WO1999019476A2

WO1999019476A2 - NON-MAMMALIAN MESODERM INDUCTION EARLY RESPONSE (nm-MIER) GENE FAMILY

Info

Publication number: WO1999019476A2
Application number: PCT/CA1998/000958
Authority: WO
Inventors: Laura Lee Gillespie; Gary David Paterno
Original assignee: Laura Lee Gillespie; Gary David Paterno
Priority date: 1997-10-10
Filing date: 1998-10-13
Publication date: 1999-04-22
Also published as: WO1999019476A3; CA2225180A1; AU9426498A

Abstract

The invention relates to a family of non-mammalian genes that are transcribed in the immediate early phase following exposure to Fibroblast Growth Factors (FGF) during mesoderm induction, termed Mesoderm Induction Immediate Early Response (MIER) genes. Defining features of the members of this family include that these genes are: a) transcribed in response to fibroblast growth factors (FGF); b) expressed within 40 minutes of FGF treatment; and c) do not require protein synthesis for transcription. There are at least eleven members within this family; as a description, the cloning and characterization of a cDNA representing a member of the nm-MIER family, er1 is presented. The invention relates generally to compositions of and diagnostic methods relating to the nm-MIER gene family, cDNA, nucleotide fragments, polypeptides coded thereby, recombinant host cells and vectors containing nm-MIER encoding polynucleotide sequences, recombinant nm-MIER polypeptides, and antibodies. By way of example, the invention discloses the cloning and functional expression of different Nm-MIER polypeptides. The invention also includes methods for using the isolated, recombinant polynucleotides, polypeptides, and antibodies directed thereto in assays designed to select substances which interact with Nm-MIER polypeptides for use in diagnostic and therapeutic applications in addition to drug design and DNA vaccination methodologies.

Description

NON-MAMMALIAN MESODERM INDUCTION EARLY RESPONSE (nm-MIER)

GENE FAMILY

FIELD OF THE INVENTION

The present invention relates to a novel family of non-mammalian immediate early response genes, the use of members ofthe family in diagnostic and therapeutic applications, in addition to drug design and vaccination protocols.

BACKGROUND OF THE INVENTION

Normal growth and differentiation of all organisms is dependent on cells responding correctly to a variety of internal and external signals. Many of these signals produce their effects by ultimately changing the transcription of specific genes. One of the major goals of developmental biologists is to define the interactions of gene products and the role they play in regulating cellular differentiation in time and space. Moreover, it is clear that inappropriate expression of many genes that control differentiation during embryonic development can lead to oncogenic transformation. Such genes include members ofthe growth factor families and components of their signal transduction pathways.

Polypeptide growth factors are members of a growing family of regulatory molecules that have been conserved throughout evolution and are known to have pleiotrophic effects which range from stimulation of cell proliferation to control of cell differentiation. Growth factors have been linked to oncogenesis as many ofthe known oncogenes have been identified as overexpressed and/or mutated forms of growth factors, growth factor receptors or components of their intracellular signal transduction pathways. Oncogenes are thought to be altered such that their product escapes the normal control mechanism(s), resulting in the signaling pathways being permanently switched on. The overall result is uncontrolled cell growth. The family of fibroblast growth factors (FGFs¹) consists of nine members related by sequence and their ability to bind heparin (1). FGFs are involved in a number of cellular activities, including mitogenesis, cell differentiation and angiogenesis (reviewed in 2). In addition, overexpression of FGF in various cell lines leads to phenotypic transformation (3-5). For example, fgf-3 was identified by its proximity to a preferred integration site of the proviral DNA of the murine mammary tumour virus (MMTV) in MMTV induced mammary carcinomas (Moore, et al, 1986), while fgf-4 was isolated from Kaposi's sarcoma by its ability to transform NIH 3T3 cells (Delli-Bovi and Basilico, 1987). Some members ofthe family were identified by their mitogenic activity such asfgf- 2, which can cause phenotypic transformation when overexpressed in cultured cells (Sasada, et al, 1988; Neufeld, et al, 1988), thus classifying them as potential oncogenes.

Most ofthe studies to date have focused on FGF's mitogenic and transforming activities, however, FGF has also been shown to act as a differentiation factor for embryonic cells (Slack et al, 1987). For example, FGFs have been shown to induce mesoderm differentiation in Xenopus embryonic tissue (6) and many ofthe initial events in the cellular response during induction are similar to those previously characterized for the FGF-mediated mitogenic response. During mesoderm induction,

FGF binds to high affinity cell surface receptors (7) which in turn become phosphorylated on tyrosine (8). The phosphorylated FGF receptor (FGFR) forms a signaling complex by binding a number of intracellular substrates (9) which results in activation of several well-characterized signaling pathways. For instance, protein kinase C becomes activated during FGF-induced mesoderm differentiation (8) as does MAPK (10).

Previously, growth and differentiation had been thought to be mutually exclusive, i.e. when a cell begins to differentiate, it stops dividing. Thus, the elucidation ofthe mechanisms that regulate the differentiation process may provide may provide valuable information about the molecular signals that are important for arresting cell growth. Further research in this field will contribute to an understanding of how growth factors, such as FGF function during early embryonic development to regulate patterning of mesodermal tissues and highlight differences in the cellular response during growth, differentiation and oncogenesis. It is therefore hoped that by elucidating the molecular mechanisms by which genes regulate developmental processes during embryogenesis, it may be possible to define how misregulation of these genes can lead to cancer.

Recent research has focused on finding means for triggering the immune system to attack cancerous cells, a tactic termed immunotherapy or vaccine therapy. Because immunity is a systemic reaction, it holds the potential to eliminate all cancer cells in a patient's body, even when they migrate away from the original tumor site or reappear after years of clinical remission. One challenge is that the immune system does not always recognize cancer cells and single them out for attack. A possible solution is to tag cancer cells with certain genes rendering them more visible to the immune system, which can then destroy them.

The immune response involves many different cells and chemicals that work together to destroy in several ways invading microbes or damaged cells. In general, abnormal cells sport surface proteins, called antigens, that differ from those found on healthy cells. When the immune system is activated, B lymphocytes produce antibodies which circulate through the body and bind to foreign antigens, thereby marking the antigen bearers for destruction by other components ofthe immune system. Other cells, T lymphocytes, recognize foreign antigens as well; they destroy cells displaying specific antigens of stimulate other killer T cells to do so. B and T cells communicate with one another by way of secreted proteins, cytokines. Other accessory cells, antigen-presenting cells and dendritic cells, further help T and B lymphocytes detect and respond to antigens on cancerous or infected cells.

One theory of a means of identifying cancer cells entails the abnormal expression of genes that are normally expressed only very early in development, such as during embryogenesis. If these types of genes are not expressed in normal, healthy adult cells, but are during cancerous growth, then proteins could be expressed that could function as an antigenic marker for immune attack. Immunizing an organism with DNA coding for this antigen, could train or sensitize the immune system to attack cells expressing these antigens that are only expressed in during cancerous growth. Moreover, sensitive diagnostic means using either labelled polynucleotide probes or antibodies could be developed to detect the polynucleic acid messengers, such as mRNA, indicating the expression of these genes, hence the transformation into cancerous growth. SUMMARY OF THE INVENTION

The subject invention concems the nm-MIER non-mammalian gene family and its polynucleotide sequences which encode proteins; members of this gene family are activated in response to fibroblast growth factor (FGF) in an immediate early sequence. As an exemplary member of the nm-MIER gene family, erl is an early response gene that encodes a transcription factor found in the cell nucleus and is activated in response to FGF.

Embodiments of this invention pertaining to the nm-MIER gene family comprise:

I) genomic sequences, gene sequences and partial sequences ofthe members ofthe non- mammalian gene family; 2) isolated, synthetic nm-MLER gene sequences;

3) polynucleotide sequence probes for diagnostic use;

4) polynucleotide sequences for antisense gene therapy;

5) polynucleotide sequences for DNA vaccines;

6) polynucleotide sequences for gene replacement therapy; 7) cloning vectors comprising non-mammalian nm-MEER gene sequences;

8) antibodies to partial non-mammalian nm-MIER gene sequences;

9) antibodies to peptides encoded by nm-MIER gene sequences;

10) diagnostic kits comprising nucleic acid probes; and

I I) diagnostic kits comprising antibodies to nm-MIER proteins.

An object ofthe present invention is to provide a family of non-mammalian genes that are transcribed in the immediate early phase of mesoderm induction following exposure to FGF. In accordance with an aspect ofthe present invention there are provided cDNAs encoding members of this nm-MIER gene family.

In accordance with another aspect ofthe invention there is provided a probe to identify and isolate similar gene sequences.

In accordance with yet a further aspect ofthe invention there is provided antisense nucleotides to block expression of gene products.

In one embodiment ofthe subject invention, the proteins encoded by the genes described herein can be used to raise antibodies which in turn can be used in diagnostic or therapeutic applications.

In one aspect, the present invention provides a member ofthe nm-MIER gene family: an isolated and purified er-1 polypeptide. Preferably, the polypeptide is a recombinant polypeptide, and more preferably comprises the amino acid sequence of FIG. 1.

In another aspect, the present invention provides an isolated and purified polynucleotide that encodes a nm-MIER polypeptide. Preferably, the polynucleotide is a DNA molecule, such as an isolated and purified polynucleotide comprising the nucleotide base sequence for one member ofthe nm-Nm-MEER family, erl, shown in FIG. 1.

The present invention also contemplates an expression vector comprising a polynucleotide that encodes a Nm-MIER polypeptide. In a preferred embodiment, the polynucleotide is operatively linked to an enhancer-promoter.

Also contemplated is a recombinant cell transfected with a polynucleotide that encodes a Nm- MIER polypeptide. Preferably, the polynucleotide is under the transcriptional control of regulatory signals functional in the recombinant cell, and the regulatory signals appropriately control expression ofthe receptor polypeptide in a manner to enable all necessary transcriptional and post-transcriptional modification.

In yet another aspect, the present invention contemplates a process of preparing a Nm-MIER polypeptide, by producing a transformed recombinant cell, and maintaining the transformed recombinant cell under biological conditions suitable for the expression ofthe polypeptide. The present invention also contemplates an antibody immunoreactive with a Nm-MIER polynucleotide and/or polypeptide. The antibody may be either monoclonal or polyclonal. Preferably, the antibody is a monoclonal antibody produced by recovering the polynucleotide and/or polypeptide from a cell host, expressing the polypeptides and then preparing antibody to the polypeptide in a suitable animal host.

In still another aspect, the present invention provides a process of detecting a Nm-MIER polynucleotide and/or polypeptide, which process comprises immunoreacting the polynucleotide and/or polypeptide with an antibody ofthe present invention and a diagnostic assay kit for detecting the presence of a Nm-MLER polynucleotide and/or polypeptide in a biological sample, the kit comprising a first container means comprising a first antibody that immunoreacts with the Nm-

MIER polynucleotide and/or polypeptide. The first antibody is present in an amount sufficient to perform at least one assay.

Still further, the present invention provides a process of detecting a DNA molecule or RNA transcript that encodes a Nm-MIER polypeptide by hybridizing the DNA or RNA transcript with a polynucleotide that encodes the polypeptide to form a duplex, and then detecting the duplex.

Still further, the present invention provides a process of screening a substance for its ability to interact with members ofthe Nm-MIER family of proteins.

It is a further object ofthe present invention to provide a diagnostic marker for rapidly proliferating cells. A further aspect ofthe invention is concemed with a diagnostic kit containing antibodies to the nucleic acid ofthe invention. Yet a further aspect ofthe invention is concemed with a diagnostic kit containing antibodies to the protein encoded by the nucleic acid ofthe instant invention.

DESCRIPTION OF THE FIGURES The drawings form part ofthe present specification and are included to demonstrate certain aspects ofthe present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein:

Figure 1 presents a nucleotide and predicted amino acid sequence of one member ofthe nm-Nm-

MEER family of genes, Xenopus erl. The nucleotide sequence numbers ofthe erl cDNA are shown on the left and the amino acid sequence numbers ofthe predicted ERl protein are shown on the right. The TAA termination codon is indicated by an asterisk. Four stretches of predominantly acidic residues are underlined, the proline-rich region is in bold and two putative nuclear localization signals (NLS) are indicated by double underlines; the second NLS conforms to the consensus for a bipartite NLS.

Figure 2 shows the partial sequences of some members ofthe nm-Nm-MEER gene family.

Figure 3 is a schematic drawing indicating the different functional and regulatory domains of ERl. The identification and boundaries of these regions were determined by experimentation, or by recognizing the conservation of amino acid sequences between proteins which define a particular function using one member ofthe nm-Nm-MEER family, Xenopus. This information ofthe functional domains of erl allows for identification of important regions for the development of superior vaccines with the minimum of cross reactivity to other important proteins and to design drugs which could interfere with specific biochemical functions. Also, other uses of this SANT domain include its use to affinity-purify the DNA sequence to which ERl binds. This is also used to isolate all the genes that Nm-MEER erl regulates. The ERl consensus DNA binding sequence is predicted to be: GTTTC/GG.

Figure 4 presents an amino acid comparison of one member ofthe nm-Nm-MEER proteins, Xenopus ERl to the rat and human MTA1 and the C. elegans similar-to-MTAl protein. A, Schematic illustrating alignment ofthe predicted Xenopus ERl protein sequence with the rat and human MTA1 and the similar-to-MTAl protein from C. elegans. The N-termini were aligned and gaps (black lines) were introduced in the C. elegans and Xenopus proteins in order to align the regions of similarity (hatched ) identified by the BLAST program. White boxes indicate unique regions. B, Alignment ofthe predicted ERl amino acid sequence with the MTA1 amino acid sequences in the regions of similarity illustrated in A. Identities are indicated by the one-letter amino acid code, conservative changes are indicated by a plus sign (+) and dashes (-) indicate non-conservative changes. The amino acid sequence numbers of the ERl protein are shown on the right.

Figure 5 demonstrates that Xenopus erl, a member ofthe nm-Nm-MEER family of genes is an

FGF immediate-early response gene. A, FGF-stimulated increase in steady-state levels of Xenopus erl. Explants (5 per sample) from stage 8 Xenopus blastulae were treated for 30 min in the presence (lane 2) or absence (lane 1) of 100 ng/ml XbFGF. Total RNA was extracted and RT-PCR analysis was performed as described under "Experimental Procedures". B, FGF-stimulated increase of erl in the absence of protein synthesis. Explants were pre-incubated for 30 min with (lanes 3, 4) or without (lanes 1, 2) 5ug/ml cycloheximide; lOOng/ml XbFGF was added to the samples in lanes 2 and 4 and all samples were incubated for an additional 30 min. Extraction and analysis were performed as described in A.

Figure 6 demonstrates that expression of eri is restricted to early developmental stages in Xenopus, one exemplary member ofthe nm-Nm-MEER family of genes. A, Northern blot analysis of Xenopus erl expression. Total RNA was isolated from the following developmental stages: stage 2 (2-cell; lane 1), stage 6 (64-cell; lane 2), stage 7 (early blastula; lane 3), stage 8 (mid-blastula; lane 4), stage 12 (mid-gastrula; lane 5), stage 17 (neurula; lane 6), stage 22 (tailbud; lane 7), stage 30 (lane 8) and stage 41 (tadpole; lane 9). Northern analysis was performed as in Sambrook et al. (20) using ³²P-labeled 2.3-kb erl cDNA as a probe. The blot was stripped and re-probed with ³²P -labeled histone H4 cDNA. B, Quantitative PCR analysis of erl levels during blastula and gastrula stages of development. Total RNA was isolated at lh intervals during blastula stages, beginning at stage 7 (lane 1) and ending with stage 9 (lane 4). For gastrula stages in lanes 5-7, RNA was isolated at stages 10, 10.5 and 12, respectively, according to morphological criteria (29). RT-PCR and analysis were performed as described in the legend to Fig. 3.

Figure 7 demonstrates the nuclear localization of one member of the nm-Nm-MEER proteins, Xenopus ERl . A, Emmunoprecipitation of in vitro translation products with anti-ERl . Rabbit reticulocyte lysates programmed with erl cDNA in pcDNA3 were immunoprecipitated with either pre-immune (lane 2) or anti-ERl (lane 3) serum prepared in our laboratory. Total translation products representing one half ofthe input into each immunopreciptation are shown in lane 1. B,

ERl is localized within the nucleus in transfected NEH 3T3 cells. NTH 3T3 cells were transfected with either the pcDNA3 vector alone (top) or erl -pcDNA3 (bottom). After 48h, cells were fixed and stained with anti-ERl, as described in "Experimental Procedures".

Figure 8 demonstrates in nm-Nm-MEER studies that ERl protein is expressed during early development. Embryo extracts from stages 6.5 (lanes 1 and 5), 8 (lanes 2 and 6), 8.5 (lanes 3 and 7) and 10 (lanes 4 and 8) were subjected to SDS-PAGE, blotted and stained with anti-ERl (lanes 5-8). The blot was stripped and re-stained with pre-immune serum (lanes 1-4). The position of ERl is indicated on the right and molecular weight standards are on the left.

Figure 9 shows results from nm-Nm-MEER studies that localization of ERl to the nucleus begins during blastula stages. Albino embryos were fixed at stages 6.5 (A, B), 8 (C, D) or 8.5 (E, F) and stained with either pre-immune serum (A, C, E) or anti-ERl (B, D, F). Nuclear staining (see arrows in D) first appears in the marginal zone cells (presumptive mesoderm) of stage 8 blastulae; by stage 8.5 (one additional cell division), virtually all nuclei in the animal hemisphere are stained (F). Bar = 0.1mm.

Figure 10 shows in nm-Nm-MEER studies that ERl is concentrated in the nucleus of marginal zone cells in stage 8 blastulae. Embryos were fixed at stages 6.5 (A) or 8 (B-D), sectioned and stained with anti-ERl . (A) The nuclei (arrows) remain unstained in early cleavage stages. (B-D) At stage 8, the nuclei (arrows in B and D) in the marginal zone begin to stain for ERl while nuclei in the endoderm (B) as well as nuclei (arrows in C) in the rest ofthe animal hemisphere remain unstained. Bars = 0.1 mm in A, B and 0.02 mm in C, D.

Figure 11 presents evidence in nm-Nm-MEER studies indicating that ERl is concentrated in the nucleus of all cells in stage 10 gastrulae. Embryos were fixed at stage 10, sectioned and stained with either pre-immune (A) or anti-ERl (B). At stage 10, ERl is concentrated in the nucleus in virtually all cells ofthe three germ layers; the arrow indicates the involuting lip; ar = archenteron; blc = blastocoel. Bars = 0.1mm.

Figure 12 demonstrates in nm-Nm-MEER studies that ERl begins to disappear from the nucleus in the epidermis and brain during tailbud stages. Embryos were fixed at stage 27, sectioned and stained with either pre-immune (A) or anti-ERl (B-F). At stage 27, nuclei are stained in the endoderm (B), somites (arrows in B and E), notochord (arrows in F) as well as in most ofthe spinal cord (tailed arrows in F). Many ofthe nuclei in the brain (tailed arrows in B-D) and epidermis (arrows in C-D) are no longer stained, as illustrated by comparing the anti-ERl stained epidermis and brain in (C) with the same section incubated with a fluorescent nuclear stain (D). The black arrows in (C) mark the position ofthe nuclei identified by white arrows in (D). Bars = 0.1mm.

Figure 13 shows in nm-Nm-MEER studies that ERl is no longer concentrated in the nucleus in stage 41 tadpoles. Embryos were fixed at stage 41 , sectioned and stained with either pre-immune (A) or anti-ERl (B-D). At stage 41 , staining is absent from neural tissue (B) except for weak cytoplasmic staining in the eye (C). Staining in mesodermal tissues is exclusively cytoplasmic and is observed in somites (tailed arrows in B and bracket in D) as well as in muscle cells (black arrows in B and C). Nuclear staining is also absent in the epidermis (tailed black arrow in C) but is still observed in some ofthe endodermal cells (tailed red arrows in B). Bars = 0.1mm.

Figure 14 shows the results of studies performed using Xenopus as an example ofthe nm-Nm- MEER family to demonstrate that the N-terminus of ERl functions as a transcriptional activator. NIH 3T3 cells were transiently transfected with various GAL4-ER1 fusion constructs along with a CAT reporter plasmid. After 48h, CAT enzyme levels were measured as described in "Experimental Procedures". Vector denotes the control pM plasmid, containing only the GAL4

DNA binding domain, while the numbers indicate the amino acids of ERl encoded by each construct. The value for each construct represents the fold activation relative to the pM plasmid, averaged from 3-12 independent transfections.

Figure 15 presents a spatial expression pattern of erl in Xenopus blastula stage embryos. Blastula stage embryos were dissected into presumptive ectoderm (A), presumptive mesoderm (M) and presumptive endoderm (V) explants. The explants were analyzed for erl expression by RT-PCR. The top part ofthe diagram shows that erl expression is highest in the presumptive mesoderm and endoderm and lowest in the presumptive ectoderm. The bottom part ofthe figure shows that the levels of RNA used were equivalent in all three conditions (normalization to Histone - H4).

Figure 16 shows RT-PCR analysis for detection of erl response to inducing factors in animal cap explants from blastula stage embryos. The top part of the diagram shows erl levels and the bottom part shows normalization ofthe RNA used to Histone (H4). Lane 1 - control explants, Lane 2 - FGF treated explants, Lane 3 - Activin treated explants, Lane 4 - Vegetal (source ofthe natural inducer) treated explants. The results show that erl is upregulated in response to FGF and vegetal treatment but not to activin. Figure 17 shows blastula stage animal cap explants time course response to FGF. Lane 1 - Time 0, Lane 2 - 30 minutes FGF treatment, Lane 3 - 1 hour , Lane 4 - 2 hours, Lane 5 - 4 hours, Lane 6 - 6 hours, lane 7 - 24 hours, erl is upregulated within 30 minutes of FGF treatment and levels subsequently decrease to become undetectable by 4 hours, erl is an early response gene in the signal transduction cascade triggered by FGF.

