
rp Arg Arg Gly Asp Gly Gly his Val Gly Arg Gly Gln Gly Gly Arg
20 25 30
Arg Leu Gly Gly Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Gly Leu
_ ... . _ . . . . . , . . .. . .. . . .. . . . . . . . ... . . . . _
WO 95/30693 '2 1 8 8 4 5 0 PCI IUB9~/0574 1
16-
35 40 45
A~p Glu Ala Glu Gly Glu Arg Pro Ly- Lys Arg Gly Pro Ly5 Lys Arg
50 55 60
Ly~ Met Thr Lys Ala Arg Leu Glu Arg Ser LYJ Leu Arg Arg Gln Lya
65 70 75 80
l h Asn Ala Arg Glu Asn Arg Met His Asp Lcu Asn Ala Ala Leu Asp
~5 90 95
sn Leu Arg Lys Val Val Pro Cys Tyr Ser Lys Thr Gln Lys Leu Ser
100 105 110
Lys Ile Glu Thr Leu Arg Leu Ala Lys Asn Tyr Ile Trp Ala Leu Ser
115 120 125
Glu Ile Leu Arg Ser Gly Lys Arg Pro Asp Leu Val Ser Tyr Val Gln
130 135 140
Thr Leu Cys Lys Gly Leu Ser Gln Pro Thr Thr Asn Leu Val Ala Gl
145 150 155 160
Cys
