CA2326601A1 - Ese genes and proteins - Google Patents

Abstract

The present invention describes the identification, isolation and characterization of novel mammalian proteins encoded by the Ese1 and Ese2 genes which are involved in endocytosis, vesicular trafficking and regulation of the actin cytoskeleton. Transcripts and products of these genes are useful for detecting abnormal cellular endocytosis processes as well as for developing assay systems to find and elucidate further binding partners of the proteins, to develop therapeutics to alter/restore protein function and for the isolation and manufacture of Ese proteins.

Description

ESE GENES AND PROTEINS
Field of the Invention This invention relates to novel mammalian proteins encoded by the Ese 1 and Ese2 genes which are involved in endocytosis, vesicular trafficking and regulation of the actin cytoskeleton.
S
Background of the Invention Endocytosis via clathrin-coated pits is a multipstep process (1, 2). Clathrin heavy and light chains are brought to the plasma membrane through association with a heterotetrameric complex known as clathrin adaptor complex 2, or AP-2. At coated pits. the membrane is bent through the assembly of clathrin triskelion into a caged lattice. The GTPase Dynamin is also recruited to the neck of coated pits where it assembles into a collar for vesicle fission (4, 5). Recruitment of Dynamin to coated pits is believed to require the Amphiphysin I/II heterodimer (6), as these proteins bind Dynamin in vivo and amphiphysin proteins are required for endocytosis in yeast (7).
In addition, ectopic expression of either Amphiphysin I or iI by themselves (6), or the isolated SH3 domain of Amphiphysin I blocks endocytosis (8, 9). Recent data has revealed that the Rab~ small GTPase is required for sequestration of ligands such as transferrin and Epidermal Growth Factor into coated pits in vitro (10). The mechanism by which these components interact to regulate coated pit assembly, cargo sequestration. followed by vesicle fission is not yet understood.
From biochemical, cell biological and genetic analysis it is clear that additional components such as kinases, phosphatases, ubiquitin conjugating enzymes as well as lipid modifying enzymes are required for clathrin-coat and vesicle formation (1, 11, 12, 13, 14, 15, 16, 17, 18, 19). Indeed there is also strong evidence for a requirement of the actin cytoskeleton in endocytosis and several proteins which may facilitate this connection (1, 7, 20, 21, 22, 23, 24, 25).
The EpslS protein was discovered in a search for substrates of the Epidermal Growth Factor Receptor (26). In 1995, Benmerah et. al. reported that EpslS is constitutively associated with a-adaptin of the AP2 complex (27). The EpslS
protein has also been localized to the neck of clathrin-coated pits by immunoelectron microscopy (28, 29). Recently, two groups have used dominant inhibitory mutants of Epsl~, or antibodies against EpslS (or the related protein EpslSR), to demonstrate that Eps 15 proteins are required for endocytosis via clathrin-coated pits (30, 31 ).
EpslS contains three large structural domains (26, 32). The N-terminal third contains three copies of an EH domain (for Epsl ~ Homology domain) (32, 33). The central region of Eps 1 ~ forms an extended coiled-coil, which is followed by a complex C-SUBST7~TUTE SHEET (RULE Z6) _7_ terminus containing SH3-binding motifs (34), a large number of DPF repeats (Aspartic acid-Proline-Phenylalanine). and a-adaptin binding sequences (31, 35, 36).
The full length EpslSR protein has a similar overall organization (34). Both EpslS
and EpslSR can be alternatively spliced to produce numerous smaller proteins(37).
A protein with similar overall organization has been identified in Saccharomyces cerevisiae named Panlp. Genetic analysis of PANI has revealed that this gene is required for endocytosis and for organization of the actin cytoskeleton (23, 38). Like EpslS and EpslSR in mammals, the Panlp protein has N-terminal EH
domains followed by a central coiled-coil domain and C-terminal proline-rich sequences. A second EH domain containing protein, End3p, has also been described in S. cerevisiae which is required for endocytosis and regulation of the actin cytoskeleton (21, 39). Co-immunoprecipitation studies have shown that Panlp and End3p form a complex in vivo (40) . Indeed, overexpression of End3p can suppress the phenotype of panl-4 hypomorphic mutants, and Panlp is mislocalized in end3 mutants indicating that these proteins function together (40). Additional studies have revealed that the EH domains of Pan 1 p bind to yeast homologues of mammalian clathrin-binding proteins, AP 180 and CALM (yAP 180A and yAP 180B), through NPF
motifs (Asparagine-Proline-Phenylalanine) in the yAP180 C-termini(25). These data have led to a proposal that the Panlp:End3p complex functions as a multivalent adaptor to coordinate protein-protein interactions during endocytosis (25, 40). At least two additional proteins are predicted to bind to the Panlp:End3p complex in vivo, as strong genetic interactions have been detected between PAN l and SJL
1 (25), and between PAN 1 and RSPS (41 ). SJL 1 encodes a phosphatidylinositol polyphosphate-5-phosphatase protein which is related to mammalian synaptojanin (42) and has a C-terminal NPF motif predicted to bind to EH domains in Panlp (or End3p) (25, 43) . RSPS encodes an E3 ubiquitin-protein ligase which may bind to the C-terminal polyproline sequences in Panlp through one of its three WW domains (25).
Numerous SH3 domain containing proteins have been implicated in the regulation of endocvtosis (441. These include Amphiphysin I(451 and IT_ (6.
46. 47, 48), Rsv161/Rsv167(7), Actin Binding Protein-1(49), Endophilin/SH3P4/8/13 (50, 51 ) and Grb2 (52). Kay and coworkers have reported the isolation of several novel SH3 encoding cDNAs (537.
The present inventors have identified novel mammalian proteins containing 3 ~ both EH and SH3 domains. which have been named Ese i and Ese2. Sequence and functional analysis of the full length proteins have implicated these proteins in receptor mediated endocvtosis via clathrin coated pits and therefore the proteins have SUBSTTTLf)cE SHEET (RULE 26) been named Ese 1 and Ese2 respectively (Ese: for EH-domain and SH3 domain regulator of Endocytosis). Also identified are several mammalian alternative transcript proteins two of which are named Ese 1 L and Ese2L.
Summary of the Invention In accordance with one series of embodiments, this invention provides isolated nucleic acids corresponding to or relating to the nucleic acid sequences disclosed herein which encode the mammalian Esel and Ese2 proteins.
The invention more specifically provides isolated nucleic acids corresponding to or relating to the nucleic acid sequences disclosed herein which encode the mouse Ese 1 and Ese2 proteins.
One of ordinary skill in the art is now able to identify and isolate mammalian Ese protein genes or cDNAs which are allelic variants of the disclosed sequences or are homologues thereof, in other species, including humans. using standard hybridisation screening and PCR techniques. The mammalian polynucleotide may be in the form of DNA, genomic DNA, cDNA, mRNA and various fragments and portions of the gene sequence encoding Ese proteins.
Homologues of the mammalian Ese protein genes are generally those sequences which share at least 80% sequence identity, preferably at least 90%
sequence identity to the mammalian Ese gene sequence.
In a further embodiment, the invention provides cDNA sequences encoding marine Esel and Ese2 proteins comprising the nucleotide sequences of Sequence ID
NOS: 1, 2, 4 and 5.
In a further embodiment, the invention provides cDNA sequences encoding marine EselL and Ese2L proteins comprising the nucleotides sequences of Sequence ID NOS: 22, 23. 25 and 26.
Also provided are portions of the Ese gene sequences useful as probes or PCR
primers or for encoding fragments, functional domains or antigenic determinants of Ese proteins. The probes can be routinely used to screen and identify homologues of the Ese gene or portions thereof while primers are useful in PCR assays for the amplification of desired portions of the selected Ese gene.
The invention also provides portions of the disclosed nucleic acid sequences comprising about 10 consecutive nucleotides to nearly the complete disclosed nucleic acid sequences. The invention provides isolated nucleic acid sequences comprising sequences corresponding to at least 10, preferably 1~ and more preferably at least 20 consecutive nucleotides of the Ese genes as disclosed or enabled herein or their complements.
SUBSTTTUT'E SHEET (RULE 26) In addition. the isolated nucleic acids of the invention include any of the above described nucleotide sequences included in a vector. Expression vectors comprising the nucleotide sequences are provided along with suitable host cells transfected with such expression vectors.
In accordance with a further series of embodiments, this invention provides substantially pure mammalian Ese proteins, fragments of these proteins and fusion proteins including these proteins and fragments.
In accordance with a further series of embodiments, this invention provides substantially pure mutant mammalian Ese proteins. fragments of these proteins and fusion proteins including these mutant proteins and fragments.
In a further embodiment, the invention provides amino acid sequences encoding murine Ese 1 and Ese2 proteins comprising the amino acid sequences of Sequence ID NOS: 3 and 6.
In a further embodiment, the invention provides amino acid sequences I S encoding murine EselL and Ese2L proteins, which are alternative transcripts, comprising the amino acid sequences of Sequence ID NOS: 24 and 27.
The proteins, fragments and fusion proteins have utility, as described herein, for the preparation of polyclonal and monoclonal antibodies to murine and mammalian Ese proteins, for the identification of binding partners of the mammalian Ese proteins and for diagnostic and therapeutic methods, as described herein.
For these uses. the present invention provides substantially pure proteins, polypeptides or derivatives of polypeptides which comprise portions of the mammalian Ese amino acid sequences disclosed or enabled herein and which may vary from about 4 to amino acids to the complete amino acid sequence of the proteins. The invention provides substantially pure proteins or polypeptides comprising sequences corresponding to at least ~, preferably at least 10 and more preferably 50 or consecutive amino acids of the mammalian Ese proteins disclosed or enabled herein.
Monoclonal antibodies having suitably specific binding affinity for the antigenic regions of a mammalian Ese protein are prepared by the use of corresponding s0 hybridoma cell Lines. In addition, polvclonal antibodies may be prepared by inoculation of animals with suitable peptides which add suitable specific binding affinities for antigenic regions of an Ese protein.
In a further embodiment of the invention, a process is provided for producing mammalian Ese proteins comprising culturing one of the above described tr~i~cted ~ host cells under suitable conditions, to produce the Ese protein by expressing the DNA sequence.
SUBSTITUTE SHEET (RLZ~, ~6) _j_ The proteins of the invention may be isolated and purified by any conventional method suitable in relation to the properties revealed by the amino acid sequences of these proteins.
Alternatively, cell lines may be produced which express or over-express the Ese gene products, allowing purification of the proteins for biochemical characterisation, large-scale production, antibody production and patient therapy.
For protein expression, eukaryotic or prokaryotic expression systems may be generated in which an Ese gene sequence is introduced into a plasmid or other vector which is then introduced into living cells. Constructs in which the Ese cDNA
sequences containing the entire open reading frame is inserted in the correct orientation into an expression plasmid may be used for protein expression.
Alternatively, only portions of the sequence may be inserted. Prokaryotic or eukaryotic expression systems allow various important functional domains of the proteins to be recovered as fusion proteins and used for binding, structural and functional studies and also for the generation of appropriate antibodies.
The present invention includes effective fragments, analogues of the Ese proteins described herein. "Effective" fragments or analogues retain the activity of the described Ese proteins to regulate endocytosis, vesicular trafficking and actin dynamics. The term "analogue" extends to any functional and/or chemical equivalent of a mammalian Ese protein including mimetics and includes proteins having one or more conservative amino acid substitutions, proteins incorporation unnatural amino acids and proteins having modified side chains.
In accordance with a further embodiment of the invention. antibodies are enabled which bind specifically to the Ese proteins disclosed herein.
Polyclonal or monoclonal antibodies may be prepared using conventional methods. Antibodies may also be prepared to individual selected domains of the Ese proteins, as described herein.
In a further embodiment, the invention provides pharmaceutical compositions containing an Ese protein, fragment or mimetic thereof or a non-functional mutant Ese protein, fragment or mimetic thereof for the treatment of mammalian disorders which involve abnormal endocytosis, vesicular trafficking and actin dynamics leading to altered cellular functioning. Administration of a therapeutically active amount of a pharmaceutical composition of the present invention means an amount effective, at dosages and for periods of time necessary to achieve the desired result. The composition comprises an Ese protein and a pharmaceutically acceptable carrier.
In accordance with a further embodiment, the invention provides a method for identifying binding partners of the Ese proteins disclosed herein. Such methods in SUBSTTTUT'E SHEET (RULE 26) general include various assays including those including radiolabelling of the Ese proteins. Other methods may include but are not restricted to phage display.
affinity purification techniques, expression cloning and the yeast 2-hybrid system, as described herein.
In accordance with a further embodiment of the present invention. is a method for identifying proteins which phosphorylate Ese proteins. Such method includes known phosphorylation assays.
The identification of proteins or peptides interact with or bind to Ese proteins can provide the basis for the design of peptide antagonists or agonists of Ese protein function or for the design of peptide antagonists or agonists of Ese protein binding partners which affect Ese protein function. Further, the structure of these peptides determined by standard techniques such as protein NMR or x-ray crystallography can provide the structural basis for the design of improved small molecule drugs.
In accordance with a further embodiment, the present invention also provides 1 ~ for the production of mouse models or transgenic non-human animal models for the study of mammalian Ese gene function, for the screening of candidate pharmaceutical compounds, for the creation of in vitro mammalian cell cultures which express the Ese proteins or in which an Ese gene has been inactivated by knock-out deletion, and for the evaluation of potential therapeutic interventions.
The invention enables a transgenic animal, including a transgenic insect, wherein the genome of the animal or of an ancestor of the animal has been modified by introduction of a transgene comprising mammalian Ese genes under the transcriptional control of tissue restricted regulatory elements including the mouse mammary-tumour virus long term repeat sequences.
Transgenic animals with inappropriate expression of Ese proteins may be examined for phenotypic changes, for example abnormal cellular development or abnormal cellular signalling, vesicular trafficking and actin dynamics and may be used to screen for compounds with potential as pharmaceuticals. Compounds which provide reversal of the phenotypic changes are candidates for development as pharmaceuticals.
Transgenic animals in accordance with the invention can be created by introducing a DNA sequence encoding a selected Ese protein either into embryonic stem (ES) cells of a suitable animal such as a mouse, by transfection or microinjection, or into a germ line or stem cell by a standard technique of oocvte 3~ microinjection. Sue methrds of producing animal models are tulle described in the literature.
SUBSTITUTE SHEET Rs'LE Z6) _'7_ In accordance with another aspect of the present invention is a method for screening a candidate compound for effectiveness as an antagonist of an Ese protein comprising:
(a) providing an assay method for determining the endocytotic regulatory capacity of an Ese protein; and (b) determining the endocytotic regulatory capacity of the Ese protein in the presence or absence of the candidate compound, wherein a reduced level of endocytosis in the presence of the candidate compound indicates antagonist activity of the compound.
In accordance with another aspect of the present invention is a method for treating in a mammal a disorder associated with an undesired level of endocytotic activity of an Ese protein comprising administering to the mammal an effective amount of a substance selected from the group consisting of:
(a) an Ese protein antagonist;
1 ~ (b) an antibody which binds specifically to an Ese protein;
(c) an antisense strand comprising a nucleic acid sequence complementary to the sequence or fragment of the sequence and capable of hybridizing to the nucleic acid sequence encoding an Ese protein;
(d) an agent which down regulates the expression of the Ese gene encoding for an Ese protein;
(e) an antagonist of an Ese protein binding partner: and (f) an Ese agonist.
According to another aspect of the present invention is a method for suppressing in a mammal, the abnormal proliferation of a cell capable of being stimulated to proliferate by a growth factor receptor, the method comprising administering to the mammal an effective amount of a Ese protein antagonist, an Ese agonist or an antibody which binds specifically to an Ese protein.
According to yet another aspect of the present invention is a method for preventing viral infection in a mammal, said method comprising administering to the mammal an effective amount of an Ese protein antagonist, an Ese agonist or an antibody which binds specifically to an Ese protein or an Ese mutant protein not capable of regulating endocytosis.
According to a further aspect of the present invention is a method for promoting endocvtosis, vesicular trafficking and/or actin dynamics in selected cells in a mammal in need of such treatment, said method comprising administering to the mammal an effective amount of an Ese protein or an active analogue, mimic or fragment thereof.
SUBSTITUTE SHEET (RULE 26) _g_ According to a further aspect of the present invention is a method for blocking clathrin-mediated endocytosis in cultured cells or in selected cells in a mammal in need of such treatment, said method comprising overexpressing Ese 1 protein or an ~ active analogue. mimic or fragment thereof.
According to yet a further aspect of the present invention is a method for regulating endocytosis, vesicular trafficking and/or actin dynamics in cultured cells or in selected cells in a mammal in need of such treatment, said method comprising providing an Esel-Eps 15 complex and further providing a protein binding partner to I O bind to the complex to regulate components of the endocytic pathway. One such binding partner is dynamin.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood.
however, that the detailed description and the specific examples while indicating preferred 15 embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
Summary of Tables and Drawings A detailed description of the preferred embodiments are provided herein below with reference to the following tables and drawings in which:
Table 1 shows alignment of mouse Esel and Ese2 protein sequences. EH, coiled-coil and SH3 domains are as indicated.
Table 2 shows the alignment of protein sequences of the Ese family in mouse, Xenopus and Drosophila. EH and SH3 domains are indicated with overlines. Amino acid identities (bolded) and similarity are boxed.
Figure 1 shows a Northern blot demonstrating expression of Ese l and Ese2 genes in various adult tissues.
Figure 2A shows Western blots demonstrating the association of Ese and Eps 1 ~ proteins in vivo. Endogenous Ese 1 proteins were precipitated with Rabbit anti-peptide antisera a~~ax:~: :he N-terminus of Esel . Immu::a -~cipitates were trlen 3~ analyzed .e:, western blots nor the presence of Esel/2 whiz Chicken anti-Ese antisera or for co-precipitation of Eps I ~ with Rabbit antisera raised against the C-terminus of EpS 1 J.
SUBSTITUTE SHE~'~ (RULE 26) Figure 2B shows alternative transcripts from the Ese I and Ese2 genes which code for EselL and Ese2L proteins respectively, with C-terminal DBL/PH and C2 ' domains. Ese l and Ese2 sequence junctions are indicated in bold red letters.
Figure 3A shows Ese and EpslS associate in vivo. This schematic representation of association of EpslS/EpslSR with Esel in the yeast two-hybrid screen. Esel coiled-coil domain fused with the Gal4 DNA binding domain (DBD) interacted with EpslS/EpslSR Gal4 activation domain (AD) fusions. The AD-Eps 15/Eps 1 SR diagram represents the shortest interacting coding region isolated.
Figure 3B shows association of Ese and EpslS C-terminal deletion mutants.
Cos-I cells were transfected with pcDNA3Eps15, pcDNA3Ese1 or the C-terminal deletion mutants pcDNA3Ese18C and pcDNA3Eps158C-Flag as indicated. Cell lysates were immunoprecipitated with rabbit anti-Esel or rabbit anti-Epsl ~
(left panel); rabbit anti-Esel, mouse anti-Flag or rabbit anti-EpsiS (right panel).
Panels represent western blots to detect the presence of Ese 1 in each immunoprecipitation. A
90kDa Ese 1 protein exists in the third and sixth lanes on the left panel which is the C-terminally truncated Ese 1 protein which is co-immunoprecipitated in a complex with Eps 15 in the sixth lane. Also, to be noted is the precipitation of Ese 1 with anti-Flag monoclonal antibody in the sixth lane of the right panel experiment. In this case, Ese 1 has been precipitated in a complex with the C-terminally truncated Eps158C
protein.
Figure 4 shows confocal immunofluorescent micrographs of Cos cells transfected with myc-Esel (A, A'); EpslS (B); myc-Esel + Epsl~ (C, C'. C") or myc-Esel + EpslSsC (D, D' and D"). Frames C" and D' represents the overlapping images from frames C/C' and D/D', respectively. Overlap in frames C" and D"
are indicated in yellow. Scale bar is equal to 10 microns.
Figure ~A and SB show that Esel links to Dynamin and EpslS. Figure SA is a schematic representation of association between Dynamin and Esel in the yeast two-hybrid screen. Esel SH3 domains fused with the Gal4 DNA binding domain (DBD) interacted with Dynamin Gal4 activation domain (AD) fusions. The AD-Dynamin diagram represents the shortest interacting coding region isolated. Figure SB
shows confocal immunofluorescent microscopy to detect transfected mycEsel (frame a), transfected EpslS (frame d) or endogenous Dynamin (frames b and e) in transfected Cos cells. Frame c and f represents the overlapping images from frames alb and d/e respectively. Scale bar is equal to 10 microns.
Figure 6 shows Esel overexpression blocks endocytosis of Transferrin in Cos-3s 1 cells. Confocal immunofluorescent microscopy was used to detect transfected mycEse 1 (frame A), internalized FITC-labeled Transferrin (frame B).
Overlapping images are shown in frame C revealing that Ese 1 overexpression blocks clathrin-SUBSTT>cUTE SHEET (RULE 26) mediated endocvtosis of Transferrin. Mononuclear morphologically normal cells from both transfected and untransfected groups where assessed for internalization of transferrin. Scale bar is equal to 10 microns.
Figure 7 shows a proposed model for Ese regulation of Endocvtosis.
In the drawings, preferred embodiments of the invention are illustrated by way of example. It is to be expressly understood that the description and drawings are only for the purpose of illustration and as an aid to understanding, and are not intended as a definition of the limits of the invention.
Detailed Description of the Invention Seauencin~ of Murine Esel and Ese~ genes The full length murine Ese 1 cDNA was sequenced (Sequence ID NO: I ). It encodes a sequence of 1213 amino acids (Sequence ID N0:3) having a predicted molecular weight of 137 kDa. Murine Ese2 cDNA was also sequenced (Sequence ID
N0:4) and encodes a sequence of I 197 amino acids (Sequence ID N0:6) having a predicted molecular weight of 135.7 kDa.
The full length Ese proteins are predicted to encode two N-terminal EH
domains followed by a coiled-coil domain and five SH3 domains (Tables 1 and

2).
Several Esel isolated clones contain only SH3A, SH3B and SH3E domains. Indeed, the SH3~ domain was not included in the original human SH3P17 partial cDNA. In addition, expressed sequence tags from the Esel gene have been found in the public databases which skip sequences encoding individual EH domains or regions of the coiled-coil domain indicating that this gene is subject to complex alternative splicing and has the potential to encode for many distinct proteins. The predicted Ese2 protein on the other hand encodes a C-terminal extension of 45 amino acids in comparison to the human SH3P18 partial cDNA, suggesting that this gene is also alternatively spliced. The Ese proteins are 53% identical over the full length of Ese 1 (645 of 1213 residues in Esel line up with identical residues in Ese2) and are related to the Ese protein from.kenopus whic~ has recently been submitted to genebanl: ( Accession #
AF032118) and Drosophila (59) (Table 2). Xenopus Intersectin is 81 % identical to mouse Esel and 54% identical to mouse Ese2 suggesting that Intersectin is an Esel orthologue (980/1213 residues of mouse Esel and 645/1198 residues of mouse Ese2 line up with identical residues in the kenopus protein). Drosophila Dynamin associated protein. Dap160-1 is ~'_'°~ identical to both mouse Ese proteins (393/I?13 residues of mouse Esel and 387/1198 residues of mouse Ese2 line up with identical residues in the Drosophila protein i. These homologies extend over the entire length SUBSTITUTE SHEET (RULE 26) of Dap160-l, except that Dap160-1 has only four SH3 domains corresponding to the first. second, fourth and fifth SH3 domains of the mouse Ese proteins.
Additional sequence analysis reveals the presence of a very large number of potential phosphorylation sites and at least one SH3-binding consensus (60} in the N-terminus of each Ese protein.
The two EH domains of Ese 1 are highly related to the respective EH domains in Ese2 and these Ese EH domains are most closely related those found in EpslS
and Eps 1 SR, two mammalian protein which are required for endocytosis through clathrin-coated pits. EH domains have also been identified in End3 and Panlp which are yeast partners proteins required for endocytosis. The central third of both Ese proteins are predicted to encode an extended coiled-coil which is a domain typically associated with protein-protein association through dimerization or tetramerization as noted for Epsl~ (29, 54). Besides the homology between Esel and Ese2, the Esel SH3 domains are most closely related to SH3 domains from Myosin IB in Acanthamoeba (SH3A), Myosin IB in Entamoeba (SH3B), the YFR024 hypothetic yeast protein (SH3~), Myosin IB from Acanthamoeba (SH3p) and Myosin IC from Acanthamoeba (SH3E). The same homologies are noted for SH3 domains from Ese2 with the exception of SH3A which is most similar to the SH3 domain from pPIX, SH3~
which is most similar to an SH3 domain from the mouse Ray protein and SH3D which is most similar to the SH3 domain from Dictyostelium myosin IB. Additional sequence analysis reveals the presence of a very large number of potential phosphorylation sites in the Ese proteins and a single SH3 binding consensus in the N-termini of each Ese protein.
With the knowledge of the amino acid sequences for Esel and Ese2 proteins and the alternative transcripts EselL and Ese2L, there is provided in accordance with the present invention antibodies which recognize epitopes within these proteins and which can be raised to provide information on the characteristics of the protein as well as for any mutant form of these proteins. The generation of antibodies enables the visualization of the protein in mammalian cells and tissues using Western blotting as described herein. Antibodies to the Esel or Ese2 proteins also allows for the use of immunocytochemistry and immunofluorescence techniques in which the proteins are visualized directly in cells and tissues as described herein. This is most helpful in order to establish the subcellular location of the protein and the tissue specificity of the protein.

3 ~ In general. methods for the preparation of antibodies are well known. In order to prepare polyclonal antibodies. fusion proteins containing defined portions or all of SUBSTITUTE SHEET (R.ULE 26) the Ese 1 or Ese2 proteins or any of their alternative transcripts can be synthesized in bacteria by expression of corresponding DNA sequences in a suitable cloning vehicle.
The protein can then be purified, coupled to a carrier protein and mixed with Freund's adjuvant (to help stimulate the antigenic response by the rabbits) and injected into ~ rabbits or other laboratory animals. Alternatively, protein can be isolated from cultured cells expressing the protein. Following booster injections at bi-weekly intervals, the rabbits or other laboratory animals are then bled and the sera isolated.
The sera can be used directly or purified prior to use, by affinity chromatography.
The sera can then be used to probe protein extracts run on a polyacrylamide gel to identify the Ese I or Ese2 protein, alternative transcript or any mutant thereof.
Alternatively, synthetic peptides can be made to the antigenic portions of these proteins and used to innoculate the animals.
Methods to produce monoclonal antibodies which specifically recognize mammalian Ese 1 or Ese2 proteins or portions thereof, are known in the art. In general, cells actively expressing the protein are cultured or isolated from tissues and the cell extracts isolated. The extracts or recombinant protein extracts, containing the Esel or Ese2 protein, are injected in Freund's adjuvant into mice. After being injected 9 times over a three week period, the mice spleens are removed and resuspended in phosphate buffered saline (PBS). The spleen cells serve as a source of lymphocytes, some of which are producing antibody of the appropriate specificity. These are then fused with a permanently growing myeloma partner cell, and the products of the fusion are plated into a number of tissue culture wells in the presence of a selective agent such as HAT. The wells are then screened to identify those containing cells making useful antibody by ELISA. These are then freshly plated. After a period of growth, these wells are again screened to identify antibody-producing cells.
Several cloning procedures are carried out until over 90% of the wells contain single clones which are positive for antibody production. From this procedure a stable lines of clones is established which produce the antibody. The monoclonal antibody can then be purified by affinity chromatography using Protein A or Protein G Sepharose.
The Ese proteins may be isolated and purified by methods selected on the basis of properties revealed by its sequence. Purification can be achieved by protein purification procedures such as chromatography methods (gel-filtration. ion-exchange and immunoaffinity), by high-performance liquid chromatography (HPLC. RP-HPLC.
33 ion-exchange HPLC, size-exclusion HPLC, high-performance chromatofocusing and hydrophobic interaction cnomatography j or by precipitation (immunoprecipitation).
SUBSTTI'UTE SHEET (RULE 26) Polyacrylamide gel electrophoresis can also be used to isolate the Ese proteins based on the molecular weight of the protein, charge properties and hydrophobicity.
Similar procedures to those mentioned can be used to purify the protein from cells transfected with vectors containing an Ese gene (e.g. baculovirus systems, yeast expression systems and eukaryotic expression systems).
The purified proteins can be used in further biochemical analyses to establish secondary and tertiary structure which may aid in the design of pharmaceuticals to interact with the protein, alter the protein charge configuration or charge interaction with other proteins or alter its function.
The Ese proteins can also be purified from the creation of fusion proteins which are expressed and recovered from prokaryotic or eukaryotic cells. The fusion proteins can be purified by affinity chromatography based upon the fusion vector sequence. The Ese protein can then be further purified from the fusion protein by enzymatic cleavage of the fusion protein.
Expression of Ese genes In order to determine where the Ese genes are expressed Northern analysis was performed on mRNA derived from several adult mouse tissues (Figure 1 ). These genes are both widely expressed and reveal a complex pattern of alternatively spliced transcripts. The highest levels of Esel mRNA were noted in brain. heart and skeletal muscle. Brain and skeletal muscle mRNA also contain an unusually large transcript which may be as much as l5kb in length. Interestingly, these tissues express high levels of many proteins involved in synaptic vesicle recycling and endocytosis.
The Northern blot reveals the presence of many alternative mRNA transcripts for both Esel and Ese2. In addition, numerous Esel expressed sequence tags have been identified which skip sequences encoding individual EH domains or regions of the coiled-coil domain indicating that this gene has the potential to encode for many distinct proteins. Indeed, the SH3~domain was spliced out of the human SH3P17 partial cDNA described by Sparks 1996 {53). The predicted Ese2 protein on the other hand encodes a C-terminal extension of 45 amino acids in comparison to the human SH3P18 partial cDNA, revealing that this gene is also alternatively spliced.
Lung, liver and kidney express high levels of a 2.4kb Ese2 transcript which is significantly smaller than the 359Ibp sequence required to code for our full Ese2 protein as shown in Table 1 or 2, indicating that a major isoform of Ese2 has only a subset of the 5 domains described above.
SUBSTTTUTE SHEET (RULE 16) Alternative Splicing of Ese 1. Ese2 transcripts Additional transcripts from both Ese 1 and Ese2 genes have also been characterized. One of the Ese 1 clones isolated from a mouse brain cDNA
library contained an extended reading frame. PCR was used from mouse brain cDNA
libraries to identify the remaining coding sequences from this transcript. The splicing event which produces this extended protein adds an additional 501 amino acids to Esel, and codes for a DBL homology domain, a Pleckstrin homology (PH) domain and a C2 domain (Figure 2B). DBL and PH domains are found together in guanine nucleotide exchange factors for the Rho family of small GTPases. This long form of Ese 1 is therefore expected to function as an activator of Rho-GTPases, which in turn regulate the actin cytoskeleton and numerous signal transduction pathways (61 ). C2 domains on the other hand are Ca+' activated membrane binding domains and protein-protein interaction domains (62). A number of expressed sequence tages which code for a novel C2 domain fused to the sequence coding for the last 11 amino acids of Ese? have been identified. PCR was used to isolate sequences coding for alternatively spliced exon(s) which can be included C-terminal to the SH3 domains but before the stop codon. The alternatively spliced exon(s) of Ese2 also code for a DBL/PH +