Figure 18 shows Mesoderm induction by FGF in explants overexpressing ERl . This figure presents results of studies conducted using a member ofthe nm-Nm-MEER family showing that overexpression of ERl results in induction in the absence of FGF and increased sensitivity to induction by FGF and increased sensitivity to induction by FGF. Control explants require 500 pg/ml FGF to achieve 70% induction. These results demonstrate that expression of Xenopus ERl in embryonic cells is sufficient to induce mesoderm formation. When synthetic RNA for ERl is injected into Xenopus embryonic cells in isolation, it is translated into protein and this protein can direct the differentiation of these cells into mesoderm derivatives. In addition, these embryonic cells expressing recombinant ERl differentiate into mesoderm derivatives at low FGF concentrations which are insufficient to induce control cells. These data demonstrate that erl can be expressed in these cells and that the protein produced is functional.

Figure 19 shows ERl is phosphorylated on tyrosine in studies using a member ofthe nm-Nm- MEER family. These results show ERl is phosphorylated on tyrosine in Xenopus embryos. Protein extracts from blastula stage embryos undergoing mesoderm induction were immunoprecipitated with anti-ERl antibodies . These immunoprecitates were subjected to Western blotting using anti- phosphotyrosine antibodies, which recognize phosphorylated tyrosine residues in all proteins, to reveal ERl staining. The presence of an ERl specific band demonstrates that ERl can be phosphorylated on tyrosine. Tyrosine phosphorylation is important in the confrol ofthe function of many proteins. Knowing that ERl is phosphorylated on tyrosine may provide a therapeutic approach to modulate its activity by modulating its phosphorylation state.

Figure 20 presents a mammalian partial nucleotide sequence for M-Nm-MEER erl. This is the putative sequence ofthe probe used to analyze M-Nm-MEER erl expression in cancer cells lines Corresponding amino acid sequence is also presented.

Figure 21 presents results of a Southern blot analysis of human genomic DNA digested with

EcoRV, Xhol, HindEEE (lane 1), EcoRv, Xhol, EcoRl (lane 2) or EcoRV, Xhol, Aval (lane 3) and probed for the presence ofthe erl gene. Genomic DNA was purified from human cells and equivalent amounts were digested with the different combinations of restriction enzymes to cleave the DNA. This DNA was subjected to Southern blotting and probed with a cloned, radioactive erl cDNA. The detectable bands reveal the size and complexity ofthe erl gene in the human genome. DNA was digested with restriction enzymes Eco RV, Xho 1, Hind EH (lane 1), Eco RV, Xho 1,

Eco Rl (lane 2) and Eco RV, Xho 1, Aval (lane 3). This data shows that ERl is present in the mammalian cell genome as a single copy gene. This is important since it indicates that there are no similar genes, duplicated genes or gene families for ERl whose gene product could mimic erl activity and/or escape the specific therapy designed to modulate erl activity. It makes it simpler to design an effective therapy because one only has to target one gene.

Figure 22 presents nucleotide and predicted amino acid sequence of human er7. The nucleotide sequence numbers ofthe human er7 cDNA are shown on the left and the amino acid sequence numbers ofthe predicted human ERl protein are shown on the right. The TAA termination codon is indicated by an asterisk. The SANT domain is underlined, the two predicted nuclear localization signals are indicated by double underlines and the proline-rich region is shown in bold.

Figure 23 presents an amino acid comparison of the Xenopus and human ERl proteins. Alignment was performed by the National Center for Biotechnology Information Blast program. The full human ERl amino acid sequence is shown in the one-letter code with the predicted NLS indicated by double underlines, the SANT domain by a single underline and the proline-rich region in bold. Amino acid sequence numbers are indicated on the right. For Xenopus ERl, only differences in the amino acid sequence are listed in the one-letter amino acid code; identities are indicated by a dot. Dashes indicate gaps introduced by the BLAST program..

Figure 24 presents expression of erl in normal human adult and fetal tissues by dot blot analysis. Poly A+ mRNA from the human tissues listed in (A) was probed with [α-³²P] human erl3'UTRcDNA(B), then re-probed with [α-³²P] ubiquitin cDNA (C). The probe used and the length of exposure, in days (d) or hours (h), is listed on the right. Row H contains several negative controls used to determine the specificity of the hybridization signal.

Figure 25 demonstrates expression of human erl in normal breast and breast carcinoma cell lines. RT-PCR was performed on RNA extracted from normal breast cell lines: Hs574, Hs578, Hs787 (lanes 1-3) and from breast carcinoma cell lines: BT-20, BT-474, Hs578T, MCF-7, Sk-BR-3, MDA-157, MDA-231, MDA-436 and MDA-468 (lanes 4 - 12) to amplify human erl (top panel) or β-actin (bottom panel) as a control. The PCR products were analyzed on a 1% agarose gel.

Figure 26 shows results indicating upregulation of erl in human breast tumors. (A)RT-PCR was performed on RNA extracted from paraffin sections of three different breast tumour samples (lanes 1-3) to amplify erl (top panel) or β-actin (bottom panel) as a control. CDNA from normal breast tissue (N) was amplified with same primer pairs (lane 4). The PCR products were analyzed on a 1% agarose gel. (B) PCR was performed in the presence of [α-³²P] cCTP on eight different breast tumour samples (1-8) and on normal breast tissue (N) as described in (A). Labelled PCR products were electrophoresed on a 6% polyacrylamide/6M urea gel and analyzed by densitometry, as described in of Example EV. The values plotted in the histogram are the ratio of erl to β-actin densitometric values.

Figure 27 shows RT-PCR analysis of er7 expression in undifferentiated (EC) and differentiated P 19 cells (Diff) using mRNA for one member of M-Nm-MEER gene family, mouse eri, in mouse embryonal carcinoma cells before and after differentiation. mRNA was extracted from embryonal carcinoma cells (EC) and EC cells which have been induced to differentiate into adult tissues.

Embryonal carcinoma are equivalent to the cells to the early mammalian embryo in that they can replace embryonic cells to give rise to a normal, tumour-free mouse in the embryonic environment. Note that erl mRNA is highly expressed in the embryonal carcinoma cells but the level in the normal differentiated derivatives is drastically reduced when compared to the control actin mRNA. This data indicates that ERl expression is a property of mammalian embryonic cells as was demonstrated in Xenopus embryos. This evidence adds support to the determination of ERl as an embryonic FGF early response gene in mammals Moreover, these results indicate that ERl is an excellent target as a tumour-specific antigen for therapeutic agents.

Figure 28 presents expression of human ERl protein in normal breast and breast carcinoma cells lines. Protein extracts harvested from a normal breast cell line HS787 (N) and two breast carcinoma cells lines MCF-7 (Tl) and MDA-468 (T2) and equivalent amounts of protein were subjected to Western blotting using anti-ERl antibodies. Note the high levels of ERl protein in the breast carcinoma cell lines compared to the normal breast cell line. The additional higher molecular weight forms of ERl are modified by post-translational modifications including phosphorylation. This data demonstrates that the ERl protein is expressed at high levels in breast carcinoma cell lines but not in normal breast cells. This confirms that the ERl protein serves as a specific target for therapeutic agents (vaccines, drugs , antibodies) designed to specifically inhibit the growth or to kill breast cancer cells. The fact that ERl is not detectable in normal adult cells makes it a superior target to many other "tumour-specific" antigens which are often expressed in adult cells; for example, carcinoembryonic antigen (CEA) which is now in clinical trials.

Figure 29 is a Western blot showing expression of ERl protein in four different clinical human breast tumour samples. Protein was extracted from a small piece of tumour tissue and run on a Western. The blot was stained with an anti-ERl antibody ofthe present invention. The results presented in this figure demonstrate that an antibody ofthe present invention can be used as a diagnostic tool for the mammalian protein and that breast tumours express the Erl protein.

Figure 30 presents results of RT-PCR analysis of erl expression in primary cervical cells (N), immortalized (I) and transformed (T) cervical cells in experiments using a M-Nm-MEER family member, specifically showing expression of human erl mRNA in cervical cells and in cervical carcinoma cell lines. mRNA was extracted from primary cervical cells (N), immortalized cervical cells (I) and cervical carcinoma (T) cell lines. Equivalent amounts of RNA were subjected to RT-

PCR analysis to reveal the levels of ERl mRNA in these cells. Primary cervical cells are normal cells which have a limited lifespan in tissue culture. Immortalized cells are normal cervical cells which have acquired the ability for continuous growth in culture but cannot form tumours. Cervical carcinoma cell lines are transformed cells which demonstrate malignant growth in culture and form tumours. Note the increase levels of er7 mRNA in the immortalized and cervical carcinoma cells.

This data indicates that there is a differential expression of erl in cervical carcinoma versus normal cervical cells. Thus, like for breast cancers, er7 can serve as a therapeutic target for the specific inhibition of growth or the killing of cervical cancer cells. This evidence also suggests that the overexpression of ERl may be a general phenomena in many types of cancer.

Figure 31 shows Northern blot analysis of human er7 mRNA expressed in the MDA-468 breast carcinoma cell line. These results are from a Northern analysis of poly A+ RNA from MDA-468, using an er7 cDNA probe. Four transcripts are indicated by arrows. mRNA was isolated from MDA-468 cell cultures and subjected to Northern blotting using cloned, radioactive er7 cDNA as a probe. The 4 detectable bands at the indicated molecular weights represents different versions of the erl mRNA. En normal tissues we have only been able to detect extremely low levels of a single mRNA of 1.6 Kb in size which is equivalent to our cloned cDNA. These additional forms of ERl in tumours cells lines may represent alternative, mutated or tumour-specific forms of ERl mRNA which may contribute to the oncogenic phenotype and which may provide superior targets for therapeutic agents.

Figure 32 Northern blot analysis of human erl mRNA expressed in MDA-468 breast carcinoma cells at various times after exposure to epidermal growth factor (EGF). MDA-468 breast carcinoma cells were starved for 24 hours and then exposed to EGF. At the indicated times, mRNA was isolated from treated cells and equivalent amounts of mRNA were subjected to Northern blotting using cloned, radioactive erl cDNA as a probe. Note the increase in the er7 mRNA levels after 4 hour exposure to EGF relative to the levels ofthe actin control mRNA. This data reveals that ERl is an early response gene to other growth stimuli and growth factors and therefore its expression in tumours may be a general feature of the growth of all tumours.

Figure 33 demonstrates Western blot analysis of ERl protein expressed in serum starved and serum-stimulated MDA-468 breast carcinoma cells, stained with anti-ERl . MDA-468 cells were starved for 24 hours before duplicated cultures were growth-stimulated with serum containing medium for 2 hours. Protein extracts were prepared from serum-stimulated (+) and serum-starved (-) cultures and equivalent amounts of protein were subjected to Western blotting using ERl antibodies. Note the increase in the levels ofthe ERl protein species. This data confirms that ERl protein levels also increase with exposure to other growth stimuli in breast cancer cells. En this case, the stimuli are physiological blood serum components.

Figure 34 demonstrates a comparison ofthe nucleic and amino acid sequence around the start of translation of identified erl variants. These variant cDNAs were identified from human cDNA libraries and our evidence suggests that they arise from alternatively spliced precursor mRNAs. The possibility exists that these variants are characteristic ofthe neoplastic stage and could be used as a more refined target for cancer cells. The underline indicates the erl variant that is reported in Example IV. The existence of cellular variants of erl RNA suggests that there may exist tumour- specific forms of erl mRNA, as we observed in our Northern blot of breast carcinoma mRNA, and/or protein which could provide more specific targets for therapy.

Figure 35 shows expression of ERl protein in mammalian cells transfected with expression vectors containing the erl cDNA sequence. The er7 plasmid was transfected with expression vectors containing the erl cDNA sequence. The er7 plasmid was transfected in mouse N1H3T3 fibroblasts (lane 1) and rat L6 myoblasts (lane 2 and 3) using a liposome delivery system. Thus study demonstrates that our the er7 cDNA ofthe present invention which has been cloned into various expression plasmids can be transfected into cells not expressing ERl and that cDNA can be transcribed and translated into ERl protein which is detectable by our ERl antibody.

Figure 36 shows phosphorylation ofthe Xenopus ERl protein on serine and/or threonine. Extracts from Xenopus embryos were subjected to immunoprecipitation with anti-ERl antibody followed by

Western blotting. The blot was stained with a monoclonal antibody that recognizes phosphoserine and phosphothreonine. This figure shows that the ERl protein is phosphorylated in the embryo and may represent a mechanism by which ERl activity is regulated. This data suggests that er7 activity may be controlled through cellular phosphorylations at these amino acids in the protein and that therapeutic modulation of phosphorylation of er7 might regulate its activity.

Figure 37 presents evidence of downregulation of nm-Nm-MEER gene S3 by treatment with FGF. Xenopus embryonic cells were treated with FGF for 30 min, then the RNA was extracted, reverse- transcribed and subjected to PCR using primers corresponding to Nm-MEER gene S3. The primers were designed using cloned sequence from S3. S3 is the only gene that appeared to be downregulated by FGF. This is an example of a Nm-MEER gene (S3) which is downregulated by

FGF. This is the opposite of what we is observed for er7, therefore, one would argue that expression of this particular gene would stop growth. These results demonstrate that this member of the Nm-MEER family could be an excellent target for gene therapy.

Figure 38 shows the isolation ofthe cDNA for Nm-MIER gene S30 from a human library. Using primers corresponding to the Xenopus sequence, a PCR (cDNA) product ofthe predicted size for

S30 from a human library was amplified. These results demonstrate that, like er7, the human homologues of the Xenopus Nm-MIER genes can be isolated using Xenopus DNA sequences. This also serves as an example ofthe procedure to be followed to isolate the other members of the human Nm-MEER genes.

Figure 39 shows expression of ERl protein in Xenopus embryos injected with synthetic er/ RNA

(cRNA). cRNA was made by in vitro transcription of erl cDNA in the expression vector pSP64T. Fertilized eggs were microinjected with 3ng of er7 cRNA and allowed to develop for 4 hours. Embryos were fixed and stained with an anti-ERl antibody of the invention. This demonstration provides evidence that ERl protein can be expressed in the cells of this invention using the vector constructs ofthe invention. Figure 40 presents results demonstrating that antisense M-Nm-MIER er7 mRNA inhibits the growth of MDA-468 breast cancer cells. Cells transfected with the indicated constructs were selected for geneticin resistance. The identical number of MDA-468 cells or NIH 3T3 were transfected with equivalent concentrations ofthe indicated plasmids. These plasmid constructs control the expression of erl mRNA (sense), er7 antisense RNA (Antisense ) or mRNA green fluorescent protein (GFP) which serves as a confrol for transfection efficiency. The cultures were exposed to the antibiotic Geneticin which kills cell which have not taken up and expressed these plasmids and cell colonies are allowed to grow. Note that antisense M-Nm-MEER er7 RNA, which blocks the normally high levels of ERl in MDA-468 cells, inhibits the growth and recovery of these cancer cells. The recovery and growth of NEH 3T3 fibroblast cells, which represent normal cells, is not affected. This data which provides unequivocal support for the effectiveness and utility of one embodiment ofthe invention, the use of antisense er7 as a treatment for cancer. It demonstrates that antisense erl RNA can completely block the growth of breast cancer cells, which express high levels of ERl, but does not affect the growth of normal NIH 3T3 fibroblast cells which do not express detectable levels of ER 1.

Figure 41 evidences cell viability after 2d treatment with oligonucleotides. These results show treatment of human breast carcinoma cells with anti-sense erl oligonucleotides reduces the number of viable cells. This data demonstrates that antisense oligonucleotides directed against ERl can inhibit the growth of human breast cancer cells. The stastical analysis indicates that the difference in growth is stastically significant as indicated by the asterix. These results demonstrate the use of oligonucleotides in anti-sense gene therapy.

Figure 42 shows that expression of Nm-MEER gene S3 is downregulated within 30 minutes of FGF treatment. Expresion levels were measured by PCR of reverse-transcribed RNA that was extracted from untreated (CON) and treated (FGF) Xenopus embryo explants. This histogram represents densitometric measurements ofthe PCR products and provides additional evidence that the Nm-MEER genes ofthe present invention are regulated by FGF and thus members ofthe family we have defined.

Figure 43 shows that expression of Nm-MEER gene S17 is upregulated within 30 minutes of FGF treatment. This histogram represents densitometric measurements ofthe PCR products and provides additional evidence that the Nm-MEER genes ofthe present invention are regulated by FGF and thus members ofthe family we have defined.

Figure 44 shows the expression of Nm-MIER gene S30 is upregulated within 30 minutes of FGF treatment. This histogram represents densitometric measurements ofthe PCR products and provides additional evidence that the Nm-MIER genes ofthe present invention are regulated by FGF and thus members ofthe family we have defined.

Figure 45 demonstrates the results from the expression and purification of Xenopus ERl protein from bacterial cells transformed with er7 cDNA in a bacterial expression vector. These results demonstrate that recombinant ERl protein can be expressed and purified from bacterial sources. This provides a way to make large quantities of ERl protein which may be used for vaccine protection other potential therapy.

Figure 46 demonstrates stimulation of an immune response with ERl peptides or er7 DNA. Rabbits were immunized with an ERl peptide conjugated to keyhole limpet hemocyanin or erl cDNA in a mammalian expression vector. Antiserum and pre-immune serum from the same rabbits were titrated in a twofold serial dilution (columns 1 - 12) and tested in a standard ELISA assay using full-length ERl protein as the antigen. Positives appear as grey /black and the pre-immune serves to indicate the background level ofthe assay. Positive reactions in the immune serum at higher dilutions than the pre-immune (arrows) demonstrates the presence of anti-ERl antibodies. The results of these studies provides evidence that the DNA based ERl constructs can be injected into animals and taken up by animal cells. The plasmids can direct the synthesis of ERl protein and this protein can elicit an immune response in the animal to generate and anti-ERl antibodies. These results provide evidence that DNA based constructs can be used as a vaccine against ERl protein. Well established principles of immunology extend these findings to indicate that such an antibody response means that immune system will react to cells expressing ERl . In this application ofthe present invention, the only cells expressing ERl would be the cancer cells.

Figure 47 shows that Nm-MEER gene sl7 is upregulated by FGF. Xenopus embryo explants were incubated in the absence (1) or presence (+) of FGF for 30 min, RNA was extracted, reverse- transcribed and subjected to PCR using primers for the Nm-MEER gene S 17. These results present additional data to support the characterization that FGF can regulate Nm-MEER genes and provides a defining feature for genes belonging to this family. Figure 48 demonstrates that Nm-MIER gene S16 is upregulatd by FGF. Xenopus embryo explants were incubated in the absence (-) or presence (+) of FGF for 30 min, RNA was extracted, reverse-transcribed and subjected to PCR using primers for the Nm-MIER gene SI 6. These results provide additional data for FGF regulation of Nm-MIER genes to support the characterization of a family of genes that are upregulated by FGF.

Figure 49 shows the isolation from a human cDNA library of partial cDNAs representing human Nm-MIER genes by PCR using Xenopus sequence primers. PCR was performed at low stringency to allow for possible mismatch between the Xenopus and human sequences.

Figure 50 presents the partial sequence of mouse erl cDNA nucleic acid sequence with corresponding amino acid sequence

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a family of non-mammalian genes that are transcribed in the immediate early phase following exposure to FGF during mesoderm induction, termed Mesoderm Induction Early Response (Nm-MIER) genes. Defining features ofthe members of this family include that these genes are a) transcribed in response to FGF; b) are expressed within 40 minutes of FGF treatment; and c) do not require protein synthesis for transcription. There are at least eleven members within this family.

The unique polynucleotide sequences ofthe subject invention include Nm-MEER gene sequences which encode the Nm-MEER proteins, as well as sequences which drive the expression of these proteins.

As an exemplary member ofthe Nm-MEER gene family, erl is an early response gene that encodes a transcription factor found in the cell nucleus and is activated in response to FGF. The gene is overexpressed in breast carcinoma and cervical carcinoma cell lines and possibly in general in all cancer cell lines. Erl is also overexpressed in an abnormal T-cell subset (CD28-) whose numbers increase with disease progression in AIDS patients. This CD28- subset also increases in chronic inflammatory disorders. Therefore this gene and its product are potential targets for diagnosis and treatment of various cancers as well as immune disorders such as AEDS. The ultimate targets of these signal transduction pathways are the immediate-early genes. To date, very few FGF immediate-early genes have been identified (11, 12). Accordingly, we have utilized the differential display methodology (13) to isolate cDNAs representing such genes

Definitions and Abbreviations

The term "Nm-MEER" refers to Mesoderm Induction Immediate Early Response genes, their nucleic acid transcription products and translated protein products. Defining features ofthe members of this family include that the genes are a) transcribed in response to fibroblast growth factors (FGF); b) are expressed within 40 minutes of FGF treatment; and c) do not require protein synthesis for transcription. There are at least eleven members within this family; one member is erl . The non-mammalian Nm-MEER genes and proteins will be referred to using the abbreviation, nm-

Nm-MEER. The mammalian Nm-MEER genes and proteins will be referred to using the abbreviation M-Nm-MEER.

The Nm-MEER genes and polypeptides ofthe present invention are not limited to a particular non- mammalian source. As disclosed herein, the techniques and compositions ofthe present invention provide, for example, the identification and isolation of sources from non-mammalian cancerous cell lines. Thus, the invention provides for the general detection and isolation ofthe genus of Nm-MEER genes and polypeptides from a variety of sources. It is believed that a number of species of the family of Nm-MIER genes and polypeptides are amenable to detection and isolation using the compositions and methods ofthe present invention.

Polynucleotides and polypeptides ofthe present invention are prepared by standard techniques well known to those skilled in the art. Such techniques include, but are not limited to, isolation and purification from tissues known to contain these genes and polypeptides, and expression from cloned DNA that encodes such polypeptides using transformed cells.

In one embodiment ofthe invention, the biological activity ofthe Nm-MEER proteins ofthe subject invention can be reduced or eliminated by administering an effective amount of an antibody to each ofthe Nm-MEER proteins. Alternatively, the activity ofthe Nm-MEER proteins can be controlled by modulation of expression ofthe Nm-MEER protein. This can be accomplished by, for example, the administration of antisense DNA. As used herein, the terms "nucleic acid" and "polynucleotide sequence" refer to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally-occurring nucleotides. The polynucleotide sequences include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into protein. The polynucleotide sequences include both full-length sequences as well as shorter sequences derived from the full-length sequences. It is understood that a particular polynucleotide sequence includes the degenerate codons ofthe native sequence or sequences which may be introduced to provide codon preference in a specific host cell. Allelic variations ofthe exemplified sequences also come within the scope ofthe subject invention. The polynucleotide sequences falling within the scope ofthe subject invention further include sequences which specifically hybridize with the exemplified sequences under stringent conditions. The nucleic acid includes both the sense and antisense strands as either individual strands or in the duplex.

The terms "hybridize" or "hybridizing" refer to the binding of two single-stranded nucleic acids via complementary base pairing.

The phrase "hybridizing specifically to" refers to binding, duplexing, or hybridizing of a molecule to a nucleotide sequence under stringent conditions when that sequence is present in a preparation of total cellular DNA or RNA.

The term "stringent conditions" refers to conditions under which a probe will hybridize to its target sub-sequence, but not to sequences having little or no homology to the target sequence. Generally, stringent conditions are selected to be about 5. degree. C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a complementary probe. Typically, stringent conditions will be those in which the salt concentration is at least about 0.1 to 1.ON Na ion concentration at a pH of about 7.0 to 7.5 and the temperature is at least about 60. degree. C. for long sequences (e.g., greater than about 50 nucleotides) and at least about 42. degree. C. for shorter sequences (e.g., about 10 to 50 nucleotides).

The terms "isolated" or "substantially pure" when referring to polynucleotide sequences encoding the Nm-MIER proteins or fragments thereof refers to nucleic acids which encode Nm-MEER proteins or peptides and which are no longer in the presence of sequences with which they are associated in nature.

The terms "isolated" or "substantially purified" when referring to the proteins ofthe subject invention means a chemical composition which is essentially free of other cellular components. It is preferably in a homogenous state and can be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein which is the predominant species present in a preparation is substantially purified. Generally, a substantially purified or isolated protein will comprise more than 80% of all macromolecular species present in the preparation. Preferably, the protein is purified to represent greater than 90% of all macromolecular species present. More preferably, the protein is purified to greater than 95%, and most preferably the protein is purified to essential homogeneity, wherein other macromolecular species are not detected by conventional techniques.

The phrase "specifically binds to an antibody" or "specifically immunoreactive with," when referring to a protein or peptide, refers to a binding reaction which is determinative ofthe presence ofthe protein in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bound to a particular protein do not bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein. See Harlow and Lan (1988) for a description of immunoassay formats and conditions that could be used to determine specific immunoreactivity. The subject invention further concerns antibodies raised against the purified Nm-MEER molecules or their fragments.

The term "biological sample" as used herein refers to any sample obtained from a living organism or from an organism that has died. Examples of biological samples include body fluids, tissue specimens, and tissue cultures lines taken from patients.

The term "recombinant DNA" or "recombinantly-produced DNA" refers to DNA which has been isolated from its native or endogenous source and modified either chemically or enzymatically to delete naturally-occurring flanking nucleotides or provide flanking nucleotides that do not naturally occur.

Flanking nucleotides are those nucleotides which are either upstream or downstream from the described sequence or sub-sequence of nucleotides.

The term "recombinant protein" or "recombinantly-produced protein" refers to a peptide or protein produced using cells that do not have an endogenous copy of DNA able to express the protein. The cells produce the protein because they have been genetically altered by the introduction of an appropriate nucleic acid sequence. The recombinant protein will not be found in association with proteins and other subcellular components normally associated with the cells producing the protein.

It is well known that DNA possesses a fundamental property called base complementarity. In nature, DNA ordinarily exists in the form of pairs of anti-parallel strands, the bases on each strand projecting from that strand toward the opposite strand. The base adenine (A) on one strand will always be opposed to the base thymine (T) on the other strand, and the base guanine (G) will be opposed to the base cytosine (C). The bases are held in apposition by their ability to hydrogen bond in this specific way. Though each individual bond is relatively weak, the net effect of many adjacent hydrogen bonded bases, together with base stacking effects, is a stable joining ofthe two complementary strands. These bonds can be broken by treatments such as high pH or high temperature, and these conditions result in the dissociation, or "denaturation," ofthe two strands. If the DNA is then placed in conditions which make hydrogen bonding ofthe bases thermodynamically favorable, the DNA strands will anneal, or "hybridize," and reform the original double stranded DNA. If carried out under appropriate conditions, this hybridization can be highly specific. That is, only strands with a high degree of base complementarity will be able to form stable double stranded structures. The relationship ofthe specificity of hybridization to reaction conditions is well known. Thus, hybridization may be used to test whether two pieces of DNA are complementary in their base sequences. It is this hybridization mechanism which facilitates the use of probes ofthe subject invention to readily detect and characterize DNA sequences of interest.

As those of ordinary skill in the art will appreciate, any of a number of different nucleotide sequences can be used, based on the degeneracy ofthe genetic code, to produce the Nm-MIER proteins described herein. Accordingly, any nucleotide sequence which encodes the Nm-MIER proteins described herein comes within the scope of this invention and the claims appended hereto. Also, as described herein, fragments ofthe Nm-MIER proteins are an aspect ofthe subject invention so long as such fragments retain the biological activity so that such fragments are useful in therapeutic and/or diagnostic procedures as described herein. Such fragments can easily and routinely be produced by techniques well known in the art. For example, time-controlled Bal31 exonuclease digestion ofthe full-length DNA followed by expression ofthe resulting fragments and routine screening can be used to readily identify expression products having the desired activity.

Polynucleotide Probes

In addition, PCR-amplified DNA may serve as a hybridization probe. In order to analyze DNA using the nucleotide sequences ofthe subject invention as probes, the DNA can first be obtained in its native, double-stranded form. A number of procedures are currently used to isolate DNA and are well known to those skilled in this art.

One approach for the use ofthe subject invention as probes entails first identifying by Southern blot analysis of a DNA library all DNA segments homologous with the disclosed nucleotide sequences. Thus, it is possible, without the aid of biological analysis, to know in advance the presence of genes homologous with the polynucleotide sequences described herein. Such a probe analysis provides a rapid diagnostic method.

One hybridization procedure useful according to the subject invention typically includes the initial steps of isolating the DNA sample of interest and purifying it chemically. For example, total fractionated nucleic acid isolated from a biological sample can be used. Cells can be treated using known techniques to liberate their DNA (and/or RNA). The DNA sample can be cut into pieces with an appropriate restriction enzyme. The pieces can be separated by size through electrophoresis in a gel, usually agarose or acrylamide. The pieces of interest can be transferred to an immobilizing membrane in a manner that retains the geometry ofthe pieces. The membrane can then be dried and prehybridized to equilibrate it for later immersion in a hybridization solution. The manner in which the nucleic acid is affixed to a solid support may vary. This fixing ofthe DNA for later processing has great value for the use of this technique in field studies, remote from laboratory facilities.

The particular hybridization technique is not essential to the subject invention. As improvements are made in hybridization techniques, they can be readily applied.

As is well known in the art, if the probe molecule and nucleic acid sample hybridize by forming a strong non-covalent bond between the two molecules, it can be reasonably assumed that the probe and sample are essentially identical. The probe's detectable label provides a means for determining in a known manner whether hybridization has occurred.

The nucleotide segments ofthe subject invention which are used as probes can be synthesized by use of DNA synthesizers using standard procedures. In the use ofthe nucleotide segments as probes, the particular probe is labeled with any suitable label known to those skilled in the art, including radioactive and non-radioactive labels. Typical radioactive labels include .sup.32 P, .sup.35 S, or the like. A probe labeled with a radioactive isotope can be constructed from a nucleotide sequence complementary to the DNA sample by a conventional nick translation reaction, using a DNase and DNA polymerase. The probe and sample can then be combined in a hybridization buffer solution and held at an appropriate temperature until annealing occurs.

Thereafter, the membrane is washed free of extraneous materials, leaving the sample and bound probe molecules typically detected and quantified by autoradiography and/or liquid scintillation counting. For synthetic probes, it may be most desirable to use enzymes such as polynucleotide kinase or terminal transferase to end-label the DNA for use as probes.

Non-radioactive labels include, for example, ligands such as biotin or thyroxine, as well as enzymes such as hydrolases or perixodases, or the various chemiluminescers such as luciferin, or fluorescent compounds like fluorescein and its derivatives. The probes may be made inherently fluorescent as described in International Application No. WO93/16094. The probe may also be labeled at both ends with different types of labels for ease of separation, as, for example, by using an isotopic label at the end mentioned above and a biotin label at the other end.

The amount of labeled probe which is present in the hybridization solution will vary widely, depending upon the nature ofthe label, the amount ofthe labeled probe which can reasonably bind to the filter, and the stringency ofthe hybridization. Generally, substantial excesses ofthe probe will be employed to enhance the rate of binding ofthe probe to the fixed DNA. Various degrees of stringency of hybridization can be employed. The more severe the conditions, the greater the complementarity that is required for duplex formation. Severity can be controlled by temperature, probe concentration, probe length, ionic strength, time, and the like. Preferably, hybridization is conducted under stringent conditions by techniques well known in the art, as described, for example, in Keller and Manak, 1987.

Duplex formation and stability depend on substantial complementarity between the two strands of a hybrid, and, as noted above, a certain degree of mismatch can be tolerated. Therefore, the nucleotide sequences ofthe subject invention include mutations (both single and multiple), deletions, insertions ofthe described sequences, and combinations thereof, wherein said mutations, insertions and deletions permit formation of stable hybrids with the target polynucleotide of interest. Mutations, insertions, and deletions can be produced in a given polynucleotide sequence in many ways, and these methods are known to an ordinarily skilled artisan. Other methods may become known in the future.

The known methods include, but are not limited to:

(1) synthesizing chemically or otherwise an artificial sequence which is a mutation, insertion or deletion ofthe known sequence;

(2) using a nucleotide sequence ofthe present invention as a probe to obtain via hybridization a new sequence or a mutation, insertion or deletion ofthe probe sequence; and

(3) mutating, inserting or deleting a test sequence in vitro or in vivo.

It is important to note that the mutational, insertional, and deletional variants generated from a given probe may be more or less efficient than the original probe. Notwithstanding such differences in efficiency, these variants are within the scope ofthe present invention.

Thus, mutational, insertional, and deletional variants ofthe disclosed nucleotide sequences can be readily prepared by methods which are well known to those skilled in the art. These variants can be used in the same manner as the instant probe sequences so long as the variants have substantial sequence homology with the probes. As used herein, substantial sequence homology refers to homology which is sufficient to enable the variant to function in the same capacity as the original probe. Preferably, this homology is greater than 50%; more preferably, this homology is greater than 75%; and most preferably, this homology is greater than 90%. The degree of homology needed for the variant to function in its intended capacity will depend upon the intended use ofthe sequence. It is well within the skill of a person trained in this art to make mutational, insertional, and deletional mutations which are designed to improve the function of the sequence or otherwise provide a methodological advantage.

It is well known in the art that the amino acid sequence of a protein is determined by the nucleotide sequence ofthe DNA. Because ofthe redundancy ofthe genetic code, i.e., more than one coding nucleotide triplet (codon) can be used for most ofthe amino acids used to make proteins, different nucleotide sequences can code for a particular amino acid.

The amino acid sequence ofthe proteins ofthe subject invention can be encoded by equivalent nucleotide sequences encoding the same amino acid sequence of the protein. Accordingly, the subject invention includes probes which would hybridize with various polynucleotide sequences which would all code for a given protein or variations of a given protein. En addition, it has been shown that proteins of identified structure and function may be constructed by changing the amino acid sequence if such changes do not alter the protein secondary structure (Kaiser and Kezdy, 1984).

En one aspect, the present invention provides an isolated and purified polynucleotide that encodes a Nm-MEER polypeptide. In a preferred embodiment, a polynucleotide ofthe present invention is a DNA molecule. Even more preferably, a polynucleotide ofthe present invention encodes a polypeptide comprising the amino acid residue sequence of Er-1, a member ofthe Nm-MEER family (FIG. 1). Most preferably, an isolated and purified polynucleotide ofthe invention comprises the nucleotide base sequence of FIG. 1.

As used herein, the term "polynucleotide" means a sequence of nucleotides connected by phosphodiester linkages. Polynucleotides are presented herein in a 5' to 3' direction. A polynucleotide ofthe present invention may comprise about several thousand base pairs. Preferably, a polynucleotide comprises from about 100 to about 10,000 base pairs. Preferred lengths of particular polynucleotides are set forth hereinafter.

A polynucleotide ofthe present invention may be a deoxyribonucleic acid (DNA) molecule or ribonucleic acid (RNA) molecule. Where a polynucleotide is a DNA molecule, that molecule may be a gene or a cDNA molecule. Nucleotide bases are indi cated herein by a single letter code: adenine (A), guanine (G), thymine (T) and cytosine (C).

A polynucleotide ofthe present invention may be prepared using standard techniques well-known to one of skill in the art. The preparation of a cDNA molecule encoding an erl polypeptide of the present invention is described hereinafter in the examples. A polynucleotide may also be prepared from genomic DNA libraries using, for example, lambda phage technologies

In another aspect, the present invention provides an isolated and purified polynucleotide that encodes a Nm-MEER polypeptide, where the polynucleotide is preparable by a process comprising the steps of constructing a library of cDNA clones from a cell that expresses the polypeptide; screening the library with a labelled cDNA probe prepared from RNA that encodes the polypeptide; and selecting a clone that hybridizes to the probe.

A further aspect of the claimed invention are antibodies that are raised by immunization of an animal with a purified protein or polynucleotides ofthe subject invention. Both polyclonal and monoclonal antibodies can be produced using standard procedures well known to those skilled in the art using the proteins ofthe subject invention as an immunogen (see, for example, Monoclonal Antibodies: Principles and Practice, 1983; Monoclonal Hybridoma Antibodies: Techniques and Applications, 1982; Selected Methods in Cellular Emmunology, 1980; Immunological Methods, Vol. II, 1981; Practical Immunology, and Kohler et al., 1975).

The proteins ofthe subject invention include those which are specifically exemplified herein as well as related proteins which, for example, are immunoreactive with antibodies which are produced by, or are immunologically reactive with, the proteins specifically exemplified herein.

The proteins described herein can be used in therapeutic or diagnostic procedures.

Probes

In another aspect, DNA sequence information provided by the present invention allows for the preparation of relatively short DNA (or RNA) sequences having the ability to specifically hybridize to gene sequences ofthe selected polynucleotide disclosed herein. In these aspects, nucleic acid probes of an appropriate length are prepared based on a consideration of a selected nucleotide sequence, e.g., a sequence such as that shown in FIG. 1. The ability of such nucleic acid probes to specifically hybridize to a polynucleotide encoding a Nm-MIER lends them particular utility in a variety of embodiments. Most importantly, the probes may be used in a variety of assays for detecting the presence of complementary sequences in a given sample.

In certain embodiments, it is advantageous to use oligonucleotide primers. The sequence of such primers is designed using a polynucleotide ofthe present invention for use in detecting, amplifying or mutating a defined segment of a gene or polynucleotide that encodes a Nm-MEER polypeptide from non-mammalian cells using PCR.TM. technology.

To provide certain ofthe advantages in accordance with the present invention, a preferred nucleic acid sequence employed for hybridization studies or assays includes probe molecules that are complementary to at least an about (14) to an about (70) nucleotide long stretch of a polynucleotide that encodes a Nm-MEER polypeptide, such as the nucleotide base sequences shown in FIG. 1. A size of at least 14 nucleotides in length helps to ensure that the fragment is of sufficient length to form a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than 14 bases in length are generally preferred, though, in order to increase stability and selectivity ofthe hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained, one will generally prefer to design nucleic acid molecules having gene-complementary stretches of 25 to 40 nucleotides, 55 to 70 nucleotides, or even longer where desired. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR.TM. technology of U.S. Pat. No. 4,603,102, or by excising selected DNA fragments from recombinant plasmids containing appropriate inserts and suitable restriction enzyme sites.

In another aspect, the present invention contemplates an isolated and purified polynucleotide comprising a base sequence that is identical or complementary to a segment of at least 14 contiguous bases of FIG. 1, wherein the polynucleotide hybridizes to a polynucleotide that encodes a Nm-MEER polypeptide. Preferably, the isolated and purified polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 25 to 70 contiguous bases of FEG. 1. For example, the polynucleotide of the invention may comprise a segment of bases identical or complementary to 40 or 55 contiguous bases ofthe disclosed nucleotide sequences.

Accordingly, a polynucleotide probe molecule ofthe invention may be used for its ability to selectively form duplex molecules with complementary stretches ofthe gene. Depending on the application envisioned, one employs varying conditions of hybridization to achieve varying degree of selectivity ofthe probe toward the target sequence. For applications requiring a high degree of selectivity, one typically employs relatively stringent conditions to form the hybrids. For example, one selects relatively low salt and/or high temperature conditions, such as provided by about 0.02M to about 0.15M NaCl at temperatures of about 50°C to about 70°C. Those conditions are particularly selective, and tolerate little, if any, mismatch between the probe and the template or target strand.

In some applications where it is the intention to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate a Nm-MEER polypeptide coding sequence from other cells, functional equivalents, or the like, less stringent hybridization conditions are typically needed to allow formation ofthe heteroduplex. In these circumstances, one employs conditions such as about 0.15M to about 0.9M salt, at temperatures ranging from about

20°C C. to about 55°C . Cross-hybridizing species may thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions may be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions may be readily manipulated, and thus will generally be a method of choice depending on the desired results.

In still another embodiment ofthe present invention, there is provided a isolated and purified polynucleotide comprising a base sequence that is identical or complementary to a segment of at least about 14 contiguous bases of rNm-MEER The polynucleotide ofthe invention hybridizes to rNm-MIER, or a complement of rNm-MEER. Preferably, the isolated and purified polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 25 to 70 contiguous bases of rNm-MEER. For example, the polynucleotide ofthe invention may comprise a segment of bases identical or complementary to 40 or 55 contiguous bases of rNm-MEER.

Alternatively, the present invention contemplates an isolated and purified polynucleotide that comprises a base sequence that is identical or complementary to a segment of at least about 14 contiguous bases of Nm-MEER.

The polynucleotide of the invention hybridizes to Nm-MEER, or a complement of Nm-MEER. Preferably, the polynucleotide comprises a base sequence that is identical or complementary to a segment of at least 25 to 70 contiguous bases of Nm-MEER. For example, the polynucleotide may comprise a segment of bases identical or complementary to 40 or 55 contiguous bases of Nm-

MEER.

In certain embodiments, it is advantageous to employ a polynucleotide ofthe present invention in combination with an appropriate label for detecting hybrid formation. A wide variety of appropriate labels are known in the art, including radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal.

In general, it is envisioned that a hybridization probe described herein is useful both as a reagent in solution hybridization as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances and criteria required (e.g., the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing ofthe matrix to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantified, by means ofthe label.

Polynucleotide Primers

Polymerase Chain Reaction (PCR) is a repetitive, enzymatic, primed synthesis of a nucleic acid sequence. This procedure is well known and commonly used by those skilled in this art (see Mullis, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al., 1985). PCR is based on the enzymatic amplification of a DNA fragment of interest that is flanked by two oligonucleotide primers that hybridize to opposite strands ofthe target sequence. The primers are oriented with the 3' ends pointing towards each other. Repeated cycles of heat denaturation ofthe template, annealing ofthe primers to their complementary sequences, and extension of the annealed primers with a DNA polymerase result in the amplification ofthe segment defined by the 5' ends ofthe PCR primers. Since the extension product of each primer can serve as a template for the other primer, each cycle essentially doubles the amount of DNA fragment produced in the previous cycle. This results in the exponential accumulation ofthe specific target fragment, up to several million-fold in a few hours. By using a thermostable DNA polymerase such as Taq polymerase, which is isolated from the thermophilic bacterium Thermus aquaticus, the amplification process can be completely automated.

The DNA sequences ofthe subject invention can be used as primers for PCR amplification. In performing PCR amplification, a certain degree of mismatch can be tolerated between primer and template. Therefore, mutations, deletions, and insertions (especially additions of nucleotides to the 5' end) ofthe exemplified primers fall within the scope ofthe subject invention. Mutations, insertions and deletions can be produced in a given primer by methods known to an ordinarily skilled artisan. It is important to note that the mutational, insertional, and deletional variants generated from a given primer sequence may be more or less efficient than the original sequences. Notwithstanding such differences in efficiency, these variants are within the scope ofthe present invention.

DNA Vaccines and Immunotherapy

Tumor Associated Antigens

Certain members ofthe Nm-MEER family of proteins are normally expressed during embryogenesis. Thus, the proteins should not be present in mature or adult cells. Of these proteins that are not present in adult cells, those that do appear can form the basis of a cancer-antigen indicating a cell that has turned cancerous. This can be determined, for example, by screening using a labelled nucleic acid probe indicating the presence of mRNA for the Nm-MIER proteins, that is not present at the same level in normal, healthy cells. In the alternative, labelled antibodies can be used to detect Nm-MIER protein as an antigenic determinant of cancerous growth. These types of results are presented in Figures 2-5.

Vaccines

In a preferred embodiment, the invention relates to specific DNA vaccines and methods of treating cancer using the immune system and/or providing protective immunity to mammals and/or non- mammals. "Protective immunity" conferred by the method ofthe invention can elicit humoral and/or cell-mediated immune responses to cancerous growth, but more importantly interferes with the activity, spread, or growth of a cell that has become cancerous and has begun to express Nm- MEER nucleic acids and/or proteins following a subsequent challenge after vaccination.