domain cassette which can be included within the Ese2 transcript (Figure 2B}.
As the Ese genes are subject to complex alternative splicing to produce proteins with novel predicted functions (eg. regulation of the cytoskeleton and membrane-binding), the alternative proteins are named as modifications of Ese.
The Esel and Ese2 long forms described in Figure 2B are called EselL and Ese2L, respectively. In the event that specific domains are spliced out from the transcripts coding for Ese 1 and Ese2 proteins as shown in Table 2, then the name is listed as an EseB variant. The protein encoded by the spliced variant in the original SH3P
17 clone is designated as Ese 1 bS~ to indicate removal of SH3~.
In an embodiment of the present invention the knowledge of the Ese l and Ese2 gene sequences and their expression in heterologous cell systems can be used to demonstrate structure-function relationships as well as provide for cell lines for the purposes of drug screening. Ligating the Ese 1 or Ese2 cDNA sequence into a piasmid expression vector to transfect cells is a useful method to test the proteins influence on various cellular biochemical parameters including the identification of substrates, binding partners as well as activators and inhibitors of the proteins. Plasmid expression vectors containing either the entire, or portions thereof, Ese 1 or Ese2 can 3~ ;:e used ir. rr: ~.~itro mutagenesis experiments which will identify portions of the protein crucial for regulatory function.
The Ese 1 or Ese2 cDNA sequence (or Ese 1 L and Ese2L cDNA sequencel can SUBSTITLTfE SHE'.-' (RULE 26) be manipulated in studies to understand the expression of the gene and its product, to achieve production of large quantities of the protein for functional analysis, for antibody production, and for patient therapy. The changes in the sequence may or may not alter the expression pattern in terms of relative quantities, tissue-specificity and functional properties. Partial or full-length cDNA sequences which encode for the Esel or Ese2 protein (or alternative transcripts thereofj, modified or unmodified, may be Iigated to bacterial expression vectors. E. coli can be used using the polymerase/promoter system using two plasmids or by labeling of plasmid-encoded proteins, or by expression by infection with M13 Phage mGPI-2. E. coli vectors can also be used with Phage lamba regulatory sequences, by fusion protein vectors (eg.
IacZ and trpE), by maltose-binding protein fusions, and by glutathione-S-transferase fusion proteins.
Alternatively, the Ese 1 or Ese2 protein or alternative transcripts thereof can be expressed in insect cells using baculoviral vectors, or in mammalian cells using vaccinia virus. For expression in mammalian cells, the cDNA sequence may be ligated to heterologous promoters, such as the simian virus (SV40) promoter in the pSV2 vector and introduced into cells, such as COS cells to achieve transient or long term expression. The stable integration of the chimeric gene construct may be maintained in mammalian cells by biochemical selection, such as neomycin, 6418 and purimycin.
Any of the Esel or Ese2 cDNA sequences can be altered using procedures such as restriction enzyme digestion, fill-in with DNA polymerase, deletion by exonuclease, extension by terminal deoxynucleotide transferase, ligation of synthetic or cloned DNA sequences, site-directed sequence alteration with the use of specific oligonucleotides together with PCR.
The cDNA sequence or portions thereof, or a mini gene consisting of a cDNA
with an intron and its own promoter, is introduced into eukaryotic expression vectors by conventional techniques. These vectors permit the transcription of the cDNA
in eukaryotic cells by providing regulatory sequences that initiate and enhance the transcription of the cDNA and ensure its proper splicing and polyadenylation.
The endogenous Ese 1 or Ese2 gene promoter can also be used. Different promoters within vectors have different activities which alters the level of expression of the cDNA. In addition, certain promoters can also modulate function such as the glucocorticoid-responsive promoter from the mouse mammary tumor virus.
Some of the vectors listed contain selectable markers or neo bacterial genes that permit isolation of cells by chemical selection. Stable long-term vectors can be maintained in cells as episomal, freely replicating entities by using regulatory SUBSTTTU7CE SHEET (RULE 26) elements of viruses. Cell lines can also be produced which have integrated the vector into the genomic DNA. In this manner, the gene product is produced on a continuous basis.
Vectors are introduced into recipient cells by various methods including calcium phosphate. electroporation, lipofection, DEAE dextran, microinjection, or by protoplast fusion. Alternatively, the cDNA can be introduced by infection using viral vectors.
Eukaryotic expression systems can be used for many studies of the Ese 1 or Ese2 gene and gene products) including determination of proper expression and post translational modifications for full biological activity, identifying regulatory elements located in the 5' region of the Esel or Ese2 gene and their role in tissue resulation of protein expression, production of large amounts of the normal and mutant protein for isolation and purification, to use cells expressing the Ese 1 or Ese2 protein or alternative transcripts thereof as a functional assay system for antibodies generated against the protein or to test effectiveness of pharmacological agents. or as a component of a signal transduction system, to study the function of the normal complete protein, specific portions of the protein, or of naturally occurring and artificially produced mutant proteins.
Using the techniques mentioned, the expression vectors containing the Esel or Ese2 cDNA sequence or portions thereof can be introduced into a variety of mammalian cells from other species or into non-mammalian cells. It is understood that the cDNA sequences for use in the present invention include those sequences disclosed herein encoding Ese 1, Ese 1 L, Ese2 and Ese2L proteins.
The recombinant cloning vector, according to this invention, comprises the selected DNA of the DNA sequences of this invention for expression in a suitable host. The DNA is operatively linked in the vector to an expression control sequence in the recombinant DNA molecule so that Ese 1 or Ese2 proteins or alternative transcripts thereof can be expressed. The expression control sequence may be selected from the group consisting of sequences that control the expression of genes of prokaryotic or eukaryotic cells and their viruses and combinations thereof.
The expression control sequence may be selected from the group consisting of the lac system. the trp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the control region of the fd coat protein, early and late promoters of SV40, promoters derived from polyoma. adenovirus, retrovirus.
3~ baculovirus, simian virus, 3-phosphoglycerate kinase promoter. yeast acid phosphatase promoters, yeast alpha-mating factors and combinations thereof.
The host cell which may be transfected with the vector of this invention may SL:": _...~~.E SHEET (RULE 26) be selected from the group consisting of E.coli, pseudomonas. bacillus subtillus.
bacillus stearothermophilus. or other bacili; other bacteria, yeast, fungi, insect, mouse or other animal, plant hosts, or human tissue cells.
Subcellular localisation of Ese proteins To determine the subcellular localization of Ese 1, a myc-epitope tagged version of this protein (mycEsel ) was engineered. This tagged protein was expressed in Cos cells and detected by confocal immunofluoresence using the mouse anti-myc monoclonal antibody 9E 10 (Figure 4). Interestingly, the transfected Ese 1 protein is highly concentrated into circular domains (Figure 4A and 4A') which are present throughout transfected cells (compare single optical section in Figure 4A with the projection of ail sections through the same cell shown in 4A'). In some optical sections rings of fluorescent staining surrounding a non-staining area were observed suggesting that the Esel induced structures are vesicles rather than inclusion bodies (data not shown and Figure 6). Esel overexpressed in IOTl/2, BHK and Hela cells using the vaccinia virus T7 expression system is also localized to large circular domains (data not shown). This concentration of ectopically expressed Ese 1 contrasts with the localization of EpslS in transiently transfected cells, EpslS being dispersed throughout the cell (Figure 4B). Esel and EpslS proteins form a complex in vivo and yet localize to distinct subcellular compartments in transfected Cos cells. We therefore determined the localization of both mycEsel and EpslS in Cos cells co-transfected with both genes. In co-transfected cells, mycEse 1 is still found in circular domains (Figure ~1C). Interestingly, the transfected EpslS is now also partially co-localized with Ese 1 in the same circular domains (Figure 4C' and C").
The C-terminal third of EpslS contains several regions which are required for association with a-adaptin of the AP2 clathrin adaptor complex (31 ). It has been shown that this region of EpslS is not required for its association with Esel (Figure