The DNA vaccines ofthe invention are transcription units containing DNA encoding a Nm-MEER polypeptide or protein. En the method ofthe present invention, a DNA vaccine is administered to a mammal and/or a non-mammal as a mode of therapy, and/or in whom protective immunization is desired. An object ofthe invention is to provide an immune response and protective immunity to a mammal and/or a non-mammal using a DNA vaccine encoding a Nm-MEER protein as it has the potential of achieving high levels of protection in the virtual absence of side effects. Such DNA vaccines are also stable, easy to administer, and sufficiently cost-effective for widespread distribution.

An object ofthe invention is to provide protective immunity to an inoculated host. If the inoculated host is a female mammal and/or a non-mammal, an object ofthe invention is to provide protection in the offspring of that female. The invention features a DNA vaccine containing a Nm-MEER DNA transcription unit (i.e., an isolated nucleotide sequence encoding a Nm-MEER-encoded protein or polypeptide). The nucleotide sequence is operably linked to transcriptional and translational regulatory sequences for expression ofthe Nm-MEER-coded polypeptide in a cell of a mammal and/or a non-mammal. Preferably the polypeptide encoded by the DNA vaccine ofthe invention is a sequence belonging to

Nm-MIER. Preferably, the nucleotide sequence encoding the polypeptide is contained in a plasmid vector.

The DNA vaccines can be administered to mammal (and/or a non-mammal) such as humans expressing tumor associated antigens, such as the erl protein.

The DNA vaccines ofthe invention are preferably contained in a physiologically acceptable carrier for in vivo administration to a cell of a mammal and/or a non-mammal. Administration ofthe DNA vaccines ofthe invention provide an immune response or protective immunity.

The invention also features a method of providing an immune response and protective immunity to a mammal and/or a non-mammal against cancerous growth of cells expressing such a tumor associated antigen. The method includes administering to a cell of a mammal and/or a non-mammal, a DNA transcription unit encoding a desired Nm-MIER-encoded antigen operably linked to a promoter sequence. Expression ofthe DNA transcription unit in the cell elicits a humoral immune response, a cell-mediated immune response, or both against the cell expressing the protein product ofthe DNA transcription unit, the tumor associated antigen, which in this invention would be a Nm- MIER-encoded antigen.

The promoter operably linked to the DNA transcription unit is of nonretroviral or retroviral origin. Preferably the promoter is the cytomegalovirus immediate-early enhancer promoter. The desired Nm-MEER-encoded antigen encoded by the DNA transcription unit is one ofthe members ofthe Nm-MIER family, demonstrated to be expressed at significantly high levels only in cancerous cells in the mature organism.

The DNA transcription unit ofthe method ofthe invention is preferably contained in a physiologically acceptable carrier and is administered to the mammal and/or a non-mammal by routes including, but not limited to, inhalation, intravenous, intramuscular, intraperitoneal, intradermal, and subcutaneous administration. The DNA transcription unit in a physiologically acceptable carrier can also be administered by being contacted with a mucosal surface ofthe mammal and/or a non-mammal.

Preferably, administration is performed by particle bombardment using gold beads coated with the DNA transcription units ofthe invention. Preferably, the gold beads are 1 .mu.m to 2 .mu.m in diameter. The coated beads are preferably administered intradermally, intramuscularly, by organ transfection, or by other routes useful in particle bombardment and known to those of ordinary skill in the art.

The term "immune response" refers herein to a cytotoxic T cells response or increased serum levels of antibodies to an antigen, or to the presence of neutralizing antibodies to an antigen, such as a Nm-MIER-encoded protein. The term "protection" or "protective immunity" refers herein to the ability ofthe serum antibodies and cytotoxic T cell response induced during immunization to protect (partially or totally) against cells expressing such tumor associated antigen. That is, a mammal and/or a non-mammal immunized by the DNA vaccines ofthe invention will experience an immune attack on cancerous cells expressing such tumor associated antigen.

The term "promoter sequence" herein refers to a minimal sequence sufficient to direct transcription.

Also included in the invention is an enhancer sequence which may or may not be contiguous with the promoter sequence. Enhancer sequences influence promoter-dependent gene expression and may be located in the 5' or 3' regions ofthe native gene. Expression is constitutive or inducible by external signals or agents. Optionally, expression is cell-type specific, tissue-specific, or species specific.

By the term "transcriptional and translational regulatory sequences" is meant nucleotide sequences positioned adjacent to a DNA coding sequence which direct transcription or translation of a coding sequence. The regulatory nucleotide sequences include any sequences which promote sufficient expression of a desired coding sequence and presentation ofthe protein product to the inoculated mammalian (and/or a non-mammalian) immune system such that protective immunity is provided.

By the term "operably linked to transcriptional and translational regulatory sequences" is meant that a polypeptide coding sequence and minimal transcriptional and translational controlling sequences are connected in such a way as to permit polypeptide expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s). En the present invention, polypeptide expression in a target mammalian and/or a non-mammalian cell is particularly preferred.

The term "isolated DNA" means DNA that is free ofthe genes and other nucleotide sequences that flank the gene in the naturally-occurring genome ofthe organism from which the isolated DNA of the invention is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequences.

A preferred embodiment of this invention relates to a method of providing protective immunity to mammal and/or a non-mammal. Protective immunity ofthe invention elicits humoral and/or cell-mediated immune responses. According to the present invention, a DNA transcription unit is administered to a mammal and/or a non-mammal in whom immunization and protection is desired.

DNA Transcription Units

A DNA transcription unit is a polynucleotide sequence, bounded by an initiation site and a termination site, that is transcribed to produce a primary transcript. As used herein, a "DNA transcription unit" includes at least two components: (1) antigen-encoding DNA, and (2) a transcriptional promoter element or elements operatively linked for expression ofthe antigen-encoding DNA. Antigen-encoding DNA can encode one or multiple antigens, such as antigens from two or more different proteins. The DNA transcription unit can additionally be inserted into a vector which includes sequences for expression ofthe DNA transcription unit.

A DNA transcription unit can optionally include additional sequences such as enhancer elements, splicing signals, termination and polyadenylation signals, viral replicons, and bacterial plasmid sequences. En the present method, a DNA transcription unit (i.e., one type of transcription unit) can be administered individually or in combination with one or more other types of DNA transcription units.

DNA transcription units can be produced by a number of known methods. For example, DNA encoding the desired antigen can be inserted into an expression vector (see, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d, Cold Spring Harbor Laboratory Press (1989)). With the availability of automated nucleic acid synthesis equipment, DNA can be synthesized directly when the nucleotide sequence is known, or by a combination of polymerase chain reaction (PCR), cloning, and fermentation. Moreover, when the sequence ofthe desired polypeptide is known, a suitable coding sequence for the polynucleotide can be inferred.

The DNA transcription unit can be administered to an individual, or inoculated, in the presence of adjuvants or other substances that have the capability of promoting DNA uptake or recruiting immune system cells to the site ofthe inoculation. It should be understood that the DNA transcription unit itself is expressed in the host cell by transcription factors provided by the host cell, or provided by a DNA transcription unit.

The "desired antigen" can be any antigen or combination of antigens from encoded by a Nm-MEER gene. The antigen or antigens can be naturally occurring, or can be mutated or specially modified. The antigen or antigens can represent different forms, such as subgroups (clades), or subtypes. These antigens may or may not be structural components of a protein encoded by a Nm-MEER gene. The encoded antigens can be translation products or polypeptides. The polypeptides can be of various lengths, and can undergo normal host cell modifications such as glycosylation, myristoylation, or phosphorylation. In addition, they can be designated to undergo intracellular, extracellular, or cell-surface expression. Furthermore, they can be designed to undergo assembly and release from cells.

Administration of DNA Transcription Units

A vertebrate can be inoculated through any parenteral route. For example, an individual can be inoculated by intravenous, intraperitoneal, intradermal, subcutaneous, inhalation, or intramuscular routes, or by particle bombardment using a gene gun. Muscle is a useful site for the delivery and expression of DNA transcription unit-encoded polynucleotides, because animals have a proportionately large muscle mass which is conveniently accessed by direct injection through the skin. A comparatively large dose of polynucleotides can be deposited into muscle by multiple and/or repetitive injections, for example, to extend therapy over long periods of time. Muscle cells are injected with polynucleotides encoding immunogenic polypeptides, and these polypeptides are presented by muscle cells in the context of antigens ofthe major histocompatibility complex to provoke a selected immune response against the immunogen (see, e.g., Feigner, et al. WO90/11092, herein incorporated by reference).

The epidermis is another useful site for the delivery and expression of polynucleotides, because it is conveniently accessed by direct injection or particle bombardment. A comparatively large dose of polynucleotides can be deposited in the epidermis by multiple injections or bombardments to extend therapy over long periods of time. In immunization strategies ofthe invention, skin cells are injected with polynucleotides coding for immunogenic polypeptides, and these polypeptides are presented by skin cells in the context of antigens ofthe major histocompatibility complex to provoke a selected immune response against the immunogen.

In addition, an individual can be inoculated by a mucosal route. The DNA transcription unit can be administered to a mucosal surface by a variety of methods including DNA-containing nose-drops, inhalants, suppositories, microsphere encapsulated DNA, or by bombardment with DNA coated gold particles. For example, the DNA transcription unit can be administered to a respiratory mucosal surface, such as the nares or the trachea.

Any appropriate physiologically compatible medium, such as saline for injection, or gold particles for particle bombardment, is suitable for introducing the DNA transcription unit into an individual.

Nm-MIER Polypeptides

In one embodiment, the present invention contemplates an isolated and purified Nm-MEER polypeptides such as Er-1 polypeptide. Preferably, a Nm-MEER Polypeptide ofthe invention is a recombinant polypeptide. Preferably, an exemplary Nm-MEER polypeptide ofthe present invention comprises an amino acid sequence of FEG. 1.

Polypeptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a single letter or a three letter code.

Modifications and changes may be made in the structure of a polypeptide ofthe present invention and still obtain a molecule having Nm-MEER-like characteristics. For example, certain amino acids may be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide's biological functional activity, certain amino acid sequence substitutions may be made in a polypeptide sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a polypeptide with like properties.

The importance ofthe hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art (Kyte and Doolittle, 1982). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

Et is believed that the relative hydropathic character ofthe amino acid determines the secondary structure ofthe resultant polypeptide, which in turn defines the interaction ofthe polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid may be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within .+-.2 is preferred, those which are within .+-.1 are particularly preferred, and those within .+-.0.5 are even more particularly preferred.

Substitution of like amino acids may also be made on the basis of hydrophilicity, particularly where the biological functional equivalent polypeptide or peptide thereby created is intended for use in immunological embodiments. U.S. Pat. No. 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a polypeptide, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property ofthe polypeptide.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5. +-.1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid may be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide. In such changes, the substitution of amino acids whose hydrophilicity values are within .+-.2 is preferred, those which are within .+-.1 are particularly preferred, and those within .+-.0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various ofthe foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine (See Table 1, below). The present invention thus contemplates functional or biological equivalents of a Nm-MEER polypeptide as set forth above.

Biological or functional equivalents of a polypeptide may also be prepared using site-specific mutagenesis. Site-specific mutagenesis is a technique useful in the preparation of second generation polypeptides, or biologically functional equivalent polypeptides or peptides, derived from the sequences thereof, through specific mutagenesis of the underlying DNA. As noted above, such changes may be desirable where amino acid substitutions are desirable. The technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more ofthe foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence ofthe desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides ofthe deletion junction being traversed.

Typically, a primer of about 14 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction ofthe sequence being altered.

TABLE 1

Original Exemplary Residue Substitutions

Ala Gly; Ser

Arg Lys

Asn Gin; His

Asp Glu

Cys Ser

Gin Asn

Glu Asp

Gly Ala

His Asn; Gin lie Leu; Val

Leu He; Val

Lys Arg

Met Leu; Tyr

Ser Thr

Thr Ser

Trp Tyr

Tyr Trp; Phe

Val He; Leu

The technique of site-specific mutagenesis is generally well-known in the art (Adelman et al., 1983).

As will be appreciated, the technique typically employs a phage vector which may exist in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage (Messing et al., 1981). These phage are commercially available and their use is generally known to those of skill in the art.

En general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector which includes within its sequence a DNA sequence which encodes all or a portion ofthe Nm-MEER polypeptide sequence selected. An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically, for example, by the method of Crea, et al., (1978). This primer is then annealed to the singled-stranded vector, and extended by the use of enzymes such as the I lenow fragment of E. coli polymerase 1, to complete the synthesis ofthe mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells such as E. coli cells and clones are selected which include recombinant vectors bearing the mutation. Commercially available kits come with all the reagents necessary, except the oligonucleotide primers.

Expression Vectors

In an alternate embodiment, the present invention provides expression vectors comprising a polynucleotide that encodes a Nm-MEER polypeptide. Preferably, an expression vector ofthe present invention comprises a polynucleotide that encodes a polypeptide comprising an amino acid residue sequence of one ofthe members ofthe Nm-MEER gene family, eg. erl as in FIG. 1. More preferably, an expression vector ofthe present invention comprises a polynucleotide comprising a nucleotide base sequence of FIG. 1. Even more preferably, an expression vector ofthe invention comprises a polynucleotide operatively linked to an enhancer-promoter. More preferably still, an expression vector ofthe invention comprises a polynucleotide operatively linked to a prokaryotic promoter. Alternatively, an expression vector of the present invention comprises a polynucleotide operatively linked to an enhancer-promoter that is a eukaryotic promoter, and the expression vector further comprises a polyadenylation signal that is positioned 3' ofthe carboxy-terminal amino acid and within a transcriptional unit ofthe encoded polypeptide.

A promoter is a region of a DNA molecule typically within about 100 nucleotide pairs in front of (upstream of) the point at which transcription begins (i.e., a transcription start site). That region typically contains several types of DNA sequence elements that are located in similar relative positions in different genes. As used herein, the term "promoter" includes what is referred to in the art as an upstream promoter region, a promoter region or a promoter of a generalized eukaryotic RNA Polymerase EE transcription unit.

Another type of discrete transcription regulatory sequence element is an enhancer. An enhancer provides specificity of time, location and expression level for a particular encoding region (e.g., gene). A major function of an enhancer is to increase the level of transcription of a coding sequence in a cell that contains one or more transcription factors that bind to that enhancer. Unlike a promoter, an enhancer may function when located at variable distances from transcription start sites so long as a promoter is present.

As used herein, the phrase "enhancer-promoter" means a composite unit that contains both enhancer and promoter elements. An enhancer-promoter is operatively linked to a coding sequence that encodes at least one gene product. As used herein, the phrase "operatively linked" means that an enhancer-promoter is connected to a coding sequence in such a way that the transcription of that coding sequence is controlled and regulated by that enhancer-promoter. Means for operatively linking an enhancer-promoter to a coding sequence are well known in the art. As is also well known in the art, the precise orientation and location relative to a coding sequence whose franscription is controlled, is dependent inter alia upon the specific nature ofthe enhancer-promoter. Thus, a TATA box minimal promoter is typically located from about 25 to about 30 base pairs upstream of a transcription initiation site and an upstream promoter element is typically located from about 100 to about 200 base pairs upstream of a transcription initiation site. In contrast, an enhancer may be located downstream from the initiation site and may be at a considerable distance from that site.

An enhancer-promoter used in a vector construct of the present invention may be any enhancer-promoter that drives expression in a cell to be transfected. By employing an enhancer-promoter with well-known properties, the level and pattern of gene product expression may be optimized.

A coding sequence of an expression vector is operatively linked to a franscription terminating region. RNA polymerase transcribes an encoding DNA sequence through a site where polyadenylation occurs. Typically, DNA sequences located a few hundred base pairs downstream ofthe polyadenylation site serve to terminate franscription. Those DNA sequences are referred to herein as transcription-termination regions. Those regions are required for efficient polyadenylation of transcribed messenger RNA (RNA). Transcription-terminating regions are well-known in the art. A preferred transcription-terminating region used in an adenovirus vector construct of the present invention comprises a polyadenylation signal of SV40 or the protamine gene.

An expression vector comprises a polynucleotide that encodes a Nm-MEER polypeptide. Such a polynucleotide is meant to include a sequence of nucleotide bases encoding a Nm-MEER polypeptide sufficient in length to distinguish said segment from a polynucleotide segment encoding a non-M-erl polypeptide. A polypeptide ofthe invention may also encode biologically functional polypeptides or peptides which have variant amino acid sequences, such as with changes selected based on considerations such as the relative hydropathic score ofthe amino acids being exchanged. These variant sequences are those isolated from natural sources or induced in the sequences disclosed herein using a mutagenic procedure such as site-directed mutagenesis.

An expression vector ofthe present invention comprises a polynucleotide that encodes a polypeptide comprising an amino acid residue sequence of FIG. 1. An expression vector may include a Nm-MIER polypeptide-coding region itself or any of the Nm-MEER polypeptides noted above or it may contain coding regions bearing selected alterations or modifications in the basic coding region of such a Nm-MIER polypeptide.

Alternatively, such vectors or fragments may code larger polypeptides or polypeptides which nevertheless include the basic coding region. In any event, it should be appreciated that due to codon redundancy as well as biological functional equivalence, this aspect ofthe invention is not limited to the particular DNA molecules corresponding to the polypeptide sequences noted above.

Exemplary vectors include the non-mammalian expression vectors ofthe pCMV family including pCMV6b and pCMVόc (Chiron Corp., Emeryville, Calif). In certain cases, and specifically in the case of these individual non-mammalian expression vectors, the resulting constructs may require co-transfection with a vector containing a selectable marker such as pSV2neo. Via co-transfection into a dihydrofolate reductase-deficient Chinese hamster ovary cell line, such as DG44, clones expressing opioid polypeptides by virtue of DNA incorporated into such expression vectors may be detected.

A DNA molecule ofthe present invention may be incorporated into a vector using standard techniques well known in the art. For instance, the vector pUC18 has been demonstrated to be of particular value. Likewise, the related vectors M 13mp 18 and M 13mp 19 may be used in certain embodiments ofthe invention, in particular, in performing dideoxy sequencing.

An expression vector ofthe present invention is useful both as a means for preparing quantities of the Nm-MEER polypeptide-encoding DNA itself, and as a means for preparing the encoded polypeptide and peptides. It is contemplated that where Nm-MEER polypeptides ofthe invention are made by recombinant means, one may employ either prokaryotic or eukaryotic expression vectors as shuttle systems. However, in that prokaryotic systems are usually incapable of correctly processing precursor polypeptides and, in particular, such systems are incapable of correctly processing membrane associated eukaryotic polypeptides, and since eukaryotic Nm-MEER polypeptides are anticipated using the teaching ofthe disclosed invention, one likely expresses such sequences in eukaryotic hosts. However, even where the DNA segment encodes a eukaryotic Nm-

MEER polypeptide, it is contemplated that prokaryotic expression may have some additional applicability. Therefore, the invention may be used in combination with vectors which may shuttle between the eukaryotic and prokaryotic cells. Such a system is described herein which allows the use of bacterial host cells as well as eukaryotic host cells.

Where expression of recombinant Nm-MEER polypeptides is desired and a eukaryotic host is contemplated, it is most desirable to employ a vector such as a plasmid, that incoφorates a eukaryotic origin of replication.

Additionally, for the puφoses of expression in eukaryotic systems, one desires to position the Nm- MEER encoding sequence adjacent to and under the control of an effective eukaryotic promoter such as promoters used in combination with Chinese hamster ovary cells. To bring a coding sequence under control of a promoter, whether it is eukaryotic or prokaryotic, what is generally needed is to position the 5' end ofthe translation initiation side ofthe proper translational reading frame ofthe polypeptide between about 1 and about 50 nucleotides 3 ' of or downstream with respect to the promoter chosen. Furthermore, where eukaryotic expression is anticipated, one would typically desire to incoφorate into the transcriptional unit which includes the Nm-MEER polypeptide, an appropriate polyadenylation side.

The pCMV plasmids are a series of expression vectors of particular utility in the present invention.

The vectors are designed for use in essentially all cultured cells and work extremely well in

SV40-transformed simian COS cell lines. The pCMVl, pCMV2, pCMV3, and pCMV5 vectors differ from each other in certain unique restriction sites in the polylinker region of each plasmid. pCMV4 differs from the other four plasmids in containing a translation enhancer in the sequence prior to the polylinker. While they are not directly derived from the pCMVl-pCMV5 series of vectors, the functionally similar pCMV6b and pCMVόc vectors are commercially available (Chiron Coφ., Emeryville, Calif.) and are identical except for the orientation of the polylinker region which is reversed in one relative to the other. The universal components ofthe pCMV vectors are as follows: The vector backbone is pTZ18R (Pharmacia, Piscataway, N.J.), and contains a bacteriophage fl origin of replication for production of single stranded DNA and an ampicillin (amp)-resistance gene. The CMV region consists of nucleotides -760 to +3 ofthe powerful promotor-regulatory region ofthe human cytomegalovirus (Towne stain) major immediate early gene (Thomsen et al., 1984; Boshart et al., 1985). The human growth hormone fragment (hGH) contains transcription termination and poly-adenylation signals representing sequences 1533 to 2157 of this gene (Seeber-lg, 1982). There is an Alu middle repetitive DNA sequence in this fragment. Finally, the SV40 origin of replication and early region promoter-enhancer derived from the pcD-X plasmid (HindEEI to PstI fragment) described in (Okayama et al., 1983). The promoter in this fragment is oriented such that transcription proceeds away from the CMV/hGH expression cassette.

The pCMV plasmids are distinguishable from each other by differences in the polylinker region and by the presence or absence ofthe translation enhancer. The starting pCMVl plasmid has been progressively modified to render an increasing number of unique restriction sites in the polylinker region. To create pCMV2, one of two EcoRI sites in pCMVl were destroyed. To create pCMV3, pCMVl was modified by deleting a short segment from the SV40 region (StuI to EcoRI), and in so doing made unique the PstI, Sail, and BamHI sites in the polylinker. To create pCMV4, a synthetic fragment of DNA corresponding to the 5'- untranslated region of a mRNA transcribed from the CMV promoter was added C. The sequence acts as a translational enhancer by decreasing the requirements for initiation factors in polypeptide synthesis (Jobling et al., 1987; Browning et al, 1988). To create pCMV5, a segment of DNA (Hpal to EcoRI) was deleted from the SV40 origin region of pCMVl to render unique all sites in the starting polylinker.

The pCMV vectors have been successfully expressed in simian COS cells, mouse L cells, CHO cells, and HeLa cells. In several side by side comparisons they have yielded 5- to 10-fold higher expression levels in COS cells than SV40-based vectors. The pCMV vectors have been used to express the LDL receptor, nuclear factor 1, G.sub.s .alpha, polypeptide, polypeptide phosphatase, synaptophysin, synapsin, insulin receptor, influenza hemagglutinin, androgen receptor, sterol 26-hydroxylase, steroid 17- and 21 -hydroxylase, cytochrome P-450 oxidoreductase, .beta.-adrenergic receptor, folate receptor, cholesterol side chain cleavage enzyme, and a host of other cDNAs. It should be noted that the SV40 promoter in these plasmids may be used to express other genes such as dominant selectable markers. Finally, there is an ATG sequence in the polylinker between the HindlH and PstI sites in pCMU that may cause sper-lious translation initiation. This codon should be avoided if possible in expression plasmids. A paper describing the construction and use ofthe parenteral pCMVl and pCMV4 vectors has been published (Anderson et al., 1989b).

Transfected Cells

In yet another embodiment, the present invention provides recombinant host cells transformed or transfected with a polynucleotide that encodes an Nm-MEER polypeptide, as well as transgenic cells derived from those transformed or transfected cells. Preferably, a recombinant host cell ofthe present invention is transfected with a polynucleotide of FIG. IC or FIG. ID. Means of transforming or transfecting cells with exogenous polynucleotide such as DNA molecules are well known in the art and include techniques such as calcium-phosphate- or DEAE-dextran- mediated transfection, protoplast fusion, electroporation, liposome mediated transfection, direct microinjection and adenovirus infection (Sambrook et al., 1989).

The most widely used method is transfection mediated by either calcium phosphate or DEAE-dextran. Although the mechanism remains obscure, it is believed that the transfected DNA enters the cytoplasm ofthe cell by endocytosis and is transported to the nucleus. Depending on the cell type, up to 90% of a population of culter-led cells may be transfected at any one time.

Because of its high efficiency, transfection mediated by calcium phosphate or DEAE-dextran is the method of choice for studies requiring transient expression ofthe foreign DNA in large numbers of cells. Calcium phosphate-mediated fransfection is also used to establish cell lines that integrate copies ofthe foreign DNA, which are usually arranged in head-to-tail tandem arrays into the host cell genome.

In the protoplast fusion method, protoplasts derived from bacteria carrying high numbers of copies of a plasmid of interest are mixed directly with cultured cells. After fusion ofthe cell membranes (usually with polyethylene glycol), the contents ofthe bacterium are delivered into the cytoplasm ofthe cells and the plasmid DNA is transported to the nucleus.

Protoplast fusion is not as efficient as transfection for many ofthe cell lines that are commonly used for transient expression assays, but it is useful for cell lines in which endocytosis of DNA occurs inefficiently. Protoplast fusion frequently yields multiple copies ofthe plasmid DNA tandomly integrated into the host chromosome. The application of brief, high- voltage electric pulses to a variety of mammalian and plant cells leads to the formation of nanometer-sized pores in the plasma membrane. DNA is taken directly into the cell cytoplasm either through these pores or as a consequence of the redistribution of membrane components that accompanies closer- le ofthe pores. Electroporation may be extremely efficient and may be used both for transient expression of cloned genes and for establishment of cell lines that carry integrated copies ofthe gene of interest. Electroporation, in contrast to calcium phosphate-mediated fransfection and protoplast fusion, frequently gives rise to cell lines that carry one, or at most a few, integrated copies ofthe foreign DNA.

Liposome transfection involves encapsulation of DNA and RNA within liposomes, followed by fusion ofthe liposomes with the cell membrane. The mechanism of how DNA is delivered into the cell is unclear but transfection efficiencies may be as high as 90%.

Direct microinjection of a DNA molecule into nuclei has the advantage of not exposing DNA to cellular compartments such as low-pH endosomes. Microinjection is therefore used primarily as a method to establish lines of cells that carry integrated copies of the DNA of interest.

The use of adenovirus as a vector for cell transfection is well known in the art. Adenovirus vector-mediated cell transfection has been reported for various cells (Stratford-Perricaudet et al., 1992).

A transfected cell may be prokaryotic or eukaryotic. Preferably, the host cells ofthe invention are eukaryotic host cells. More preferably, the recombinant host cells ofthe invention are COS-1 cells. Where it is of interest to produce Nm-MEER polypeptides, cultured or human cells are of particular interest.

En another aspect, the recombinant host cells ofthe present invention are prokaryotic host cells. Preferably, the recombinant host cells ofthe invention are bacterial cells ofthe DH5. alpha.. TM. (GelBCa BRL, Gaithersber-lg, Md.) strain of E. coli. In general, prokaryotes are preferred for the initial cloning of DNA sequences and constructing the vectors useful in the invention. For example,

E. coli K12 strains may be particularly useful. Other microbial strains which may be used include E. coli B, and E. coli X1776 (ATCC No. 31537). These examples are, of coer-lse, intended to be illustrative rather than limiting. In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli may be transformed using pBR322, a plasmid derived from an E. coli species (Bolivar et al, 1977). pBR322 contains genes for amp and tetracycline resistance and thus provides easy means for identifying transformed cells.

The pBR322 or other microbial plasmid or phage must also contain, or be modified to contain, promoters which may be used by the microbial organism for expression of its own polypeptides.

Those promoters most commonly used in recombinant DNA construction include the .beta.-lactamase (penicillinase) and .beta.-galactosidase (.beta. -Gal) promoter systems (Chang et al., 1978; Itaker-la et al., 1977; Goeddel et al, 1979; Goeddel et al., 1980) and a tryptophan (TRP) promoter system (EPO Appl. Publ. No. 0036776; Siebwenlist et al., 1980). While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published, enabling a skilled worker to introduce promoters functional into plasmid vectors (Siebwenlist et al., 1980).

In addition to prokaryotes, eukaryotic microbes, such as yeast may also be used. Saccharomyces cerevisiae or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used (Stinchcomb et al., 1979; Kingsman et al., 1979; Tschemper et al., 1980). This plasmid already contains the frpL gene which provides a selection marker for a mutant sfrain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, 1977). The presence ofthe frpL lesion as a characteristic ofthe yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

Suitable promotor sequences in yeast vectors include the promoters for 3 -phosphogly cerate kinase

(Hitzeman et al., 1980) or other glycolytic enzymes (Hess et al., 1968; Holland et al, 1978) such as enolase, gly ceraldehyde-3 -phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3 -phosphogly cerate mutase, pyruvate kinase, trios ephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, the termination sequences associated with these genes are also introduced into the expression vector downstream from the sequences to be expressed to provide polyadenylation ofthe mRNA and termination. Other promoters, which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Any plasmid vector containing a yeast-compatible promoter, origin or replication and termination sequences is suitable.

In addition to microorganisms, cultures of cells derived from multicellular organisms may also be used as hosts. In principle, any such cell culture may be employed, whether from mammalian and/or a non-mammalian culture. However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in tissue culture has become a routine procedure in recent years (Kruse and Peterson, 1973). Examples of such useful host cell lines are AtT-20, VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, BHK, COSM6, COS-7, 293 and MDCK cell lines. Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located upstream ofthe gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences.

For use in mammalian cells, the control functions on the expression vectors are often derived from viral material. For example, commonly used promoters are derived from polyoma, Adenovirus 2,

Cytomegalovirus (CMV) and most frequently Simian Virus 40 (SV40). The early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et al., 1978). Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the HindEII site toward the Bgll site located in the viral origin of replication. Further, it is also possible, and often desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems.

An origin of replication may be provided with by construction ofthe vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV,

CMV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.

Preparing a Recombinant Nm-MIER Polypeptide

In yet another embodiment, the present invention describes a process of preparing an Nm-MEER polypeptide comprising transfecting cells with a polynucleotide that encodes an Nm-MEER polypeptide to produce a transformed host cell; and maintaining the transformed host cell under biological conditions sufficient for expression ofthe polypeptide. Preferably, the transformed host cell is a eukaryotic cell. Even more preferably, the polynucleotide transfected into the transformed cells comprises a nucleotide base sequence of F1G1. Most preferably transfection is accomplished using a hereinbefore disclosed expression vector.

A host cell used in the process is capable of expressing a functional, recombinant Nm-MEER polypeptide. A variety of cells are amenable to a process ofthe invention, for instance, yeasts cells, human cell lines, and other eukaryotic cell lines known well to those ofthe art.

Following transfection, the cell is maintained under culture conditions for a period of time sufficient for expression of an Nm-MEER polypeptide. Culture conditions are well known in the art and include ionic composition and concentration, temperature, pH and the like. Typically, transfected cells are maintained under culture conditions in a culture medium. Suitable medium for various cell types are well-known in the art. In a preferred embodiment, temperature is from about 20°C. to about 50°C, more preferably from about 30°C. to about 40°C, and even more preferably, about 37°C.

pH is preferably from about a value of 6.0 to a value of about 8.0, more preferably from about a value of about 6.8 to a value of about 7.8, and most preferably, about 7.4. Osmolality is preferably from about 200 milliosmols per liter (mosm/L) to about 400 mosm/1 and, more preferably from about 290 mosm/L to about 310 mosm/L. Other biological conditions needed for transfection and expression of an encoded protein are well-known in the art.

Transfected cells are maintained for a period of time sufficient for expression of an Nm-MEER polypeptide. A suitable time depends inter alia upon the cell type used and is readily determinable by a skilled artisan. Typically, maintenance time is from about 2 to about 14 days. Recombinant Nm-MIER polypeptide is recovered or collected either from the transfected cells or the medium in which those cells are cultured. Recovery comprises isolating and purifying the Nm- MEER polypeptide. Isolation and purification techniques for polypeptides are well-known in the art and include such procedures as precipitation, filtration, chromatography, electrophoresis and the like.

Antibodies

En still another embodiment, the present invention provides an antibody immunoreactive with an Nm-MEER polypeptide (e.g., one which is specific for Nm-MEER polypeptide). Preferably, an antibody ofthe invention is a monoclonal antibody. Preferably, an Nm-MEER polypeptide comprises an amino acid residue sequence of FIG. . Means for preparing and characterizing antibodies are well-known in the art (See, e.g., "Antibodies: A Laboratory Manual", E. Howell and D. Lane, Cold Spring Harbor Laboratory, 1988).

Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide or polynucleotide ofthe present invention, and collecting antisera from that immunized animal. A wide range of animal species may be used for the production of antisera. Typically an animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster or a guinea pig. Because ofthe relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.

As is well-known in the art, a given polypeptide or polynucleotide may vary in its immunogenicity. It is often necessary therefore to couple the immunogen (e.g., a polypeptide or polynucleotide) ofthe present invention) with a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin may also be used as carriers.

Means for conjugating a polypeptide or a polynucleotide to a carrier protein are well-known in the art and include glutaraldehyde, m-maleimidobencoyl-N- hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.

As is also well-known in the art, immunogencity to a particular immunogen may be enhanced by the use of non-specific stimulators ofthe immune response known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant, incomplete Freund's adjuvants and aluminum hydroxide adjuvant.

The amount of immunogen used ofthe production of polyclonal antibodies varies inter alia, upon the nature ofthe immunogen as well as the animal used for immunization. A variety of routes may be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal. The production of polyclonal antibodies is monitored by sampling blood ofthe immunized animal at various points following immunization. When a desired level of immunogenicity is obtained, the immunized animal may be bled and the serum isolated and stored.

In another aspect, the present invention contemplates a process of producing an antibody immunoreactive with an Nm-MIER polypeptide comprising the steps of (a) transfecting a recombinant host cell with a polynucleotide that encodes an Nm-MIER polypeptide; (b) culturing the host cell under conditions sufficient for expression ofthe polypeptide; (c) recovering the polypeptide; and (d) preparing an antibody to the polypeptide. Preferably, the host cell is transfected with a polynucleotide of FIG 1. The present invention also provides an antibody prepared according to the process described above.

A monoclonal antibody ofthe present invention may be readily prepared through use of well-known techniques such as those exemplified in U.S. Pat. No. 4,196,265. Typically, a technique involves first immunizing a suitable animal with a selected antigen (e.g., a polypeptide or polynucleotide ofthe present invention) in a manner sufficient to provide an immune response.

Rodents such as mice and rats are preferred animals. Spleen cells from the immunized animal are then fused with cells of an immortal myeloma cell. Where the immunized animal is a mouse, a preferred myeloma cell is a murine NS-1 myeloma cell.

The fused spleen/myeloma cells are cultured in a selective medium to select fused spleen/myeloma cells from the parental cells. Fused cells are separated from the mixture of non-fused parental cells, for example, by the addition of agents that block the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both pNm-MEERines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a soNm-MEERce of nucleotides. Where azaserine is used, the media is supplemented with hypoxanthine.

This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants for reactivity with an antigen-polypeptides. The selected clones may then be propagated indefinitely to provide the monoclonal antibody.

By way of specific example, to produce an antibody ofthe present invention, mice are injected intraperitoneally with between about 1 to about 200 μg of an antigen comprising a polypeptide of the present invention. B lymphocyte cells are stimulated to grow by injecting the antigen in association with an adjuvant such as complete Freund's adjuvant (a non-specific stimulator ofthe immune response containing killed Mycobacterium tuberculosis). At some time (e.g., at least two weeks) after the first injection, mice are boosted by injection with a second dose ofthe antigen mixed with incomplete Freund's adjuvant.

A few weeks after the second injection, mice are tail bled and the sera tirered by immunoprecipitation against radiolabeled antigen. Preferably, the process of boosting and titering is repeated until a suitable titer is achieved. The spleen ofthe mouse with the highest titer is removed and the spleen lymphocytes are obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5 x 10⁷ to 2 x 10⁸ lymphocytes.

Mutant lymphocyte cells known as myeloma cells are obtained from laboratory animals in which such cells have been induced to grow by a variety of well-known methods. Myeloma cells lack the salvage pathway of nucleotide biosynthesis. Because myeloma cells are tumor cells, they may be propagated indefinitely in tissue culture, and are thus denominated immortal. Numerous cultured cell lines of myeloma cells from mice and rats, such as murine NS-1 myeloma cells, have been established.

Myeloma cells are combined under conditions appropriate to foster fusion with the normal antibody-producing cells from the spleen ofthe mouse or rat injected with the antigen/polypeptide ofthe present invention. Fusion conditions include, for example, the presence of polyethylene glycol. The resulting fused cells are hybridoma cells. Like myeloma cells, hybridoma cells grow indefinitely in culture. Hybridoma cells are separated from unfused myeloma cells by culturing in a selection medium such as hypoxanthine-aminopterin-thymidine (HAT) medium. Unfused myeloma cells lack the enzymes necessary to synthesize nucleotides from the salvage pathway because they are killed in the presence of aminopterin, methotrexate, or azaserine. Unfused lymphocytes also do not continue to grow in tissue culture. Thus, only cells that have successfully fused (hybridoma cells) may grow in the selection media.

Each ofthe surviving hybridoma cells produces a single antibody. These cells are then screened for the production ofthe specific antibody immunoreactive with an antigen/polypeptide ofthe present invention. Single cell hybridomas are isolated by limiting dilutions of the hybridomas. The hybridomas are serially diluted many times and, after the dilutions are allowed to grow, the supernatant is tested for the presence ofthe monoclonal antibody. The clones producing that antibody are then cultured in large amounts to produce an antibody ofthe present invention in convenient quantity.

By use of a monoclonal antibody ofthe present invention, specific polypeptides and polynucleotide ofthe invention may be recognized as antigens, and thus identified. Once identified, those polypeptides and polynucleotide may be isolated and purified by techniques such as antibody-affinity chromatography. In antibody-affinity chromatography, a monoclonal antibody is bound to a solid substrate and exposed to a solution containing the desired antigen. The antigen is removed from the solution through an immunospecific reaction with the bound antibody. The polypeptide or polynucleotide is then easily removed from the substrate and purified.

Pharmaceutical Compositions

In a preferred embodiment, the present invention provides a pharmaceutical composition comprising an Nm-MEER polypeptide and a physiologically acceptable carrier. More preferably, a pharmaceutical composition comprises an Nm-MEER polypeptide comprising an amino acid residue sequence of FIG. . Alternatively, pharmaceutical compositions include a polynucleotide that encodes an Nm-MEER polypeptide and a physiologically acceptable carrier. An example of a useful pharmaceutical composition includes a polynucleotide that has the nucleotide sequence of FIG.

A composition ofthe present invention is typically administered parenterally in dosage unit formulations containing standard, well-known nontoxic physiologically acceptable carriers, adjuvants, and vehicles as desired. The term parenteral as used herein includes intravenous, intramuscular, intraarterial injection, or infusion techniques.

Injectable preparations, for example sterile injectable aqueous or oleaginous suspensions, are formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.

Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. En addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this puφose any bland fixed oil may be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Preferred carriers include neutral saline solutions buffered with phosphate, lactate, Tris, and the like. Of course, one purifies the vector sufficiently to render it essentially free of undesirable contaminant, such as defective interfering adenovirus particles or endotoxins and other pyrogens such that it does not cause any untoward reactions in the individual receiving the vector construct. Means of purifying the vector may involve the use of buoyant density gradients, such as cesium chloride gradient centrifugation.

A carrier may also be a liposome. Means for using liposomes as delivery vehicles are well-known in the art (See, e.g., Gabizon et al., 1990; Ferruti and Tanzi, 1986; Ranade, 1989).

A transfected cell may also serve as a carrier. By way of example, a liver cell may be removed from an organism, transfected with a polynucleotide ofthe present invention using methods set forth above and then the transfected cell returned to the organism (e.g., injected intravascularly).

Detecting Nm-MIER Encoding Polynucleotides and Nm-MIER Polypeptides

Alternatively, the present invention provides a process of detecting an Nm-MIER polypeptide, wherein the process comprises immunoreacting the polypeptide with an antibody prepared according to a process described above to form an antibody-polypeptide conjugate, and detecting the conjugate.

In yet another embodiment, the present invention contemplates a process of detecting a messenger RNA transcript that encodes an Nm-MIER polypeptide, wherein the process comprises (a) hybridizing the messenger RNA transcript with a polynucleotide sequence that encodes an Nm- MEER polypeptide to form a duplex; and (b) detecting the duplex. Alternatively, the present invention provides a process of detecting a DNA molecule that encodes an Nm-MEER polypeptide, wherein the process comprises (a) hybridizing a DNA molecule with a polynucleotide that encodes an Nm- MEER polypeptide to form a duplex; and (b) detecting the duplex.

Screening Assays

In yet another aspect, the present invention contemplates a process of screening substances for their ability to interact with an Nm-MIER polypeptide comprising the steps of providing an Nm-MIER polypeptide, and testing the ability of selected substances to interact with the Nm-MIER polypeptide.

Utilizing the methods and compositions ofthe present invention, screening assays for the testing of candidate substances such as agonists and antagonists of Nm-MEERs may be derived. A candidate substance is a substance which potentially may interact with or modulate, by binding or other intramolecular interaction, an Nm-MEER polypeptide. In some instances, such a candidate substance will be an agonist ofthe polypeptide and in other instances may exhibit antagonistic attributes when interacting with the polypeptide. In other instances, such substances may have mixed agonistic and antagonistic properties or may modulate the Nm-MIER in other ways.

Recombinant polypeptide expression systems ofthe present invention possess definite advantages over tissue-based systems. Such a method ofthe present invention makes it possible to produce large quantities of Nm-MIERs for use in screening assays. More important, however, is the relative purity ofthe polypeptides provided by the present invention. A relatively pure polypeptide preparation for assaying a protein-protein interaction makes it possible to use elutive methods without invoking competing, and unwanted, side-reactions. Cloned expression systems such as those ofthe present invention are also useful where there is difficulty in obtaining tissue that satisfactorily expresses a particular polypeptide. Cost is another very real advantage, at least with regard to the microbial expression systems ofthe present invention. For antagonists in a primary screen, microorganism expression systems ofthe present invention are inexpensive in comparison to prior art tissue-screening methods.

Traditionally, screening assays employed the use of crude polypeptide preparations. Typically, animal tissue slices thought to be rich in the polypeptide of interest was the source ofthe polypeptide. Alternatively, investigators homogenized the tissue and used the crude homogenate as a polypeptide source. A major difficulty with this approach is the provision that the tissue contain only a single polypeptide type being expressed. The data obtained therefore could not be definitively correlated with a particular polypeptide. With the recent cloning of polypeptide sub-types and sub-sub-types, this difficulty is highlighted. A second fundamental difficulty with the fraditional approach is the unavailability of human tissue for screening potential drugs. The traditional approach almost invariably utilized animal polypeptides. With the cloning of human polypeptides, there is a need for screening assays which utilize human polypeptides.

With the availability of cloned polypeptides, recombinant polypeptide screening systems have several advantages over tissue based systems. A major advantage is that the investigator may now confrol the type of polypeptide that is utilized in a screening assay. Specific polypeptide sub-types and sub-sub-types may be preferentially expressed and its interaction with a ligand may be identified. Other advantages include the availability of large amounts of polypeptide, the availability of rare polypeptides previously unavailable in tissue samples, and the lack of expenses associated with the maintenance of live animals.