4). In order to test whether Eps 15 function may be required for Ese localization, mycEsel and Eps158C have been co-transfected. Interestingly, mycEsel localization is still partially colocalized with Eps158C (Figure 4D, D' and D") but is no longer found to be concentrated within circular structures, indicating that Ese 1 requires EpslS function for distinctive subcellular localization in transfected cells.
In addition, these data suggest that the Esel:EpslS complex may require association with AP2 which binds to the EpslS C-terminus in order to form the large circular domains in Esel transfected cells.
SUBSTTTUTE SHEET (RULE 26) Identification of bindin~partners of Ese proteins In order to identify Ese partners, a systematic analysis was initiated of each domain for binding partners using the yeast two hybrid system. The central coiled-coil domain of Esel from amino acid 330 to 732 was fused to the GAL4 DNA binding ~ domain and transformed into the Y 190 reporter strain together with plasmid cDNA
libraries from several tissue sources. Yeast colonies were selected for growth on Histidine minus plates in the presence of 40mM 3-amino triazole to select for interaction between library encoded GAL4 activation domain fusions and the DNA binding domain Esel coiled-coil bait. Yeast colonies which survived selection for expression of the His3 gene where also tested for induction of the integrated LacZ
gene which is GAL4 responsive in Y190. Prey plasmids were recovered from 37 positive yeast colonies which were selected for expression of both His3 and LacZ.
Several classes of cDNA were recovered in this screen. One class included Eps 1 ~
which was isolated twice and Eps 1 SR which was isolated from four yeast colonies.
Interestingly these clones were all partial cDNA fusions which minimally included C-terminal sequences from the central coiled-coil domain to the 3'UTR (Figure 3A). In the case of EpslS, the positive clones start from amino acid 306 and 376 whereas in Eps 1 ~R the N-terminal boundary of clones were amino acid 4, 10, 222 and 386.
These data indicate that minimal sequences required for interaction between the Ese 1 coiled-coil domain and EpsiS(R) include either or both of the central coiled-coil and/or the C-terminal third of these proteins.
The Ese proteins were analyzed in vivo to test for their association with Epsl~
or EpslSR. Polyclonal antisera were raised in chickens against a GST fusion containing the C-terminus of Esel from amino acid 665 to the stop codon. This region of Esel contains all five of the SH3 domains. In addition, polyclonal antisera were generated in rabbits against a peptide representing the first 21 amino acids of Esel. Cell lysates were prepared from A431, PC12, MDCK and Hela cells which represent cell lines from several distinct tissue types and species. The rabbit anti-peptide antisera were used to precipitate Ese 1 from each lysate and precipitates were analyzed by western blotting using the chicken anti-Ese sera. In each cell line the presence of several specific bands in the range of 150kDa were observed which were precipitated in the absence but not in the presence of the peptide to which the sera was generated (Figure 3A). Thus the Esel protein is expressed in many tissue culture cells and runs in a range consistent with the predicted molecular wei;~=~~ of 137kDa. The 3~ same samples were also analyzed for co-immunoprecipitation of EpslS
proteins. In each case multiple Eps 15 proteins were detected which co-purify with Ese 1.
The anti-Esel peptide antisera and commercially available anti-EpslS antisera which were SUBSTITUTE SHEET (RULE 26) used were raised against epitopes which are not shared by these proteins indicating that Ese l and Eps 15 are constitutively associated partners in vivo. This association is reminiscent of the previously detected complex between two EH domain containing yeast proteins, End3p and Panlp.
Esel and EpslS proteins both contain central coiled-coil motifs. In addition, Esel contains multiple C-terminal SH3 domains while EpslS contains SH3-binding motifs. In order to map the regions of each protein which are required for their association in vivo, C-terminal truncations of each (Eps158C and EselBC) were generated. Full length EpslS was co-transfected into Cos-1 cells together with either full length Ese 1 or C-terminally truncated Ese 1 sC. Cell lysates were precipitated with either rabbit anti-Epsl~ or with rabbit anti-Esel, and precipitates were western blotted with chicken anti Ese 1 antisera. Interestingly, the C-terminally truncated Ese 1 protein was efficiently immunoprecipitated in a complex with Eps 15 using anti-Eps 15 sera.
In a reciprocal experiment Cos-1 cells were transfected with Esel alone or together with Eps 15 or C-terminally truncated Eps 15 which had been Flag-epitope tagged (Eps 158C-Flag). Cell lysates were prepared and immunoprecipitated with either rabbit anti-Esel, mouse anti-Flag, or rabbit anti-EpslS antibodies. These immunoprecipitations were also western blotted to analyze for the presence of Esel (Figure 3B). The anti-Flag antibody efficiently precipitated Esel from cells expressing Flag-tagged EpsI~SC indicating that the C-terminally truncated EpslS
protein can bind to Ese 1 in vivo. The Ese l and Eps I 5 proteins are therefore associated through interaction of their central coiled-coil regions and do not require the presence of SH3 and SH3-binding motifs in their respective C-termini.
In addition to the identification of Epsl~ and EpslSR in the yeast two hybrid screen with the coiled-coil domain bait of Esel, the following were also identified as Ese binding proteins: TSG101 (accession #U52945), meningioma expressed antigens 6/11 (accession #U94780 for mea6), p-tropomyosin, rabaptin5 (accession #D86066), Adora2a (accession #Y13345), Ll lipid binding protein {accession #K02109) and nttmerous cytokeratins and laminins. Novel genes identified in this screen are detailed below.
Also performed was a yeast two hybrid screen using amino acids 66~-1213 of Esel as bait. This screen led to the isolation of the following clones which produced GAL4 Activation domain fusions which bound to this SH3 domain bait of Ese 1.
The Ese-binding proteins identified in our SH3 screen were the cbl-b oncoprotein (accession #U26712). Dynamin II (accession #L31398), KIAA0268 (accession #D87742), Jerky (accession #U35730), hnRNP-K (accession #L29769), SAP49 SUBSTTTLJTE SHEET (RULE 26) (accession #L3~013) and SOS-1 (accession #Z11574). Novel genes identified in this screen are detailed below and several novel clones as outlined below.
These results demonstrate that the novel Ese genes and the proteins that they encode function in a complex with Eps 1 ~ proteins to regulate endocytosis together. In addition, this complex contains binding sites for numerous other proteins.
Furthermore. with the identification of several potential phosphorylation sites on the Ese proteins, these results also suggest that whether or not complexed with Epsl ~, Ese proteins are involved in intracellular signalling processes which are likely to lead to altered cellular activity. Many Ese partners have been identified in these studies. Also revealed is a novel method to identify more Ese partners. Yeast cells have a complex formed by two EH domain proteins (Panlp and End3p) which regulates both endocytosis as well as the actin cytoskeleton. Indeed, the Eps 15 protein has been reported to regulate both endocytosis and the actin cytoskeleton ( 1 ). As Eps 15 and Ese function together. and Ese contain many protein-protein interaction surfaces on this complex, strongly suggesting that the Ese proteins and their binding proteins are critical regulators of Eps I S:Ese functions in vivo.
Ese and Dvnamin Yeast two-hybrid screens using baits composed of the GAL4 DNA-binding domain fused to individual SH3 domains of Ese 1 were performed to identify Ese partners which bind the SH3s of this multi-domain protein. Initial screens with SH3B
and SH3C domain fusions resulted in the artifactual isolation of many proline-rich fragments which did not represent real Ese partners. Screens were then done with GAL4 fusions containing all f ve SH3 domains from amino acid 66~ to 1213.
Forty one His3+/LacZ+ colonies from such screens were selected for further analysis.
Six colonies were found to encode fragments of the Dynamin II gene and two encoded fragments of Dynamin I (Figure SA). All Dynamin II clones had 5' start sites between amino acid 252 and 278 and terminated within the 3' UTR. The two Dynamin I clones were identical and contained a small coding rregion from amino acid 673 to the C-terminus. Dynamin sequences contained within each Esel-interacting clone therefore minimally code for the proline-rich motifs (Figure SA) which are known to bind SH3 domains in vitro (44). This result is consistent with the interaction between Dap 160 and Dynamin that has been recently described in D.-::~sophila (59). To determine the significance of Esel:Dynamin binding, functional 3s in:~ractions between these proteins were tested for. MycEsel was transfected into Cos cells. The subcellular locaiizatie~: . . ~ynamir: ,~ Lse i overexpressina cells was analyzed by staining for endogenous Dvnamin and the mvc tag on Ese 1. In many SUBSTITUTE SHE: LE 26) Ese 1 transfectants, endogenous Dynamin was recrutied to the Ese 1 staining vesicles and both proteins were co-localized (Figure 5B, panel a. b and c). These results demonstrate that Ese proteins bind Dynamin and can regulate its subcellular localization. Given that Ese 1 functions in a complex with Eps 15 and Ese can regulate the subcellular distribution of Dynamin, it was investigated whether EpslS
also co-localized with Dynamin. EpslS transfected into Cos cells was diffusely localized (Figure 4). Endogenous Dynamin was not concentrated and was therefore not visable in Eps 15 transfected cells (data not shown). When Ese l and Eps 15 were co-transfected however, Eps 15 and endogenous Dynamin are colocalized on the Ese 1 induced circles (Figure 5, panels d, a and fJ indicating that all three proteins co-localize at these structures.
Ese and Epsin Family Proteins In order to identify additional Ese partners, a fusion between GAL4-DBD and the N-terminal EH domain region of Esel (amino acids I-393) was generated.
This fusion protein was also used in yeast two hybrid screens with several GAL4 activation domain cDNA libraries. A total of 1 i His3+/LacZ+ colonies were identified as containing library encoded fusion proteins which interact with the Ese 1 EH-domain bait. Two of these clones coded for mouse homologues of the Epsin protein which was recently identified on the basis of its affinity for EpslS, and a-adaptin and is required for clathrin-mediated endocytosis (63). The two Epsin clones code for C-terminal fragments from amino acids 403 and 470 respectively. One of the Ese 1 EH-domain interacting clones which was obtained coded for a C-terminal fragment of a novel Epsin-family protein (63, 64). This cDNA has been independently isolated and named Ibp-2 in accession #AF057286. The Ibp2 clone which has been identified includes amino acids 326 to 509 of the partial coding sequence in Genbank.
Each Epsin family protein which has been isolated contains a C-terminal region which codes for three copies of an NPF motif. This is also the region of Epsin which binds to EpslS. It has previously been shown that AP-2 can independently bind both Epsin and Eps 15. It is now demonstrated that the C-terminus of Epsin family proteins can bind not only to the EH domains of Eps 15 but also to the EH domains of Ese 1.
These data suggest that either multiple Epsin proteins exist in the Ese:EpslS
complex or the interaction between individual proteins in various AP-2:Epsin:EpslS:Ese:Dynamin complexes may be subject to dynamic rearrangement during clathrin coated pit formation. invagination and scission. Also identified was a mammalian homologue of drosopnila, SINA, seven in ansentia.
SUBSTITUTE SHEET (RULE 26) Ese 1 Overexpression Blocks Endocvtosis Overexpression of Pan 1 p in yeast induces phenotypes which are identical to those observed in pant loss-of fucntion mutants (65) suggesting that by altering the relative ratios of Panlp to its numerous partners, the function of higher order Panlp complexes may be blocked. As the Ese:Eps 15 complex contains many protein-protein interaction domains which bind partners including Dynamin, Epsin and AP-2, it was thought that overexpressed Esel may disrupt the formation of higher order complexes between Ese proteins and partners. It was therefore tested whether clathrin-mediated endocytosis was functional in myc Esel overexpressing cells. Cos-1 cells were transfected with myc-tagged Esel, and 48 hrs post transfection, FITC-labelled Transferrin was added to cultures for 30 minutes. Cells were then fixed and analyzed for expression of mycEsel and for internalization of Transferrin. As shown in Figure 6, transfected cells do not internalize transferrin in contrast to their untransfected neighbors. Cell counts in a representative experiment indicate that 96% of Ese I
overexpressing cells do not internalize labeled transferrin (N=46) whereas 100% of untransfected cells were capable of clathrin-mediated endocytosis (N=100). It appears that overexpression of Dynamin II may overnde the Ese 1 induced endocytic block.
These data indicate that overexpression of Ese 1 blocks endocytosis and this may be through sequestration of Dynamin or other Ese 1 partners into non-productive binary complexes during endocytosis or recycling of the tranfernn receptor.
Overexpressed Ese 1 would therefore be functioning as a dominant inhibitory protein through recruitment of partners into non-productive complexes which do not contain all of the necessary components for endocytosis to proceed.
Taken together, these studies demonstrate the identification and isolation of novel mammalian Ese proteins encoded by novel Ese genes which are involved in the regulation of endocytosis via clathrin-coated pits, vesicular trafficking and actin dynamics. These studies also demonstrate the identification and isolation of two alternative transcripts of the genes. named Ese 1 L and Ese2L. The process of endocytosis including receptor-mediated endocytosis as well as pinocytosis or non-eceptor mediated endocytosis. The novel Ese proteins of the present invention appear to function to regulate endocvtosis involving the formation of ciathrin-coated pits by the polymerization of clathrin into a lattice along the cytosolic face of a region of membrane causing the region to expand inward. Ultimately, the pit pinches off from the membrane. and the clathrin cage is completed thus producing a coated vesicle.
Through the binding of Esp 1 ~ to form an Ese-Eps I 5 complex via a central binding SUBSTTTUTE SHEET (RULE 26) _73_ region. the complex can recruit other proteins such as, Epsin, AP-2 and dynamin and promote coated vesicle formation and perhaps their transport to appropriate locations within the cell and release of internalized proteins and/or molecules (Figure 7). The SH3 C-terminal domains of the Ese proteins can bind and interact with several other proteins leading to a host of protein-protein interactions involved in subcellular trafficking and signalling. The Ese proteins may be activated or inactivated via phosphorylation of the proteins at numerous phosphoryiation sites by the action of activated receptors on cell surfaces.
Due to the fact that the Ese proteins appear to be a key central player in the complex process of endocytosis involving protein-protein interactions and intracellular signalling, these proteins are most likely involved in a myriad of clinical conditions and processes which are very likely to include but not be limited to regulation of endocytosis (as described above), cell division and cancer (EpslS and cbl are oncoproteins), cell migration (regulation of the actin cytoskeleton is required for many forms of cell migration), cell polarity, plane of cell division and cell fate specification (EpslS binds to Numb in vivo which is required for these processes (43), RNA localization (several RNA binding proteins have been identified in the present screens) and viral infection and life cycle (EpslS binds to RAB a cellular cofactor for HIV Rev (43)).
With respect to viral infection Ese proteins may play an important role and thus may be a target for developing therapeutic strategies against viral infection and virally-induced disease states. HIV is known to alter endocytosis of several important cell surface molecules including CD4 and MHC antigens. HIV-NEF has been demonstrated to bind to SH3 domains. NEF induces clathrin coated pit formation. As NEF binds SH3 domains and induces endocytosis, it is possible that Ese proteins may bind to NEF and are involved in NEF function. Therapeutic strategies to provide treatment for viral infection and virally induced disease states may therefore include the inhibition of Ese-NEF interactions, antibodies or other agents directed against Ese complexes to inhibit endocytosis and in this manner inhibit viral infection and virally induced disease states. It is also expected that many types of viruses will interact with the mufti-component EpslS-Ese complex.
Synaptic transmission and abnormal or altered synaptic transmission as seen in various nervous disorders may also be a target for the therapeutic use of Ese proteins and/or antagonists. The EpslS:Ese complex has been demonstrated to regulate endocvtosis, is highly expressed in the brain and Ese binds to Jerky; a protein required to prevent epilepsy in mice (57). Futhermore, Ese proteins are highly expressed not onlv in the brain but also in the heart and in skeletal muscle which are tissues SUBSTITUTE SHEET (RULE Z6) _2~_ involving high levels of synaptic transmission. This suggests that Ese protein may be used in the treatment of nervous system disorders involving altered synaptic transmission.
Receptor-mediated cell signaling such as seen with several different types of growth factors also involves endocytosis. Over-expression, mutation or over-stimulation of growth factor receptors has been demonstrated to lead to abnormal cell division and growth as seen in cancer. For example, EGF is a potent mitogen for many epithelial cells and EGF receptor activation is known to stimulate intracellular kinase pathways leading to cell proliferation. Such activity may play a role in cancer progression.By altering the rate of endocytosis by targeting Ese proteins, the cell proliferative effect of growth factor receptor stimulation may be counteracted.
Abnormal cell division and cell migration is seen in several diseases and involves the cell cytoskeleton. The intracellular cytoskeleton is highly organized and consists of microtubules, microfilaments and intermediate filaments acting as an internal reinforcement in the cytoplasm of a cell. Together these structures associate in a regular and defined manner which is regulated by extracellular signals and may transduce plasma membrane signals by association with other proteins or by second messengers. The Ese-EpslS complex is very likely to regulate the cytoskeleton by analogy to the role of Panlp:End3p in regulation of yeast cytoskeleton.
Furthermore, endocytosis is known to involve a rearrangement of the intracellular cvtoskeleton.
Cell division and migration require the continual rearrangement of the intracellular cytoskeleton. Therefore, abnormal patterns of cell division and migration may involve altered Ese function and altered endocytosis. The Ese proteins or the genes may therefore be used to alter regulation of endocytosis or the association of the Ese proteins with the cytoskeleton and may restore cell division and migration to normal levels and patterns.
Tissue development also involves the continual remodeling of the cytoskeletal network along with its associated proteins. Developmental diseases can occur as a result of abnormal remodeling of the cytoskeleton leading to altered intracellular signaling. As Ese proteins are likely to be involved with both the cvtoskeleton and intracellular signaling they may also be directly involved in the development of certain developmental diseases and therefore may be a target for therapeutic treatment of such diseases. ~c° rroteins may also be involved in normal development including shat of stem celi~ . a:e self renewing cells that divide to produce differentiated 3 ~ daughter cells in var:us tissues. As Ese proteins are associated with the cvtoskeleton .~ gay play a part in the formation of certain types of differentiated cells through w-.;~nirioning of RNA and proteins such as nuMb durin~~ cell division.
SUBSTTTLTTE SHEET (RULE 26) T_ rans~enic Animal Models The creation of transgenic animal models for abnormal endocytotic function characterized by altered Esel or Ese2 activity is important to the understanding of the function of these proteins in intracellular signaling and for the testing of possible therapies for abnormal endocytosis involving protein-protein interactions and intracellular signalling and leading to various clinical conditions. In general, techniques of generating transgenic animals are widely accepted and practiced There are several ways iri which to create an animal model in which the Ese 1 or Ese2 gene expression or function is altered. One could simply generate a specific mutation in the mouse Ese 1 or Ese2 gene as one strategy. Secondly a wild type human Esel or Ese2 gene and/or a humanized marine gene could be inserted into the animals genome by homologous recombination. It is also possible to insert a mutant (single or multiple) human gene as genomic or minigene construct using wild type or mutant or artificial promoter elements. More commonly, and most preferred in the present invention, knock-out of the endogenous marine genes may be accomplished by the insertion of artificially modified fragments of the endogenous gene by homologous recombination. The modifications include insertion of mutant stop codons, the deletion of DNA sequences, or the inclusion of recombination elements (lox p sites) recognized by enzymes such as Cre recombinase. Gene knockout produces homozygous mutant mice, which show symptoms or phenotype similar to those exhibited by a human.
In general. for gene knock-out, embryonic stem cells heterozygous for a knockout mutation in a gene of interest (ie. Ese 1 or Ese2 gene) and homozygous for a marker gene (eg. coat colour) are transplanted into the blastocoel cavity of 4.5 day embryos homozygous for an alternate market. The early embryos then are implanted into a pseudopregnant female. Some of the resulting progeny are chimeras.
Chimeric mice then are backcrossed. Intercrossing will eventually produce individuals homozygous for the disrupted allele that is, knockout mice. (Capecchi, MR.
1989.
Science.244:1299-1291).
To inactivate the Ese 1 or Ese2 mouse gene chemical or x-ray mutagenesis of mouse gametes, followed by fertilization, can be applied. Heterozygous offspring can then be identified by Southern blotting to demonstrate loss of one allele by dosage, or failure to inherit one parental allele using RFLP markers.
To create a transgenic mouse, a mutant or normal version of the human Esel or Ese2 gene can be inserted into a mouse germ line using standard techniques of oocyte microinjection or transfection or microinjection into stem cells.
Alternatively, SUBSTITUTE SHEET (RULE 26) if it is desired to inactivate or replace the endogenous Ese I or Ese2 gene.
homologous recombination using embryonic stem cells may be applied.
For oocyte injection, one or more copies of a mutant or normal Esel or Ese2 gene can be inserted into the pronucleus of a just-fertilized mouse oocyte.
This oocvte is then reimplanted into a pseudo-pregnant foster mother. The liveborn mice can then be screened for integrants using analysis of tail DNA for the presence of transgenic Ese 1 or Ese2 gene sequences. The transgene can be either a complete genomic sequence injected as a YAC or chromosome fragment. a cDNA with either the natural promoter or a heterologous promoter, or a minigene containing ail of the coding region and other elements found to be necessary for optimum expression.
Retroviral infection of early embryos can also be done to insert the human or mouse Ese 1 or Ese2 gene. In this method, the Ese 1 or Ese2 gene is inserted into a retroviral vector which is used to directly infect mouse embryos during the early stages of development to generate a chimera, some of which will lead to germline transmission.
Homologous recombination using stem cells allows for the screening of gene transfer cells to identify the rare homologous recombination events. Once identified, these can be used to generate chimeras by injection of mouse blastocysts, and a proportion of the resulting mice will show germline transmission from the recombinant line. This methodology is especially useful if inactivation of the Esel or Ese2 gene is desired. For example. inactivation of the Esel or Esel gene can be done by designing a DNA fragment which contains sequences from a Ese 1 or Ese2 exon flanking a selectable marker. Homologous recombination leads to the insertion of the marker sequences in the middle of an exon, inactivating the Esel or Ese2 gene.
DNA
2~ analysis of individual clones can then be used to recognize the homologous recombination events.
This embodiment of the invention has the most significant commercial value as a mouse model for abnormal endocytotic activity and this may include disorders such as those involving abnormal cell division, cancer, abnormal cell migration, viral infection. abnormal tissue development and abnormal synaptic transmission disorders.
Theranv Gene therapy is another potential therapeutic approach for treating disorders involving abnormal endocytosis, vesicular trafficking and abnormal regulation the 3~ actin cvtoskeleton. Such disorders may include for example but not be limited to disorders such as those involving abnormal cell division, cancer, abnormal cell migration. viral infection. abnormal tissue development and abnormal synaptic SUBSTTTt'_ ."e"° SHEET (RULE 26) _7'7_ transmission disorders.
In such therapy, normal copies of a mammalian Ese gene are introduced into patients to code successfully for normal protein in several different affected cell types.
The gene must be delivered to those cells in a form in which it can be taken up and code for sufficient protein to provide effective function Retroviral vectors can be used for somatic cell gene therapy especially because of their high efficiency of infection and stable integration and expression.
The targeted cells however must be able to divide and the expression of the levels or normal protein should be high. The full length Ese gene can be cloned into a retroviral vector and driven from its endogenous promoter or from the retroviral long terminal repeat or from a promoter specific for the target cell type of interest.
Other viral vectors which can be used include adeno-associated virus, vaccinia virus. bovine papilloma virus, or a herpevirus such as Epstein-Barr virus.
Gene transfer could also be achieved using non-viral means requiring infection in vitro. This would include calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes may also be potentially beneficial for delivery of DNA
into a cell. Although these methods are available, many of these are lower efficiency.
Antisense based strategies can employed to explore mammalian Ese gene function and as a basis for therapeutic drug design. The principle is based on the hypothesis that sequence-specific suppression of gene expression can be achieved by intracellular hybridization between mRNA and a complementary antisense species.
The formation of a hybrid RNA duplex may then interfere with the processing/transport/translation and/or stability of the target Ese mRNA.
Hybridization is required for the antisense effect to occur, however the efficiency of intracellular hybridization is low and therefore the consequences of such an event may not be very successful. Antisense strategies may use a variety of approaches including the use of antisense oligonucleotides, injection of antisense RNA
and transfection of antisense RNA expression vectors. Antisense effects can be induced by control (sense) sequences, however, the extend of phenotypic changes are highly variable. Phenotypic effects induced by antisense effects are based on changes in criteria such as protein levels. Protein activity measurement, and target mRNA
levels.
Multidrug resistance is a useful model to study molecular events associated with phenotypic changes due to antisense effects, since the multidrug resistance phenotype can be established by expression of a single gene mdrl (MDR gene) encoding for P-glycoprotein.
Transplantation of normal genes into the affected area of the patient can also be useful therepy for any disease condition which includes abnormal endocytosis, SUBSTITUTE SHEET (RULE 26) _7g_ vesicular trafficking and abnormal regulation of the actin cytoskeleton. In this procedure, a normal human Ese gene is transferred into a cultivatable cell type, either exogenously or endogenously to the patient. These cells are then injected serotologically into the disease-affected tissue or tissues.
Immunotherapy is also possible for treating disorders which includes abnormal endocytosis, vesicular trafficking and abnormal regulation of the actin cytoskeleton.
Antibodies are raised to a mutant Ese protein (or a portion thereof) and are administered to the patient to bind or block the mutant protein and prevent its deleterious effects. Simultaneously, expression of the normal protein product could be encouraged. Alternatively, antibodies are raised to specific complexes between mutant or normal Ese proteins and their binding partners.
A further approach is to stimulate endogenous antibody production to the desired antigen. Administration could be in the form of a one time immunogenic preparation or vaccine immunization. An immunogenic composition may be prepared as injectables, as liquid solutions or emulsions. The Ese protein or other antigen may be mixed with pharmaceutically acceptable excipients compatible with the protein.
Such excipients may include water, saline, dextrose, glycerol, ethanol and combinations thereof. The immunogenic composition and vaccine may further contain auxiliary substances such as emulsifying agents or adjuvants to enhance effectivess. Immunogenic compositions and vaccines may be administered parenterally by injection subcutaneously or intramuscularly.
The immunogenic preparations and vaccines are administered in such amount as will be therapeutically effective, protective and immunogenic. Dosage depends on the route of administration and will vary according to the size of the host.
Screening for Disease In another embodiment of the invention the knowledge of mammalian Esel and Ese2 cDNA sequences provides for screening by conventional methods to obtain the corresponding human sequences and thus screening for various diseases involving abnormal Esel or Ese2 (or alternative transcripts thereof) in which the defect is due to a mutant Esel or Ese2 gene and thus an altered and abnormal endocytosis process involved in various disorders. Mutant forms of the protein may not be able to bind with their normal binding partners and thus endocvtosis, vesicular trafficking and/or actin dynamics are negatively affected. Such defects may include, for example.
3~ cancer. Other defects may include abnormal cell division. abnormal cell migration, viral infection. abnormal receptor signalling, abnormal tissue development and abnormal synaptic transmission disorders. People at a risk for such an abnormality or, SUBSTITUTE SHEET (RULE 26) individuals not previously known to be at risk. or people in general may be screened routinely using probes to detect the presence of a mutant Ese 1 or Ese2 gene by~ a variety of techniques. Genomic DNA used for the diagnosis may be obtained from body cells, such as those present in the blood, tissue biopsy, surgical specimen, or autopsy material. The DNA may be isolated and used directly for detection of a specific sequence or may be PCR amplified prior to analysis. RNA or cDNA may also be used. To detect a specific DNA sequence hybridization using specific oligonucleotides, direct DNA sequencing, restriction enzyme digest. RNase protection, chemical cleavage, and ligase-mediated detection are all methods which can be utilized. Oligonucleotides specific to mutant sequences can be chemically synthesized and labelled radioactively with isotopes, or non-radioactively using biotin tags. and hybridized to individual DNA samples immobilized on membranes or other solid-supports by dot-blot or transfer from gels after electrophoresis. The presence or absence of these mutant sequences are then visualized using methods such as autoradiography, fluorometry, or colorimetric reaction. Suitable PCR primers can be generated which are useful for example in amplifying portions of the subject sequence containing identified mutations.
Direct DNA sequencing reveals sequence differences between normal and mutant Esel or Ese2 DNA. Cloned DNA segments may be used as probes to detect specific DNA segments. PCR can be used to enhance the sensitivity of this method.
PCR is an enzymatic amplification directed by sequence-specific primers, and involves repeated cycles of heat denaturation of the DNA, annealing of the complementary primers and extension of the annealed primer with a DNA
polymerase. This results in an exponential increase of the target DNA.
Other nucleotide sequence amplification techniques may be used. such as lieation-mediated PCR, anchored PCR and enzymatic amplification as would be understood by those skilled in the art.
Sequence alterations may also generate fortuitous restriction enzyme recognition sites which are revealed by the use of appropriate enzyme digestion followed by gel-blot hybridization. DNA fragments carrying the site (normal or mutant) are detected by their increase or reduction in size, or by the increase or decrease of corresponding restriction fragment numbers. Genomic DNA samples may also be amplified by PCR prior to treatment with the appropriate restriction enzyme and the fragments of different sizes are visualized under UV light in the presence of 3~ ethidium bromide after gel electrophoresis. Alternatively fluorography may be employed.
Genetic testing based on DNA sequence differences may be achieved by SUBSTITUTE SHEET (RULE 26) detection of alteration in electrophoretic mobility of DNA fragments in gels.
Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. Small deletions may also be detected as changes in the migration pattern of DNA heteroduplexes in non-denaturing gel electrophoresis.
Alternatively, a single base substitution mutation may be detected based on differential primer length in PCR. The PCR products of the normal and mutant gene could be differentially detected in acrylamide gels.
Nuclease protection assays (S1 or ligase-mediated) also reveal sequence changes at specific locations. Alternatively, to confirm or detect a polymorphism restriction mapping changes ligated PCR, ASO, REF-SSCP and SSCP may be used.
Both REF-SSCP and SSCP are mobility shift assays which are based upon the change in conformation due to mutations.
DNA fragments may also be visualized by methods in which the individual DNA samples are not immobilized on membranes. The probe and target sequences may be in solution or the probe sequence may be immobilized. Autoradiography, radioactive decay, spectrophotometry, and fluorometry may also be used to identify specific individual genotypes.
According to an embodiment of the invention, the portion of the DNA
segment that is informative for a mutation, can be amplified using PCR. The DNA
segment immediately surrounding a specific mutation acquired from peripheral blood or other tissue samples from an individual can be screened using constructed oligonucleotide primers. This region would then be amplified by PCR, the products separated by electrophoresis, and transferred to membrane. Labeled probes are then hybridized to the DNA fragments and autoradiography performed.
In a further embodiment, the invention provides pharmaceutical compositions comprising Esel or Ese2 proteins or a functional analogue or mimetic of these proteins or their alternative transcripts for the treatment of certain disorders characterized by abnormal endocytosis and thus cell-signalling due to lack or absence of the proteins. Such disorders may include but are not limited to abnormal cell division, cancer. viral infection, abnormal synaptic transmission as seen in central nervous disorders and abnormal cell differentiation. Such compositions as provided herein can be appropriately packaged and targeted to specific cells and/or tissues.
Administration of a therapeutically active amount of a pharmaceutical composition of the present invention means an amount effective. at dosages and for periods of time necessary to achieve the desired result. This may also vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the Ese 1 or Ese2 proteins, peptides or alternative transcripts ( for example Ese 1 L
SUBSTT~TTE SHEET (RULE 26) and Ese2L) to elicit a desired response in the subject. Dosage regima may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
By pharmaceutically acceptable carver as used herein is meant one or more compatible solid or liquid delivery systems. Some examples of pharmaceutically acceptable carriers are sugars, starches, cellulose and its derivatives, powdered tragacanth, malt, gelatin, collagen, talc, stearic acids, magnesium stearate, calcium sulfate, vegetable oils, polyols, agar, alginic acids, pyrogen-free water, isotonic saline, phosphate buffer. and other suitable non-toxic substances used in pharmaceutical formulations. Other excipients such as wetting agents and lubricants.
tableting agents, stabilizers, anti-oxidants and preservatives are also contemplated.
The compositions described herein can be prepared by known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable carrier. Suitable carriers and formulations adapted for particular modes of administration are described, for example. in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985). On this basis the compositions include, albeit not exclusively, solutions of the substance in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
The pharmaceutical compositions of the invention may be administered therapeutically by various routes such as by injection or by oral, nasal, buccal, rectal, vaginal, transdermal or ocular routes in a variety of formulations, as is known to those skilled in the art.
The present invention also enables the analysis of factors affecting the expression of the Ese 1 or Ese2 gene in humans or in animal models. The invention further provides a system for screening candidate compounds for their ability to turn on or turn off expression of the Ese 1 or Ese2 gene or to screen compounds which are binding partners of these proteins.
For example, a cell culture system can be used to identify compounds which activate production of Esel or Ese2 proteins or, once Esel or Ese2 production has been activated in the cells, they can be used to identify compounds which lead to suppression or switching off of Esel or Ese2 protein production. Compounds thus identified are useful as therapeutics in conditions where Esel or Ese2 production is deficient or excessive.
SUBST'ITUT'E SHEET (RULE 26) Compounds can also be screened in culture for their ability to overcome the effect of Ese protein expression in cell culture, tissue culture or in an animal model.
The present invention enables also a screening method for compounds of therapeutic utility as antagonists of the biological activity, binding activity. of Ese 1 or Ese2 proteins and their binding partners. Those skilled in the art will be able to devise a number of possible screening methods for screening candidate compounds for Ese 1 or Ese2 protein antagonism.
A screening method may also be based on binding to the Ese 1 or Ese2 protein.
Such competitive binding assays are well known to those skilled in the art.
Once binding has been established for a particular compound. a biological activity assay is employed to determine agonist or antagonist potential.
Cell-free assays can also be readily designed by those skilled in the art to monitor and measure endocvtosis, vesicular trafficking and actin dynamics.
To summarize, Ese proteins, Ese complexes including Eps 15, Eps 1 SR and 1 ~ manv of the proteins identified in the screens as well as others identified through similar screens can be targeted for use in therapies to treat diseases including cancer, viral infection based diseases, developmental diseases due to altered cell fate specification and/or division as well as neurological diseases and diseases of altered cell migration and other diseases due to defects in the actin cytoskeleton.
EXAMPLES
The examples are described for the purposes of illustration and are not intended to limit the scope of the invention.
Methods of molecular genetics, protein and peptide biochemistry and immunology referred to but not explicitly described in this disclosure and examples are reported in the scientific literature and are well known to those skilled in the art.
Material and Methods Ese cloning and plasmids High Stringency screenine was used to isolate the two mouse Ese cDNAs by previously described methodology (58). Esel was cloned from an adult mouse brain cDNA library using a PCR product from nt 1707-2197 of the coding sequence as probe. Ese2 was cloned from a mixed tissue adult mouse cDNA library using a mixture of three probes EST#583881 (Research Genetics Inc.), EST#652549 (Research Genetics Inc.) and a PCR product from nt 2712 to nt 3456 of the Ese2 COQIIla sequence.
SUBSTITUTE SHEET (RULE 26) The Ese 1 sequence was obtained from a single clone, whereas the Ese2 reading frame was predicted from the overlap of two cDNA clones. The DBL/PH/C2 region of Ese 1 L was obtained using PCR with an upstream primer designed from sequences within the DBL/PH domain region:
GAAGGAGAACTCAGACCGGCTGGAGTGGAT (this sequence was obtained from one partial EselL clone which we had isolated from a mouse brain cDNA
library).
This upstream primer was paired with downstream primers for the vector. The DBL/PH/C2 region of Ese2L was obtained using PCR with upstream and downstream primers flanking the site in Ese2 where sequence divergence had been noted within an EST clone (upstream Ese2 sequence: GACAGAGGAGCGGTACATGGA and downstream Ese2 sequence: AGCTCCCCTGGTTCTGGCTTC). The mouse EpslS
cDNA was generated through a combination of high stringency library screening with Est sequences from the Eps 15 gene and rt PCT according to established methods.
pcDNA3Esel:
Full length Esel was cloned into the Notl site of pcDNA3 (Invitrogen Inc.).
The Esel cDNA includes 53 nucleotides of 5' UTR plus a natural NotI site and nucleotides of 3'UTR plus a small region of polylinker including a NotI site.
~cDNA3m c The 5' end of pcDNA3Ese1 from the EcoRI site in the pcDNA3 polylinker to the start codon was replaced with the DNA sequence GAATTCAGAACCATGGAACAAAAGCTTATTTCTGAAGAAGACTTGGGGCCC
ATG: where the first underline corresponds to an EcoRI site which was fused into the pcDNA3 EcoRI site and the extended underlined sequence codes for a myc-epitope tag.
This is followed by nine nucleotides which code for glycine, proline and the natural Ese 1 start codon. This sequence was joined to the sequences coding for amino acids (the remainder of Ese 1 ). The new start codon in this tagged Ese 1 construct is bolded.
The 3' end of Esel in this vector is the same as in pcDNA3Ese1 above.
gcDNA3EselsC:
The C-terminus of Ese 1 was removed by replacing all sequences from nt 2209 of the coding sequence to the XhoI site in pcDNA3Ese1 with TGACTCGAG where the stop codon is in bold and the XhoI site is underlined. This construct codes for amino acids 1-736 of Ese 1.
pCDNA3Eps15:
SUBSTTTUT'E SHEET (RULE 26) This plasmid was constructed from four pieces. It contains the full length EpslS. The 5'UTR of this construct has been constructed to be GGATCCACCATG
where a BamHI site is underlined and the start codon is bolded. This BamHI
site was fused to the BamHI site in pcDNA3. The 3'UTR in this vector is 204 nt of the mouse natural 3'UTR fused to a short cloning linker ending in the sequence AAGCTTGGGCCC where an ApaI site is underlined; this ApaI site was fused to the ApaI site in pcDNA3.
pcDNA3Epsl SsC:
This vector is the same as pcDNA3Epsl5 except that sequences downstream from and including mouse EpslS coding nucleotide 1500 have been replaced with CCTGGATTACAAGGATGATGATGACAAATGACTCGAG where the first underlined sequence codes for the Flag-epitope. an inframe stop codon is bolded and an XhoI site is underlined. This XhoI site was fused to the polylinker in pcDNA3.
The resulting plasmid encodes amino acids 1-501 of mouse EpslS fused to a C-terminal Flag epitope. The 5' end of EpslS in this construct is as indicated above for pcDNA3Eps15.
pGBT9Ese 1 cc:
The Esel sequence coding for amino acids 330 to 732 were fused directly to GAATTC (EcoRI site) on the 5' end and to TAGGATCC (stop codon followed by a BamHI site) on the 3' end. This fragment was cloned into EcoRI/BamHI digested pGBT9 in frame with the GAL4 DBD.
pGBT9Ese 1 A3.3 This plasmid encodes the bait for our SH3 screen. It encodes all five SH3 domains from amino acid 665-1213 and was subcloned into pGBT9 on an EcoRI
fragment which fuses the Ese 1 SH3 region in frame with the DBD of GAL4.
PGBT9EselNterm:
This plasmid codes for the N-terminal 393 amino acids of Esel, including both EH domains. It was subcloned into pGBT9 on an EcoRI/SaII fragment.
Yeast 2-Hybrid Screening 3 5 pGBT9Ese 1 ce was transformed into S. cerevisiae strain Y 190 using 45%
PEG4000, 100 mM LiAc. 1 U mi'~t Tris-H~~i tpH 7.5:i as per standard proto;:va.
Cells were plated onto Sc-Trp drop-out media. Single colonies were isolated and axpression SUBSTT>CUTE SHEET (RULE Z6) of bait was analyzed by Western Blot using antibodies against the GAL4 DBD. A
clone expressing the bait fusion was used to inoculate a 100 mL Sc-Trp liquid culture grown overnight at 30°C. Cells were then re-inoculated into YPD at a density of 5x106 cells/mL and grown at 30°C until the titer reached 2x10 cells/mL.
Cells were pelleted, resuspended in 50 mL 100 mM LiAc and incubated for 10 min at 30°C.
Once again the cells were pelleted, resuspended in 20 mL PLA [35% PEG, 100 mM
LiAc, 2 mg/mL salmon sperm DNA] containing 30 ~g plasmid library (cloned in pAD-GAL4) incubated at 30°C for 30 min.; then heat shocked at 42°C for 40 min., pelleted, resuspended in water and plated onto [Sc-Trp-Leu-His+40 mM 3-AT].
Plates were incubated at 30°C until colonies were formed. Colonies were picked, patched and grown at 30°C overnight on Whatman filter paper /aid on top of Sc-Trp-Leu-His plates. Filters were submerged in liquid nitrogen for 15 seconds then placed on top of blotting paper soaked in Z-buffer + X-gal. ~-galactosidase activity was measured by the appearance of blue colour. Plasmids from p-galactosidase positive colonies were 1 S shuttled to bacteria by electroporation and isolated for sequencing.
Northern Blot analysis A multiple tissue northern blot (Clontech) was prehybridized in 5 mL of ExpressHyb Solution (Clontech) at 68°C for 30 min. Probe was added at 1x106 cpm/mL for 1 hr. The Blot was washed twice (2X SSC, 0.05% SDS) at room temperature, twice (O.1X SSC, 0.1% SDS) at 50°C and then exposed to film overnight.
Antibodies and Western Blot analysis Western blot analysis was performed according to standard protocols. Briefly, cultured cell lines or 48 hours post tranfection Cos-1 cells where washed with PBS
and lysed in one ml of cold lysis buffer (50 mM Hepes pH 7.4, 150 mM NaCI, 10%
glycerol, 1 % Triton X 100, 1 mM EGTA, 1.5 mM MgCI?, 10 mM NaF, 10 mg ml-I
aprotinin, 1 mM PMSF, 10 mg ml-~ leupeptin, 1 mM Na3V04). Supernatants were clarified by centrifugation and immunoprecipitated with specific antiserum as indicated. Antigen-Antibody complexes where purified on anti-rabbit agarose or anti-mouse agarose (Sigma Chemical Co.) Samples were run on 7.5% PAGE gels and transferred to nitrocellulose membranes. Filters were blocked in 5% dry milk powder/0.05% Tween 20/PBS. washed in 1% dry milk powder/0.05% Tween 20/PBS, 3~ and probed with the appropriate antisera at lmgiml in wash buffer (In the case of chicken anti-Esel we used lOpg/ml to probe western blots). Probed filters were SUBSTITUTE SHEET (RULE 26) further washed. probed again with I /5000 dilution of horseradish peroxidase conjugated anti-rabbit IgG, anti-mouse IgG antibodies (Amersham), or anti-chicken-IgY (Zymed); washed and signal detected using the Enhanced Chemiluminescence detection system as per manufacturers instructions (Amersham).
Rabbit anti-Ese 1 antisera was raised against a peptide of the following sequence: MAQFPTPFGGSLDVWAITVEE. The antisera was affinity purified over the same peptide (Research Genetics). This peptide was also used at S~g to compete for the S~g of antibody per immunoprecipitation reaction. Chicken anti-Esel antisera was raised against a fusion protein between GST and amino acids 665-1213 of mouse Ese 1. This sera was cleared of antibodies reacting against GST by incubation with glutathione s-transferase on glutathione agarose beads.
Rabbit antibodies have also been raised against a peptide encoding the first twenty one amino acids of mouse Ese2. These antibodies were affinity purified and used to immunoprecipitate endogenous Ese 1 containing protein complexes from cultured PC 12 cells. A protein of approximately 130 kDa was co-immunoprecipitated in complex with Ese2. This protein was isolated from a silver stained gel slice, digested with trypsin and the resulting peptide fragments were analyzed using both MALDI-TOF and Q-TOF Mass Spectrometers. Taken together, these mass spec analyses revealed that the Ese-2 binding protein in question is a Rho-Family exchange protein variably known as KIAA0362, Dbs and Ost. The gene encoding KIAA0362, Dbs and Ost proteins is subject to complex alternative splicing but each protein is predicted to contain the DBL/PH exchange domain. Thus Ese proteins are found in complex with exchange proteins for the Rho-family of small GTP-binding proteins.
2~
Monoclonal antibodies against Ese proteins were produced by immunizing mice with a GST fusion protein encoding the Esel C-terminus (from amino acid to the stop codon) according to standard protocols. After multiple injections, the mouse spleens were removed and resuspended in phosphate buffered saline (PBS).
The spleen cells served as a source of lymphocytes. These lymphocytes were fused with a perm:~_nentiy growin~~ myeloma partner cell, and the products of the fusion were plated into 96we11 plates in the presence of selective media. The culture supernatents were then screened by ELISA to identify those containing cells which were secreting anti-Ese antibodies. A histidine tagged version of the EselC-terminus was used in 3~ these ELISA screens to ensure that anti-Ese antibodies were detected rather than aIILiUOdIeS directed against the GST
SUBSTITUTE SHEET (RULE 26) portion of our original immunogen. From this procedure we isolated several independent stable hybridoma cloned lines which secrete anti-Ese antibodies.
The monoclonal antibodies have then been purified on protein A/G columns as per standard protocols and demonstrated to bind Ese protein.
Immunofluorescence For Immunofluorescent staining, Cos-1 cells were typically plated at a density of 2x10' per 22x50mm coverglass and transfected with 2.Spg of plasmid using Superfect (Qiagen Inc.). After 2 hours, the cells were washed with 10%FBS in Iscove's Media and fed with fresh 10%FBS in Iscove's Media. Two days later, these cultures where fixed with cold methanol for 30 minutes at room temperature.
Cultures were washed three times 10 minutes with Phosphate buffered saline (PBS), blocked for one hour at room temperature with 1%BSA in PBS and then incubated with primary antibody in blocking solution for one hour, also at room temperature.
Slides were then washed three times 10 minutes with PBS, incubated with secondary antibody/l% BSA/PBS in the dark for one hour at room temperature. Finally.
slides were washed three times 10 minutes in the dark at room temperature and mounted using Dabco anti-fade solution (Sigma Chemical Co.). Slides were analyzed on the confocal microscope using the 63x objective and optical filters to separate signals on each channel.
For primary antibodies Mouse anti-myc monoclonal 9E 10 ( 1 O~g/ml: Santa Cruz Biotech.), Rabbit anti-myc (S~g/ml: Upstate Biotechnology Inc.), Rabbit anti-Eps 1 ~ antisera #C20 ( 1 p,g/ml: Santa Cruz Biotech), Rabbit anti-Flag epitope antisera (Sp.g/ml: Zymed) and mouse anti-Dynamin I #D25520, which recognizes Dynamin in Cos-1 cells by both immunoprecipitation and western blotting (data not shown]
(20~g/ml: Transduction labs, Inc.) were used. As secondary antibodies we used FITC-labelled goat anti-mouse ( 1:80 dilution) and Texas Red-labelled goat anti-rabbit antibodies (1:100 dilution) (Jackson Immuno Research Laboratories Inc.).
Endogenous Eps 1 S staining above background in non-transfected cells was undetected. Prominent cytoplasmic Epsl ~ staining in transfected cells obscured the detection of clathrin-coated pit associated EpslS at the membrane (Figure 4B).
In Figure ~B panel a and b, Dynamin staining with both primary and secondary' antibodies was performed first. MycEsel was then stained for using biotinylated 9E10 followed by streptavidin conjugated to Texas Red. Following application of the secondary antibody to stain for Dynamin. alt further incubations and washes were SUBSTITUTE SHEET (RULE 26) performed under dark condition. Endocytosis assays were performed as previously described (6, 9).
Mouse Ese 1 cDNA - Sequence ID NO: I
CGGCACGAGGAGGAGTGGAGCGGCGCGGGAGGGCGCGCAGCTTGGTTGC
TCCGTAGTACGGCGGCTCGCAAGGGAGCATCCCGAGCGGGCTCCGGGACG
GCCGGGAGGCAGGCAGGCGGGCGGGCGGGGATGGTGTGCGCGGCTGCGG
ACTCGGCGTTCCTCGCGCGGCGTGCGGGCTGCACTGATTTGTGTGAGGGG
CGGCCGCGCGCACCCGCCCGGAGATGAGGCGTCGATCAGCAAGGTGAAC
GTAATAGAACCATGGCTCAGTTTCCCACACCTTTCGGTGGTAGCCTGGATG
TCTGGGCCATAACTGTGGAGGAAAGGGCCAAGCATGACCAGCAGTTCCTT
AGCCTGAAGCCGATAGCGGGATTTATTACTGGTGATCAAGCGAGGAACTT
TTTTTTCCAATCTGGGTTACCTCAGCCTGTCTTAGCACAAATATGGGCGCT
AGCGGACATGAATAACGATGGAAGGATGGATCAAGTGGAATTTTCCATAG
CCATGAAGCTTATCA.AACTGAAGCTACAAGGATATCAGCTCCCCTCCACA
CTTCCCCCTGTCATGAAACAGCAACCAGTGGCTATTTCCAGTGCACCAGCA
TTTGGTATAGGAGGGATTGCTAGCATGCCACCACTCACAGCTGTTGCTCCT
GTGCCAATGGGCTCCATTCCAGTTGTTGGAATGTCTCCACCCTTAGTATCT
TCTGTCCCTCCAGCAGCAGTGCCTCCCCTGGCTAACGGGGCTCCTCCCGTC
ATACAGCCTCTGCCTGCGTTTGCGCATCCTGCAGCCACATGGCCAAAGAG
TTCTTCCTTCAGCAGATCTGGTCCAGGGTCACAATTAAACACTAAGTTACA
GAAGGCACAATCATTCGATGTCGCCAGCGCCCCTCCAGCAGCAGAATGGG
CTGTGCCTCAGTCATCAAGGCTGAAATACAGGCAGTTATTCAACAGCCAC
GACAAA.ACTATGAGTGGACACTTAACAGGTCCCCAGGCAAGAACTATTCT
CATGCAATCAAGTTTACCCCAGGCTCAGCTGGCTTCAATATGGAATCTTTC
TGACATTGATCAAGATGGAAAACTCACTGCAGAAGAATTTATCCTAGCTA
TGCACCTAATTGATGTTGCCATGTCTGGTCAGCCACTGCCGCCCGTCCTGC
CTCCAGAATACATCCCTCCTTCCTTCAGAAGAGTTCGCTCCGGCAGTGGGA
TGTCCGTCATAAGCTCTTCTTCTGTGGATCAGAGGCTGCCTGAGGAGCCGT
CGTCAGAGGATGAGCAGCAGCCAGAGAAG.aAACTGCCTGTGACATTTGAA
GATAAGAAGCGGGAGAACTTCGAGCGAGGCAGTGTGGAGCTGGAGAAGC
GCCGCCAAGCGCTCTTGGAGCAGCAGCGCAAAGAGCAGGAGCGGTTGGC
TCAGCTGGAGCGCGCCGAGCAGGAGAGGAAAGAGCGGGAGCG~'~aGGAG
CAGGAGGCCAAGCGGCAGCTGGAGCTGGAGAAGCAGCTGGAG:-.
GGGAGCTGGAGCGGC.-':~CGAGAGGAGGAGAGGAGGAAGGAGATCGAGA
GG~. ~=~ GAGGCCGCA~.-L~CGGGAACTGGAAAGGCAGCGACAACTTGAATG
GG=-:-..~~~rGAACCGGAGACAGGAACTCCTGAATCAGAGGAACAAGGAGCAG
SUBSTITUTE SHEET (RULE 26) GAGGGCACCGTGGTCCTGAAGGCAAGGAGGAAGACTCTGGAGTTTGAGTT
AGAAGCTCTGAATGACAAAAAGCATCAGCTAGAAGGAAAACTTCAGGAT
ATCAGGTGTCGACTGGCAACCCAGAGGCAAGAAATTGAGAGCACGAACA
AGTCTAGAGAGCTAAGAATTGCTGAAATCACCCACTTACAGCAGCAGTTG
CAGGAATCTCAGCAAATGCTTGGAAGACTTATTCCAGAGAAACAGATACT
CAGTGACCAGTTAAAACAAGTCCAGCAGAACAGTTTGCATAGAGACTCGC
TTCTTACCCTCAAAAGAGCCTTGGAAGCAAAGGAGCTGGCCCGGCAGCAG
CTCCGGGAGCAGCTGGACGAGGTGGAGAGAGAGACCAGGTCAAAGCTGC
AGGAGATTGATGTTTTCAACAACCAGCTGAAGGAACTGAGAGAGATACAT
AGCAAACAGCAACTCCAGAAGCAGAGGTCCCTGGAGGCAGCGCGACTGA
AGCAGAAAGAGCAGGAGAGGAAGAGCCTGGAGTTAGAGAAGCAAAAGG
AAGACGCTCAGAGACGAGTTCAGGAAAGGGACAAGCAATGGCTGGAGCA
TGTGCAGCAGGAGGAGCAGCCACGCCCCCGGAAACCCCACGAGGAGGAC
AGACTGAAGAGGGAAGACAGTGTCAGGAAGAAGGAGGCGGAAGAGAGA
GCCAAGCCGGAAATGCAAGACAAGCAGAGTCGGCTTTTCCATCCGCATCA
GGAGCCAGCTAAGCTGGCCACCCAGGCACCCTGGTCTACCACAGAGAAAG
GCCCGCTTACCATTTCTGCACAGGAGAGTGTAAAAGTGGTATATTACCGA
GCGCTGTACCCCTTTGAATCCAGAAGTCACGATGAGATCACCATCCAGCC
AGGAGATATAGTCATGGTGGATGAAAGCCAGACTGGAGAGCCAGGATGG
CTTGGAGGAGAGCTGAAAGGGAAGACGGGATGGTTCCCTGCAAACTATGC
AGAAAAGATTCCAGAAAATGAGGTTCCCACTCCAGCCAAACCAGTGACCG
ATCTGACATCTGCCCCTGCCCCCAAACTGGCTCTGCGTGAGACCCCTGCTC
CTTTGCCAGTGACCTCTTCTGAGCCCTCCACAACCCCCAACAACTGGGCAG
ACTTCAGTTCCACGTGGCCCAGCAGCTCAAACGAGAAGCCAGAAACGGAC
AACTGGGATACGTGGGCGGCTCAGCCTTCTCTGACCGTACCTAGTGCTGG
CCAGTTACGGCAGAGATCAGCCTTTACCCCAGCCACAGCCACTGGCTCCT
CCCCATCTCCCGTCCTGGGCCAGGGTGAAAAGGTGGAAGGGCTACAAGCG
CAAGCCCTGTATCCCTGGAGAGCCA,AA.AAAGACAACCACTTAAATTTTAA
CAAAAGTGACGTCATCACCGTTCTGGAACAGCAAGACATGTGGTGGTTTG
GAGAAGTTCAAGGTCAGAAGGGTTGGTTCCCCAAGTCTTACCrTGAAACTC
ATTTCAGGGCCCGTAAGGAAATCCACAAGCATCGATACTGGCCCTACTGA
AAGTCCTGCTAGTCTAAAGAGAGTGGCTTCCCCGGCCGCCAAGCCAGCCA
TTCCCGGAGAAGAGTTTATTGCCATGTACACATACGAGAGTTCTGAGCAA
GGAGATTTAACCTTTCAGCAAGGGGATGTGATTGTGGTTACCAAGAAAGA
3~ TGGTGACTGGTGGACGGGAACGGTGGGCGACAAGTCCGGAGTCTTCCCTT
CTAACTATGTGAGGCTTAAA(sATTCAGAGGGCTCTGGAACTGCTGGGAAA
ACAGGGAGTTTAGGAAAAAAACCTGAAATTGCCCAGGTTATTGCTTCCTA
SUBSTIT1UTE SHEET (RULE 26) CGCTGCTACTGGTCCCGAACAACTCACCCTGGCTCCTGGGCAGCTGATTCT
GATCCGGAAAAAGAACCCAGGTGGATGGTGGGAAGGAGAACTGCAAGCT
CGAGGGAAAAAGCGCCAGATAGGGTGGTTTCCAGCAAATTATGTCAAACT
TCTAAGCCCCGGAACAAGCAAAATCACCCCAACTGAGCTACCCAAGACCG
S CAGTGCAGCCAGCAGTGTGCCAGGTGATCGGGATGTACGATTACACCGCC
CAGAACGATGACGAACTAGCCTTCAGCAAAGGCCAGATCATCAACGTCCT
CAACAAGGAGGACCCGGACTGGTGGAAAGGAGAAGTCAGTGGGCAAGTT
GGGCTCTTCCCATCCAATTATGTAAAGCTGACCACAGACATGGACCCCAG
CCAGCAATGAATCATATGTTGTCCATCCCCCCCTCAGGCTTGAAAGTCCTC
AAAGAGACCCACTATCCCATATCACTGCCCAGAGGGATGATGGGAGATGC
AGCCTTGATCATGTGACTTGCAGCATGATCACCTACTGCCTTCTGAGTAGA
AGAACTCACTGCAGAGCAGTTTACCTCATTTGACCTTAGTTGCATGTGATC
GAAATGTCTGAGTCACTGCGTGCAGAGGCAGAAGCAAATTGCAGAACTGC
ACAGGGTGGTGGGTCCTTTTGGGGCTTTCCTAGTCACTCAGACTGACCGGC
CCCGCCTTCACACGGGCGCTTTCAATAGTTTTAAGATTATTTTTAAATGTG
TATTTTAGCCTTTTAATAAAAATCTCAATCAATTACTTCTTTGCCTATTTTG
GTTTTACAAAAACACCCACTATCAAGGAGTGCCTGTCTGCGGACGATTAA
AATGCTGTTCCGGGCGTACCGTAAACTGAGAGCTTGCTGTACCTTTGCCGT
TTGTCCAGTGTTCCCAACCACATTGTGTAGTTTGGGGCTGTTCCCTGCCGT
AGAGCACAGAGGAGATGGGTGTACCTGTTTTGAAAATGTGTATGTAGACT
GAGCCTGACTATGGAAGGGGTTATGCTTGTCTGTGACCATCACGTGTACCT
GTCGCGCATGTACCATCTGTACCGAAGAAGTAGCTCTTCCTCCATGGCTAA
ACCCACCACCGTGTACAGTGCTCTCATCTACTGCATTCATTTTACTTTGCA
CAGTGACCTTGTAGCCACCTGAGGAAGCACCCATGTTTCCGTTTGGTCTCA
GATGTACCTAGTTGTGCCCGTGTTTTGTTTTTATTTTTCAATCTGGCATGTC
TTCACACCATAAACTAGTAAGACGCCAACTGCCCAGGCGGTTACGATCAT
CAGTACCCACCGTCTTAGTCTCTGTTACGTGAAGTTTATTCCAGTTGCTTTT
TATGGAATATCTTGAACAAGTAATCTTCTTGACAAGAAAGAATGTATAGA
AGTCTCCCTGCAATTAATTTCCCAGTGTTTACATTTTTTAACTAGACTGTGG
GGGTTGCTACAGATTAATATGAAATGGCGCTCCTGGTCCGTGTGTGTGTT.A
ACTTGTGCTGTAGCTGAAGCCGTGTGTCCTTAGATATTAGTTGGAAGTCGG
GAAGAGAATTCGATATCAAGCTT
Mouse Ese I coding sequence - Sequence ID N0:2 ATGGCTi~AGTTTCCCACACCTTTCGGTGGTAGCCTGGATGTCTGGGCCATA
ACTGTGGAGGAAAGGGCCAAGCATGACCAGCAGTTCCTTAGCCTGAAGCC
GATAGCGGGATTTATTACTGGTGATCAAGCGAGGAACTTTTTTTTCCAATC
SUBSTITUTE SHEET (RULE 26) TGGGTTACCTCAGCCTGTCTTAGCACAAATATGGGCGCTAGCGGACATGA
ATAACGATGGAAGGATGGATCAAGTGGAATTTTCCATAGCCATGAAGCTT
ATCAAACTGAAGCTACAAGGATATCAGCTCCCCTCCACACTTCCCCCTGTC
ATGAAACAGCAACCAGTGGCTATTTCCAGTGCACCAGCATTTGGTATAGG
AGGGATTGCTAGCATGCCACCACTCACAGCTGTTGCTCCTGTGCCAATGG
GCTCCATTCCAGTTGTTGGAATGTCTCCACCCTTAGTATCTTCTGTCCCTCC
AGCAGCAGTGCCTCCCCTGGCTAACGGGGCTCCTCCCGTCATACAGCCTCT
GCCTGCGTTTGCGCATCCTGCAGCCACATGGCCAAAGAGTTCTTCCTTCAG
CAGATCTGGTCCAGGGTCACAATTAAACACTAAGTTACAGAAGGCACAAT
CATTCGATGTCGCCAGCGCCCCTCCAGCAGCAGAATGGGCTGTGCCTCAG
TCATCAAGGCTGAAATACAGGCAGTTATTCAACAGCCACGACAAAACTAT
GAGTGGACACTTAACAGGTCCCCAGGCAAGAACTATTCTCATGCAATCAA
GTTTACCCCAGGCTCAGCTGGCTTCAATATGGAATCTTTCTGACATTGATC
AAGATGGAAAACTCACTGCAGAAGAATTTATCCTAGCTATGCACCTAATT
GATGTTGCCATGTCTGGTCAGCCACTGCCGCCCGTCCTGCCTCCAGAATAC
ATCCCTCCTTCCTTCAGAAGAGTTCGCTCCGGCAGTGGGATGTCCGTCATA
AGCTCTTCTTCTGTGGATCAGAGGCTGCCTGAGGAGCCGTCGTCAGAGGA
TGAGCAGCAGCCAGAGAAGAAACTGCCTGTGACATTTGAAGATAAGAAG
CGGGAGAACTTCGAGCGAGGCAGTGTGGAGCTGGAGAAGCGCCGCCAAG
CGCTCTTGGAGCAGCAGCGCAAAGAGCAGGAGCGGTTGGCTCAGCTGGA
GCGCGCCGAGCAGGAGAGGAAAGAGCGGGAGCGCCAGGAGCAGGAGGC
CAAGCGGCAGCTGGAGCTGGAGAAGCAGCTGGAGAAGCAGCGGGAGCTG
GAGCGGCAGCGAGAGGAGGAGAGGAGGAAGGAGATCGAGAGGCGCGAG
GCCGCA.AAACGGGAACTGGAAAGGCAGCGACAACTTGAATGGGAACGGA
ACCGGAGACAGGAACTCCTGAATCAGAGGAACAAGGAGCAGGAGGGCAC
CGTGGTCCTGAAGGCAAGGAGGAAGACTCTGGAGTTTGAGTTAGAAGCTC
TGAATGACAAAAAGCATCAGCTAGAAGGAAA.ACTTCAGGATATCAGGTGT
CGACTGGCAACCCAGAGGCAAGAAATTGAGAGCACGAACAAGTCTAGAG
AGCTAAGAATTGCTGAAATCACCCACTTACAGCAGCAGTTGCAGGAATCT
CAGCA.AATGCTTGGAAGACTTATTCCAGAGAAACAGATACTCAGTGACCA
GTTAAAACAAGTCCAGCAGAACAGTTTGCATAGAGACTCGCTTCTTACCC
TCAAAAGAGCCTTGGAAGCAAAGGAGCTGGCCCGGCAGCAGCTCCGGGA
GCAGCTGGACGAGGTGGAGAGAGAGACCAGGTCAAAGCTGCAGGAGATT
GATGTTTTCAACAACCAGCTGAAGGAACTGAGAGAGATACATAGCAAACA
3~ GCAACTCCAGAAGCAGAGGTCCCTGGAGGCAGCGCGACTGAAGCAGAAA
GAGCAGGAGAGGAAGAGCCTGGAGTTAGAGAAGCAAAAGGAAGACGCTC
AGAGACGAGTTCAGGAAAGGGACAAGCAATGGCTGGAGCATGTGCAGCA
SUBSTITUTE SHEET (RULE 26) GGAGGAGCAGCCACGCCCCCGGAAACCCCACGAGGAGGACAGACTGAAG
AGGGAAGACAGTGTCAGGAAGAAGGAGGCGGAAGAGAGAGCCAAGCCG
GAAATGCAAGACAAGCAGAGTCGGCTTTTCCATCCGCATCAGGAGCCAGC
TAAGCTGGCCACCCAGGCACCCTGGTCTACCACAGAGAAAGGCCCGCTTA
CCATTTCTGCACAGGAGAGTGTAAA.AGTGGTATATTACCGAGCGCTGTAC
CCCTTTGAATCCAGAAGTCACGATGAGATCACCATCCAGCCAGGAGATAT
AGTCATGGTGGATGAAAGCCAGACTGGAGAGCCAGGATGGCTTGGAGGA
GAGCTGAAAGGGAAGACGGGATGGTTCCCTGCAAACTATGCAGAAAAGA
TTCCAGAAAATGAGGTTCCCACTCCAGCCAAACCAGTGACCGATCTGACA
TCTGCCCCTGCCCCCAAACTGGCTCTGCGTGAGACCCCTGCTCCTTTGCCA
GTGACCTCTTCTGAGCCCTCCACAACCCCCAACAACTGGGCAGACTTCAGT
TCCACGTGGCCCAGCAGCTCAAACGAGAAGCCAGAAACGGACAACTGGG
ATACGTGGGCGGCTCAGCCTTCTCTGACCGTACCTAGTGCTGGCCAGTTAC
GGCAGAGATCAGCCTTTACCCCAGCCACAGCCACTGGCTCCTCCCCATCTC
CCGTCCTGGGCCAGGGTGAAAAGGTGGAAGGGCTACAAGCGCAAGCCCT
GTATCCCTGGAGAGCCAAAAAAGACAACCACTTAAATTTTAACAAAAGTG
ACGTCATCACCGTTCTGGAACAGCAAGACATGTGGTGGTTTGGAGAAGTT
CAAGGTCAGAAGGGTTGGTTCCCCAAGTCTTACGTGAA.ACTCATTTCAGG
GCCCGTAAGGAAATCCACAAGCATCGATACTGGCCCTACTGAAAGTCCTG
CTAGTCTAAAGAGAGTGGCTTCCCCGGCCGCCAAGCCAGCCATTCCCGGA
GAAGAGTTTATTGCCATGTACACATACGAGAGTTCTGAGCAAGGAGATTT
AACCTTTCAGCAAGGGGATGTGATTGTGGTTACCAAGAAAGATGGTGACT
GGTGGACGGGAACGGTGGGCGACAAGTCCGGAGTCTTCCCTTCTAACTAT
GTGAGGCTTAAAGATTCAGAGGGCTCTGGAACTGCTGGGAAAACAGGGA
GTTTAGGAA.AAAAACCTGAAATTGCCCAGGTTATTGCTTCCTACGCTGCTA
CTGGTCCCGAACAACTCACCCTGGCTCCTGGGCAGCTGATTCTGATCCGGA
AAAAGAACCCAGGTGGATGGTGGGAAGGAGAACTGCAAGCTCGAGGGAA
AAAGCGCCAGATAGGGTGGTTTCCAGCAAATTATGTCAAACTTCTAAGCC
CCGGAACAAGCAA.AATCACCCCAACTGAGCTACCCAAGACCGCAGTGCA
GCCAGCAGTGTGCCAGGTGATCGGGATGTACGATT.ACACCGCCCAGAACG
ATGACGAACTAGCCTTCAGCAAAGGCCAGATCATCAACGTCCTCAACAAG
GAGGACCCGGACTGGTGGAAAGGAGAAGTCAGTGGGCAAGTTGGGCTCTT
CCCATCCAATTATGTAAAGCTGACCACAGACATGGACCCCAGCCAGCAAT
GAATCATATGTTGTCCATCCCCCCCTCAGGCTTGAAAGTCCTCAA~GAGAC
3~ CCACTATCCCATATCACTGCCC_-'.G:~GGGATGA
Mouse Ese 1 protein - Sequence ID N0:3 SUBSTITUTE SHEET (RULE 26) MAQFPTPFGGSLDV WAITVEERAKHDQQFLSLKPIAGFITGDQARNFFFQSGL
PQPVLAQIWALADMNNDGRMDQVEFSIAMKLIKLKLQGYQLPSTLPPVMKQ
QPVAISSAPAFGIGGIASMPPLTAVAPVPMGSIPVVGMSPPLVSSVPPAAVPPL
ANGAPPVIQPLPAFAHPAATWPKSSSFSRSGPGSQLNTKLQKAQSFDVASAPP
AAEWAVPQSSRLKYRQLFNSHDKTMSGHLTGPQARTILMQSSLPQAQLASIW
NLSDIDQDGKLTAEEFILAMHLIDVAMSGQPLPPVLPPEYIPPSFRRVRSGSGM
SVISSSSVDQRLPEEPSSEDEQQPEKKLPVTFEDKKRENFERGSVELEKRRQAL
LEQQRKEQERLAQLERAEQERKERERQEQEAKRQLELEKQLEKQRELERQRE
EERRILEIERREAAKRELERQRQLEWERNRRQELLNQRNKEQEGTVVLKAR.R
KTLEFELEALNDKKHQLEGKLQDIRCRLATQRQEIESTNKSRELRIAEITHLQQ
QLQESQQMLGRLIPEKQILSDQLKQVQQNSLHRDSLLTLKRALEAKELARQQ
LREQLDEVERETRSKLQEIDVFNNQLKELREIHSKQQLQKQRSLEAARLKQKE
QERKSLELEKQKEDAQRRVQERDKQWLEHVQQEEQPRPRKPHEEDRLKRED
SVRKKEAEERAKPEMQDKQSRLFHPHQEPAKLATQAPWSTTEKGPLTISAQE
SVKVVYYRALYPFESRSHDEITIQPGDIVMVDESQTGEPGWLGGELKGKTGW
FPANYAEKIPENEVPTPAKPVTDLTSAPAPKLALRETPAPLPVTSSEPSTTPNN
WADFSSTWPSSSNEKPETDNWDTWAAQPSLTVPSAGQLRQRSAFTPATATGS
SPSPVLGQGEKVEGLQAQALYPWRAKKDNHLNFNKSDVITVLEQQDMWWF
GEVQGQKGWFPKSYVKLISGPVRKSTSIDTGPTESPASLKRVASPAAKPAIPGE
EFIAMYTYESSEQGDLTFQQGDVIVVTKKDGDWWTGTVGDKSGVFPSNYVR
LKDSEGSGTAGKTGSLGKKPEIAQVIASYAATGPEQLTLAPGQLILIRKKNPGG
WWEGELQARGKKRQIGWFPANYVKLLSPGTSKITPTELPKTAVQPAVCQVIG
MYDYTAQNDDELAFSKGQIINV LNKEDPDW WKGEV SGQV GLFPSNYVKLTT
DMDPSQQ
Mouse Ese2 cDNA - Sequence ID N0:4 CCCTTCCTTTCCTTTTTTTGTGTTCGCCTTCGGCCGTGCCGGCTGAGAGCCC
AGCAGCCGTGACAGGCTGCGCAACAGGTTCGCTGCGGCCGGCCTGACGAC
TGACCCGGCGGCGGCGGCCGCGGCACGGCAGGGTCTTCCCGGAGCTTGGC
CGCGCCCACGCGCCGGTGTCGAGGAGCGCGCGGGGTCGCGCCGGGACGT
GCGCGAGGCGCCAGATGGCTGAGAGCTGCAAGAAGAAGTCAGGATCATG
ATGGCTCAGTTTCCCACAGCGATGAATGGAGGGCCAAATATGTGGGCTAT
TACATCTGAAGAACGTACTAAGCATGATAAACAGTTTGATAACCTCAAAC
3~ CTTCAGGAGGTTACATAACAGGTGATCAAGCCCGTACTTTTTTCCTACAGT
CAGGTCTGCCGGCCCCGGTTTTAGCTGAAATATGGGCCTTATCAGATCTGA
ACAAGGATGGGAAGATGGACCAGCAAGAGTTCTCTATAGCTATGAAACTC
SUBSTTTUTE SHEET (RULE 26) ATCAAGTTAAAGTTGCAGGGCCAACAGCTGCCTGTAGTCCTCCCTCCTATC
ATGAAACAACCCCCTATGTTCTCTCCACTAATCTCTGCTCGTTTTGGGATG
GGAAGCATGCCCAATCTGTCCATTCATCAGCCATTGCCTCCAGTTGCACCT
ATAGCAACACCCTTGTCTTCTGCTACGTCAGGGACCAGTATTCCTCCCCTA
ATGATGCCTGCTCCCCTAGTGCCTTCTGTTAGTACATCCTCATTACCAAAT
GGAACTGCCAGTCTCATTCAGCCTTTATCCATTCCTTATTCTTCTTCAACAT
TGCCTCATGCATCATCTTACAGCCTGATGATGGGAGGATTTGGTGGTGCTA
GTATCCAGA.AGGCCCAGTCTCTGATTGATTTAGGATCTAGTAGCTCAACTT
CCTCAACTGCTTCCCTCTCAGGGAACTCACCTAAGACAGGGACCTCAGAG
TGGGCAGTTCCTCAGCCTTCAAGATTAAAGTATCGGCAAAAATTT AATAGT
CTAGACAAAGGCATGAGCGGATACCTCTCAGGTTTTCAAGCTAGAAATGC
CCTTCTTC AGTCAAATCTCTCTCAAACTCAGCTAGCTACTATTTGGACTCT
GGCTGACATCGATGGTGACGGACAGTTGAAAGCTGAAGAATTTATTCTGG
CGATGCACCTCACTGACATGGCCAA.AGCTGGACAGCCACTACCACTGACG
TTGCCTCCCGAGCTTGTCCCTCCATCTTTCAGAGGGGGAAAGCAAGTTGAT
TCTGTTAATGGAACTCTGCCTTCATATCAGAAAACACAAGAAGAAGAGCC
TCAGAAGAAACTGCCAGTTACTTTTGAGGACAAACGGAAAGCCAACTATG
AACGAGGAAACATGGAGCTGGAGAAGCGACGCCAAGTGTTGATGGAGCA
GCAGCAGAGGGAGGCTGAACGCAAAGCCCAGAAAGAGAAGGAAGAGTG
GGAGCGGAA.ACAGAGAGAACTGCAAGAGCAAGAATGGAAGAAGCAGCTG
GAGTTGGAGAAACGCTTGGAGAAACAGAGAGAGCTGGAGAGACAGCGGG
AGGAAGAGAGGAGAAAGGAGATAGAAAGACGAGAGGCAGCAAAACAGG
AGCTTGAGAGACAACGCCGTTTAGAATGGGAAAGACTCCGTCGGCAGGAG
CTGCTCAGTCAGAAGACCAGGGAACAAGAAGACATTGTCAGGCTGAGCTC
CAGAAAGAAAAGTCTCCACCTGGAACTGGAAGCAGTGAATGGAAA.ACAT
CAGCAGATCTCAGGCAGACTACAAGATGTCCAAATCAGAAAGCAAACAC
AAA.AGACTGAGCTAGAAGTTTTGGATAAACAGTGTGACCTGGAAATTATG
GAAATCAAACAACTTCAACAAGAGCTTAAGGAATATCAAAATAAGCTTAT
CTATCTGGTCCCTGAGAAGCAGCTATTAAACGAAAGAATTAAA.AACATGC
AGCTCAGTAACACACCTGATTCAGGGATCAGTTTACTTCATAAAAAGTCA
TCAGAAAAGGAAGAATTATGCCAAAGACTTAAAGAACAATTAGATGCTCT
TGAAAA.AGAAACTGCATCTAAGCTCTCAGAAATGGATTCATTTAACAATC
AGCTGAAGGAACTCAGAGAAAGCTATA.ATACACAGCAGTTAGCCCTTGAA
CAACTTCATAA.AATCAAACGTGACAAATTGAAGGAAATCGAAAGAAAAA
GATTAGAGCAAATTCAAAAAAAGAAACTAGAAGATGAGGCTGCAAGGAA
AGCAAAGCAAGGAAAAGAAAACTTGTGGAGAGAAAGTATTAGAAAGGAA
GA.AGAGGAAAAGCAAAAACGACTCCAGGAAGAAA.AGTCACAGGACAAA
SUBSTITUTE SHEET (RULE 26) ACTCAAGAAGAGGAACGAA.AAGCTGAGGCAAAACAAAGTGAGACAGCCA
GTGCTTTGGTGAATTACAGAGCACTGTACCCTTTTGAAGCAAGAAACCA'1' GATGAGATGAGTTTTAGTTCTGGGGATATAATTCAGGTTGATGA.AAAAAC
TGTAGGAGAGCCTGGTTGGCTTTATGGTAGTTTTCAGGGAAAGTTTGGCTG
GTTCCCCTGCAACTATGTAGAAAAAGTGCTGTCAAGTGAAA.AAGCTCTGT
CTCCTAAGAAGGCCTTACTTCCTCCTACAGTGTCTCTCTCTGCTACCTCAA
CTTCTTCCCAGCCACCAGCATCAGTGACTGATTATCACAATGTATCCTTCT
CAAACCTTACTGTTAATACAACATGGCAGCAGAAGTCAGCTTTTACCCGC
ACTGTGTCCCCTGGATCTGTGTCCCCCATTCACGGACAGGGGCAGGCTGTA
GAAAACCTGAAAGCCCAGGCCCTTTGTTCCTGGACGGCAAAGAAGGAGA
ACCACCTGAACTTCTCAAAGCACGACGTCATCACTGTCCTGGAGCAGCAG
GAAA.ACTGGTGGTTTGGGGAGGTGCACGGAGGAAGAGGATGGTTCCCCA
AGTCTTATGTCAAGCTCATTCCTGGGAATGAAGTACAGCGAGGAGAGCCA
GAAGCTTTGTATGCAGCTGTGACTAAGAAACCTACCTCCACAGCCTATCC
AGTTACCTCCACAGCCTATCCAGTTGGAGAAGACTACATTGCACTTTATTC
ATACTCAAGTGTAGAGCCCGGGGATTTGACTTTCACTGAAGGTGAAGAAA
TTCTAGTGACCCAGAAAGATGGAGAGTGGTGGACAGGAAGTATTGGAGA
GAGAACTGGAATCTTCCCGTCCAACTACGTCAGACCAAAGGATCAAGAGA
ATTTTGGGAATGCTAGCAAATCTGGAGCATCAAACA,AAAAACCCGAGATC
GCTCAAGTAACTTCAGCATATGCTGCTTCAGGGACTGAGCAGCTCAGCCTT
GCGCCAGGACAGTTAATATTAATCTTAAAGAAAAACACAAGCGGGTGGTG
GCAAGGAGAGCTACAGGCCAGAGGGAAGAAACGACAGAAGGGATGGTTT
CCTGCCAGCCATGTAAAGCTGCTAGGTCCAAGCAGTGAAAGAACCATGCC
TACTTTTCACGCTGTATGTCAAGTGATTGCTATGTATGACTACATGGCGAA
TAACGAAGATGAGCTCAATTTCTCCAAAGGACAGCTGATTAATGTTATGA
ACAAAGATGACCCTGACTGGTGGCAAGGAGAAACCAATGGTCTGACTGGT
CTCTTTCCTTCAAACTATGTTAAGATGACAACAGACTCAGATCCAAGTCAA
CAGTGGTGTGCTGACCTCCAAGCCCTGGACACAATGCAGCCTACGGAGAG
GAAGCGACAGGGCTACATTCACGAGCTCATTCAGACAGAGGAGCGGTAC
ATGGACGACCTGCAACTTTTTGAACA,AAAAACTCTCCTTTGAGGGCCTGG
GGAAGCCAGAACCAGGGGAGCTGCCCACAAGGCTGGGTCTAAAGACAGA
TTTTGCTCTCCCAGGACAGAGGAGCATCACATCGGCTTCATCCATCCAAAC
AAGCCACACTCGCTGGGCCTGGTATTTTATTGCACCACTAAAATTGCTAGC
AATCTATGCAAACATGATCTTTTTAAACAAACGCCACAGCACAGTGCCTT
3~ GTACTAGTGTTAACCTGTTCAGCTGTGTTAGATGCCAGGGTTTCCATTTTC
AGGGCTATAAAAGTATTATGTGGGAAATGAGACATCAGACCACCGGACGT
TACCACTTGGCAAATCTGTCCACTGTGGAGTTGGTGATGTTGGAACCATTC
SUBSTITUTE SHEET (RULE I6) CACACTATGTGACCTCTGCTGGGTCACACACTCAGGAGGTGAAGGGCTGA
GATGAAATGCTGCAGCCTTGGGGCTTGTGCAGCCTGATACTGAAATAGCA
TCCACTTGTGCACTGAATAAATAGAAACTTGATCGTTTTATTCTGACTAGA
TATTATCATTCTCTGCTAAGACAATATAGTTTGAAATATTATAGTTTGAAT
ATAAGGAGGAAAGCTTGATGTACTTTAAATATACTGTGAACTCTAATAAT
GTGGGGATATTTTTCAACTTTAATTTTCTTAAGTATAAATTATTTATGTAAA
TTCTTTGTTTTGCATATTTCATAGAACATGCATCTTTAAGCTTTATCATTGC
CAACAATGTACAGAAAGAGAATAAAAGTATAAGTTTATGAATGTAAAAA
AA
Mouse Ese2 coding sequence - Sequence ID NO:S
ATGGCTCAGTTTCCCACAGCGATGAATGGAGGGCCAAATATGTGGGCTAT
TACATCTGAAGAACGTACTAAGCATGATAAACAGTTTGATAACCTCAAAC
CTTCAGGAGGTTACATAACAGGTGATCAAGCCCGTACTTTTTTCCTACAGT
CAGGTCTGCCGGCCCCGGTTTTAGCTGAAATATGGGCCTTATCAGATCTGA
ACAAGGATGGGAAGATGGACCAGCAAGAGTTCTCTATAGCTATGAAACTC
ATCAAGTTAAAGTTGCAGGGCCAACAGCTGCCTGTAGTCCTCCCTCCTATC
ATGAAACAACCCCCTATGTTCTCTCCACTAATCTCTGCTCGTTTTGGGATG
GGAAGCATGCCCAATCTGTCCATTCATCAGCCATTGCCTCCAGTTGCACCT
ATAGCAACACCCTTGTCTTCTGCTACGTCAGGGACCAGTATTCCTCCCCTA
ATGATGCCTGCTCCCCTAGTGCCTTCTGTTAGTACATCCTCATTACCAAAT
GGAACTGCCAGTCTCATTCAGCCTTTATCCATTCCTTATTCTTCTTCAACAT
TGCCTCATGCATCATCTTACAGCCTGATGATGGGAGGATTTGGTGGTGCTA
GTATCCAGAAGGCCCAGTCTCTGATTGATTTAGGATCTAGTAGCTCAACTT
CCTCAACTGCTTCCCTCTCAGGGAACTCACCTAAGACAGGGACCTCAGAG
TGGGCAGTTCCTCAGCCTTCAAGATTAAAGTATCGGCAAA.AATTTAATAGT
CTAGACAAAGGCATGAGCGGATACCTCTCAGGTTTTCAAGCTAGAAATGC
CCTTCTTCAGTCAAATCTCTCTCAAACTCAGCTAGCTACTATTTGGACTCT
GGCTGACATCGATGGTGACGGACAGTTGAAAGCTGAAGAATTTATTCTGG
CGATGCACCTCACTGACATGGCCAAAGCTGGACAGCCACTACCACTGACG
TTGCCTCCCGAGCTTGTCCCTCCATCTTTCAGAGGGGGAAAGCAAGTTGAT
TCTGTTAATGGAACTCTGCCTTCATATCAGAAAACACAAGAAGAAGAGCC
TCAGAAGAAACTGCCAGTTACTTTTGAGGACAAACGGAAAGCCAACTATG
AACGAGGAAACATGGAGCTGGAGAAGCGACGCCAAGTGTTGATGGAGCA
GCAGCAGAGGGAGGCTGAACGCAAAGCCCAGAAAGAGAAGGAAGAGTG
GGAGCGGAAACAGAGAGAACTGCAAGAGCAAGAATGGAAGAAGCAGCTG
GAGTTGGAGAAACGCTTGGAGAAACAGAGAGAGCTGGAGAGACAGCGGG
SUBSTITUTE SHEET (RULE 26) AGGAAGAGAGGAGAAAGGAGATAGAAAGACGAGAGGCAGCAAAACAGG
AGCTTGAGAGACAACGCCGTTTAGAATGGGAAAGACTCCGTCGGCAGGAG
CTGCTCAGTCAGAAGACCAGGGAACAAGAAGACATTGTCAGGCTGAGCTC
CAGAAAGAAAAGTCTCCACCTGGAACTGGAAGCAGTGAATGGAAAACAT
CAGCAGATCTCAGGCAGACTACAAGATGTCCAAATCAGAAAGCAAACAC
AAA.AGACTGAGCTAGAAGTTTTGGATAAACAGTGTGACCTGGAAATTATG
GAAATCAAACAACTTCAACAAGAGCTTAAGGAATATCA.A.AATAAGCTTAT
CTATCTGGTCCCTGAGAAGCAGCTATTAAACGAAAGAATTAAAAACATGC
AGCTCAGTAACACACCTGATTCAGGGATCAGTTTACTTCATAA.AAAGTCA
TCAGAA.A.AGGAAGAATTATGCCAAAGACTTAAAGAACAATTAGATGCTCT
TGAAAAAGAAACTGCATCTAAGCTCTCAGAAATGGATTCATTTAACAATC
AGCTGAAGGAACTCAGAGAAAGCTATAATACACAGCAGTTAGCCCTTGAA
CAACTTCATA.A.AATCAAACGTGACAAATTGAAGGAAATCGAAAGAAA.AA
GATTAGAGCAAATTCAAAAAAAGAAACTAGAAGATGAGGCTGCAAGGAA
AGCAAAGCAAGGAAAAGAAAACTTGTGGAGAGAAAGTATTAGAAAGGAA
GAAGAGGAAAAGCAAAAACGACTCCAGGAAGAAA.AGTCACAGGACAAA
ACTCAAGAAGAGGAACGAAAAGCTGAGGCAAAACAAAGTGAGACAGCCA
GTGCTTTGGTGAATTACAGAGCACTGTACCCTTTTGAAGCAAGAAACCAT
GATGAGATGAGTTTTAGTTCTGGGGATATAATTCAGGTTGATGAAAAAAC
TGTAGGAGAGCCTGGTTGGCTTTATGGTAGTTTTCAGGGAAAGTTTGGCTG
GTTCCCCTGCAACTATGTAGAAAAAGTGCTGTCAAGTGAAAAAGCTCTGT
CTCCTAAGAAGGCCTTACTTCCTCCTACAGTGTCTCTCTCTGCTACCTCAA
CTTCTTCCCAGCCACCAGCATCAGTGACTGATTATCACAATGTATCCTTCT
CA..~1ACCTTACTGTTAATACAACATGGCAGCAGAAGTCAGCTTTTACCCGC
ACTGTGTCCCCTGGATCTGTGTCCCCCATTCACGGACAGGGGCAGGCTGTA
GAAAACCTGAAAGCCCAGGCCCTTTGTTCCTGGACGGCAAAGAAGGAGA
ACCACCTGAACTTCTCAAAGCACGACGTCATCACTGTCCTGGAGCAGCAG
GAAAACTGGTGGTTTGGGGAGGTGCACGGAGGAAGAGGATGGTTCCCCA
AGTCTTATGTCAAGCTCATTCCTGGGAATGAAGTACAGCGAGGAGAGCCA
GAAGCTTTGTATGCAGCTGTGACTAAGAAACCTACCTCCACAGCCTATCC
AGTTACCTCCACAGCCTATCCAGTTGGAGAAGACTACATTGCACTTTATTC
ATACTCAAGTGTAGAGCCCGGGGATTTGACTTTCACTGAAGGTGAAGAAA
TTCTAGTGACCCAGAAAGATGGAGAGTGGTGGACAGGAAGTATTGGAGA
GAGAACTGGAATCTTCCCGTCCAACTACGTCAGACCAAAGGATCAAGAGA
ATTTTGGGAATGCTAGCAAATCTGGAGCATCAAACAAAAAACCCGAGATC
GCTCAAGTAACTTCAGCATATGCTGCTTCAGGGACTGAGCAGCTCAGCCTT
GCGCCAGGACAGTTAATATTAATCTTAAAGAAAAACACAAGCGGGTGGTG
SUBSTITUTE SHEET (RULE 26) GCAAGGAGAGCTACAGGCCAGAGGGAAGAAACGACAGAAGGGATGGTTT
CCTGCCAGCCATGTAAAGCTGCTAGGTCCAAGCAGTGAAAGAACCATGCC
TACTTTTCACGCTGTATGTCAAGTGATTGCTATGTATGACTACATGGCGAA
TAACGAAGATGAGCTCAATTTCTCCAAAGGACAGCTGATTAATGTTATGA
ACAAAGATGACCCTGACTGGTGGCAAGGAGAAACCAATGGTCTGACTGGT
CTCTTTCCTTCAAACTATGTTAAGATGACAACAGACTCAGATCCAAGTCAA
CAGTGGTGTGCTGACCTCCAAGCCCTGGACACAATGCAGCCTACGGAGAG
GAAGCGACAGGGCTACATTCACGAGCTCATTCAGACAGAGGAGCGGTAC
ATGGACGACCTGCAACTTTTTGAACAAAAAACTCTCCTTTGA
mouse Ese2 - Sequence ID N0:6 MAQFPTAMNGGPNMWAITSEERTKHDKQFDNLKPSGGYITGDQARTFFLQS
GLPAPVLAEIWALSDLNKDGKMDQQEFSIAMKLIKLKLQGQQLPVVLPPIMK
QPPMFSPLISARFGMGSMPNLSIHQPLPPVAPIATPLSSATSGTSIPPLMMPAPL
VPSVSTSSLPNGTASLIQPLSIPYSSSTLPHASSYSLMMGGFGGASIQKAQSLID
LGSSSSTSSTASLSGNSPKTGTSEWAVPQPSRLKYRQKFNSLDKGMSGYLSGF
QARNALLQSNLSQTQLATIWTLADIDGDGQLKAEEFILAMHLTDMAKAGQPL
PLTLPPELVPPSFRGGKQVDSVNGTLPSYQKTQEEEPQKKLPVTFEDKRKANY
ERGNMELEKRRQVLMEQQQREAERKAQKEKEEWERKQRELQEQEWKKQLE
LEKRLEKQRELERQREEERRKEIERREAAKQELERQRRLEWERLRRQELLSQK
TREQEDIVRLSSRKKSLHLELEAVNGKHQQISGRLQDVQIRKQTQKTELEVLD
KQCDLEIMEIKQLQQELKEYQNKLIYLVPEKQLLNERIKNMQLSNTPDSGISLL
HKKSSEKEELCQRLKEQLDALEKETASKLSEMDSFNNQLKELRESYNTQQLA
LEQLHKIKRDKLKEIERKRLEQIQKKKLEDEAARKAKQGKENLWRESIRKEEE
EKQKRLQEEKSQDKTQEEERKAEAKQSETASALVNYRALYPFEARNHDEMS
FSSGDIIQVDEKTVGEPGWLYGSFQGKFGWFPCNYVEKVLSSEKALSPKKALL
PPTVSLSATSTSSQPPASVTDYHNVSFSNLTVNTTWQQKSAFTRTVSPGSVSPI
HGQGQAVENLKAQALCSWTAKKENHLNFSKHDVITVLEQQENWWFGEVHG
GRGWFPKSYVKLIPGNEVQRGEPEALYAAVTKKPTSTAYPVTSTAYPVGEDY
IALYSYSSVEPGDLTFTEGEEILVTQKDGEWVv'TGSIGERTGIFPSNYVRPKDQE
NFGNASKSGASNKKPEIAQVTSAYAASGTEQLSLAPGQLILILKKNTSGWWQ
GELQARGKKRQKGWFPASHVKLLGPSSERTMPTFHAVCQVIAMYDYMANN
EDELNFSKGQLINVMNKDDPDWWQGETNGLTGLFPSNYVKMTTDSDPSQQ
WCADLQALDTMQPTERKRQGYIHELIQTEERYMDDLQLFEQKTLL
3s Mouse Ese2 alternative transcript partial cDNA sequence - Sequence ID N0:7 SUBSTITUTE SHEET (RULE Z6) CCGTCTTCCACATTTCCCACATTGATCGTGTGTACACACTCCGAACAGACA
ACATCAACGAGAGGACGGCCTGGGTCCAGAAGATCAAGGGTGCCTCAGA
GCAGTACATCGACACTGAGAAGAAGAAACGGGAAAAGGCTTACCAAGCC
CGTTCTCAAAAGACTTCAGGTATTGGGCGTCTGATGGTGCATGTCATTGAA
GCTACAGAATTAAAAGCCTGCAAACCAAACGGGAAAAGTAATCCATACTG
TGAAGTCAGCATGGGCTCCCAAAGCTATACCACCAGGACCCTGCAGGACA
CACTAAACCCCAAGTGGAACTTCAACTGCCAGTTCT'TCATCAAGGATCTTT
ACCAGGACGTTCTGTGTCTCACTATGTTTGACAGAGACCAGTTTTCTCCAG
ATGACTTCTTGGGTCGTACTGAAGTTCCAGTGGCAAAA.ATCCGAACAGAA
CAGGAAAGCAAAGGCCCCACCACCCGCCGACTACTACTGCACGAAGTCCC
CACTGGAGAAGTCTGGGTCCGCTTTGACCTGCAACTTTTTGAACA.AAAAA
CTCTCCTTTGAGGGCCTGGGGAAGCCAGAACCAGGGGAGCTGCCCACAAG
GCTGGGTCTAAAGACAGATTTTGCTCTCCCAGGACAGAGGAGCATCACAT
GGCTTCATCCATCAAACAGCCACACTCGCTGGGCCTGTATTTTATTGCACA
CTAAATTGCTAGCAATCTATGCAAACATGATCTTT
Mouse Ese2 alternative partial protein containing C2 membrane-binding domains -Sequence ID N0:8 VFHISHIDRVYTLRTDNINERTAWVQKIKGASEQYIDTEKKKREKAYQARSQK
TSGIGRLMVHVIEATELKACKPNGKSNPYCEVSMGSQSYTTRTLQDTLNPKW
NFNCQFFIKDLYQDVLCLTMFDRDQFSPDDFLGRTEVPVAKIRTEQESKGPTT
RRLLLHEVPTGEV WVRFDLQLFEQKTLL
Novel Ese-coiled-coil interacting clones:
Mouse homoloeue of C07E3.1 protein (clone 651: - Sequence ID N0:9 GAATTCGGCACGAGGGCTGAGAGAAGCGGACTCCGAGGACTCTGATGCTG
AAGAGAAGCCTGTTAAGCAGGAGGACTTCCCGAAGATTTAGGACCAAAG
AAGTTAAAGACGGGTGGCAATTTTAAGCCCAGCCAGAAAGGCTTTTCAGG
AGGAACCAAGTCCTTCATGGACTTTGGCAGCTGGGAGAGACACACGAAAG
GGATCGGGCAGAAGCTGCTGCAGAAGATGGGCTACGTCCCTGGGCGTGGC
CTGGGGAAGAACGCACAGGGGATCATCAACCCCATCGAAGCCAAACAGA
GAAA.AGGCAAGGGAGCCGTGGGGGCCTATGGCTCGGAGAGGACCACTCA
GTCTCTGCAGGACTTCCCCGTGGCCGACTCGGAAGAGGAGGCAGAAGAGG
AGTTTCAGAAGGAGCTGAGCCAATGGAGGAAAGACCCCAGCGGGAGCAA
GAAGA.AGCC.A.AAGTACTCTTAC ~AGACTGTGGAGGAGCTGAAGGCCAAG
GGCAGGGTCAGCAAGAAGCTCACAGCACCTCAGAAGGAACTGTCTCAGGT
CAAGGTGATCGACATGACAGGCCGGGAGCAGAAGGTGTACTACAGCTAC
SUBSTTTU'TE SHEET (RULE 26) AGCCAAATCAGCCACAAGCACAGCGTGCCCGATGAAGGGGTGCCATTGCT
GGCGCAGCTGCCCCCCACAGCCGGCAAGGAAGCCAGGATGCCGGGCTTTG
CACTGCCTGAGCTGGAGCACAACCTGCAGCTGCTCATTGAGCGCACGGAG
CAGGAGATCATCCAGAGCGACCGGCAGCTCCAGTATGAGCGGGACATGGT
GGTCAGCCTGTCGCATGAGCTGGAGAAGACGGCCGAGGTTCTTGCACATG
AGGAGCGTGTCATCTCTAACCTCAGCAAGGTGCTGGCCCTGGTGGAGGAA
TGTGAGCGCCGCATGCAGCCCCATGGCACCGACCCCCTCACTCTGGATGA
GTGTGCCCGCATCTTTGAGACACTACAGGACAAGTATTATGAGGAGTACC
GCCTGGCGGACCGCGCAGACCTCGCTGTGGCCATTGTCTACCCGCTCGTG
AAGGACTACTTTAAGGATTGGCACCCCTCGAGGG
Mouse partial C07E3.1 protein (clone 65): - Sequence ID NO:10 GTKSFMDFGS WERHTKGIGQKLLQKMGYVPGRGLGKNAQGIINPIEAKQRKG
KGAVGAYGSERTTQSLQDFPVADSEEEAEEEFQKEI,SQWRKDpSGSKK.KPKY
SYKTVEELKAKGRVSKKLTAPQKELSQVKVIDMTGREQKVYYSYSQISHKHS
VPDEGVPLLAQLPPTAGKEARMPGFALPELEHNLQLLIERTEQEIIQSDRQLQY
ERDMVVSLSHELEKTAEVLAHEERVISNLSKVLALVEECERRMQPHGTDPLT
LDECARIFETLQDKYYEEYRLADRADLAVAIVYPLVKDYFKDWHPSR
Mouse Novel cDNA clone 42/ Est accession #W~9719 and #AA915044 - Sequence ID NO:11 CATGGCGGCGGCTGCGGAGGGCGTCCCGGCGACGCGACGGAGGACGAGC
CACCTCGAGATGATGCTGCGGTGGAGACAGCCGAGGAAGCAAAGGAGC
Mouse Novel cDNA clone 70~ - Sequence ID N0:12 CTTGAGTCTACTGAAAATACCCTGCAGGAAGCTACATCATCCATGTCTTTG
ATGACCCAATTTGAACAGGAAGTATCTGGCCTCCAAAGACCATACGTGAT
ATTGAGACTAGCGAAGAGATGC
Mouse Novel cDNA clone 83/Est accession #AA589041 and V~'98708; - Sequence ID
N0:13 GAATTCGGCACGAGGGAGTCTGGTTCTGGAA.AGCCGACAGAAGCTGAGCT
TGTCAACTTAGATTTCTTGGGAGATTTGGATGTTCCCTGTATCTGCCCCACC
CCTGTGTGTCTGAGCTCGAGTCTCTCTGCTGGACTATGG
3~
I~lovel Ese-SH3 interacting clones:
Mouse YNKl partial cDNA~ - Seauence ID N0:14 SUBSTITUTE SHEET (RULE 26) -~l-CTTTACGAGCAGAGGGAGCCAAATTCAGAGCCGTTTTAGATAAAGCTGTG
CAAGCGGATGGACAGGTGAAGGAGCGCTACCAGTCCCATCGAGACACCA
TCGCACTTCTGTGTAAGCCGGAGCCAGAGCTGAATGCTGCCATCCCCTCTG
CTAACCCAGCAAAGACCATGCAGGGCAGCGAGGTTGTAAGTGTCTTAAAG
TCCTTATTATCAAATCTTGATGAAATCAAGAAGGAAAGAGAGAGTCTTGA
GAATGACCTGAAGTCAGTGAATTTTGACATGACAAGCAAGTTTTTGACAG
CTCTGGCCCAAGATGGCGTGATAAATGAGGAGGCTCTCTCTGTCACTGAG
CTGGATCGGATCTATGGCGGTCTAACAAGTAAAGTTCAAGAGTCTCTGAA
GAAACAAGAGGGACTTCTA,AAAAATATACAGGTCTCACACCAAGAATTCT
CCA.AA.ATGAAGCAATCTAACAACGAGGCTAACTTGAGAGAAGAAGTTCTG
AAGAACCTAGCAACTGCGTATGACAACTTTGTTGAGCTTGTAGCTAACTTG
AAGGAGGGCACAAAGTTTTACAATGAGCTGACTGAGATCCTGGTCAGGTT
CCAGAACAAATGCAGTGACATAGTGTTTGCACGGAAGACAGAAAGAGAC
GAGCTCTTGAAGGATCTGCAGCAGAGCATTGCCAGAGAGCCCAGCGCTCC
TTCAATCCCTCCTCCAGCCTATCAGTCCTCCCCAGCAGCGGGGCATGCAGC
AGCGCCTCCAACTCCAGCCCCAAGAACCATGCCGCCTGCTAAGCCCCAGC
CTCCAGCCCGGCCTCCACCTCCTGTGCTTCCTGCAAACCGAGTTCCTCCTG
CTTCTGCTGCTGCTGCCCCTGCAGGCGTGGGGACGGCTTCAGCAGCGCCG
CCACAGACCCCTGGCTCTGCTCCCCCGCCACAGGCTCAGGGACCACCATA
CCCTACCTATCCAGGATATCCCGGGTATTGCCAAATGCCCATGCCCATGGG
CTACAACCCCTACGCATATGGCCAGTACAATATGCCGTACCCACCGGTGT
ATCACCAGAGCCCCGGACAGGCTCCATACCCAGGACCCCAGCAGCCTACC
TACCCCTTCCCTCAGCCCCCGCAGCAGTCCTACTATCCACAGCAGTAACGC
TGCCACGTGCTGCTGGTTCAGATCAGAGCGACAGGACAGCAGCTGCCACC
AGCTCTAAGCCACGCTCTGGCCACTCGAGAGTATCTTGCTCTATTGATTGC
TGTGGATGATTTCTGTCTGTGGCTAAAGCCGAAGGCTGGGCCCCACCTCCA
CATTTGATCGCACTCGTGAGATTCTGCTGCTGTTGCAGTATAAACGCTAGC
TATAATAGCATTTGAAAAAAATTACAGTTCCATAAAATGCTGAAAATGAG
AAATTAAACCTGCAAGTGAAACATTTGAAATTAGCATACTTTATAAGATG
CAGTTGGGACAAAGATGGCTTAAGTACTGATATTTAAGGAAAAAGTTTTC
TTTCTCTTTTGGTTTATTGATTTAGTTTAATTTCTATTATGATATTTTGCATA
ATCAAGGCATTGTAAATCTTATAATTTAAAAATAAATTACTTACGAACAGT
TGTCATTGTTATGTTTTGTCATTGATTCTCATTGCTGTCTAGTTCCTTTCTG
GTATTAGCCTCTCCTTCTGTATGTTCACAGGCTCCATTACTGTGTTGAATTG
~ CGTGACGTCAGGTGAGCAGTCAGGGAGGGCTGCTCTGCGGACGCCAAGCG
CACACCAGCTTGTCTCAGGCTCAGCAGTCAGCTCATCTGGACATTTCTATT
TAAAAGTCCTTTAATGTGGAAGATACACACAATTGTTACCAAAGGTTCTTC
SUBSTITUTE SHEET (RULE 26) _j7_ CAATTAATTTTACAATTTAAA.AAGTATGTATTAATGTTTTATTGTTAGATTT
TCCAAAAAAATGATGCAAATTCTGGTAATATTCATTTCCCTCACCCATAAT
TTGGTTAAAATGAGTAGTTTTAGCCATACAGTCTCATCTGCTGTGGAGGAA
CCTGGAGAAAGTCCCCTGTGCCTTTCTAGCCCTTGGGTTCTATTCTTATCCT
GCAATGTCTACTGCACAGTGTGTTTGAGCAGATCCTAACCCTCCTTTTACA
GTTTCTTCTTCTTACTTCTTTATTCTTTTTGTGGCTCCTGAAATCTGAGGTTA
TTTTGTAATTCAGGAGCATGCAGGACAATTGTTGGGACATGTGCCTAGTCC
GGAATACAGCCCAGGACAGCAAGGAGATGCGTCCTGCACCAGGAAGCCG
TGCAGGCAGGAGCTGTCCAAGGTCCCGGCGGCTCTGCCTGTGTGAGGCAG
GAGAATGAGCAGATTCCCTAATCTATGTTCTCGAAGTTTAATGCTGATGTT
GTCTTGCCTTATCCTCATTTAACTGATACTGTCACCCAGTCCACCTTTGCTC
TCATTGCAAAGTGATAGTGTAATTTCAAATGTAAGACTGAAGATACGATT
GTAAAAGGGAGTAAACTGGTTTAAACGTGTTATTCTAAAGCACCTTACTTT
GTTGTTGTATGCAGAA.AACACAGATGCGCTAATTCAGTATAAATGACTGA
TTGCCTGGAATTTGGACGTTGGCTTAA.AGTCCGATAGCTAAACCTTGGCAA
AACATAACAAACATTTCATTGCTCAGCCTCAGTGCTCTGGAGTATTCAGTG
TATGAGACAGGTTTATTTGAGTCCTCTGTAAATGGCATTTGAATTTTATAT
TCTCCCCTCCCGAGTATCTTATAAGACATCCCCTGAGTTAGGGAGTTCCCA
GACTGCTACTCTATTCCTTATGAATGCAAAACAACCACCAATAGAACAAA
AAAAAAAA,~~AAAAACTCGAG
Mouse YNK 1 partial coding cDNA - Sequence ID NO:15 CTTTACGAGCAGAGGGAGCCAAATTCAGAGCCGTTTTAGATAAAGCTGTG
CAAGCGGATGGACAGGTGAAGGAGCGCTACCAGTCCCATCGAGACACCA
TCGCACTTCTGTGTAAGCCGGAGCCAGAGCTGAATGCTGCCATCCCCTCTG
CTAACCCAGCAAAGACCATGCAGGGCAGCGAGGTTGTAAGTGTCTTAAAG
TCCTTATTATCAAATCTTGATGAAATCAAGAAGGAAAGAGAGAGTCTTGA
GAATGACCTGAAGTCAGTGAATTTTGACATGACAAGCAAGTTTTTGACAG
CTCTGGCCCAAGATGGCGTGATAAATGAGGAGGCTCTCTCTGTCACTGAG
CTGG:ATCGGATCTATGGCGGTCTAAC AAGTAAAGTTCA:AGAGTCTCTG AA
GAAACAAGAGGGACTTCTAAAAAATATACAGGTCTCACACCAAGAATTCT
CCAAAATGAAGCAATCTAACAACGAGGCTAACTTGAGAGAAGAAGTTCTG
AAGAACCTAGCAACTGCGTATGACaaCTTTG~'.'TGAGCT'TGTAGCTAACTTG
AAGGAGGGCACAAAGTTTTACAATG:~GCTG~ii~TGAGATCCTGGTCAGGTT
~5 CCAG_ _-~.CAA.4TGCAGTG_~r'ATAGTGTTTGCACGGAAGACAGAAAGAUAC
GAGC~~:TTGAAGG= i~GCAGCAGAGCATTGCCAGAGAGCCCAGCGCTCC
TTC~-=..TCCCTCC''~ ~CCT-'.TCAGTCCTCCCCAGCAGCGiri GC:'-1TGCAGC
jUBSTITUTE SHEET (RITLE 26) -$3-AGCGCCTCCAACTCCAGCCCCAAGAACCATGCCGCCTGCTAAGCCCCAGC
CTCCAGCCCGGCCTCCACCTCCTGTGCTTCCTGCAAACCGAGTTCCTCCTG
CTTCTGCTGCTGCTGCCCCTGCAGGCGTGGGGACGGCTTCAGCAGCGCCG
CCACAGACCCCTGGCTCTGCTCCCCCGCCACAGGCTCAGGGACCACCATA
CCCTACCTATCCAGGATATCCCGGGTATTGCCAAATGCCCATGCCCATGGG
CTACAACCCCTACGCATATGGCCAGTACAATATGCCGTACCCACCGGTGT
ATCACCAGAGCCCCGGACAGGCTCCATACCCAGGACCCCAGCAGCCTACC
TACCCCTTCCCTCAGCCCCCGCAGCAGTCCTACTATCCACAGCAGTAA
Mouse YNK1 partial protein - Sequence ID N0:16 LRAEGAKFRAVLDKAVQADGQVKERYQSHRDTIALLCKPEPELNAAIPSANP
AKTMQGSEVVSVLKSLLSNLDEIKKERESLENDLKSVNFDMTSKFLTALAQD
GVINEEALSVTELDRIYGGLTSKVQESLKKQEGLLKNIQV SHQEFSKMKQSNN
EANLREEVLKNLATAYDNFVELVANLKEGTKFYNELTEILVRFQNKCSDIVFA
1$ RKTERDELLKDLQQSIAREPSAPSIPPPAYQSSPAAGHAAAPPTPAPRTMPPAK
pQPPARPPPPVLPANRVPPASAA.AAPAGVGTASAAPPQTPGSAPPPQAQGPPY
PTYPGYPGYCQMPMPMGYNPYAYGQYNMPYPPVYHQSPGQAPYPGPQQPT
YPFPQPPQQSYYPQQ
Mouse novel cDNA clone 4: - Sequence ID N0:17 GGTCTTGGCTAGAATTTTAAATTTCTTCTCATTTGAGTAAAATGTTGCATTC
TGAAGTCCCATGCTACCTGAAGTTGCATTTGGAGTCCCAAGCTACTGGAAT
GTTTATATGTGACCGTTTCCCAGGAGGCTTACACTGCAGAAGGAAGAATG
AATCTAGGTGAGGTGGGCAGCTGCTTGGCAGTCCTCTCTTGTGCCCCAACT
2$ GTAAACCAGATAGAAATGTTCAGGGGAGGATACTTTCATTATTGTGGTTTG
TAGTGTTAAGATGATTGCTTCTGCCTTGGAAATACCTCAAGCTGTTCTTAT
TTAACAGGTAAGTGACTGAGTATAATATTCCAGAAAAATTTGAAATCCTA
ATTTCTTCCATATTTCATTAAATTTTTTGCATACAGGTCTAACAAATATGGA
TATGTATACACATCCTCTTTAATGAAGGTATTATTTTGGTTACTTTTCCTAA
GATATACCTTAAAAGATGTTCTATACATTTCCTACTTAAATTCTGGGGGAT
TTGGAGTATGTACATGATAAAAAAGATTATAATATATCGATTGAAGTTACT
TTATTTTCTAATTAGAATTATTTTAATAGTCCTTTATTGAATAAGTGCTGTA
ATTTGTTTGCTATGAGACTTATTCCTGATGTGAATGTAAATTATTTTTCCAC
ATGCATGA.AAAAATGTATGTACTAATCAGAGTTGTCTCCATTGCATTGAAA
3 $ TTACTTGTTTTG.~ACTAAAGTAACTCATATTTATGTAGTAGAATGCTTATG
TTTTCAGACTTTGTAATGATTTCCTTTGGATGTATTTTAAATCAATCGGTCT
GGGTAACATATCAGTTTAGATTAATATGTGCTTAAAAGAAGAAAAA.A.ATT
SUBSTIT'L1TE SHEET (RULE 26) -~4-CAATGGTTCATAGTAGAAATGTGCCACACTTAAATAAGCTCTGTATGACAT
GAAATTCTGTTAAAACATTGTAATTCATGGTGACTTTTAACTTATAAAA.AT
ACTACTTGCACGGGTTACTTGATTTATGGATATATGAAAACTTCTCAGGAC
GAAAGTTCTTCTTTCTCTAGAACTATTCTTCTGTCGGTCATGCAGAATGCT
GTTATTCTGAAAAGTGTCCCTGTTGCATATGATGGTCACTTTATTTGGGGG
GATTCTTCATAAGATGTGAGATGTTGATGCCAGTCTTTCCCAAGTAAGTGC
TCGT~.AAAAAGGACTACTAACTAGCCTGCATCTGTCTCTAACTGGGACCA
AGGGGTCTGCTGAAGGAAACTGAAGAGCTCTAACATTTTCACAGCTTGGA
GAAGATAGAATCTTTAAAAGTACAACTGAAGCTTGATCTATTTTACAAGT
GCATTGATGGCCCCTGTCCTTCTCTGGTTCCTGTCATTTGAAACCAACTCCT
GTTGTAAATAGGAAGAATATGGGACATTCATATTTAAGAA.A.ATTTGATGT
CATTAGGTGACTAAGTAGAAGGCTTAGAAAAATGTATTCATTTGCAAGTA
TTTTGGCACAAGAAATTTTCCAACTGAATAGTAAGCAAAAGCTAAGTTGTT
TCATTGAAATCATAAGGCAGTTTAAGATAAACTGGAGAAGATAACTGTTC
TAATAGAGGATAATCGAATTGATTGTCAAGTGGATGTTATTTATTGGATAG
TGACAGAGTTTATTTGTAACCTTAATTATATTAAAAGTTATTCTGTTAGGA
TGTTTTGTATTAATAAACGTGAACAAAATT C
TCGAGGG
Mouse Novel cDNA clone 8/47/52: - Sequence ID N0:18 GAGAAGGCGGCCTGCCGCAGCGGGACAACCTAGAGCGCGACGTGGAGGC
GCGTAGCGGAGCTGGAGCAACTGCGCACCGAGGTGGATGTGCGCATTAGC
GCNNTGGACACCTGCGTCAAGGCCAAGTCGCTGCCAGCCGTCCCGCCGAG
AGTCTCAGGCCCACCCCCGAACCCTCCACCCATTGATCCAGCTAGCCTGG
AGGAATTCAAGAA.AAGGATCCTGGAGTCTCAGCGGCTCCCTGTAGTCAAC
CCTGCWGCCCAACCCAGCGGTTGAGRACCCAGCTGCCGCAGGACGCTGG
GTGCCAGAATCGCCCACCTGTGGATGGGGGCAGCCAGGTGCCCACAGTGC
TGGACACCCGCCGTGCCTGCCGGCAGCCTCCACCCCCAGCGCCTTCTCTGG
CACCCCTTCACTGTCCCSTGCATCCCCRCCATTCSSCASWSASKGGATTTAA
GGCACACACAGCTGTGAGATGACTTCACATCGACCCCTTGTGCAGTGACC
CGGATGGTGCCCCACCCACACATGAAGCACCCACAGCTCAGCTGCCACCC
TAGGCAACTCCTCCGGTTTCCTATCACTCTGCTCCTGACCCGGGAGGTGAG
AACAGGAAGCCCAGCCTTCAGCTCCCTTGGGAGTTTCCAGCCTCCCTCTTA
AAGGCCACTAGGGTTTCCAGATCCTATTTGAGAGTCTCCAGGCCTCCCCTG
3~ AAGGGTTCTAGCCA.CCACGCCCACAGGATTCCCATTAGGTTTTAAAGTCTT
TTCCAGAGTCCGCTGGTTCCCCTCCTCCTCACAAGGAAGGGCCTCAATTGT
AGATGAGCGTTCCGGGTGGATCTTAGAGCCCTAGAGGGAGGCTTTTGCTT
SUBSTITLTT'E SHEET (RULE 26) GTARCCCCCTAAAGATATTACTGGCACA~TAATAA.ATATGAAAGTCCTTTG
AAA
GTTGGACACTGCGCAAATGGGGCTCTCCATGGACCGCAGCCCATACGCCC
GCACGGGGGACCAGCAGCGCGGCTCTGGTTCTACCTGCGCTATTTCTTCCT
CTTCGTGTCGCTCA'I'TCAGTTCCTCATCATCCTGGGCCTGGTCCTCTTCATG
ATCTATGGCAATGTGCACGCCACCACTGAGTCCAGCCTGCGCGCCACGGA
GATCCGCGCCGACAGCCTGTACAGCCAGGTGGTTCJGACTATCGGCCTCAC
AGGCTAACCTGAGCAAACAGCTGAACATCAGCTTGCTTGTCAAGGAAACA
GTCATGCAGCAACTGTTGACTACGCGACGTGAGATGGAGCGCATC.AACGC
CAGCTTCCGCCAGTGCCAAGGCGACCTGATCACCTACATAAACTATAATC
GCTTCATCGCCGCTATCATCCTGAGCGAGAAGCAGTGCCAGGAAC.AGCTG
AAGGAGGTCAACAAGACCTGCGAACTTTACTCTTCr'1AGCTGGGAGAAAAA
GTT.~~AGACACTGGAGATGGAGGTGGCCAAGGAGAAGGCAGTGTGCTCCA
AGGACAAGGAGAGCCTGCTGGCAGGAAAGCGGCAGACGGAAGAGCAGCT
l~ GGAGGCCTGTG
Mouse Novel cDNA clone I8/2~: - Sequence ID NO:I9 Fend of partial clone TGTGCGCCGCCTCTAGAACTAGTGGATCCCCCGGGCCTGCAGGAATTCCG
GCACGt'1CGGCCGAGCGCCGCGGACCACCCGCGGCTGCCCGCCGAGCCGTC
GAC,~.TGTGGGGGGACTGGGGTGGGAGCGGCCGGAGCAGCGCCAGGTACC
CGGGCGCGCAGAACCATGGCTCTCGCTCGCCTGTCCTGACCTGGCTTGCTC
GCCCCACCGAAGAATGTCAGCCAAGTCCAAGGGGAACCCTCCTCGTCCTC
CGCAGCCGAGGGACCGCCGGCAGCCTCCA.AAACCAAGGTGAAGGAGCAG
ATCAAGATCATAGTGGAGGATCTGGAATTAGTCCTGGGCGACCTGAAGGA
CGTGGCCAAAGAACTTAAGGAGGTGGTTGACCAGATTGAC.ACCCTGACCT
CTGATCTACAGCTGGAAGATGAGATGACCGACAGCTCCAAAACAGACACT
CTGAACAGCAGCTCCAGTGGGACAACAGCCTCCAGCATAGAGAAGATCA
AAGAACAGGCCAATGCTCCCCTCATTAAACCTCCAGCACACCCGTCTGCT
ATCCTGACTGTCCTGAGAAAGCCAAACCCTCCACCGCCTCCTCCAAGGTTG
ACACCCGTGAGGTGTGAAGAGCCTCAGAGAGTGGTGCCGACTGCCAACCC
TGTAAAGACCAATGGCACTCTTCTGCGGAATGGAGGCTTAGCGGGGAGGC
CCAACAAAATTCCAAATGGAG
~ 3' end - Sequence ID N0:20 CTCGAGTTTTTTTTTTTTTTTTTTTTTTTTTTTCATTATTTACTATTAT'TTATT
GACATATTTCCAAAGCTCAAAATATTTTATTATACATATAGTTGAAC.ATAT
SUBSTT1CIJT'E SHEET (RULE 26) GTTTCAAATTGTATAGTATAGA.AAATAAACTTTTTTGTAGTGTCCTCAGCA
TTTCATGATGCAAAACTATTGACAAACATCTTTAGAAAAAT'AATAAAA1'A
GTCCTTCGGTATTAAAATTCTTATTAAAAAGCATTAGATCAAAGGGAGAA
CTATGACATCATCAATGCATAGATGAGATAGGCATGAATGGAATGAGTTG
CCCTGGCTTTATCAACAAATCAAAATATCTGACATCCCAGCTCTT'AT'AATA
GACCAAAATACTTGGAATCAGAAGGTCACAGTTTGTTTTAGGTCAATCAC
AAAAAAATAAAATTCATTCATACTTTCTCAATTTTCCGCAGTTTCTGATGA
TGGAACATAGAAAACAATGTACGTCCAGGACAGAGGCGCTACTCTGCATA
CTTACCACGTGATTTTTTATGCCACTTTGTTGAATGCAGATTAATATATTTG
GGCTTTTTATTGCTTGAGTAGAAAGTGCTCATTACTTATTATTTTACGTTTA
TCATATAGAAAATTAAAAACAAACAGAACGTTTTCTTAAATGGCAGATAT
CACACTGTGGTAGTGGTGGATTTCCTCAGGATGGTCTTCTGTGGTTTTGGT
GCAGCGGGAGGAGGCACGGTTGCAGGTGTGGGAGGGGGGAAACTGTTAC
TGTGGCTTATTCCCAGTCCCCCATTTTCTAATGGGAAAT
Mouse Novel cDNA clone 95/ EST accession #AA 1 19951: - Sequence ID NO:? 1 GCACAGCCCCCCTCCATCCTGAAGAAAACCTCAGCGTATGGGCCTCCAGC
TTCGGGCCGTGTCTATCCTTCCTCTCCTGGGACATGGTGTTCCCCGCTTGCC
CCCCTGGCAGAAAACCG
?0 Mouse Esel I. cDNA: Sequence ID NO:??
CGGCACGAGGAGGAGTGGAGCGGCGCGGGGAGGGCGCGCACJCTTGGTTG
CTCCGTAGT,aCGGCGGCTCGCAAGGGAGC.ATCCCCJAGCGGGCTCCGGGAC
?5 GGCCGGGAGGCAGGCAGGCGGGCGGGCGGGGATGGTGTGCGCGGCTGCG
GACTCGGCGTTCCTCGCGCGGCGTGCGGGCTGCACTGATTTGTGTGAGCTG
GCGGCCGCGCGCACCCGCCCGGAGATGAGGCGTCGATCAGCAAGGTGAA
CGTAATAGAACCATGGCTCAGTTTCCCACACCTTTCGGTGGTAGCCTGGAT
GTCTGGGCCATAACTGTGGAGGAAAGGGCCAAGCATGACCAGCAGTTCCT