Screening assays ofthe present invention generally involve determining the ability of a candidate substance to bind to the polypeptide and to affect the activity ofthe polypeptide, such as the screening of candidate substances to identify those that inhibit or otherwise modify the polypeptide's function. Typically, this method includes preparing recombinant polypeptide polypeptide, followed by testing the recombinant polypeptide or cells expressing the polypeptide with a candidate substance to determine the ability ofthe substance to affect its physiological function. In preferred embodiments, the invention relates to the screening of candidate substances to identify those that affect the enzymatic activity ofthe human polypeptide, and thus can be suitable for use in humans. A screening assay provides a polypeptide under conditions suitable for the binding of an agent to the polypeptide. These conditions include but are not limited to pH, temperature, tonicity, the presence of relevant cofactors, and relevant modifications to the polypeptide such as glycosylation or prenylation. It is contemplated that the polypeptide can be expressed and utilized in a prokaryotic or eukaryotic cell. The host cell expressing the polypeptide can be used whole or the polypeptide can be isolated from the host cell. The polypeptide can be membrane bound in the membrane ofthe host cell or it can be free in the cytosol ofthe host cell. The host cell can also be fractionated into sub-cellular fractions where the polypeptide can be found. For example, cells expressing the polypeptide can be fractionated into the nuclei, the endoplasmic reticulum, vesicles, or the membrane surfaces ofthe cell.

pH is preferably from about a value of 6.0 to a value of about 8.0, more preferably from about a value of about 6.8 to a value of about 7.8, and most preferably, about 7.4. En apreferred embodiment, temperature is from about 20°C to about 50°C, more preferably, from about 30°C to about 40°C, and even more preferably about 37°C. Osmolality is preferably from about 5 milliosmols per liter (mosm/L) to about 400 mosm/1, and more preferably, from about 200 milliosmols per liter to about 400 mosm/1 and, even more preferably from about 290 mosm L to about 310 mosm/L. The presence of cofactors can be required for the proper functioning ofthe polypeptide. Typical cofactors include sodium, potassium, calcium, magnesium, and chloride. En addition, small, non-peptide molecules, known as prosthetic groups may also be required. Other biological conditions needed for polypeptide function are well-known in the art.

It is well-known in the art that proteins can be reconstituted in artificial membranes, vesicles or liposomes. (Danboldt et al., 1990). The present invention contemplates that the polypeptide can be incoφorated into artificial membranes, vesicles or liposomes. The reconstituted polypeptide can be utilized in screening assays.

It is further contemplated that a polypeptide ofthe present invention can be coupled to a solid support, e.g., to agarose beads, polyacrylamide beads, polyacrylic beads or other solid matrices capable of being coupled to polypeptides. Well-known coupling agents include cyanogen bromide (CNBr), carbonyldiimidazole, tosyl chloride, and glutaraldehyde.

In a typical screening assay for identifying candidate substances, one employs the same recombinant expression host as the starting source for obtaining the polypeptide, generally prepared in the form of a crude homogenate. Recombinant cells expressing the polypeptide are washed and homogenized to prepare a crude polypeptide homogenate in a desirable buffer such as disclosed herein. In a typical assay, an amount of polypeptide from the cell homogenate, is placed into a small volume of an appropriate assay buffer at an appropriate pH. Candidate substances, such as agonists and antagonists, are added to the admixture in convenient concentrations and the interaction between the candidate substance and the polypeptide is monitored.

Where one uses an appropriate known substrate for the polypeptide, one can, in the foregoing manner, obtain a baseline activity for the recombinantly produced polypeptide. Then, to test for inhibitors or modifiers ofthe polypeptide function, one can incoφorate into the admixture a candidate substance whose effect on the polypeptide is unknown. By comparing reactions which are carried out in the presence or absence ofthe candidate substance, one can then obtain information regarding the effect ofthe candidate substance on the normal function ofthe polypeptide.

Accordingly, this aspect of the present invention will provide those of skill in the art with methodology that allows for the identification of candidate substances having the ability to modify the action of Nm-MIER polypeptides in one or more manner.

Additionally, screening assays for the testing of candidate substances are designed to allow the determination of structure-activity relationships of agonists or antagonists with the polypeptides, e.g., comparisons of binding between naturally-occurring hormones or other substances capable of interacting or otherwise modulating with the polypeptide; or comparison of the activity caused by the binding of such molecules to the polypeptide.

In certain aspects, the polypeptides ofthe invention are crystallized in order to carry out x-ray crystallographic studies as a means of evaluating interactions with candidate substances or other molecules with the Nm-MIER polypeptide. For instance, the purified recombinant polypeptides of the invention, when crystallized in a suitable form, are amenable to detection of infra-molecular interactions by x-ray crystallography.

The recombinantly-produced Nm-MIER polypeptide may be used in screening assays for the identification of substances which may inhibit or otherwise modify or alter the function ofthe polypeptide. The use of recombinantly-produced polypeptide is of particular benefit because the naturally-occurring polypeptide is present in only small quantities and has proven difficult to purify. Moreover, this provides a ready source of polypeptide, which has heretofore been unavailable.

A screening assay ofthe invention, in preferred embodiments, conveniently employs an Nm-MIER polypeptide directly from the recombinant host in which it is produced. This is achieved most preferably by simply expressing the selected polypeptide within the recombinant host, typically a eukaryotic host, followed by preparing a crude homogenate which includes the enzyme. A portion ofthe crude homogenate is then admixed with an appropriate effector ofthe polypeptide along with the candidate substance to be tested. By comparing the binding ofthe selected effector to the polypeptide in the presence or absence ofthe candidate substance, one may obtain information regarding the physiological properties ofthe candidate substance.

There are believed to be a wide variety of embodiments which may be employed to determine the effect ofthe candidate substance on the polypeptides ofthe invention, and the invention is not intended to be limited to any one such method. However, it is generally desirable to employ a system wherein one may measure the ability ofthe polypeptide to bind to and or be modified by the effector employed in the presence of a particular substance.

The detection of an interaction between an agent and a polypeptide may be accomplished through techniques well-known in the art. These techniques include but are not limited to centrifugation, chromatography, electrophoresis and spectroscopy. The use of isotopically labeled reagents in conjunction with these techniques or alone is also contemplated. Commonly used radioactive isotopes include ³H, ¹⁴C, ²²Na, ³²P, ³⁵S, ⁵Ca, ⁶⁰Co, ¹²⁵I, and I. Commonly used stable isotopes include ²H, ¹³C, ¹⁵N, and ¹⁸O.

For example, if an agent binds to the polypeptide ofthe present invention, the binding may be detected by using radiolabeled agent or radiolabeled polypeptide. Briefly, if radiolabeled agent or radiolabeled polypeptide is utilized, the agent-polypeptide complex may be detected by liquid scintillation or by exposure to x-ray film.

When an agent modifies the polypeptide, the modified polypeptide may be detected by differences in mobility between the modified polypeptide and the unmodified polypeptide through the use of chromatography, electrophoresis or centrifugation. When the technique utilized is centrifugation, the differences in mobility is known as the sedimentation coefficient. The modification may also be detected by differences between the spectroscopic properties ofthe modified and unmodified polypeptide. As a specific example, if an agent covalently modifies a polypeptide, the difference in retention times between modified and unmodified polypeptide on a high pressure liquid chromatography (HPLC) column may easily be detected. Alternatively, the spectroscopic differences between modified and unmodified polypeptide in the nuclear magnetic resonance (NMR) spectra may be detected. Or, one may focus on the agent and detect the differences in the spectroscopic properties or the difference in mobility between the free agent and the agent after modification of the polypeptide.

When a secondary polypeptide is provided, the agent-polypeptide-secondary polypeptide complex or the polypeptide-secondary polypeptide complex may be detected by differences in mobility or differences in spectroscopic properties as described above. The interaction of an agent and a polypeptide may also be detected by providing a reporter gene. Well-known reporter genes include β-Gal, chloramphenicol (Cml) transferase (CAT) and luciferase The reporter gene is expressed by the host and the enzymatic reaction ofthe reporter gene product may be detected.

In one example, a mixture containing the polypeptide, effector and candidate substance is allowed to incubate. The unbound effector is separable from any effector/polypeptide complex so formed. One then simply measures the amount of each (e.g., versus a control to which no candidate substance has been added). This measurement may be made at various time points where velocity data is desired. From this, one determines the ability ofthe candidate substance to alter or modify the function ofthe polypeptide.

Numerous techniques are known for separating the effector from effector/polypeptide complex, and all such methods are intended to fall within the scope ofthe invention. Use of thin layer chromatographic methods (TLC), HPLC, spectrophotometric, gas chromatographic/mass spectrophotometric or NMR analyses. It is contemplated that any such technique may be employed so long as it is capable of differentiating between the effector and complex, and may be used to determine enzymatic function such as by identifying or quantifying the substrate and product.

Screening Assays for Nm-MIER Polypeptides The present invention provides a process of screening a biological sample for the presence of an Nm-MIER polypeptide. A biological sample to be screened may be a biological fluid such as extracellular or intracellular fluid, a cell, a tissue extract, a tissue homogenate or a histological section. A biological sample may also be an isolated cell (e.g., in culture) or a collection of cells such as in a tissue sample or histology sample. A tissue sample may be suspended in a liquid medium or fixed onto a solid support such as a microscope slide.

In accordance with a screening assay process, a biological sample is contacted with an antibody specific for a Nm-MIER polypeptide whose presence is being assayed. Typically, one mixes the antibody and the Nm-MIER polypeptide, and either the antibody or the sample with the Nm-MDER polypeptide may be affixed to a solid support (e.g., a column or a microtiter plate). Optimal conditions for the reaction may be accomplished by adjusting temperature, pH, ionic concentration, etc.

Ionic composition and concentration may range from that of distilled water to a 2 molar solution of NaCl. Preferably, osmolality is from about 100 mosmols/1 to about 400 mosmols/1, and more preferably, from about 200 mosmols/1 to about 300 mosmols/1. Temperature preferably is from about 4°C. to about 100°C, more preferably from about 15°C to about 50°C, and even more preferably from about 25°C to about 40°C. pH is preferably from about a value of 4.0 to a value of about 9.0, more preferably from about a value of 6.5 to a value of about 8.5, and even more preferably, from about a value of 7.0 to a value of about 7.5. The only limit on biological reaction conditions is that the conditions selected allow for antibody-polypeptide conjugate formation and that the conditions do not adversely affect either the antibody or the Nm-MEER polypeptide.

Incubation time varies with the biological conditions used, the concentration of antibody and polypeptide and the nature ofthe sample (e.g., fluid or tissue sample). Means for determining exposure time are well-known to one of ordinary skill in the art. Typically, where the sample is fluid and the concentration of polypeptide in that sample is about 10^"10 M, exposure time is from about

10 min to about 200 min.

Nm-MIER polypeptide in the sample is determined by detecting the formation and presence of antibody-Nm-MIER polypeptide conjugates. Means for detecting such antibody-antigen (e.g., polypeptide polypeptide) conjugates or complexes are well-known in the art and include such procedures as centrifugation, affinity chromatography and the like, binding of a secondary antibody to the antibody-candidate polypeptide complex. Detection may be accomplished by measuring an indicator affixed to the antibody. Exemplary and well-known such indicators include radioactive labels (e.g., ³²P, ¹²⁵I, ¹⁴C), a second antibody or an enzyme such as horse radish peroxidase. Methods for affixing indicators to antibodies are well-known in the art. Commercial kits are available.

Screening Assay for Nm-MIER Antibody

The present invention provides a process of screening a biological sample for the presence of antibodies immunoreactive with a Nm-MIER polypeptide (i.e., Nm-MIER antibody). In accordance with such a process, a biological sample is exposed to an Nm-MEER polypeptide under biological conditions and for a period of time sufficient for antibody-polypeptide conjugate formation and the formed conjugates are detected.

Screening Assay for a Polynucleotide Encoding A Nm-MIER Polypeptide

A DNA molecule and, particularly a probe molecule, may be used for hybridizing as oligonucleotide probes to a DNA source suspected of possessing an Nm-MIER polypeptide encoding polynucleotide or gene. The probing is usually accomplished by hybridizing the oligonucleotide to a

DNA source suspected of possessing such a polypeptide gene. In some cases, the probes constitute only a single probe, and in others, the probes constitute a collection of probes based on a certain amino acid sequence or sequences ofthe Nm-MIER polypeptide and account in their diversity for the redundancy inherent in the genetic code.

A suitable source of DNA for probing in this manner is capable of expressing Nm-MIER polypeptides and may be a genomic library of a cell line of interest. Alternatively, a soer-lce of DNA may include total DNA from the cell line of interest. Once the hybridization process ofthe invention has identified a candidate DNA segment, one confirms that a positive clone has been obtained by fer-lther hybridization, restriction enzyme mapping, sequencing and/or expression and testing.

Alternatively, such DNA molecules may be used in a number of techniques including their use as: (1) diagnostic tools to detect normal and abnormal DNA sequences in DNA derived from patient's cells; (2) means for detecting and isolating other members ofthe Nm-MIER family and related polypeptides from a DNA library potentially containing such sequences; (3) primers for hybridizing to related sequences for the per-lpose of amplifying those sequences; and (4) primers for altering the native Nm-MERDNA sequences; as well as other techniques which rely on the similarity ofthe DNA sequences to those ofthe Nm-MIER DNA segments herein disclosed.

As set forth above, in certain aspects, DNA sequence information provided by the invention allows for the preparation of relatively short DNA (or RNA) sequences (e.g., probes) that specifically hybridize to encoding sequences ofthe selected Nm-MIER gene. In these aspects, nucleic acid probes of an appropriate length are prepared based on a consideration ofthe selected Nm-MEER encoding sequence (e.g., a nucleic acid sequence such as shown in FIG. . The ability of such nucleic acid probes to specifically hybridize to Nm-MIER encoding sequences lend them particular utility in a variety of embodiments.

Most importantly, the probes are useful in a variety of assays for detecting the presence of complementary sequences in a given sample. These probes are useful in the preparation of mutant species primers and primers for preparing other genetic constructions.

To provide certain ofthe advantages in accordance with the invention, a preferred nucleic acid sequence employed for hybridization studies or assays includes probe sequences that are complementary to at least an about 14 to about 40 or so long nucleotide stretch ofthe Nm-MIER encoding sequence, such as shown in FIG. A size of at least 14 nucleotides in length helps to ensNm-MIERe that the fragment is of sufficient length to form a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than 14 bases in length are generally preferred, though, to increase stability and selectivity ofthe hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of about 14 to about 20 nucleotides, or even longer where desired. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR.TM. technology of U.S. Pat. No. 4,603,102,, or by introducing selected sequences into recombinant vectors for recombinant production.

Accordingly, a nucleotide sequence ofthe present invention may be used for its ability to selectively form duplex molecules with complementary stretches ofthe gene. Depending on the application envisioned, one employs varying conditions of hybridization to achieve varying degrees of selectivity ofthe probe toward the target sequence. For applications requiring a high degree of selectivity, one typically employs relatively stringent conditions to form the hybrids. For example, one selects relatively low salt and/or high temperature conditions, such as provided by about 0.02M to about 0.15M NaCl at temperatures of about 50°C to about 70°C. Such conditions are particularly selective, and tolerate little, if any, mismatch between the probe and the template or target strand.

Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate Nm-MIER coding sequences from related species, functional equivalents, or the like, less stringent hybridization conditions are typically needed to allow formation of the heteroduplex. Under such circumstances, one employs conditions such as from about 0.15M to about 0.9M salt, at temperatures ranging from about 20°C to about 55°C. Cross-hybridizing species may thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions may be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions may be readily manipulated, and thus will generally be a method of choice depending on the desired results.

En certain embodiments, it is advantageous to employ a nucleic acid sequence ofthe present invention in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In preferred embodiments, one likely employs an enzyme tag such a urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmentally undesirable reagents. In the case of enzyme tags, calorimetric indicator substrates are known which may be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.

In general, it is envisioned that the hybridization probes described herein are useful both as reagents in solution hybridization as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the sample containing test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions depend inter alia on the particular circumstances based on the particular criteria required (depending, for example, on the G+C content, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing ofthe hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantified, by means ofthe label.

Assay Kits

In another aspect, the present invention contemplates a diagnostic assay kit for detecting the presence of Nm-MIER polypeptide in a biological sample, where the kit comprises a first container containing a first antibody capable of immunoreacting with Nm-MEER polypeptide, with the first antibody present in an amount sufficient to perfonn at least one assay. An assay kit ofthe invention further optionally includes a second container containing a second antibody that immunoreacts with the first antibody. The antibodies used in the assay kits of the present invention may be monoclonal or polyclonal antibodies. For convenience, one may also provide the first antibody affixed to a solid support. Additionally, the first and second antibodies may be combined with an indicator, (e.g., a radioactive label or an enzyme).

The present invention also contemplates a diagnostic kit for screening agents for their ability to interact with an Nm-MEER. Such a kit will contain an Nm-MEER ofthe present invention. The kit may further contain reagents for detecting an interaction between an agent and a polypeptide ofthe present invention. The provided reagent may be radiolabeled. The kit may also contain a known radiolabeled agent that binds or interacts with a polypeptide ofthe present invention.

The present invention provides a diagnostic assay kit for detecting the presence, in a biological sample, of a polynucleotide that encodes an Nm-MIER polypeptide, the kits comprising a first container that contains a second polynucleotide identical or complementary to a segment of at least about 14 contiguous nucleotide bases of a polynucleotide of FIG.

In another embodiment, the present invention contemplates a diagnostic assay kit for detecting the presence, in a biological sample, of an antibody immunoreactive with an Nm-MIER polypeptide, the kits comprising a first container containing an Nm-MIER polypeptide that immunoreacts with the antibody, with the polypeptide present in an amount sufficient to perform at least one assay. The reagents ofthe kit may be provided as a liquid solution, attached to a solid support or as a dried powder. When the reagent is provided in a liquid solution, the liquid solution is an aqueous solution. When the reagent provided is attached to a solid support, the solid support may be chromatograph media or a microscope slide. When the reagent provided is a dry powder, the powder may be reconstituted by the addition of a suitable solvent. The solvent may also be included in the kit.

Process of Modifying the Function of a Nuclear Polypeptide using Nm-MIER

In another aspect, the present invention provides a process of altering the function of a nuclear polypeptide. In accordance with that process, a nuclear polypeptide is exposed to an Nm-MIER of the present invention. A preferred nuclear polypeptide used in such a process is the same as set forth above and includes nuclear polypeptides for thyroid hormone, vitamin D, retinoic acid and the like. Preferred Nm-MEERs and their corresponding DNA sequences are shown in FEG.

The present invention provides DNA segments, purified polypeptides, methods for obtaining antibodies, methods of cloning and using recombinant host cells necessary to obtain and use Nm-

MIERs. Accordingly, the present invention concems generally compositions and methods for the preparation and use of Nm-MIERs.

Nm-MIER Genes and Isoforms in Other Organisms

Er- 1 may be considered as a member of a subfamily of early response polypeptides that may include Mtal . It is probable that Er-1 isoforms are also encoded by multiple genes. Since nuclear polypeptides usually have a high homology, the sequences of Nm-MIER may be used as probes to screen cDNA libraries. Considering the fact that different isoforms of nuclear polypeptides may have different tissue distribution patterns and may be expressed to different extents in different tissues, the Nm-MIER may used as a probe to screen genomic libraries for genes encoding Nm-MEBR isoforms.

The present invention also provides cDNA libraries which are useful for screening of additional Nm-MIER isoforms. Using the nucleotide sequences ofthe present invention, it is possible to determine structural and genetic information (including restriction enzyme analysis and DNA sequencing) concerning these positive clones. Such information will provide important information concerning the role of these isoforms in vivo and in vitro. Nm-MIER sequence information may be used to analyze Nm-MIER cDNAs and Nm-MIER-like gene sequences in other organisms. Using PCR.TM. techniques, restriction enzyme analysis, and DNA sequencing, the structure of these Nm-

MIER-like isoform genes may be determined with relative facility.

The following examples illustrate preferred embodiments ofthe invention. Certain aspects ofthe following examples are described in terms of techniques and procedures found or contemplated by the present inventors to work well in the practice of the invention. These examples are exemplified through the use of standard laboratory practices of the inventor.

It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice ofthe invention, and thus may be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes may be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLES

As an example of one embodiment of a member ofthe Nm-MIER family, we have utilized the polymerase chain reaction (PCR)-based differential display methodology (Liang and Pardee, Science 257, 967-969, 1992) to identify a novel transcript whose expression levels increased in

Xenopus embryo explants during mesoderm induction by fibroblast growth factor (FGF). The PCR product was used to clone a 2.3-kb cDNA representing this transcript, which we have named erl (early response 1). The er7 cDNA contained a single open reading frame (ORF) predicted to encode a protein of 493 amino acid residues. A database homology search revealed that the predicted ERl amino acid sequence contains three regions of similarity to the rat and human proteins encoded by the metastasis-associated gene, mtal, and two regions of similarity to the C. elegans similar-to-mtai sequence. The FGF-induced increase in er7 steady-state levels was not dependent on de novo protein synthesis, demonstrating that er7 is an immediate-early gene. Northern blot analysis revealed a single 2.8-kb mRNA that was observed predominantly during the initial cleavage and blastula stages of Xenopus development, with little or no detectable mRNA during subsequent development. Quantitative PCR analysis of early developmental stages showed that erl peaked during late blastula. Computer-assisted analysis ofthe predicted ERl amino acid sequence revealed two putative nuclear localization signals, four highly acidic regions clustered at the N-terminus and a proline-rich region located near the C-terminus. Subcellular localization by immunocytochemistry revealed that the ERl protein was targeted exclusively to the nucleus. Transactivation assays, using various regions of ERl fused to the DNA binding domain of GAL4, demonstrated that the N-terminal acidic region is a potent trans activator. These data suggest that ERl may function as a transcription factor.

EXAMPLE 1

Embryos and mesoderm induction

Xenopus laevis were purchased from Nasco. Embryos were obtained and cultured as in (14). The recombinant Xenopus bFGF (XbFGF) used for induction was prepared as in (15). Animal pole explants (animal caps) were induced to form mesoderm as described (9), and animal caps were treated for 30 min prior to RNA extraction. For inhibition of protein synthesis during induction, animal caps were pre-treated for 30 min with 5ug/ral cycloheximide (Sigma), cultured with or without FGF for an additional 30 min, then processed for PCR analysis, as described below. Protein synthesis was measured in parallel samples by including 2uCi/ul of ³⁵S-methionine in the culture medium and ³⁵S-incoφoration into TCA precipitable material was determined according to Clemens (16).