T T T TTTTCCAATCTGGGTTACCTCAGCCTGTCTTAGCACAAt'1TATGGGCGC
TAGCGGACATGAATAACGATGGAAGGATGGATCAAGTGGAATTTTCCATA
GCCATGAAGCTTATCAAACTGAAGCTACAAGGATATCAGCTCCCCTCCAC
ACTTCCCCCTGTC.ATG~'1AACAGCAACC.4GTGGCT'~T~I'TCC:1GTGCACC.~GC
ATTTGGTATAGGAGGGATTGCTAGCATGCC.ACC.ACTCAC.aGCTGTTGC : CC
TGTGCCAATGGGCTCCATTCCAC',TTGTTGGAA rt~uCTCCACCCTT.? G-f',A'I'C
TTCTGTCCCTCCAGCAGCAGTGCCTCCCCTGGCTAACGGGGCTCCTCCCGT
SUBSTITUTE SHEET (RULE 26) _;'7_ CATACAGCCTCTGCCTGCGTTTGCGCATCCTGCAGCCACATGGCCAAAGA
GTTCTTCCTTCAGCAGATCTGGTCCAGGGTCACAATTAAACACT.a.aGTTAC
AGAAGGCACAATCATTCGATGTCGCCAGCGCCCCTCCAGCAGCAGAATGG
GCTGTGCCTCAGTCATCAAGGCTGAAATACAGGCAGTTATTCAACAGCCA
CGACAAAACTATGAGTGGACACTTAACAGGTCCCCAGGCAAGAACTATTC
TCATGCAATCAAGTTTACCCC.aGGCTCAGCTGGCTTCAATATGGAATCTTT
CTGACATTGATCAAGATGGAAAACTCACTGCAGAAGAATTTATCCTAGCT
ATGCACCTAATTGATGTTGCCATGTCTGGTCAGCCACTGCCGCCCGTCCTG
CCTCCAGAA'rACATCCCTCCTTCCTTCAGAAGAGTTCGCTCCGGCAGTGGG
.ATGTCCGTCATAAGCTCTTCTTCTGTGGATCAGAGGCTGCCTGAGGAGCCG
TCGTCAGAGG.aTGAGCAGCAGCCAGAGAAGAAACTGCCTGTGACA'rTTGA
AGATAAGA.aGCGGGAGAACTTCGAGCGAGGCAGTGTGGAGCTGGAGAAG
CGCCGGC,aAGCGCTCTTGGAGCAGCAGCGCAAAGAGCAGGAGCGGTTGG
CTC,aGCTGG,aGCGCGCCGAGCAGGAGAGGAAAGAGCGGGAGCGCCAGGA
I~ GCAGGAGGCCAAGCGGC.aGCTGGAGCTGGAGAAGCAGCTGGAGAAGCAG
CGGGAGCTGGAGCGGCAGCGAGAGGAGGAGAGGAGGAAGGAGATCGAG
AGGCGCGAGGCCGCAAAACGGGAACTGGAAAGGCAGCGACAACTTGAAT
GGGAACGGAACCGGAGACAGGAACTCCTGAATCAGAGGAACAAGGAGCA
GGAGGGCACCGTGGTCCTGAAGGCAAGGAGGAAGACTCTGGAGTTTGAGT
?0 TAGt~AGCTCTGAATGACAAAAAGCATCAGCTAGAAGGAAAACTTC,aGGAT
ATCAGGTGTCGACTGGCAACCCAGAGGCAAGAAATTGAGAGCACGAACA
AGTCTAGAGAGCTAAGAATTGCTGAAATCACCCACTTACAGCAGCAGTTG
CAGGAATCTCAGCAAATGCTTGGAAGACTTATTCCAGAGAAACAGr~.TACT
CAGTGACCAGTTAAAACAAGTCCAGCAGAACAGTTTGCATAGAGACTCGC
?5 TTCTTACCCTCAAAAGAGCCTTGGAAGCAAAGGAGCTGGCCCGGCAGCAG
CTCCGGGAGCAGCTGGACGAGGTGGAGAGAGAGACCAGGTCA.aAGCTGC
AGGAGATTGATGTTTTCAACAACCAGCTGAAGGAACTGAGAGAG.aTACAT
AGCAAACAGCAACTCCAGAAGCAGAGGTCCCTGGAGGCAGCGCG,aCTGA
AGCAGAAAGAGCAGGAGAGGAAGAGCCTGGAGTTAGAGAAGCAAAAGG

TGTGCAGCAGGAGGAGCAGCCACGCCCCCGGAAACCCCACGAGGAGGAC
AGACTGAAGAGCiGAAGACAGTGTCAGGAAGAAGGAGGCGGAAGAGAGA
GCCAAGCCGGAAATGCAAGACAAGCAGAGTCGGCTTTTCCATCCGCATCA
GGAGCCAGCTAAGCTGGCCACCCAGGCACCCTGGTCTACCACAGAGAAACJ
GCCCGCTT.ACCATTTCTGC.aC.aGGAGAGT'GT.aAAAGTC'~GTATATTACCG.a GCGC~I'GTACCCCTTTGAA~I~CC~~GAAGTC.aCG.ATGAG:~~i C.aC.'C:"~TCC.-1GCC
AGGAG ATATAGTCATGG'~GGATGAAAGCCAGAC'rGGAGAGCC:rGG,-~TGG
SUBSTITUTE SHEET (RULE 26) _~g_ CTTGGAGGAGAGCTGAAAGGGAAGACGGGATGGTTCCCTGCAAACTATGC
AGA.aAAGATTCCAGAAAATGAGGTTCCCACTCCAGCCAAACC,~GTGACCCJ
ATCTGACATCTGCCCCTGCCCCCA.aACTGGCTCTGCGTGfIGACCCCTGCTC
CTTTGCCAGTGACCTCTTCTGAGCCCTCCACAACCCCC.aACAACTGGGCAG
.aCTTCAGTTCC.aCGTGGCCCAGCAGCTCAAACGAGAAGCCAGAAACGGAC
AACTGGGATACGTGGGCGGCTCAGCCTTCTCTGACCGTACCTAGTGCTGG
CCAGTTACGGCAGAGATCAGCCTTTACCCCAGCCACAGCCACTGGCTCCT
CCCCATCTCCCGTCCTGGGCCAGGGTGAAAAGGTGGAAGGGCTACAAGCG
CAAGCCCTGTATCCCTGGAGAGCCAAAAAAGACAACCACTTAAATTTTAA
CAA.a.aGTGACGTCATCACCGTTCTGGAACAGCAAGAC.=1TGTGGTGGTTTG
GAG.a.aGTTCAAGGTCAGAAGGGTTGGTTCCCCAAGTCTT'ACGTGAAACTC
ATTTCAGGGCCCGTAAGGAAATCCACAAGCATCGAT.aCTGGCCCTACTGA
AAGTCCTGCTAGTCTAAAGAGAGTGGCTTCCCCGGCCGCCAAGCCAGCCA
TTCCCGGAGAAGAGTTTATTGCCATGTACACATACGAGAGTTCTGAGCAA
1 ~ GGAGATTTAACCTTTCAGCAAGGGG.a'rGTGATTGTGGTT.aCCAAGAAAGA
TGGTGACTGGTGGACGGGAACGGTGGGCGACAAGTCCGGAGTCTTCCCTT
CTAACTATGTGAGGCTTAAAGATTCAGAGGGCTCTGGAACTGCTGGGAAA
ACAGGGAGTTTAGGAAAAAAACCTGAAATTGCCCAGGTTATTGCTTCCTA
CGCTGCTACTGGTCCCGAACAACTCACCCTGGCTCCTGGGCAGCTG.aTTCT
GATCCGGAAAAAGAACCCAGGTGGATGGTGGGAAGG.aGAACTGCA.AGCT
CGAGGGA.AAA.aGCGCCAGATAGGGTGGTTTCCAGCAAATTATGTC.aAACT
TCTAAGCCCCGGAACAAGCAAAATCACCCCAACTGAGCTACCCAAGACCG
CAGTGCAGCC.aGC AGTGTGCC.aGGTGATCGGGATGT:aCGATTAC.aCCGCC
CAG.aACGATGACGAACTAGCCTTCAGCAAAGGCCAGATCATCAACGTCCT
?5 CAACAAGGAGGACCCGGACTGGTGGAAAGGAGAAGTC.aGTGGGC.aAGTT
GGGCTCTTCCCATCCAATTATGTAAAGCTGACCACAGACATGGACCCCAG
CCAGCAATGGTGCTCAGACCTGCATCTCTTAGATATGCTGACCCCGACTGA
GAGGAAGCGGCAAGGCTACATCCATGAACTCATTGTCACGGAGGAGAACT
ACGTGAACGACTTGCAGCTGGTCACAGAGATCTTTCAGAAACCCCTGACG
~~0 GAGTCTGAGCTGCTG.ACAGAA,A:~AGAGGTT'GCTATG.aTTTTTGTTAACTG
GAAGGAGCTGATCATGTGTAATATCAAACTGCTGAAAGCGCTGAGAGTCC
GCAAGAAGATGTCTGGGGAGAAGATGCCGGTGAAGATGATTGGCG.aCAT
CCTG.aGCGCCCAGCTGCCGCACATGCAGCCTT~.AC A.'T'~."CGCTTCTGC.aGCTG
CCAGCTCA.aTGGGGCTGCCCTCATC C.AGCAGA.aoJ.~~CGGACGAGGCTCC:aG
AC ~'CA='1GG:1GTTCG T ~' =. '.4-~G?,CTGGCAATGGACCCTCGGTGCA.a.AGGA
ATGCCTCTGTCCAGCT:-:.-~TACTGAAGCCTATGCAGCGTGTCACAAGATAC
CCGCTG.a TC.aTTAAA:4ACATCCTCr('~.a.AAACACTCCTG.~G.=1.ACCATCCA<;:~
SUBSTITUTE SHEET (RULE 26) CCACAGCCACCTGAAGCATGCCCTGGAAAAGGCCiGAGGAGCTGTGCTCCC
AGGTGAACGAGGGAGTTCGAGAGAAGGAGAACTCAGACCGGCTGGAGTG
GATCCAAGCCCACGTGCAGTGTGAAGGCCTTTCTGAGCA.aCTGGTGTTCA
ATTCAGTGACCAACTGCTTGGGACCACCICAAGTTTCTGCACAGCGGGAAG
CTCTACA,aGGCCAAGAGCAATAAAGAACTGTATGGCTTCCTCTTCAACGA
CTTCCTCCTGCTGACCCAAATCACAAAGCCCTTAGGCTCTTCCGGCACCGA
CAAAGTCTTCAGCCCCAAATCTAACCTTCAGTATAAA,aTGTACAAAACGC
CCATTTTCTTAAATGAGGTTCTAGTAAAATTGCCCACGGACCCTTCTGGAG
ATGAGCCTATCTTCCACATTTCCCACATCGACCGGGTCTACACCCTCCGAG
CAGAGAGCATAAATGAGAGGACTGCCTGGGTGCAGAAAATCAAGGCGGC
GTCTGAGCTCTACATAGAGACGGAGAAAAAGAAGCGAGAGAAGGCGTAC
CTGGTCCGTTCCCAGCGGGCGACCGGTATTGGAAGGTTG.aTGGTGAACGT
GGTAGAAGGCATTGAGC~I'GAAGCC.'CTGTCGGTCACATGGAAAGAGCAACC
CGTACTGTGAGGTGACCATGGGCTCTCAGTGCCACATCACCAAGACAATC
CAGGACACGCTAAACCCCAAGTGGA.aTTCTAACTGCCAGTTCTTCATCAG
:aGACCTGGAGCAGGAGGTTCTCT'GCATCACAGTGTTTGAGAGGGACCAGT
TCTCGCCTGATGATTTTTTGGGTCGGACAGAGATCCGAGTGGCCGACATCA
AGAAAGACCAGGGCTCCAAGGGGCCGGTTACGAAGTGTCTCCTGCTGCAT
GAGGTCCCCACGGGAGAGATTGTCiGTCCGCCTTGACCTGCAGTTGTTTGAT
?0 GAGCCGTAGCAGCCCTGCGATGATCGTAGATGACTTCCTCCTCA.aGGCCC
CGTGCGGGCGTGCTGTCTGGTGGTCAGCCTCAGAGCAACGGGG,aTGAAGC
AAAGACGAAGCCCCTCGAGGCTGCTAGGAGTCGTTCTCGACAATCCTGCC
CTTCAAACCATCiTCTCATTTTATGAATCCAAATTCTC'~'TTTCCTTTGCTCTC
CCTATGGTCTCATCATGGCTTCTAGAGTCTCTGAAATCTGTGACCTTTAAC
?5 TAGGTTCCATTGGGAGCCTGGCTCCTTCCCTGGGCTGGAGGTGTGGGTCTG
GTTTCTATAAAATAGATTATAAACTCGAGAATCACTAGT
Mouse Esel L codin~:Sequence ID NO:?3 ATGGCTCAGTTTCCCACACCTTTCGGTGGTAGCCTGGATGTCTGGGCCATA