EXAMPLE 2

Differential display

RNA was extracted from induced or uninduced animal caps using the NETS protocol (17). Reverse transcription (RT) and polymerase chain reaction (PCR) were performed as in (13) with the following primers: 5'-T_nAC-3' and 5'-CTGATCCATG-3'. PCR products were separated on a 6% polyacrylamide/6M urea gel; the gel was dried and the products visualized by autoradiography. Differentially expressed bands were excised and the PCR products eluted from the gel in l00ul ofH₂O. EXAMPLE 3

cDNA cloning and sequencing of erl from Xenopus embryos

Eluted PCR products were cloned into the pCR_π vector using the TA cloning kit (Invifrogen). The sequence for both strands ofthe initial er7 PCR product and all subsequent cDNA inserts was determined as in (14). A 2.3-kb er7 cDNA was isolated from a stage 8 Xenopus (lZAP II) cDNA library (14), using primers designed according to the er7 sequence (5'-TCCGTTACACCAGGATGTAG-3'; 5'-GGCTGAAATTCCAGTT GGTA-3'; 5'-GCATCAGCTGCAGATCAAGG-3'; 5'-GTTTAAGAAAGGGC-AGTTCG-3') and the lZAP vector sequence (5'-GCTCGAAATTAACCCTCACTAAAG-3'; 5'-GGTACCTAATA CGACTCACTATAGGG-3 '). The cDNA was cloned into ρCR_π and the sequence determined and verified by sequencing several clones on both strands.

EXAMPLE 4

Quantitative PCR and Northern analysis

Quantitative PCR analysis was performed as described in (18) with the following modifications: RNA was prepared as in (19); l/8th ofthe RT sample was added to a 50ul PCR reaction and the annealing temperature was 56°C. Histone H4 was used as a control with forward (F) and reverse (R) primers as described (18) and the primer sequences for er7 were as above. The PCR products were analyzed in the linear range for amplification, determined empirically (18) to be 19 cycles for histone H4 and 24 cycles for erl. Quantitation by densitometry was performed as described in (19) with normalization to histone H4. Northern analysis was carried out as described in (20), using the 2.3-kb erl or histone H4 cDNA as a probe.

EXAMPLE 5

Immunocytochemistry and protein analysis

Anύ-Xenopus ERl antiserum was prepared by immunizing rabbits as (9) with a C-terminal synthetic peptide (CIKRQRMDSPGKEST) of the predicted ERl protein sequence. Coupled in vitro transcription-translation, immunoprecipitation and SDS-PAGE were performed as in (9). For immunocytochemistry, NIH 3T3 cells were transfected with either pcDNA3 (Envitrogen) or er7-pcDNA3. After 48h, the cells were processed for immunocytochemistry as in (19), using a 1:50 dilution ofthe anti-ERl antiserum.

EXAMPLE 6

Plasmid construction and transient transactivation assays

NIH 3T3 cells (ATCC) were maintained in Dulbecco's modified Eagle's medium plus 10% calf serum and transfected with Lipofectamine according to the manufacturer's directions (Life Technologies, Enα). The expression vectors used in this assay were engineered to contain various portions of ERl fused to the GAL4 DNA binding domain ofthe pM plasmid (Clontech) and are named according to the amino acids of ERl that each encodes. Specific primers incoφorating 5' and 3' Bglll sites (ER 1-493, ER 176-493) or a 5 'EcoRI and a 3' BamHl site (ER 1-175, ER 1-25) were used to amplify PCR fragments encoding the appropriate amino acids. The digested PCR fragments were inserted into the complementary sites ofthe pM plasmid and all plasmids were sequenced to verify the junctions and the erl sequence and to ensure the proper reading frame. ER 1-98 and ERl -57 were generated by digesting the ER 1 - 175 construct with PstI or PvuII, respectively, and re-ligating the cut vector.

0.5ug of a CAT reporter plasmid (pG5CAT, Clontech) was cotransfected into 3X10⁵ cells with 1.Oug of either the pM vector alone, or one of the pM-er7 fusion constructs. After 48h, cell extracts were prepared and assayed for CAT enzyme using a CAT Elisa kit (Boehringer Mannheim) according to the manufacturer's directions.

Results and Conclusions

In our efforts to elucidate the molecular mechanisms of FGF-induced mesoderm differentiation in Xenopus, we employed the PCR-based differential display method (13) to identify and characterize genes that are expressed early during the cellular response to FGF. RNA was isolated and reverse-transcribed from five individual sets of 30 min FGF-treated or control animal pole explants

(animal caps) from Xenopus blastulae. PCR products from the five sets were separated on a 6% polyacrylamide/urea gel. Only those bands that were differentially expressed in all five sets were chosen for further analysis. A total of eleven differentially expressed bands were identified and one of these was eluted from the gel, cloned and sequenced. A search ofthe database for similarity to known sequences revealed that this cDNA represented a novel Xenopus gene, which we have named erl (early esponse 1).

The sequence ofthe erl PCR product was used to obtain a 2.3-kb cDNA from a.Xenopus blastula library (14). This cDNA consisted of a single 1497-bp open reading frame (ORF), bracketed by a 214-bp 5 '-untranslated region which contained several stop codons in all three frames and a 626-bp 3 '-untranslated region (Fig. 1). The ATG initiation codon is predicted to be at nucleotides 233-235, as this site is positioned within a Kozak consensus sequence for the start of translation (21), with a purine in the -3 position and a G in the +4 position. The ORF is predicted to encode a protein of 493 amino acids, beginning at nucleotide 233 and ending with an in-frame TAA stop codon at position 1712 (Fig. 1).

Computer-assisted analysis ofthe deduced amino acid sequence using MOTIFS and PSORT software programs predicts that ERl does not contain an N-terminal signal sequence for transfer into the endoplasmic reticulum or a hydrophobic domain characteristic of transmembrane proteins. However, ERl does contain two potential nuclear localization signals (NLS): RRPR and

KKSERYDFFAQQTRFGKKK (Fig. 1); the latter conforms to the consensus sequence for a bipartite NLS (22). ERl also contains a proline-rich sequence near the C-terminus which corresponds to the PXXP motif found in all high affinity SH3-domain binding ligands (23). The N-terminus of ERl includes several highly acidic stretches (Fig. 1), characteristic ofthe acidic activation domains of many transcription factors (24).

A database homology search using the National Center for Biotechnology Information BLAST Network Service revealed that ERl contains three regions of similarity to the product ofthe rat metastasis-associated gene, tα7 (25) (Fig. 4), a gene that was isolated by differential cDNA library screening and whose expression was associated with a metastatic phenotype. mtal encodes a 703 amino acid, 79kDa polypeptide of unknown function that contains a putative SH3 binding domain near the C-terminus. ERl also displays similarity to the human MTAl (accession no. U35113) and to the C. elegans MTAl-like sequence (accession no. U41264) (Fig. 4). Within the regions of similarity, the percent amino acid similarity ranged from 46% to 64%, however, the overall percent similarity was only 13.0%, 14.0% and 15.6% to the rat, human and C. elegans sequences, respectively. To investigate whether er7 represents the Xenopus homolog of mtal or simply a related protein, we screened by RT-PCR a human breast carcinoma cell line, MDA-468 (26), for erl -related sequences. We obtained a partial human cDNA clone"spanning sequence inside and outside the regions of similarity shown in Fig. 2; this sequence displays 91% overall similarity to erl at the amino acid level (data not shown). The existence of a human gene product that is distinct from human mtal and that shows a high degree of similarity to erl suggests that erl and mtal are not homologs, but possibly related members of a family of proteins or simply proteins containing some ofthe same functional domains.

Verification that the steady-state levels of er7 were increased in response to FGF during mesoderm induction in vitro was performed by quantitative PCR after a 30 min treatment with FGF, using histone H4 as an internal standard (Fig. 5A). In several independent experiments, densitometric analysis revealed that erl levels ranged from three- to four- fold higher in FGF-treated samples, after normalization to histone H4. These data confirm that erl levels were increased by treatment with FGF and demonstrate that the increase in er7 occurs early during the cellular response to FGF.

The possibility that er7 is an immediate-early gene was investigated further. By definition, transcription of immediate-early genes is a rapid response and is not dependent on de novo protein synthesis. The FGF-induced increase in er7 levels was measured in the presence or absence of

5ug/ml cycloheximide. Cycloheximide inhibited 90% of ³⁵S-methionine incoφoration into TCA-precipitable material (data not shown) but did not prevent the FGF-induced increase in erl levels (Fig. 5B), demonstrating that er7 is an immediate-early gene.

Northem analysis of the temporal pattern of er7 expression during embryonic development revealed a single erl mRNA (Fig. 6A). The estimated 2.8-kb size of the message was slightly larger than that of the cDNA clone, but this is probably due to the presence of a poly-A tail, erl was detectable during initial cleavage stages, prior to the start of zygotic transcription which occurs at mid-blastula transition (27), indicating that erl is a maternally derived mRNA. Densitometric analysis revealed that steady-state levels of er7 were relatively constant during early cleavage stages (stages 2, 6, 7; Fig. 6A, lanes 1-3), increased slightly at blastula stage (Fig. 6A, lane 4), then decreased sixfold during gastrula, neurula and tailbud stages (stages 12, 17, 22; Fig. 6A, lane 5-7) and remained below detectable levels during subsequent development (stages 30 and 41; Fig. 6A, lanes 8 and 9). Mesoderm induction, a process in which FGF is known to play a pivotal role, takes place during blastula stages. Therefore, we examined erl levels at lh time intervals during blastula and gastrula stages, using a quantitative PCR assay (18). er/ expression levels were shown to increase twofold from early blastula (stages 7-8; Fig. 6B, lanes 1-2) to late blastula stages (stages 8-9; Fig. 6B, lanes 3-4), followed by a fivefold decrease at gastrulation (stage 10; Fig. 6B, lane 5).

Our sequence analysis revealed two putative nuclear localization signals, suggesting that ERl is targeted to the nucleus. We investigated the subcellular localization ofthe ERl protein using a polyclonal anti-ERl antibody to stain transfected NEH 3T3 cells expressing ERl. This antibody, directed against a synthetic C-terminal peptide, recognizes full-length ERl protein synthesized in vitro (Fig. 7A, lane 3) and specifically stains the nuclei of cells expressing ERl (Fig. 7B). Cells transfected with the pcDNA3 vector alone (Fig. 7B) as well as pcDNA3-er7 transfected cells stained with pre-immune serum (not shown) gave similar patterns and showed no specific nuclear staining.

The fact that ERl is targeted to the nucleus and that its N-terminus contains stretches of acidic residues characteristic of acidic activation domains (25), suggests that ERl may function as a franscription factor. We investigated this possibility by testing the transactivation potential of various regions ofthe ERl protein. Constructs, containing different portions of er/ fused to the GAL4 DNA binding domain, were used along with a CAT reporter plasmid in transient transfections.

Assays of CAT enzyme levels revealed that, although full-length ERl did not activate transcription, the N-terminal region (ER 1175), containing all four acidic stretches (Fig. 1), stimulated transcription 10-fold (Fig. 6). The complementary C-terminal portion, ER 176-493, on the hand, had no transactivational activity. It is unclear why full-length ERl was unable to stimulate franscription, but one possible explanation is that fusion of ERl to GAL4 may alter the tertiary structure ofthe ERl protein, affecting its activity A similar observation was made with the ETS franscription factor ER81 which, when fused to the GAL4 DNA binding domain, lost its ability to activate transcription (28).

Interestingly, deletion ofthe N-terminal region to produce a construct containing only the first three acidic stretches (ER 1 -98), resulted in a much more potent transactivator that stimulated transcription 80-fold (Fig. 14). This suggests that a negatively acting domain is located between amino acids 99-176. Further truncation ofthe N-terminus to generate ER 1-57 and ER 1-25 completely abolished transactivation. These results demonstrate that the ERl protein contains regions with transcription transactivating activity and that ERl has the potential to function as a transcription factor. An important aspect ofthe present invention is the use of Er-1 as a means of detecting and diagnosing early response polypeptide mutations. The present work suggests that Er-1 plays a regulatory role in FGF interaction with mesodermal cells, and as such, Er-1 may play a critical role in regulating growth and differentiation signal transduction. Therefore, by determining the function and expression of Er-1 in subjects, it is possible to detect abnormal early response hormone function in these patients.

By studying Er-1 mRNA and Er-1 levels in cells from normal individuals and patients with cancer of AIDS, one may determine the effect(s) of Er-1 on these cells. DNA isolated from blood cells or fibroblasts of these patients may also be screened for possible mutations in the Er-1 gene. The present invention has determined the DNA sequence ofthe human and rat Er-1 genes. Exons of Er- 1 have been amplified by PCR.TM. techniques and analyzed by nucleotide sequencing, restriction fragment length polymoφhism (RFLP) and single stranded conformational polymoφhisms.

The inventors have detected Er-1 in Xenopus embryos by immunocytochemical staining, and found that Er-1 expression varies with tissue and stages of development. Thus, levels of Er-1 may be related to developmental or cellular processes.

Although the make-up ofthe natural response elements for Er-1, in the control regions of various genes is undoubtedly more complex than the synthetic er-1 sequences used in this study, the interaction of Er-1 with FGF and EGF well as Er-1 modulation of gene transactivation suggest a mechanism in which a number of nuclear polypeptides of this subfamily, possibly including some yet to be discovered, interact in a composite fashion to yield a net transcriptional activity in the cell nucleus for a given response element. This net transcriptional activity is also dependent upon the presence of polypeptide ligands and the particular structure ofthe response element.

Because numerous modifications and variations in the practice ofthe present invention are expected to occur to those skilled in the art, only such limitations as appear in the appended claims should be placed thereon.

All ofthe compositions and methods disclosed and claimed herein may be made and executed without undue experimentation in light ofthe present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spirit and scope ofthe invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept ofthe invention as defined by the appended claims.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing ofthe present invention, the preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incoφorated by reference. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages ofthe invention will be apparent from the detailed description, and from the claims.

EXAMPLE 7

A Spatial Expression Pattern of One Member ofthe Non-Mammalian Nm-MIER Family

One cell stage embryos were injected with erl RNA or control RNA and animal pole explants were dissected at blastula stage. The explants were cultured in either salt solution alone (not shown) or at subthreshold concentrations of FGF . The results show that erl is able to trigger low levels of mesoderm induction (data not shown) and that at subthreshold concentrations of FGF, much higher levels of mesoderm induction is observed than in embryos injected with control RNA.

One member the nόn-mammalian Nm-MIER family of genes, Early Response gene 1 (erl) (as demonstrated in Figures 1 and 3), which has been cloned in our laboratory (Paterno et al. J. Biol. Chem. 272(41): 25591-25595, 1997) is upregulated within 30 minutes of FGF treatment and levels subsequently decrease to become undetectable (Figure 17). erl is also upregulated in response to FGF and vegetal cells, the source ofthe natural inducer, but not activin (a member ofthe TGFβ family of growth factors) (Figure 16). Here we show the spatial expression pattern of erl (Figure 15) and the expression of ERl in early development (Figures 9 and 11). Figures 17 and 13 show a time course response of FGF in blastula stage animal cap explants, in particular that erl induces animal cap explants to form mesodermal derivatives at subthreshold concentrations of FGF. Thus, the results of this example demonstrate that: 1) erl is an immediate-early response gene to FGF which is upregulated within 30 minutes of FGF treatment; 2) erl is upregulated in response to FGF and vegetal inducer but not to activin; 3) ERl expression is spatial and temporal with localization to the nuclei of presumptive mesodermal and ectodermal cells and maximum expression at mid to late blastula stages; and 4) erl is able to autoinduce mesoderm formation and can trigger mesoderminduction at subthreshold levels of FGF.

EXAMPLE 8

Sequence Listing

The following is a copy of the genbank listing. The accession no. is AFO 15454.

LOCUS AF015454 2339 bp mRNA VRT 07-SEP-1997

DEFINITION Xenopus laevis ERl mRNA, complete eds.

ACCESSION AFO 15454

KEYWORDS

SOURCE African clawed frog.

ORGANISM Xenopus laevis

Eukaryotae; mitochondrial eukaryotes; Metazoa; Chordata;

Vertebrata; Amphibia; Batrachia; Anura; Mesobatrachia; Pipoidea;

Pipidae; Xenopodinae; Xenopus.

REFERENCE 1 (bases 1 to 2339) FEATURES Location/Quali fiers source 1..2339

/organism="Xenopus laevis"

/dev_stage="blastula stage embryo" gene 1..2339

/note="immediate early gene"

/gene="erl"

CDS 233..1714

/gene="erl" /function="potential franscription factor" /note="nuclear protein; some similarity to mtal" /codon_start=l /product="ERl" /translation=

"MAEPSLRTASPGGSAASDDHEFEPSADMLVHEFDDEQTLEEEEMLEGEVNFTSEIEHLE RESEMPIDELLRLYGYGSTVPLPGEEDEEDMDNDCNSGCSGEIKDEAIKDSSGQEDETQS SNDDPTPSFTCRDVREVIRPRRCKYFDTNHEEEEESEDDEDYVPSEDWKKEEMVGSMFQ AEEPVGICKYRETEKVYENDDQLLWNPEYVMEERVIDFLNEASRRTCEERGLDAIPEGSH IKDNEQALYEHVKCNFDTEEALRRLRFN VKAAREELSVWTEEECRNFEQGLKAYGKDF

HLIQANKVRTRSVGECVAFYYMWKKSERYDFFAQQTRFGKKKYNLHPGVTDYMDRL LDESESATSSRAPSPPPTTSNSNTSQSEKEDCTASNNTQNGVSVNGPCAITAYKDEAKQ GVHLNGPTISSSDPSSNETDTNGYNRENVTDDSRFSHTSGKTDTNPDDTNERPIKRQRM DSPGKESTGSSEFSQEVFSHGEV" BASE COUNT 806 a 383 c 513 g 637 1

ORIGEN 1 ttgcatcagc tgcagatcaa ggttaaaata tatatatcag aagaatacac aaataattaa 61 attaaatgtc tcaaacaact ccttccatat gaaggcctct ctgtacctgt gcagcgtttt 121 tcaaaacaga gcaaggaatt catacattac aaatatattt gttgtgtcat aagctacaga 181 gaaagttata gtgaaaccaa caaaacataa atgacccgtc agtacggcaa acatggcgga

241 gccttcactc aggaccgcaa gcccaggtgg ctcggctgca tcagatgacc atgagtttga 301 gccatcagct gacatgcttg ttcatgaatt tgatgatgaa caaacgttgg aagaagagga 361 gatgctggag ggagaagtca acttcacttc agaaatagag caccttgaaa gagaaagtga 421 aatgccaatt gatgaattat tgcgactcta tggttatggc agtacagtgc cactaccagg 481 agaagaagat gaggaggata tggataatga ttgtaacagt ggctgcagtg gagaaataaa

541 ggatgaagct attaaggact cttcaggaca ggaagatgaa acacagtctt caaatgatga 601 tcctactcca tcttttacat gtagagatgt acgagaagta atccgtccac gtcggtgcaa 661 gtattttgat acaaatcatg aaatagaaga ggagtctgag gatgatgagg attatgtacc 721 ttcagaagat tggaaaaagg aaattatggt gggatccatg ttccaggctg aaattccagt 781 tggtatttgc aaatacagag aaacagagaa agtatatgaa aatgatgatc agctcctctg

841 gaatccagaa tatgtaatgg aagaaagagt aatagacttc ttaaatgagg catccagaag 901 gacttgtgaa gagagagggc tagatgctat tcctgaagga tcccacataa aggacaatga 961 gcaggcccta tatgaacatg taaaatgcaa ttttgacaca gaagaggcat tgagaagact 1021 aagatttaat gtcaaagccg ccagagaaga actttccgtt tggactgaag aagaatgtag 1081 aaattttgag caaggtctaa aagcttatgg caaagatttc cacttgattc aggctaacaa

1141 ggtaaggaca aggtctgttg gagaatgtgt ggcattctac tacatgtgga aaaaatcaga

1201 acgttatgac ttctttgccc aacaaacacg atttggaaaa aagaagtata atctacatcc

1261 tggtgtaacg gattacatgg atcgtctttt ggatgaaagc gaaagtgcta cctccagcag 1321 ggcgccatct cccccaccaa ctacctccaa cagcaatact agtcaatccg aaaaggagga

1381 ctgtacagcc agtaacaaca ctcagaatgg agtttctgtg aatggcccat gtgcaataac

1441 tgcatacaaa gatgaagcca aacaaggggt gcatttaaat ggacctacta taagtagcag

1501 tgatccctct tcgaatgaaa ccgacaccaa tgggtataat cgtgaaaatg ttacggacga

1561 ttccagattt agtcatacaa gtggaaaaac tgacacaaat ccagatgata caaacgaaag 1621 gccaataaaa aggcaacgta tggacagccc tgggaaggaa agtacaggat catctgaatt

1681 ctctcaggaa gtgttttcac atggagaggt ttaaacattt tgcagatttg agggaaaaca

1741 cattttgggg ggatgaagaa atttctgggg atcttggaac ttcagggttt attaaattat

1801 ttagcaagtt atttttttgt attatttttc tatttgtccc atgcacattt gagccccaca

1861 gaagagtgaa atatrttgtg tagttgaata gtgaaatttt tgaagccctc tggaaaagta 1921 agcagccttg cagatattca gcctatgcct gaatgcagtt tggctttacg ttatcattcg

1981 ttacatgaag aaggatcttt aaatagaaaa agaattgttc cagaatatgt ctgcagtgtt

2041 gttgcagtgg aaaatattaa ccctgaaagt tgttggtatg atttttttta ggtaggtgtt

2101 aagaataaac caaatgaggt ttgtgtatgt aatttattga catcaatgat gtctttccta

2161 ttcttatctg ggctgaaaaa gatacattct gtatttttcc agatctcttt gtagcctttg 2221 aaagattttt acattatcta tgttttgatc gaactgcctt tcttaacaaa gcttgtataa

2281 ttttcttaac ttgtacagtt gataaacttt tattatgaaa aggaaaaaaa aaaaaaaaa

EXAMPLE 9

Differential Nuclear Localization of ERl Protein During Embryonic Development in Xenopus laevis

The erl gene is a novel fibroblast growth factor (FGF)-regulated immediate-early gene, first isolated from Xenopus blastulae, that encodes a nuclear protein with potent transcription fransactivational activity (Patemo et al, 1997). This example presents the expression pattern ofthe ERl protein during Xenopus embryonic development. ERl protein is present in the early embryo but doesn't begin to appear in the nucleus until mid-blastula stage. The first cells to show nuclear localization of ERl are the presumptive mesodermal cells of the stage 8 blastula. ERl gradually becomes localized to the nucleus ofthe remaining cells, first in the presumptive ectoderm and finally, in the presumptive endoderm such that by late blastula, all nuclei in the animal hemisphere are stained. By early gastrula, nuclear staining is ubiquitous. During subsequent development, ERl protein gradually disappears from the nuclei of various tissues. In tailbud stages, ERl begins to disappear from the nucleus of ectodermally-derived tissues, such as epidermis and brain, while remaining localized in the nucleus of endodermal cells and of mesodermal tissues, such as somites and notochord. In tadpoles, ERl is no longer detectable in the nucleus of any cells, except for a few endodermal cells. Cytoplasmic staining, on the other hand, is observed in some mesodermal tissues, including somites and muscle cells. Neural tissue is largely unstained except for weak cytoplasmic staining in the eye.