GA'I'AGCGGGATTTATTACTGGTGATCAAGCGAGGAACTTTTTTTTCCAATC
TGGGTTACCTCAGCCTGTCTTAGCACAAATA'I~GGGCGCTAGCGGACATGA
ATAACGATGGAAGGATGGATCAAGTGGAATTTTCCATAGCCATGAAGCTT
,aTCAAACTGAAGC TACA,aGCJATA~I~CAtiCTCCCCTCCACACTTCCCC:CTGTC
,5 :~TGAAACAGCAACCAGTGGCTATTTCCAGTGCACCAGCATTTGGTATAGG
aGGGATTGCTAGCATGCCACCACTCACAGCTGTTGCTCCTGTGCC,aATGG
GCTCCATTCCAGTTGTTGGAATGTCTCCACCCTTAGTATCTTCTGTCCCTCC
SUBSTITUTE SHEET (RULE 26) AGCAGCAGTGCCTCCCCTGGCTAACGGGGCTCCTCCCG~rCATACAGCCTCT
GCCTGCGTTTGCGCATCCTGCAGCCACATGGCCAAAGAGTTCTTCCTTCAG
CAGATCTGGTCCAGGGTCACAATTAAACACTAAGTTACAGAAGGCACAAT
CATTCGATGTCGCCAGCGCCCCTCCAGCAGCAGAATGGGCTGTGCCTCAG
TCATCAAGGCTGAAATACAGGCAGTTATTCAACAGCCACGACAAAACTrIT
GAGTGGACACTTAACAGGTCCCCAGGCAAGAACTATTCTCATGCAATCAA
GTTTACCCCAGGCTCAGCTGGCTTCAATATGGAATCTTTCTGACATTGATC
AAGATGGAAAACTCACTGC.AGAAGAATTTATCCTAGCTATGCACCTAATT
GATGTTGCCATGTCTGGTCAGCCACTGCCGCCCGTCCTGCCTCCAGAATAC
ATCCCTCCTTCCTTCAGAAGAGTTCGCTCCGGCAGTGGGATGTCCGTCATA
AGCTCTTCTTCTGTGGATCAGAGGCTGCCTGAGGAGCCGTCGTCAGAGGA
TGAGCAGCAGCCAGAGAAGAAACTGCCTGTGACATTTGAAGATAAGAAG
CGGGAGAACTTCGAGCGAGGCAGTGTGGAGCTGGAGAAGCGCCGGCAAG
CGCTCTTGGAGCAGCAGCGCAAAGAGCAGGAGCGGTTGGCTCAGCTGGA
l~ GCGCGCCGAGCAGGAGAGGAAAGAGCGGGAGCGCCAGGAGCAGGAGGC
CAAGCGGCAGCTGGAGCTGGAGAAGCAGCTGGAGAAGCAGCGGGAGCTG
GAGCGGCAGCGAGAGGAGGAGAGGAGGAAGGAGATCGAGAGGCGCGAG
GCCGCAAAACGGGAACTGGAAAGGCAGCGACAACTTGAATGGGAACGGA
ACCGGAGACAGGAACTCCTGAATCAGAGGAACAAGGAGCAGGAGGGCAC
?0 CGTGGTCCTGAAGGCAAGGAGGAAGACTCTGGAGTTTGAGTTAGAAGCTC
TGAATGACAAAAAGCATCAGCTAGAAGGAAAACTTCAGGATATCAGGTGT
CGACTGGCA.4CCCAGAGGCAAGAAATTGAGAGCACGAACAAGTCTAGAG
AGCT,AAGAATTGCTGAAATCACCCACTTACAGCAGCAGTTGCAGGAATCT
CAGCAAATGCTTGGAAGACTTATTCCAG AGAAACAGAT.aCTCAGTG.aCCA
GTTAAAACAAGTCCAGCAGAACAGTTTGCATAGAGACTCGCTTCTTACCC
TCAAAAGAGCCTTGGAAGC.~1AAGGAGCTGGCCCGGCAGC,AGCTCCGGGA
GCAGCTGGACGAGGTGGAGAGAGAGACCAGGTCAAAGCTGCAGGAGATT
GATGTTTTCAACAACCAGCTGAAGGAACTGAGAGAGATACATAGCAAACA
GCAACTCCAGAAGCAGAGGTCCCTGGAGGCAGCGCGACTGAAGC,-~GAAA
:0 GAGCAGCr,AG.AGGA~'~GAGCCTGGAGTTAGAG.AAGCA,AA:'1GGAAGACGCTC
AGAGACGAGTTCAGGAAAGGGACAAGCAATGGCTGGAGCATGTGCAGCA
GGAGGAGCAGCCACGCCCCCGGAAACCCCACGAGGAGGACAGACTGAAG
AGGGA.4G,ACAGTGTCAGCJA.AGAAGGAGGCGGA.AGAGAGAGCC,a:aGCCG
G,-~AAT~J~ AAGACAAGCAGAGTCGGCTTT'TCCAT~~~vGCATC.AGGAGCCACrC
>> TAAGCTGGCCACCCAGGCACCCTGGTCTACCACAGAGAAAGGCCCGCTTA
CCATTTCTGC.ACAGGAGAGTGTAAAAGTGGTATATTACCGAGCGCTGTAC
CCCTTTGAATCCAGAAGTCACGATGAGATCACCATCCAGCCAGGAGATAT
SUBSTTTUTE SHEET (RULE 26) AGTC.aTGGTGGATGAAAGCCAGACTGCJAGAGCCAGGATGGCTTGGAGGA
GAGCTGA.aAGGGAAGACGGGATGGTTCCCTGC,aAACT,aTGC,aGAAAAGA
TTCCAGAAAATGAGGTTCCCACTCCAGCCAAACCAGTGACCGATCTGACA
TCTGCCCCTGCCCCCAAACTGGCTCTGCGTGAGACCCCTGCTCCTTTGCCA
> GTGACCTCTTCTGAGCCCTCCACAACCCCCAACAACTGGGCAGACTTCAGT
TCCACGTGGCCCAGCAGCTCAAACGAGAAGCCAGAAACGGACAACTGGG
ATACGTGGGCGGCTCAGCCTTCTCTGACCGTACCTAGTGCTGGCCAGTTAC
GGCAGAGATCAGCCTTTACCCCAGCCACAGCCACTGGCTCCTCCCCATCTC
CCGTCCTGGGCCAGGGTGAAAAGGTGCiAAGGGCTACAAGCGCAAGCCCT
GTATCCCTGGAGAGCCAAAAAAGACAACCACTTAAATTTTAACAAAAGTG
ACGTCATC.aCCGTTCT'GGAACAGCAAGACATGTGCiTGGTTTGGAGAAGTT
CAAGGTCAGA,aGGGTTGGTTCCCCaACrTCTTACGTGAAACTCATTTCAGG
GCCCGTAAGG.aAATCCACAACiCA'I~CGATACTGGCCCTACTGAAAGTCCTG
CTAGTCTAAAGAGAGTGGCTTCCCCGGCCGCCAAGCCAGCCAT'CCCCGGA
1 ~ GAAGAGTTTATTGCCATGTACACATACGAGAGTTCTGAGCAAGGAGATTT
AACCTTTCAGCAAGGGGATGTGATTGTGGTTACCAAGAAAGATGGTGACT
GGTGGACGGGAACGGTGGGCGACAAGTCCGGAGTCTTCCCTTCTAACTAT
GTGAGGCTTAAAGATTCAGAGGGCTCTGGAACTGCTGGGAAAACAGGGA
GTTTAGGAAAAAAACCTGAAATTGCCCAGGTTATTGCTTCCTACGCTGCTA
?0 CTGGTCCCGAACAACTCACCCTGGCTCCTGGGC.aGCTGATTCTGATCCGGA
AAAAGAACCCAGGTGGATGGTGGCJAAGGAGAACTGCAAGCTCGAGGGAA
AAAGCGCCAGATAGGGTGGTTTCCAGCAAATTATGTCAAACTT'CTAAGCC
CCGGAACAAGCAAAATC.ACCCCAACTGAGCTACCCAAGACCGCAGTGCA
GCC.aGCAGTGTGCCAGGTGATCGGGATGT,aCGATTACACCGCCC:aGAACG
2~ ATGACGAACTAGCCTTCAGCAAAGGCCAGATCATCAACGTCCT'CAACAAG
GAGGACCCGG.aCTGGTGGAAAGGAGAAGTCAGTGGGCAAGTTGGGCTCTT
CCCATCCAATTATGTAAAGCTGACCACAGACATGGACCCCAGCCAGCAAT
GGTGCTCAGACCTGCATCTCTTAGATATGCTGACCCCGACTGAGAGGAAG
CGGCAAGGCT.aCATCCATGAACTCATTGTCACGGAGGAGAACTACGTGAA

AGCTGCTGACAGAAAAAGAGGTTGCTATGATTTTTGTTAACTGGAAGGAG
CTGA'I'CATGTGTAATATCAAACTGCTGAAAGCGCTGAGAGTCCGCAAGAA
GATGTCTGGGG.AGAAGATGCCGGTGAAGATGATTGGCGACATCCTGAGCG
CCCAGCTGCCGCACATGCAGCCTTACATCCGC:TTCTGC ACiCTGCC AGCTCA
.~ ATGGGGCTGC:CCTC.aTCC:aGCAGAAGACGG:aCGAGGC'rCCAGACTTCAA(J
G:~GTTCG'hC,a,=~AAei.aCTGGCAATGGACCCTCGGTGCA~AGGAA-I~GC.'CTCZ~
GTCC.aGCTTT:aTACTGAAGCCT.aTGt:AGC GTGTCACAAG.aT,aCCCGCTGAT
SUBSTTTUTE SHEET (RULE 26) -6?-CATTAAAAACATCCTGGAAAACACTCCTGAGAACCATCCAGACC.aCAGCC
.aCCTGAAGC,aTGCCCTGGAAAAGGCGGAGGAGCTGTGCTCCCAGGTGAAO
GAGGGAGTTCGAGAGAAGGAGAACTCAGACCGGCTGGAGTGGATCCAAG
CCCACGTGCAGTGTGAAGGCCTTTCTGAGCAACTGGTGTTCAATTCAGTGA
CCAACTGCTTGGGACCACGCAAGTTTCTGCACAGCGGGAAGCTCT~~CAAG
GCCAAGAGC.aATAAAGAACTGTATGGCTTCCTCTTCAACGACTTCCTCCTG
CTGACCCAAATCACAAAGCCCTTAGGCTCTTCCGGCACCGACAAAGTCTT
CAGCCCCAA.AT'CTAACCTTCAGTATAAAATGTACAAAACGCCCATTTTCTT
AAATGAGGTTCTAGTAAAATTGCCCACGGACCCTTCTGGAGATGAGCCTA
TCTTCCACATTTCCCACATCGACCGGGTCTACACCCTCCGAGCAGAGAGCA
TAA.ATGAGAGGACTGCCTGGGTGCAGAAAATCAAGGCGGCGTCTGAGCTC
TACATAGAG,aCGGAGAAAAAGAAGCGAGAGAAGGCGTACCTGGTCCGTT
CCCAGCGGGCGACCGGTAT'TGGAAGGTTGATGGTGAACGTGGTAGAGGCA
TTGAGCTGAAGCCCTGTCGGTCACATGGAAAGAGCAACCCGT.aCTGTGAG
l~ GTGACCATGGGCTCTCAGTGCCACATCACCAAGACAATCCAGGACACGCT
AAACCCCAAGTGGAATTCTAACTGCCAGTTCTTCATCAGAGACCTGGAGC
AGGAGGTTCTC'I'GC.ATCACAGTGTTTGAGAGGGACCAGTTCTCGCCTGAT
GATTTTTTGGGTCGGACAGAGATCCGAGTGGCCGACATCAAGAAAGACCA
GGGCTCCAAGGGGCCGGTTACGAAGTGTCTCCTGCTGCATGAGGTCCCCA
?0 CGGGAGAG.aTTGTGGTCCGCCTTGACCTGCAGTTGTTTGATGAGCCGTAG
Murine Ese 1 L protein: Sequence ID NO: 2~
MAQFPTPFGGSLDV WAITVEERAKHDQQFLSLKPIAGFITGDQARNFFFQSGL
PQPVLAQIWALADMNNDGRMDQVEFSIAMKLIKLKLQGYQLPSTLPPVMKQ
2~ QPVAISSAPAFGIGGIASMPPLTAVAPVPMGSIPVVGMSPPLVSSVPPAAVPPL
ANGAPPVIQPLPAFAHPAATWPKSSSFSRSGPGSQLNTKLQKAQSFDVASAPP
AAEWAVPQSSRLKYRQLFNSHDKT'MSGHLTGPQARTILMQSSLPQ:~QLASI W
NLSDIDQDGKLTAEEFILAMHLIDVAMSGQPLPPVLPPEYIPPSFRRVRSGSGM
SVISSSSVDQRLPEEPSSEDEQQPEKKLPVTFEDKKRENFERGSVELEKRRQAL
?0 LEQQRKEQERLAQI_ERA'1E(~'rRhERERQEQEAKRQLELEKQLEKQRE=LERQRE
EERRKEIERREAAKRELERQRQLEWERNRRQELLNQRNKEQEGTVVLKARR
KTLEFELEALNDKKHQLEGKLQDIRCRLATQRQEIESTNKSRELRIAEITHLQQ
QLQESQQMLGRLIPEKQI LSDQLKQVQQNSLHRDSLLTLKR.ALEAKEI. ARQQ
LREQLDEVERETRSKLQEIDVFNNQLKELREIHSKQQL.(~KQRSLE.AARLKQKL
QERKSLELFKQKEDAQRRVQERDKQWLE~IVQQEEQPRPRKPHEEDISLi~nE~
SVRKKE.aEERaKPEMQDKQSRLFHPHt~cPf~KLATQAPWS i~TE.IW ,'LTIS:a~>E
SVKVVY~'RALYPFESRSI-IDEITIQPGDIV'MVDESQTGEPGU'LGGELKGKTGt~' SUBSTITUTE SHEET (RULE 26) -6s-t.L'~,DFSSTWPSSSNEKPETDNWDTWAAQPSLTVPSAGQLRQRSAFTPATATG~
SPSPVLGQGEKVEGLQAQALYPWRAKKDNHLNFNKSDVITVI_EQQDMWWF
GEVQGQKGWFPKSYVKLISGPVRIhSTSID'I'GPTESPASLKRVASPAAKPAIPGE
EFIAM1'TYESSEQGDLTFQQGDVIVVTKKDGDWWTGTVGDKSGVFPSNYVR
LKDSEGSGTAGKTGSLGKKPEIAQVIASYAATGPEQLTLAPGQLILIRKKNPGG
WVVEGELQARGKKRQIGWFPANYVKLLSPGTSKITPTELPKTAVQPAVCQVIG
ItI~'DYTAQNDDELAFSKGQIINVLNKEDPDWWKGEVSGQVGLFPSNYVKLTT
DMDPSQQWCSDLHLLDML'rPTERKRQUYIHELIVTEENYVNDLQLVTEIFQK
PLTESELLTEKEVAMIFVNWKELIMCNIKLLKrILRVRKKMSGEKMPVIuWIGD
ILSAQLPHMQPYIRFCSCQLNGAALIQQKTDEAPDFKEFVKRLAMDPRCKGM
PLSSFILKPMQRVTRYPLIIKNILENTPENHPDHSHLKHALEKAEELCSQVNEG
VREKENSDRLEWIQAHVQCEGLSEQLVFNSVTNCLGPRhFLHSGKL~'KAKSN
KELYGFLFNDFLLLTQITKPLGSSGTDKVFSPKSNLQYKMYKTPIFLNEVLVKL
l~ PTDPSGDEPIFHISI-IIDRVYTLRAESINERT.aWVQKIKAASELYIETEKKKREKA
~'LVRSQRATGIGRLM VNV V EGIELKPCRSI-IGKSNPYCEVTMGSQCHITKTIQD
TLNPKWNSNCQFFIRDLEQEVLCITVFERDQFSPDDFLGRTEIRVADIKKDQGS
KGPVTKCLLLHEVPTGEIVVRLDLQLFDEP
?0 '~-Iouse Ese2L cDNA: Sequence ID NO:?>
CCCTTCCTTTCCTTTTTTTGTGTTCGCCTTCGGCCGTGCCGGCTG.aGAGCCC
.aGCAGCCGTGACrIGGCT~GCGCAACAGGTTCGC'rGCGGCCGGCCTGACGAC
TGACCCGGCGGCGGCGGCCGCGGCACGGCAGGCiTCTTCCCGGAGCTTGGC
CGCGCCCACGCGCCGGTGTCGAGGAGCGCGCGGGGTCGCGCCGGGACGT
''~ GCGCGAGGCGCCAGATGGCTGAGAGCTGCAAGAAGAAGTCAGGATCATG
ATGGCTCAGTTTCCCACAGCGATGAATGGAGGGCCAAATATGTGGGCTAT
T.aCATCTGAAGAACGTACTAAGCATGATAAAC AGTTTG.ATAACCTCAAAC
CTTCAGGAGGTT,aCATAACAGGTGATCAAGCCCGTACTTTTTTCCTACAGT
CAGGTCTGCCGGCCCCGGTTTTAGCTGAAATATGGGCCTTATCAGATCTGA

.aTCAAGTTAAAGTTGCAGGGCCAACAGCTGCCTGTAGTCCTCCCTCCTATC
ATGAAACAACCCCCTATGTTCTCTCCACTAATCTCTGCTCGTTTTGGGATG
GGAAGCATGCCCAATCTGTCCATTCATCAGCCATTGCCTCCAGTTGCACCT
AT.aGCA,ACACCCTTGTCTTCTGCTACGTCAGGGACCAG~I':~TTCCTCCCCTA
aTGATGCCTGCTCCCCTeGTGCCTTC'I~G'1'TAGT.:~CATCCTC:1'rTACC.aA:J,T
GGA.aC TGCC AGTCTC.-vTTC.aGCC'rTTATCCa-I"I~CCTT:'~TTCTTCTTC.a.aC.-'~T
TCiCCTC.aTGCATC; aTCTTAC AGC CTG A'I~GATGC~GAGGr~TTTGCJTGG'I~GCT!~
SUBSTITUTE SHEET (RULE 26) -6=~-GTATCCAGAAGGCCCAGTCTCTGATTGATTTAGGATCTAGTAGCTCAACTT
CCTCAACTGCTTCCCTCTCAGGGAACTCACCTAAGACAGGGACCTCAGAG
TGGGCAGTTCCTCAGCCTTCAAGATTAAAGTATCGGCAAAAATTTAATAGT
CTAGACAAAGGCATGAGCGGATACCTCTCAGGTTTTCAAGCTAGAAATGC
CCTTCTTCAGTCAAATCTCTCTCAAACTCAGCTAGCTACTATTTGGACTCT
GGCTGACATCGATGGTGACGGACAGTTGAAAGCTGAAGAfITTTATTCTGG
CGATGCACCTCACTGACATGGCCAAAGCTGGACAGCCACTACC.4CTGACG
TTGCCTCCCGAGCTTGTCCCTCCATCTTTCAGAGGGGGAAAGCAAGTTGAT
TCTGTTAATGGAACTCTGCCTTCATATCAGAAAACACAAGAAGAAGAGCC
TCAGAAGAAACTGCC,AGTTACTTTTGAGGACAAACGGAAAGCCAACTATG
AACGAGGAA.ACATGGAGCTGGAGAAGCGACGCCAAGTGTTGATGGAGCA
GCAGCAGAGGGAGGCTGAACGCAAAGCCCAGAAAGAGAAGGAAGAGTG
GGAGCGGAAACAGAGAGAACTGCAAGAGCAAGAATGGAAGAAGCAGCTG
GAGTTGGAGAAACGCTTGGAGAAACAGAGAGAGCTGGAGAGACAGCGGG
1~ AGGAAGAGAGGAGAAAG(iAGATAGAAAGACGAGAGGCAGCAAAACAGG
AGCTTGAGAGACAACGCCGTTTAGAATGGGAAAGACTCCGTCGGCAGGAG
CTGCTCAGTCAGAAGACCAGGGAACAAGAAGACATTGTCAGGCTGAGCTC
CAGAAAGAAAAGTCTCCACCTGGAACTGGAAGCAGTGAATGGAAAACAT
CAGCAGATCTCAGGCAGACTACAAGATGTCCAAATCAGAA.4GCAAACAC
?O aAA.aGACTGAGCTAGAAGTTTTGGATAAACAGTGTGACCTGG.AAATT.ATG
GAAATCAAAC AACTTCAACAACIAGCTTAAGGAATATCAAAATAAGCTTAT
CTATCTGGTCCCTGAGAAGCAGCTATTAAACGAAAGAATTAAAAACATGC
AGCTCAGTAAC ACACCTGATTCAGGGATC AGTTTACTTCATAAAAAGTCA
TCAGAAAAGGAAGAATTATGCCAAAGACTTAAAGAAC.4ATT,AGATGCTCT
TGAAAAAGAAACTGCATCTAAGCTCTCAGAAATGGATTCATTTAACAATC
AGCTGAAGGAACTCAGAGAAAGCTATAATACACAGCr'1GTTAGCCCTTGAA
CAACTTCATAAAATCAAACGTGACAAATTGAAGGAAATCGAAAGAAAAA
GATTAGAGCAAATTCAAAAAAAGAAACTAGAAGATGAGGCTGCAAGGAA
AGCAAAGCAAGGAAAAGAAAACTTGTGGAGAGAAAGTATTAGAAAGGAA
~~J G.AAGAGGAAAAGC.AAAAACGACTCCAGGAAGAAAAGTCACAGGACAAA
ACTCAAGAAGAGGAACGAAAAGCTGAGGCAAAACAAAGTGAGACAGCCA
GTGCTTTGGTGAATTACAGAGCACTGTACCCTTTTGAAGCAAGAAACCAT
GATGAGATGAGTTTTAGTTCTGGGGATATAATTCAGGTTGATGAAAAAAC
'~ iTAGGAG;~GCCTGGTTGGCTTTATGGTAGTTTTCAGGGA;'~AGTTTGGCTCI
;, teTCCCCTGCAACTATGT.aG~~.AAAGTGCTGTCAAGTGAAAAAGCTCTGT
CTCCTAAGAAGGCCTTACTTCCTCCTACAGTGTCTCTCTCTGCTACCTCA.a CTTCTTCCCAGCCACCAGCATCAGTG.4CTGATTA1'CACAATGTATCCTTCT
-sZJBSTITiTTE SHEET (RULE 26) -6~-CAAACC'1'TACTGTTAATACAACATGGCAGCAGAAGTCAGCTTTTACCCGC
ACTGTGTCCCCTGGATCTGTGTCCCCCATTCACGGACAGGGGCAGGCTGTA
GAAAACCTGAAAGCCCAGGCCCTTTGTTCCTGGACGGCAAAGAAGGAGA
ACCACCTGAACTTCTCAAAGCACGACGTCATCACTGTCCTGGAGCAGCAG
GAAAACTGGTGGTTTGGGGAGGTGCACGGAGGAAGAGGATGGTTCCCCA
AGTCTTATGTCAAGCTCATTCCTGGGAATGAAGTACAGCGAGGAGAGCCA
GAAGCTTTGTATGCAGCTGTGACTAAGAAACCTACCTCCACAGCCTATCC
AGTTACCTCCACAGCCTATCCAGTTGGAGAAGAC'I'ACATTGCACTTTATTC
ATACTCAAGTGTAGAGCCCGGGGATTTGACTTTCACTGAAGGTGAAGAAA
TTCTAGTGACCCAGAAAGATGGAGAGTGGTGGACAGGAAGTATTGGAGA
GAGAACTGGAATCTTCCCGTCCAAC.TACGTCAGACCAAAGGATCAAGAGA
ATTTTGGGAATGCTAGCAAATCTGGAGCATCAAACAAAAAACCCGAGATC
GCTCAAGTAACTTCAGCATATGCTGCTTC AGGGACTGAGCAGCTCAGCCTT
GCGCCAGGACAGTTAATATTAATCTTAAAGAAAAACACA.AGCGGGTGGTG
l~ GCAAGGAGAGCTACAGGCCAGAGGGAAGAAACGACAGAAGGGATGGTTT
CCTGCCAGCCATGTAAAGCTGCTAGGTCCAAGCAGTGAAAGAACCATGCC
TACTTTTCACGCTGTATGTCAAGTGATTGCTATGTATGACTACATGGCGAA
TAACGAAGATGAGCTCAATTTCTCCAAAGGACAGCTGATTAATGTTATGA
ACAAAGATGACCCTGACTGGTGGCAAGGAGAAACC.4ATGGTCTGACTGGT
CTCTTTCCTTCAAACTATGTTAAGATGACAACAGACTCAGATCCA.AGTCAA
CAGTGGTGTGCTCiACCTCCAAGCCCTGGACACAATGC,AGCCTACGGAGAG
GAAGCGACAGGGCTACATTCACGAGCTCATTCAGACAGAGGAGCGGTAC
ATGGACGACGACCTGCAGCTGGTCATCGAGG'rCTTCCAGAAACGGATGGC
TGf~GGAAGGCTTCCTCACTGAAGCAGACATGGCTCTGATCTTTGTGAACT
GGAAAGAGCTCATCATGTCCAACACGAAGCTGCTGAGGGCCTTGCGGGTG
AGGAAGAAGACTGGGGGTGAGAAGATGCCAGTTCAGATGATTGGAGACA
TCCTGGCGGCAGAC~CTGTCCCACATGCAGGCCTACATCCGCTTCTGCAGCT
GTCAGCTTAATGGGGCAACCCTGTTACAGCAGAAGACAGACGAGGACAC
GGACTTCAAGGAATTTCTAAAGAAGTTGGC ATCAGACCCACGATGCAAAG
GGATGCCCCTCTC'CAGCTTCCTGCTGAAGCCCATGCAGAGGA~rC.~CTCGCT
ACCCGCTGCTCATCCGAAGTAT'CCTGGAGAACACTCCACAGAGTCATGTT
GACCACTCCTCCCTGAAGCTGGCCCTAGAACGTGCTGAGGAGCTGTGCTC
TCAGGTGAACGAGC~GAGTCCGGGAGAAGGAAAATTCAGACCGGCTGGAG
TGG.aTCCAGGCAC AC GTGC,AGTGCGAAGGCTTGGCAGAGCAACTTATTTT
3~ CAACTCCCTCACCAACTGCCTGGGCCCCCGGAAGCTTCTGCACAGCGGGA
AGCTGTAC.AAGACCAAGAGCAATAAGGAGCTGCACGCCTTCCTCTTC.AAC
GACTTCCTGCTGCTCACC~I~ACCTGGTCAGGCAGTTTGCCGCCGCCTCTGGC
SUBSTITUTE SHEET (RULE 26) CACGAGAAGCTCTTCAACTCCAAGTCCAGTGCTCAGTTCCGGATGTACAA
AACGCCCATTTTCCTGAATGAAGTGTTGGTGA.aACTTCCCACAGACCCTTC' CGGCGATGAGCCCGTCTTCCACATTTCCCACATTGATCGTGTGT,~1CACACT
CCGAACAGACAACA'I'CAACGAGAGGACGGCCTGGGTCCAGAAGATCAAG
GGTGCCTC.aGAGCAGTACATCGACACTGAGAAGAAG,AAACGGGA.a.aAGG
CTTACCAAGCCCGTTCTCAAAAGACTTCAGGTATTGGGCGTCTGATGGTGC
ATGTCATTG,aAGCTACAGAATTAAAAGCCTGCAAACCA,aACGGGAAAAGT
AATCCATACTGTGAAGTCAGCATGGGCTCCC.aAAGCTATACCACCAGGAC
CCTGCAGGACACACTAAACCCCAAGTGGAACTTCAACTGCCAGTTCTTCA
TCAAGGATCTTTACCAGGACGTTCTG'rGTCTCACTATGTTTGACAGAGACC
AGTTTTCTCCAGATGACTTCTTGGGTCGTACTGAAGTTCCAGTGGCAAAAA
TCCC~AACAGAACAGGAAAGC,aAAGGCCCCACCACCCGCCGACTACTACTG
C,aCGAAGTCCCCACTGGAGAAGTCTGGGTCCGCTTTGAC CTGC,aACTTTTT
GAACAAAAAACTCTCCTTTGAGGGCCTGGGGAAGCCAGAACCAGGGGAG
is CTGCCCACA.aGGCTGGGTCTAAAGACAGATTTTGCTCTCCCAGGACAGAG
GAGCATCACATGGCTTCATCCATCAAACAGCCACACTCGCTGGGCCTGTA
TTTTATTGC.aCACTAAATTGCTAGCAATCTATGCAAACATGATCTTTTAAA
CAAACGCCACAGCACAGTGCCTTGTACTAGTGTTAACCTGTTCAGCTGTGT
TAGATGCC.aGGGTTTCCATTTTCAGGGCTATAAAAGTATTATGTGGAAATG
AGGCATCrIGACCACCGGACGTTACCACTTGGCAAATCTGTCCACTGTGGA
GTTGGTGATGTTGGAACCATTCCACACTATGTGACCTCTGCTGGGTCACAC
ACTCAGGAGGTGAAGGGCTGAGATGAAATGCTGCAGCCTTGGGGCTTGTG
CAGCCTG,aTACTGAAATAGCATCCACTTGTGCACTGAATAAATAGAAACT
TGATCGTTTTATTCTGACTAGATATTATCATTCTCTCJCTA AGACA.aTATAGT
2~ TTGAAATATT.aTAGTTTGAATATAAGGAGGAAAGCTTGATGTACTTTAAAT
ATACTGTGAaCTCTAATAATGTGGGGATATTTTTCAACTTTAATTTTCTTAA
GTATAA,aTTATTTATGTAAATTCTTTGTTTTGCATATTTCATAGAAC.aTGCA
TCTTTAAGCTTTATCATTGCCAACAATGTACAGAAAGAGAATAAAAGTAT
AAGTTTATGA,aTGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA~a 3 (>
Murine Ese?L codin~_: Sequence ID N(): 26 ATGGCTCAGTTTCCCACAGCGATGAATGGAGGGCCAAATATGTGGGCTAT
TACATCTGA.aG.aACGT:aCTAAGCATG,aTAAACAGTT'T'G:~T.<~ACCTCAAAC
CTTC:'~GGAGGTTACAT:IACtIGGTGATC.aAGCCCGT:'WT'TTT i TCCT.-''~C:1GT
CAGG~TCTGCCGGCCCCGCJTTTTAGCTGA..~AT:a T GGCIC C ~~-I' ~1~I C.aG:~TCTGA
ACA.aGG.aTGGGAAGATGGACCAGC.1AGAGTTCTCTAT.aGCTATGAAACTC
ATC.a.aGTTA;1:~GTTGCACiGGCC.aACAGCTGCCTGTtIGTCCTCCCTCCTATC
SUBSTT)rUTE SHEET (RULE 26) ATGrIAACAACCCCCTATGTTCTCTCCAC~I'AATCTCTGCTCGTTTTGGGATG
GGAAGCATGCCCAATCTGTCCATTCATCAGCCATTGCCTCCAGTTGCACCT
ATAGCAACACCCTTGTCTTCTGCTACGTCAGGGACCAGTATTCCTCCCCTA
AT'GATGCCTGCTCCCCTAGTGCCTTCTGTTAGTACATCCTCATTACCAAAT
GGAACTGCCAGTCTC.ATTCAGCCTTTATCCATTCCTTATTCTTCTTCAACAT
TGCCTCATGCATCATCTTACAGCCTGATGATGGGAGGATTTGGTGGTGCTA
GTATCCAGAAGGCCCAGTCTCTGATTGATTTAGGATCTAGTAGCTCAACTT
CCTCAACTGCTTCCCTCTCAGGGAACTCACCTAAGACAGGGACCTCAGAG
TGGGCAGTTCCTCAGCCTTCAAGATTAAAGTATCGGCAAAAATT'rAATAGT
CTAGACAAAGGCATGAGCGGATACCTCTCAGGTTTTCAAGCTAGAAATGC
CCTTCTTCAGTCAAATCTCTCTCAAACTCAGCTAGCTACTATTTGGACTCT
GGCTGACATCGATGGTGACGGACAGTTCJAAAGCTGAAGAATTTATTCTGG
CGA~I~GCACCTCACTGACATGGCCAAAGCTGGACAGCCACTACCACTG
ACGTTGCCTCCCGAGCTTGTCCCTCCATCTTTCAGAGGGGGAAAGCAAGTT
1 ~ GATTCTGTTA.~TGGAACTCTGCCTTCATAZ'CAGAAAAC ACAAGAAGAAGA
GC CTCAGAAGAAAC7'GCCAG~rTACTTTTGAGGACAAACGGAAAGCCAACT
ATGAACGAGGAAACATGGAGCTGGAGAAGCGACGCCAAGTGTTGATGGA
GCAGCAGCAGAGGGAGGCTGAACGCAAAGCCCAGAAAGAGAAGGAAGA
GTGGGAGCGGAAACAGAGAGAACTGCAAGAGCAAGAATGGAAGAAGCA
?0 GC~rGGAGTTGGAGAAACGCTTGGAGAAACAGAGAGAGCTGGAGAGACAG
CGGGAGGAAGAGAGGAGAAAGGAGATAGAAAGACGAGAGGCAGCAAAA
CAGGAGCTTGAGAGACAACGCCGTTTAGAATGGGAAAGACTCCGTCGGCA
GGAGCTGCTCAGTC.AG,aAGACCAGGGAACAAGAAGACATTGTC,aGGCTG
AGCTCCAGAAAGAAAAGTCTCCACCTGGAACTGGAAGCAGTGAATGGAf'1 ?5 AACATCAGCAGATCTC.~GGCAGACTACAAGATGTCCAAATCAGAAAGCA
AACACAAAAGACTGAGCTAGAAGTTTTCiGATAAACAGTGTGACCTGGAAA
TTATGGAAATCAAACAACTTCAACAAGAGCTTAAGGAATATCAAAATAAG
CTTATCTATCTGGTCCCTGAGAAGCAGCTATTAAACGAAAGAATTAAAAA
CATGCAGCTCAGTAACACACCTGATTCAGGGATCAGTTTACTTCATAAAA
>0 AGTC.aTCAGAAAAGCiAAGAATTA~1'GCC'AAAGACTTAAAGAAC.AATTAGAT
GCTC~rTGAAAAAGAAACTGCATCT.AAGC TCTCAGAAATGGATTCATTTAA
CAATCAGCTGAAGGAACTCAGAGAAAGCTATAATACACAGCAGTTAGCCC
TTGAACAACTTCATAAAATCAAACGTGACAAATTGAAGGAA.ATCGAAAG.A
AAAAG.ATTAGAGCAAATTC.AA AAAAAGAAACTAG,AAGATG,AGGCTGCAA
GGA:~AGCAAAGCA=~GGAAAAGA.~A.4CTTGTCiGAGAG,aAAGTATTAGAAA
GGA:~GAAG AGGAA.u:~GCA.-~~~~waCGACTCCAGGAAGAAAAGTC.ACAGGA
SUBSTITUTE SHEET (RULE 26) CAAAACTCAAGAAGAGGAACGAAAAGCTGAGCJC.aAAACAA,aGTGAGAC:a GCC.aGTGCTTTGGTG
AATTACAGAGCAC'rGTACCCTTTTGAAGCAAGAAACCATGATGAGATGAG
TTTTAGTTCTGGGGATATAATTCAGGTTGATGAAA.aAACTGTAGGAGAGC
CTGGTTGGCTTTATGGTAGTTTTCAGGGAAAGTTTGGCTGGTTCCCCTGCA
ACTATGTAGAAAAAGTGCTGTCAAGTGAAAAAGCTCTGTCTCCT.a.aGAAG
GCCTTACTTCCTCCTACAGTGTCTCTCTCTGCTACCTCAACTTCTTCCCAGC
CACCAGCATCAGTGACTGATTATCACAATGTATCCTTCTCAAACCTTtICTG
TTAATACAACATGGCA
IO GCAGAAGTCAGCTTTTACCCGCACTGTGTCCCCTGGATCTGTGTCCCCCAT
TCACGGACAGGGGCAGGCTGTAGAAAACCTGAAAGCCCAGGCCCTTTGTT
CCTGGACGGCAAAGAAGGAGAACCACCTGAACTTCTC.aAAGCACGACGTC
ATCACTGTCCTGGAGCAGCAGGAAAACTGGTGGTTTGGGGAGGTGC,aCGG
AGGAAGAGGATGGTTCCCCAAGTCTTATGTCAAGC'rCATTCCTGGGAATG
1 ~ :aAGTACAGCG,aGGAGAGCCAGAAGCTTTGTATGCAGCTGTGACT.aAGAAA
CCT.aCCTCCACAGCCTATCCAGTTACCTCCACAGCCTATCCAGTTGGAGAA
GACTACATTGCACTTTATTCATACTCAAGTGTAGAGCCCGGGGATTTGACT
TTCACTGAAGGTGAAGAAATTCTAGTGACCCAGAAAGATGGAGAGTGGTG
GAC.aGGAAGTATTGGAGAGAGAACTGGAATCTTCCCGTCCAACT:aCGTCA
?0 GACCAAAGGATCAAGAG.aATTTTGGGAATGCTAGCAAATCTGGAGCATCA
AAC.aAAAAACCCGAGATCGCTCAAGTAACTTCAGCATATGCTGCTTCAGG
GACTGAGCAGCTCAGCCTTGCGCCAGGACAGTTAATATTAATCTT.aAAGA
AA.aACACAAGCGGGTGGTGGCAAGGAGAGCTACAGGCCAGAGGG:aAGAA
ACGACAGAAGGGATGGTTTCCTGCCAGCCATGTAAAGCTGCTAGGTCCAA
GCAGTGAAAGAACCATGCCTACTTTTCACGCTGTATGTCAAGTGATTGCTA
TGTATG.aCTACATGGCGAATAACGAAGATGAGCTCAATTTCTCC.a.a AGGA
CAGCTGATTAATGTTATGAACAAAGATGACCCTGACTGGTGGCA.aGGAGA
AACCAATGGTCTGACTGGTCTCTTTCCTTCAAACTATGTTAAGATG,aCAAC
AGACTCAGATCCAAGTCAACAGTGGTGTGCTGACCTCCA:aGCCCTGGACA
~0 CA.aTGC_AGCCZ'ACGGAG:~GGAAGCGACAGCIGCT.aCATTC.aCGAGCTCATT
CAGACAGAGGAGCGGTACATGGACGACGACCTGCAGCTGGTCATCGAGG
TCTTCCAGAAACGGATGGCTGAGGAAGGCTTCCTCACTGAAGC.aGACATG
GCTCTGATCTTTGTGA.ACTGG,aAAG.aGCTCATC:1TGTCC:1ACACG:~1GCT
GCTGAGGGCCTTGC(iGGTGAGGA:aGA.aGACTCICTGGGTG.AGAAG.aTGCC.a 3s E.~~TTCAGAT:.'_',-:=~TTGG.~GAC.~ T CC'I'CJGCi~C~C.aGAGCTGTCCCACATGCAGGC
CTAC ATCCGC'I'TCTGCAGC~I'GTC AGCTTA:~~t~GGGGCAACCCTGTT.aCAGCA
GAAGA r~ aGACG:aCiGACACGGACTTCA.aGGA:~TTTCT.a_~AGAAGTTGGCAT
SUBSTTTIJTE SHEET (RULE 26) CAGACCCACGATGCAAAGGGATGCCCCTCTCCAGCTTCCTGC'rGAAGCCC
ATGCAGAGGATCACTCGCT,aCCCGCTGCTCATCCGAAGTATCCTGGAGA.a CACTCCACAGAGTCATGTTGACCACTCCTCCCTGAAGCTGGCCCTAGAAC
GTGCTGAGGAGCTGTGCTCTCAGGTGAACGAGGGAGTCCGGGAGAAGGA
AAATTCAGACCGGCTGGAGTGGATCCAGGCACACGTGCAGTGCGAAGGCT
TGGCAGAGCAACTTATTTTCAACTCCCTCACCAACTGCCTGGGCCCCCGGA
AGCTTCTGC.aCAGCGGGAAGCTGTACAAGACCAAGAGCAATAAGGAGCT
GCACGCCTTCCTCTTCAACGACTTCCTGCTGCTCACCTACCTGGTCAGGCA
GTTTGCCGCCGCCTCTGGCCACGAGAAGCTCTTCAACTCCAAGTCCAGTGC
TCAGTTCCGGATGTACAAAACGCCCATTTTCCTGAATGAAGTGTTGGTGAa ACTTCCCACAGACCCTTCCGGCGATGAGCCCGTCTTCCACATTTCCCACAT
TGATCGTGTGTACACACTCCGAACAGACAACATCAACGAGAGGACGCJCCT
GGGTCCAGAAGATCAAGGGTGCCTCAGAGCAGTACATCGAC.aCTGAGAA
GAAGAAACGGGAAAAGGC'rTACCAAGCCCGTTCTCAAAAGACTTCAGGTA
I ~ TTGGGCGTCTGATGCJTGCATGTCATTGAAGCTACAGAATTAAAAGCCTGC
AAACCAAACGGGAAAAGTAATCC.aTACTGTGAAGTCAGCATGGGCTCCCA
AAGCTATACCACCAGGACCCTGCAGGACACACTAAACCCCAAGTGGAACT
TCAACTGCCAGTTCTTCATCAAGGATCTTTACCAGGACGTTCTGTGTCTCA
CTATGTTTGACAGAGACCAGTTTTCTCCAGATGACTTCTTGGGTCGTACTG
?0 AAGTTCCAGTGGCA.aAATCCGAAC,aGAACAGGAAAGCAAAGGCCCCACC
ACCCGCCGACTAC~t~ACTGCACGAAGTCCCCACTGGAGAAGTCTGGGTCCG
CTTTGACCTGCAACTTTTTGAACAAAAAACTCTCCTTTGA
ylouse Ese?L protein: Sequence ID NO:?7 2~ MAQFPTAMNGGPNMWAITSEERTKIIDKQFDNLKPSGGYITGDQARTFFLQS
GLP.aPVLAEI WALSDLNKDGKMDQQEFSIAMKLIKLKLQGQQLPVVLPPIMK
QPPMFSPLISARFGMGSMPNLSIHQPLPPVAPIATPLSSATSGTSIPPLMMPAPL
VPSVSTSSLPNGT.aSLIQPLSIPYSSSTLPHASSYSLMMGGFGGASIQKAQSLID
LGSSSSTSSTASI_.SGNSPKTGTSEWAVPQPSRLKYRQKFNSLDKGMSGYLSGF

PLTLPPELVPPSFRGGKQVDSVNGTLPSYQKTQEEEPQKKLPVTFEDKRKANY
ERGNMELEKRRQVLMEQQQREAERKAQKEKEEWERKQRELQEQEWKKQLE
LEKRLEKQRELERQREEERRKEIERREAAKQELERQRRLEWERLRRQELLSQK
TREQEDIVRLSSRKf:SLHLELLAVNGKHQQISGRLQDVQIRKQTQKTELEVLD
KQCDLEIi~~tEIKQLQ(.~ELKE~'ONKLI~'L.'~'PEKQLLNERIKNMQI_.SNTPDSGISLL
HKKSSEKEELCQRLKEQLDt'~LEIVET:~SKLSEMDSFNNQLKELRES~'?v'TQQLA
LEQLHKIKRDKLKEIERKRLEQfQKKKLF:DEAARKAKQGKENLWRESIRKEEE
SUBSTITUTE SHEET (RULE 26) EKQKRLQEEKSQDKTQEEERKAEAKQSETASALVNYRALYPFEARNHDEMS
FSSGDIIQVDEKTVGEPGWLYGSFQGKFGWFPCNYVEKVLSSEKALSPKKALL
PPTVSLSATSTSSQPPASVTDYHNVSFSNLTVNTTWQQKSAFTRTVSPGSVSPI
HGQGQAVENLKAQALCS WTAKKENHLNFSKHDVITVI_EQQENWVVFGEVHG
GRGWFPKSYVKLIPGNEVQRGEPEALYAAVTKKPTSTAYPVTSTAYPVGEDY
IALYSYSSVEPGDLTFTEGEEILVTQKDGEWWTGSIGERTGIFPSNYVRPKDQE
NFGNASKSGASNKKPEIAQVTSAYAASGTEQLSLAPGQLILILKKNTSGWWQ
GELQARGKKRQKGWFPASHVKLLGPSSERTMPTFHAVCQVIAMYD1'MANN
EDELNFSKGQLINVMNKDDPDWWQGETNGLTGLFPSNYVKMTTDSDPSQQ
WCADLQALDTMQPTERKRQGYIHELIQTEERYMDDDLQLVIEVFQKRMAEE
GFLTEADMALIFVNWKELIMSNTKLLR.ALRVRKKTGGEKMPVQMIGDILAAE
I_SHMQAYIRFCSCQLNGATLLQQKTDEDTDFKEFLKKLASDPRCKGMPLSSF
LLKPMQRITRYPLLIRSILENTPQSHVDHSSLKLALERAEELCSQVNEGVREKE
NSDRLEWIQAHVQCEGLAEQLIFNSLTNCLGPRKLLHSGKLYKTKSNKELHA
I~ FLFNDFLLLTYLVRQFAAASGHEKLFNSKSSAQFRMYKTPIFLNEVLVKLPTD
PSGDEPVFHISHIDRVYTLRTDNINERTAWVQKIKGASEQYIDTEKKKREKAY
QARSQKTSGIGRLMVHVIEATELKACKPNGKSNPYCEVSMGSQSYTTRTLQD
TLNPKWNFNCQFFIKDLYQDVLCLTMFDRDQFSPDDFLGRTEVPVAKIRTEQE
SKGPTTRRLLLHEVPTGEV WVRFDLQLFEQKTLL
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?-4. V. Moreau. J.-M. Galan. G. Devilliers. R. Haguenauer-Tsapis. B. Vv'insor.
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32. Vl'. T. Won~~. C. Schumacher. A. E. Salcini. A. Romano. P. Castaunino. P.
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41. T. Zoladek. A. Tobiasz. G. Vaduva, M. Boguta. N. C. Martin. A. I~. Hopper.
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44. I. Gout. R. Dhand. I. D. Hiles. M. J. Fry. G. Panayotou. P. Das. O.
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SUBSTITUTE SHEET (RULE 26) Ese Proteins Ese 1 MAQFPTPFGGSLDWAITVEERAKHD'WFLST KPIAG~'IT~GD ~ARNFF=
EBe2 MAQFPTAMNGGPNMWAITSEERTKHDKOFDNLKPSGGYTTGDOAF'r'FFLC
EHl ~r_P' pV=A~ T_'~'ALADM'.~1NDGRMDnVE~'SIAMKT TKLKLQGYQL pSTLPP 100 SGLPAPVLAFTj~tp~,cDLNKDGKMDO ~EFSIAMKLIKLKLQGQQLPWLPP 100 VMKQQPVAISSAPA-FGIGGIASMP---PLTAVAPV--PMGSIPWGMSP
I'_~lKQPPMFSPLISARFGMGSMPNLSIHQPLPPVAPIATPLSSATSGTSIP
pLVSS---VPpAAVPPLANGAPPVIQPLPAFAHPAATWPKSSSFSRSGPG 191 SQLNTKLQKAQSF-DVASAPPAA---------------EWAVPOSSRLKY
FG-GASIQKAQSLIDLGSSSSTSSTASLSGNSPKTGTSEWAVPOPSRLKY
EH~ R"~LFNS~DK'"h~S~GHLTGPnARTIT MOSSLp AOLASIWNLS'~ID"DGKLT X75 ROKFNS' DK'=~~SGYLSGFOARNALLOSNLS~T"LATIWTLADI~GDG'~~Lh' 298 AEEFIT_AMHL_DVAMSGQPLPPVLPPEYIPPSFRRVRSGSGMSVISSSSV
AEEFILAMHLTDMAKAGQPLPLTLPPELVPPSFR-----GGKQV---DSV
DQRLPEEPSSEDEQQPEKKLPVTFEDKKRENFERGSVELEKRRQALLEQ~;~ 375 RKEQERLAQLERAEQERKERERQEQEAKRQLELEKQLEKQRELERQREEE
QREAERKAQKEKEEWERKQRELQEQEWKKQLELEKRLEKQRELERQREEE

'-C TLEFELEALNDKKHQLEGKLQDIRCRLATQRQEIESTNKSRELRIAEITH
SLHLELEAVNGKHQQISGRLQDVQIRKQTQKTELEVLDKQCDLEIMEIKQ

L~YQELb;EYQNKLIYLVPEF~;QLLNERIh'IVMQLSNTPDSGT_SLLHKKSS-E- 567 KELARQQLREQ:.DEVERETRSKLQE=DVFNNQLKELREIHSKQQLQF~;QRS
KEELC;~~RLb:EQLDALERETASKLSEMDSFI~TIQLKELRESYNT. QQLALE~yL
SUBSTITUTE SHEET (RULE 26) TABLE 1 - Cont'd RPRKPHEEDRLKREDSVRKKEAEERAKPEMQDKQSRLFHPHQEPAKLATQ
KQK------RLQEEKSQDKTQEE~R-KAEA--KQSET-------------APWSTTEKGPLTISAQESVKZ".;'y~'~ ~' vn~ ~ cp ~Hn~T."--.pwr,T~",~,.-,._ 769 _________________ASAJ'~'''~'7,ryn~~~S~TUnFn~tcF.~:,i:TT-~'r~ 748 "'z'~'~~-~=---~'~:~~."'~~;'~ nnT~ ~'AEKIPENEVPTPAKPVTDLTSAPAPK
__ ~ _~~.~,~ D,.r.,~yEKVLSSE-- _____~LSPK
_ .~.~._ ;..,:.= _____ PSLTVPSAGQLRQRSAFTPATATGSSPSPVLGQGEKVEGLQAQA='.'~:~.'~.:~
------------QKSAF'T_'RTVSPGSV-SPIHGQGQAVENLKAQ::_ . ~
_',T.,.tm ~ --,nmr~,r~,:~~~,-.~ --,~,:nt-...'. 'LISGPVRKS

~_~' .r. ::::
_W~ -.~'!Vlr.''~'="EV:B;': ~'~'~~~'~'~'VKLIPGNEVQR 914 TSIDTGPTESPASLKRVASPAAKPAIP-GEEFT_~_?~~="~1'E===._ ;;r=m~- -GEPEALYAAVTKKPTSTAYPVTSTAYPVGEDYI a _" ~',- ~: ~~"gin ;~
~u: ~ '=V','r":
.;.~V~'r'~~'''~'RLKDSEGSGTAGKTGSLGKK 1068 -""'-' ~~~1'r'~.~ ~ =:~~'r.~.y.-RpF;DQENFGNASKSGASNKK 1 C14 PEIAQVIA:;'_' ='-.'_",?E_ _.~'-~~,---~=F~:.:.'~ nv;-:~.",,~..;.._ -~M.,. ~ ~
__ PET_AQVT~.~_-_.~.;TF'. .y~...'.. ..;,T~..~~,-'..........
'.d~'P??~TTVKLLSPGTSKT'~'PTELPKTAVQPAVCQVI:-~tf_'=':'~_'?.-T;r"~-~ 1168 ~~;~:'FPA~i-TVKLLGPSSERTMPT-_____-F . . .Q. . ~M'r~ ~~~ T~ ~~,~ 1i07 HAVC VI___.___._.?~N-~_~~
;ta : ~ ~;~- =?HIV: ~:~nrl,~,r~~:- r,,r_ -. ,r F'n_~, ~.~t.Kr mmDMDPSQQ

. _~_~~~,T,._.... _,r"~..~ ~ ~ ', _ ,-' ~ . . . . . . . : ~ r,~ = 1 '.'KM _ ~~_ S w _. _ QpL,ymt~Y nTER~;R~:,''IHEr,' YTEER='MDD'.,~LF EQKTLi., 1197 SUBSTTTUTE SHEET (RULE 26) er: _33. °s&k? ~k~? ~~3~~ '~!:~ 3~!'s ~53f~ rr3s:
._ ._ . . . . . , , < Ot~ . p r ~~
~a r x o N~:
._. . oY.z . . ~' Y G Y ~ O ~ . r . . . . , p 'a " IY a D a lu ~ ~ , a Z Y Y O O -n N n o 4 _ ~o v- NzN
O N O a < N < . ..., . . . > - Z 19 Z < ~ a U > . ~ D O-~Ot s > l w >
x s o ..~~> i> a x x - Y - O m Z m 2 Z O <
O - n < O N O n ~ ~ < 4a a ~ H - r - r w .' inia .~ .-J Om ~aOY rZaY
O, a > K ~ ~ OY 7G YI O J O a _ii~- W x a x N z -,u ~O~ , ~ aOaa u1 ~J OW
'yY'~.J~- " -~ '~ W Y IY N ~ N
..."... Y :.. a < ~~ .... ~o~ s 'aiilr:a ~a o<a ~ x - - r a ' ':r ~~ 0 0 o c .. _ a r a N ' x ' ~.':ViGN ° O O O D ~ >-~ r <
.~ ~~ OZOn NZZN
'Paso 0 D o Y Y
w.:a z a o a > ~ : r9iF,~ W
.mss, a N i N ~ _ > ~ a r a s'. z_' ~ ~ rz x .~z~. D uN ,. aNn ~eax z Y ~ :Y
I > zNZ< O
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N < ~ ~ ~~~ a Wo C ~ o ~ o a . _.
a . Y N>r N <> u< xax o~,~~ z'~z<
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N a a D ~ ~ N ~c ~ N "
i. ~~.. Nio i ~ra x o i du ~ ~ N i ~
> " a ;~Y~'i N
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N;~10 ~'Y

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y > .",. . ~ .>. o o z . i . . a ~ . - . -o < . . . . . ' ~" . x r~ZDZ ~ Y ~ : ~N ~uNYn<
n ~ ~Y
p ~ N a N ' ~ Z ~ ~ O _ Y a N 1 N . O . a Y a r Z ~ 4 O
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sa_~o so_~n zako ~a ° aa~-° aa° oa -o_s~ aa~
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"~
~~~~' ,~~,~~ ~~~ H~~ a~~~ ~~~s~~~
iaxc $~xo~' ~$xo ~iKO~' ~ =o iixo i~xo' ~~xo~' ~~xo SUBSTITUTE SHEET (RULE 26) s~a~ :a~~ 3z3a 3:"c3 g~F: s's_sn ="_sis __~s ,. . ~ '" rw ~ V...,.-_ x , x . C~ m ° ~ > ~ .~ a Y n <
on p . _..: 5 Q ~ x z . r . n<.r.- «a- ~-o 012n ~ Z ~ Q< .ni~, n4n0 00 O O n O _ ~ 2 ~ .n > O ~; O r U_< m ~ ~ r°
o is - cn » I eesoe ~ 5 o Z ~'o M : , s~
N .,r" Y ~ - ~ p .. m a~n oao aY ~».z :: ~~ ._.,_Q ~ . ao <uz Y. .. o r < - ~~ _ 0 z ~ z o < i.~. a,a i'1~.P_ '~~ a 'll',~ ldw.<
<m3 Q Z Z
Q Y Q
2 S N Z w S
0' ~ 0 °°
~'uro, . m ero ~ ,~ p N ..,- ~ _ _ M ~r < 6 Z
D = Y O > a m ~ 3 .r tn Y . r r ";~", z Zm ~ rar,u ~oHo < Y VI Y m O t7 Z
OY ><..< .,..- <Y<O ~a ~~~~> Z
a n < p n N O N Q 5 Q Y
< n o _ ~ z cm> roam O Jl N a Zn~ 'LGZ ~-~-n ~ I O s. G
.~ y/ Q N < X:
Y s.N. mo 1J . > ~ a > N . r "~ ~- - '' a a . m v c z N,re O Y . c 3~a V ~ -. .. '< ~>a . ~rm c x Wr. ~
m 'ni Y O
Y O . ° z n o ~ mn ~ '1~Y J S
aY Q < ~~' . r<~> z aano u~o z ao =m ~ o ~ r<r "", a Y
o N w o ~ o Y W . Y y a ° N
< . O _ , , v.
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NN2O > Q~Q~ . O<O
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n , a .. r m n O ~ O ~ - o n o~<~ n<n< o~o~ oYx 5 OY_.a ~ < ~ < ~
z u. m ,n :: < Q r Y Y . r . Z N N O n ~ n i w a ~Z ~ iumlu> ~.>.
o < z u~ ~ 1 ~ a ~ ~ . z (J ~: - o r o p . . r.~ 00 m m ra cu. < _ . a . a ~

p . p . ~ . p .
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or. ", z.z. . .n - ao ° ~ a ~ ,. -. ., ~, 111 yu c, 533: _~g°_ sscc $5n~ 38E3 ~3$: "s_'s_eg N_"_s_s i~is3 ~ss$ :~ g sss$ s~s$ $~ $ s<s$ -$s$ sos$
aa$o aslp oa~o Qlo aalo aa~o solo a~&-o s to a p mo 'p oop~ a i c~ a o op m a-°O
ww~~ o m m ~ _ °s-5. 5 % C 3 5. x D 5 5 % O 5 5 % D 3 5 % O 5 3 % O 5 3 % D s' 3 x G 3 3 % D
SUBSTTTUTE SHEET (RULE 26)

Claims (49)

We Claim:

1. An isolated nucleic acid comprising a nucleotide sequence encoding a mammalian Ese1 protein or a splice variant thereof.

2. The nucleic acid of claim 1, wherein said nucleotide sequence encodes a murine Ese1 protein or a splice variant thereof.

3. The nucleic acid of claim 1. wherein said nucleotide sequence encodes a human Ese1 protein or a splice variant thereof.

4. The nucleic acid sequence of claim 1. wherein said nucleic acid comprises a nucleotide sequence selected from the group consisting of a genomic sequence a cDNA sequence. a polydeoxyribonucleic acid nucleotide sequence a polyribonucleic acid nucleotide sequence an allelic variant or homologue thereof.

The nucleic acid of claim 1 encoding a protein comprising the amino acid sequence of Sequence ID No. 3 or Sequence ID No. 24.

6. The nucleic acid of claim 1 comprising the sequence of Sequence ID No. 1.
Sequence ID No.2. Sequence ID No. 22 or Sequence ID No. 23.

7. An isolated nucleic acid comprising a nucleotide sequence of at least 10 consecutive nucleotides selected from the group consisting of Sequence ID
No. 1. Sequence ID No.2, Sequence ID No. 22. Sequence ID No. 23 and a sequence complementary to any of these sequences.

8. The nucleic acid of claim 7, wherein said sequence is used as a probe or a primer.

9. A recombinant vector comprising the isolated nucleic acid of any of the preceding claims.

10. A host cell comprising the recombinant vector of claim 9.

11. A substantially pure mammalian Ese1 or Ese1 L protein.

12. A substantially pure murine Ese1 or Ese1 L protein.

13. A substantially pure human Ese1 or Ese1 L protein.

14. The protein of claim 11 wherein the protein comprises an Ese1 protein comprising the amino acid sequence of Sequence ID No. 3 or the Ese1 L
protein comprising the amino acid sequence of Sequence ID No. 24.

15. A substantially pure polypeptide comprising an amino acid sequence of at least 5 consecutive amino acid residues of Sequence ID No. 3 or Sequence ID
No. 24.

16. A substantially pure polypeptide comprising at least one functional domain of a mammalian Ese1 protein or a mammalian Ese1 L protein.

17. A substantially pure polypeptide comprising an antigenic determinant of a mammalian Ese1 protein or a mammalian Ese1 L protein.

18. An antibody which binds specifically to a polypeptide of claim 16.

19. A process for recombinantly producing murine Ese1 protein comprising culturing a host cell comprising a recombinant vector comprising the nucleic acid of claim 2, 3 or 4 under conditions whereby the Ese1 protein is expressed and isolating the Ese1 protein therefrom.

20. An isolated nucleic acid comprising a nucleotide sequence encoding a mammalian Ese1 protein or a splice variant thereof.

21. The nucleic acid of claim 20, wherein said nucleotide sequence encodes a murine Ese2 protein or a splice variant thereof.

22. The nucleic acid of claim 20, wherein said nucleotide sequence encodes a human Ese2 protein or a splice variant thereof.

23. The nucleic acid sequence of claim 20, wherein said nucleic acid comprises a nucleotide sequence selected from the group consisting of a genomic sequence a cDNA sequence a polydeoxyribonucleic acid nucleotide sequence a polyribonucleic acid nucleotide sequence an allelic variant or homologue thereof.

24. The nucleic acid of claim 20 encoding a protein comprising the amino acid sequence of Sequence ID No. 6 or Sequence ID No. 27.

25.The nucleic acid of claim 20 comprising the sequence of Sequence ID No. 4.
Sequence ID No. 5. Sequence ID No. 25 or Sequence ID No. 26.

26. An isolated nucleic acid comprising a nucleotide sequence of at least 10 consecutive nucleotides selected from the group consisting of Sequence ID
No. 4. Sequence ID No. 5. Sequence ID No. 25. Sequence ID No. 26 and a sequence complementary to any of these sequences.

27. The nucleic acid of claim 26. wherein said sequence is used as a probe or a primer.

28. A recombinant vector comprising the isolated nucleic acid of any of claims to 26.

29. A host cell comprising the recombinant vector of claim 28.

30. A substantially pure mammalian Ese2 or Ese2L protein.

31. A substantially pure murine Ese2 or Ese2L protein.

32. A substantially pure human Ese2 or Ese2L protein.

33. The protein of claim 32 wherein the protein comprises an Ese2 protein comprising the amino acid sequence of Sequence ID No. 6 or the Ese2L
protein comprising the amino acid sequence of Sequence ID No. 27.

34. A substantially pure polypeptide comprising an amino acid sequence of at least 5 consecutive amino acid residues of Sequence ID No. 6 or Sequence ID
No. 27.

35. A substantially pure polypeptide comprising at least one functional domain of a mammalian Ese2 protein or a mammalian Ese2L protein.

36. A substantially pure polypeptide comprising an antigenic determinant of a mammalian Ese2 protein or a mammalian Ese2L protein.

37. An antibody which binds specifically to a polypeptide of claim 36.

38. A process for recombinantly producing murine Ese2 protein comprising culturing a host cell comprising a recombinant vector comprising, the nucleic acid of claim 20, 21 or 22 under conditions whereby the Ese2 protein is expressed and isolating the Ese2 protein therefrom.

39. A pharmaceutical composition for the treatment of mammalian disorders which involve abnormal endocytosis leading to altered cellular functioning.
said composition comprising an active ingredient selected from the group consisting of:
a) an Ese protein selected from the group consisting of Ese1, Ese1L, Ese2, Ese2L.
b) a fragment or mimetic thereof or a non-functional mutant protein, fragment or mimetic thereof of the proteins of a): and c) a pharmaceutically acceptable carrier.

40. A method of screening a candidate compound for efficacy in treating, a disorder characterized by an abnormality in the endocytotic pathway, wherein said pathway involves an interaction between an Ese1, Ese1L, Ese2 or Ese2L
protein and a binding partner of any one of these proteins, comprising screening said candidate compound for its ability to disrupt or promote said interaction as an indication of its efficacy.

41. A method for preventing or treating a disorder in a mammal characterized by an abnormality in the endocytotic pathway, wherein said pathway involves an interaction between an Ese1, Ese1L, Ese2 or Ese2L protein and a binding partner of any one of these proteins, comprising the step of disrupting or promoting said interaction in vivo.

42. The method of claim 40 or 41, wherein said disorder is selected from the group consisting of cancer, abnormal cell division, abnormal cell migration, viral infection, abnormal receptor signalling, abnormal tissue development and abnormal synaptic transmission disorders.

43. A method for screening a candidate compound for effectiveness as an antagonist of an Ese protein selected from the group consisting of Ese1, Ese1L, Ese2 and Ese2L, said method comprising:
(a) providing an assay method for determining the endocytotic regulatory capacity of a selected Ese protein: and (b) determining the endocytotic regulatory capacity of the selected Ese protein in the presence or absence of the candidate compound, wherein a reduced level of endocytotic regulatory capacity in the presence of the candidate compound indicates effectiveness of the compound as an antagonist.

44. A method for treating in a mammal a disorder associated with an undesired level of endocytotic activity of an Ese protein selected from the group consisting of Ese1, Ese1L, Ese2 and Ese2L, said method comprising administering to the mammal an effective amount of a substance selected from the group consisting of:
(a) an Ese protein antagonist:
(b) an antibody which binds specifically to an Ese protein:
(c) an antisense strand comprising a nucleic acid sequence complementary to a sequence or fragment of the sequence represented by Sequence ID Nos. 1, 2. 4, 5, 22, 23, 25 and 26 and capable of hybridizing to the nucleic acid sequence encoding an Ese protein:
(d) an agent which down regulates the expression of an Ese gene encoding for an Ese protein:
(e) an antagonist of an Ese protein binding partner; and (f) an Ese agonist.

45. A method for suppressing in a mammal, abnormal proliferation of a cell capable of being stimulated to proliferate by a growth factor receptor, the method comprising administering to the mammal an effective amount of a Ese protein antagonist, an Ese agonist or an antibody which binds specifically to an Ese protein.
wherein the Ese protein is selected from the group consisting of Ese1, Ese1L, Ese2 and Ese2L.

46. A method for preventing viral infection in a mammal, said method comprising administering to the mammal an effective amount of an Ese protein antagonist, an Ese monist or an antibody which binds specifically to an Ese protein or an Ese mutant protein not capable of regulating endocytosis. wherein the Ese protein is selected from the group consisting of Ese1, Ese1L, Ese2 and Ese2L.

47. A method for promoting endocytosis in selected cells in a mammal in need of such treatment, said method comprising administering to the mammal an effective amount of an Ese protein or an active analogue, mimic or fragment thereof, wherein the Ese protein is selected from the group consisting of Ese1, Ese1L, Ese2 and Ese2L.

48. A method for blocking clathrin-mediated endocytosis in cultured cells or in selected cells in a mammal in need of such treatment, said method comprising overexpressing Ese1 protein or an active analogue, mimic or fragment thereof in said cells.

49. A method for regulating endocytosis in cultured cells or in selected cells in a mammal in need of such treatment, said method comprising providing an Ese1-Esps 15 complex and further binding dynamin to said complex to regulate components of the endocytic pathway.