It was demonstrated previously that erl is an immediate-early gene whose expression is activated by FGF during mesoderm induction in Xenopus and whose gene product is targeted to the nucleus (Patemo et al, 1997). In this same report, erl was shown to be a maternally-derived message whose expression is restricted to stages prior to mid-gastrula. Western blot analysis of ERl protein expression during these same stages reveals that ERl protein is detectable and that expression levels are similar for all stages examined (Fig. 1). In whole mounts and sections stained with anti-

ERl antibody, the first detectable staining is observed in the nucleus of marginal zone cells (presumptive mesoderm) of stage 8 blastulae (Fig. 2A-D and Fig. 3), even though equivalent levels of ERl protein are present at earlier stages (stage 6.5, Fig. 1; stage 2, not shown). Thus, ERl protein is present in the cells ofthe early stage embryo but does not become concentrated in the nucleus until mid-blastula stage.

As development proceeds, more nuclei become stained and by late blastula (stage 8.5-9), virtually all nuclei in the animal hemisphere are stained (Fig. 2E, F). At this stage, the nuclei in the vegetal hemisphere begin to stain and by early gastrula (stage 10), ubiquitous nuclear staining is observed (Fig. 4).

During tailbud stages, endodermal and mesodermal tissues retain their nuclear staining (Fig. 5B, E, F), however, in ectodermally-derived tissues, such as the brain and epidermis, nuclear staining begins to disappear (Fig. 5C, D). This pattern of decreasing concentration of ERl in the nucleus of various tissues continues throughout late development and by tadpole stage, nuclear staining is only observed in some endodermal nuclei (Fig. 6A, B). At this stage of development, nuclear staining is no longer detected in any ectodermally or mesodermally-derived tissue (Fig. 6B-D), however, cytoplasmic staining is observed in some mesodermal tissues (Fig. 6B-D). Neural tissue is not stained except for weak cytoplasmic staining in the eye (Fig. 6B, C).

Materials and Methods

Xenopus laevis embryos were obtained as described in Ryan and Gillespie (1994) and staged according to Nieuwkoop and Faber (1967). Antibody staining of whole-mount embryos, immunocytochemistry and nuclear staining of sectioned embryos was performed as previously described (Harland, 1991), using our d x-Xenopus ERl antibody (Patemo et al, 1997) and an alkaline phosphatase-coupled goat-anti-rabbit secondary antibody (Life Technologies, Inc.). Nuclear staining was performed by incubating the slides in a 1 : 500 dilution of a live-cell nucleic acid stain (Molecular Probes).

Extracts from embryos at different developmental stages were prepared for Western blotting as described in Ryan and Gillespie (1994). The extracts were vortexed with an equal volume of freon and total protein was precipitated out of the aqueous layer with acetone. The pellet was resuspended in sample buffer and protein measurements were performed using the Bio-Rad assay to ensure equal loading of protein. The blots were stained using the ECL system (Amersham), as described in Ryan and Gillespie (1994).

References

Harland, R.M. 1991. Meth. Cell Biol. vol. 36, pp.683-695, eds. B.K. Kay and HB. Peng, Academic Press, N.Y.

Nieuwkoop, P.D., and Faber, J. 1967. "Normal Table of Xenopus laevis." Amsterdam. Holland Paterno, G.D., et al.,1997. J.Biol.Chem. 272, 25591-25595.

Ryan, P.J., and Gillespie, L.L. 1994. Develop.Biol.166, 101-111.

EXAMPLE 10

Molecular Cloning of Human erl cDNA and its Differential Expression in Breast Tumours and Tumour-Derived Cell Lines

Based on the recently cloned and characterized nm-Nm-MIER gene, called erl, from Xenopus embryos whose expression levels were increased during mesoderm induction by fibroblast growth factor (FGF), we were able to isolate and describe the expression pattern ofthe human erl sequence. Human ERl dXenopus ERl proteins display 91% similarity; the amino acid sequence motifs, including the putative DNA-binding SANT domain, the predicted nuclear localization signals (NLS) and putative SH3 binding domain share 100% identity. er mRNA expression was negligible in all 50 normal human tissues analyzed. Examination of nine breast carcinoma-derived cell lines and eight breast tumour tissue samples by reverse transcription- polymerase chain reaction (RT-PCR) revealed that human erl was consistently expressed in all tumour cell lines and tumour tissue while remaining undetectable in normal breast cell lines and breast tissue. These data suggest that erl expression is associated with the neoplastic state in human breast carcinoma.

cDNA cloning and sequencing of human erl

Using forward (F) and reverse (R) primers 5'-TCCGTTACACCAGGATGTAG-3' (F) and 5'- GGCTGAAATTCCAGTTGGTA-3 ' (R) designed according to the Xenopus erl sequence, a 440 bp product was amplified from a human testis cDNA library (Clontech, Inc.), as described (Paterno et al., 1997). The cDNA was cloned into pCR2.1 (Invifrogen Inc.) (Patemo et al, 1997) and the sequence for both strands of this erl cDNA and all subsequent cDNA inserts was determined as in Gillespie et al. (1995). A 1.6kb cDNA was isolated from the human testis cDNA library using primers 5'-TGATCAGCTCCTGTGGGACC-3 ' (F) and 5 '-CCAAATCGTGTTTGCTGAGC-3 '

(R) designed according to the sequence ofthe 440bp human erl cDNA and the testis library vector sequence 5'-GTTCCAGATTACGCTAGCTTGGG-3' (F) and 5'-

CACAGTTGAAGTGAACTTGCGG-3' (R). The cDNAs were cloned into pCR2.1 and several clones were sequenced on both strands (Gillespie et al., 1995).

Cell lines and tumour samples

The cell lines Hs574, Hs578, Hs787, BT-20, BT-474, Hs578T, MCF-7, Sk-BR-3, MDA-157, MDA-231, MDA-436 and MDA-468 (ATCC) were cultured under conditions described by the ATCC. Breast tumour samples were fixed in formalin and embedded in paraffin using standard histological methods known to those skilled in the art.

Dot blot and PCR analysis

Dot blot analysis was carried out as described in Patemo et al. (1997) with the following modifications: the dot blot and ExpressHyb solution were purchased from Clontech, Inc. and labelled probes were made using either human erl V untranslated region (3'UTR) or ubiquitin cDNA (Clontech, Inc.).

Total RNA for PCR analysis was prepared from the cell lines as described in Yang et al. (1997) and from sections of formalin-fixed, paraffin-embedded breast tumours as in Krafft et al. (1997). cDNA from normal breast tissue was purchased from Invifrogen, Inc. RT and PCR analysis were performed as described in Patemo et al. (1997), with the following modifications: β-actin was used as a confrol; the number of cycles in labelled PCR reactions was 26 for erl and 24 for β-actin and in unlabelled reactions was 28 for both. For analysis of the cell lines, the human erl primers were those listed in section 2.1 and the β-actin primers were as follows: 5'- ATCTGGCACCACACCTTCTACAATGAGCTGCG-3' (F) and 5'-

CGTCATACTCCTGCTTGCTGATCCACATCTGC-3 ' (R). For the breast tumour samples, primer pairs were designed to amplify a sequence <200bp and of similar size for both erl and the confrol, β-actin, in order to control for the small target size generally recovered from formalin-fixed paraffin-embedded tissue (Krafft et al, 1997). The human erl primers used: 5'- CAAGGGCTGAAGGCCTATGG-3'(F) and 5'-CCAAATCGTGTTTGCTGAGC-3'(R) generated a 146bp product while the β-actin primers: 5'- ATCTGGCACCACACCTTCTACAATGAGCTGCG-3' (F) and 5'-

ATGGCTGGGGTGTTGAAGGTCTC-3' (R) generated a 142bp fragment. Densitometric analysis ofthe blot and PCR products was performed using a Canberra-Packard Chemilmager or Cyclone phosphorimager. The individual values obtained for erl were divided by the ubiquitin (blot) or β- actin (PCR) values to obtain the relative level of erl expression.

cDNA cloning of human erl

The cloning and characterization of erl, a novel immediate-early gene from. Xenopus laevis whose expression levels were increased by FGF was described recently (Patemo et al., 1997). Using primers based on the Xenopus sequence, a similar sequence was amplified from a human testis cDNA library. The full-length human cDNA consisted of a single open reading frame (ORF) of 1296bp bracketed by a 68bp 5'UTR containing an in-frame stop codon and a 210bp 3'UTR containing an 18bp poly-A tail (Fig. 22). The ORF in the human erl sequence is predicted to encode a protein of 432 amino acids (aa) (Fig. 22), producing a protein that has 61 fewer aa at the C-terminus than the Xenopus ERl (Fig. 23).

Human ERl displays 91% similarity to Xenopus ERl at the amino acid level, with stretches of 100% identity (Fig. 23), indicating that ERl is highly conserved between lower and higher vertebrates. Contained within the blocks of 100% identity are the protein sequence motifs identified previously in Xenopus ERl, namely the two predicted nuclear localization signals (NLS) and a proline-rich region corresponding to consensus for binding Src-homology 3 (SH3) domains (Fig.

23). Further sequence analysis revealed the presence of a SANT domain that is also 100% conserved between human and Xenopus ERl (Fig. 23) The SANT domain is a recently described motif (Aasland et al., 1996), identified in self-comparisons ofthe co-repressor N-CoR and found in a number of other transcription factors including SW13, ADA2 and TFIIIB. The prior art also reported a similarity between this motif and the DNA binding domain of myb-related proteins, leading them to suggest that the SANT domain is involved in DNA binding.

It was reported previously that Xenopus ERl displays limited similarity (13%) to MTA1 (Toh et al., 1994), the product ofthe rat metastasis-associated gene, a 703 amino acid, 79kDa polypeptide whose expression has been associated with the metastatic phenotype (Toh et al.,

1994; Toh et al, 1995; Toh et al., 1997). Further examination has revealed that the similarity between ERl and MTA1 lies in the SANT domain. Thus, ERl and MTA1 may belong to the same class of transcriptional regulators that share a common DNA binding motif.

Expression of erl in normal human tissues and tumour cells Initially, we examined the expression of human erl by Northem blotting. However, on a blot prepared with total RNA from eight different human tissues, human erl mRNA was barely detectable by phosphorimager analysis (data not shown). The estimated size ofthe single band observed was 1.5kb which is consistent with the size of the cloned human erl cDNA. Dot blot analysis of enriched poly A+ mRNA, a more sensitive detection procedure, confirmed that erl was expressed at very low levels in normal human tissues (Fig. 24B). In all 50 tissues examined (Fig.

24A), erl mRNA was not expressed at significant levels when compared to ubiquitin mRNA (Fig. 24C). Normalization of erl to ubiquitin levels by densitometiy revealed that expression of erl in the testis, intestinal fract (small intestine and colon), spleen, adrenal glands as well as in the adult and fetal thymus was slightly higher (1.5-2.5 times) than in the other tissues.

FGFs play a role in the pathology of a number of human cancers, therefore, it was decided to investigate the expression of erl in human tumour cell lines and tumour tissue. Examination of breast carcinoma cell lines revealed consistent expression of erl in all nine lines examined, while erl was not detectable in the three normal breast cell lines, even when the sensitive PCR method was employed (Fig. 25). On occasion, a faint band was obtained for erl in the Hs574 cell line, but not for the other two normal cell lines, Hs578 and Hs787. Many ofthe available normal breast cell lines, like Hs574, are in fact derived from histologically normal tissue surrounding a breast tumour. Thus, the results with the Hs574 cell line may either reflect a low level of erl expression in normal cells or may be indicative of a mixed population in this cell line.

Examination of erl expression in breast tumour samples by RT-PCR revealed a pattern similar to that observed for the cell lines (Fig. 26). erl mRNA was expressed in all breast tumour samples tested, albeit at variable levels (Fig. 26A, lanes 1-3; 26B, lanes 1-8), while remaining undetectable in normal breast tissue (Fig. 26A, lane 4; 26B, laneN). Expression studies demonsfrate that erl mRNA is not present at significant levels in normal human adult or fetal tissues. This is consistent with the expression pattern observed in Xenopus, where erl mRNA was only detectable by Northern blot during pre-gastrula stages of development and not in later stages (Patemo et al., 1997).

Although the function of erl is yet to be determined, its expression pattern points to a role in early embryonic development and, like many other developmental-regulated genes, overexpression in adult tissues may contribute to the neoplastic phenotype.

This example presents the cloning and expression analysis ofthe human homologue of erl. Comparison of the Xenopus and human ERl proteins reveals a high degree of conservation between lower and higher vertebrates .

Human erl mRNA expression was negligible in all noπnal tissues and breast cell lines examined, but was upregulated in breast carcinoma cell lines and in breast tumours, suggesting that expression of erl is associated with the neoplastic state in human breast carcinomas.

References Aasland, R., Stewart, A.F., Gibson, T., 1996, Trends Biochem. Sci. 21, 87-88.

Friesel, R. and Maciag, T., 1995. FASEB J. 9, 919-925.

Gillespie, L.L., Chen, G. and Patemo, G.D., 1995. J. Biol Chem. 270, 22758-22763.

Krafft, A.E., et al., 1997. Mol. Diag. 2, 217-230.

Patemo, G.D., et al.,. J. Biol. Chem. 272, 25591-25595. Toh, Y., Pencil, S.D. and Nicolson, G.L., 1994. J. Biol. Chem. 269, 22958-22963.

Toh, Y., Pencil, S.D. and Nicolson, G.L., 1995. Gene 159, 97-104.

Toh, Y., et al., 1997, Int. J. Cancer 74, 459-463.

Yang, X., et al., 1997. J. Cell. Biochem. 66, 309-321.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. nm-MIER genes, of which there are at least nine members, and partial sequences thereof, characterized in that said genes are: (a) transcribed in response to fibroblast growth factor(s) (FGF);

(b) are expressed within 40 minutes of FGF treatment; and

(c) do not require protein synthesis for transcription.

2. Substantially pure, isolated or synthetic, DNA molecules encoding one or more of the nm-MIER genes of claim 1.

3. A DNA molecule of claim 2, wherein said DNA is cDNA.

4. A DNA molecule of claim 2, having a nucleotide sequence selected from the group comprising: nm-MEER er-1, nm-MIER S3, nm-MIER S14, nm-MEER S16, nm- MIER SI 7, nm-MIER S24 or nm-MIER S30.

5. A DNA molecule having at least 90% homology to any one ofthe DNA molecules of claim 2 or 3.

6. A DNA molecule selected from the group comprising the nucleotide sequence for nm-MIER er-1; nm-MEER s3; nm-MEER sl4; nm-MEER si 6; nm-MIER si 7; nm- MEER s24; or nm-MIER s30 wherein said sequence is at least 70% homologous to the corresponding Xenopus nm-MIER gene.

7. An isolated polynucleotide comprising a member selected from the group consisting of:

(a) a polynucleotide as set forth for human nm-MIER erl ;

(b) a polynucleotide encoding the cDNA insert for human nm-MEER erl;

(c) a polynucleotide capable of hybridizing to the polynucleotide of (a), or (b); (d) a polynucleotide which is at least 90% identical to the polynucleotide of (a) or (b);

(e) a polynucleotide which is a complement of (a), or (b); and

(g) a polynucleotide fragment ofthe polynucleotide of (a), or (b).

8. The polynucleotide of Claim 7 wherein the polynucleotide is DNA.

9. A vector containing the DNA of Claim 8.

10. A host cell transformed or transfected with the polynucleotide of Claim 7.

11. A process for producing a polypeptide comprising: expressing from the host cell of Claim 10 the polypeptide encoded by said DNA.

12. A process for producing cells capable of expressing a polypeptide comprising transforming or transfecting the cells with the polynucleotide of Claim 7.

13. The polynucleotide of claim 7 wherein the polynucleotide is fused to a heterologous polynucleotide.

14. The substantially pure DNA molecule of claim 2, wherein two or more of said

DNA molecules are arranged in tandem airay in an amplicon.

15. An expression vector containing a DNA molecule of claim 2.

16. An expression vector containing the DNA molecule of claim 3.

17. An expression vector containing the DNA molecule of claim 4.

18. The expression vector of any one of claims 15, 16 or 17, wherein said vector comprises a bacterial phage in recombinant form.

19. The expression vector of any one of claims 15, 16 or 17, wherein said vector comprises a plasmid in recombinant foim.

20. The expression vector of any one of claims 15, 16 or 17 wherein said vector is selected from the group comprising pcDNA₃; pIRES; pCR_{2 1}; pSP₆₄T; or pT₇Ts

21. A host cell comprising the expression vector of claim 15.

22. A substantially pure, isolated or synthetic, RNA molecule, or partial sequences thereof, complementary to the DNA molecule of claim 2, or a portion thereof.

23. A substantially pure, isolated or synthetic, RNA molecule, or partial sequences thereof, complementaiy to the DNA molecule of claim 4, or a portion thereof.

24. A polypeptide encoded by any one ofthe DNA molecules of claim 2.

25. A polypeptide encoded by any one ofthe nucleotide sequences of claim 4.

26. A polypeptide selected from the group comprising nm-MIER ER 1; nm-MIER ER S3; nm-MIERS14; nm-MIER SI 6; nm-MIER SI 7; nm--MIER S24 or nm- MIER S30.

27. An antibody specific for a polypeptide of any one of claims 24, 25 or 26.

28. The antibody according to claim 27 which is polyclonal.

29. The antibody according to claim 27 which is monoclonal.

30. The antibody according to claim 27 which is monospecific.

31. The use of the antibody of claim 27 to identify patients expressing nm-MIER genes.

32. A DNA probe which is complementaiy to at least a portion of the DNA molecule of claim 2.

33. A DNA probe which is complementary to at least a portion ofthe nucleotide sequence of claim 4.

34. An RNA probe which is complementaiy to at least a portion ofthe DNA molecule of claim 22.

35. An RNA probe which is complementaiy to at least a portion ofthe DNA molecule of claim 23.

36. The probe of any one of claims 32, 33, 34 or 35, wherein said probe is labeled with a detectable marker.

37. The probe of claim 36, wherein said marker is radioactive.

38. The probe of claim 36 , wherein s aid marker is fluores cent.

39. The probe of claim 36, wherein said marker is biotinylated.

40. A bioassay for detection of nm-MIER gene expression comprising the steps of: a) contacting a tissue sample with a probe of claim 36, under conditions such that regions of DNA or messenger RNA (mRNA) in said tissue sample and said probe with complementary sequences will base pair so that a RNA:probe or DNA:probe complex is formed; and b) detecting the presence or absence of said RNA:probe or DNA:probe complex.

41. A bioassay for detection of nm-Nm-MIER gene expression comprising the steps of: a) contacting a tissue sample with the antibody of claim 27 under conditions such that said antibody can form a complex with said peptide in said tissue sample; and b) detecting the prese ce or absence of said antibody :peptide complex.

42. A diagnostic kit comprising the probe of claim 36 and reagents to effect formation of and detection of an RNA: DNA or RNA: RNA complex and instructions for use of said kit.

43. An immunodetection kit comprising one or more antibodies of claim 27 and reagents to effect formation of and detection of peptide: antibody complex.

44. A DNA vaccine comprising an expression vector and a nucleic acid sequence encoding one or more nm-MIER genes or fragments thereof, wherein upon administration into a mammal, said mammal is protected from cancer.

45. The DNA vaccine of claim 44, wherein the expression vector is a plasmid.

46. The DNA vaccine of claim 45, additionally comprising transcription regulatory elements operably linked to said nucleic acid sequence.

47. The DNA vaccine of claim 44, wherein said regulatory elements comprise a promoter sequence and a terminator sequence.

48. The DNA vaccine of claim 45, wherein the expression vector is a replication defective adenovirus.

49. The DNA vaccine of claim 44, additionally comprising one or more nucleic acid sequences selected from the group comprising encapsidation signals, packaging signals, tripartite leader sequences, and major late enhancer sequences.

50. The DNA vaccine of claim 44, wherein said nucleic acid sequence is selected from the group comprising cDNA, genomic DNA, or a cDNA/genomic DNA hybrid.

51. The DNA vaccine of claim 44, wherein said nucleic acid sequence further comprises a ATG codon, a Kozak motif, and/or a C-terminal stop codon.

52. A host cell that has been transformed or transfected by the DNA vaccine of claim 50.

53. A composition comprising (i) a DNA vaccine comprising an expression vector and a nucleic acid sequence encoding one or more M-MIER genes or partial sequences thereof, wherein upon administration into a mammal free from cancer, said mammal is protected from developing cancer; and (ii) a pharmaceutically acceptable carrier, buffer, solvent, or diluent.

54. A host cell that has been transformed or transfected by the DNA vaccine of claim 44.

55. A kit for the administration of a DNA vaccine, wherein said DNA vaccine comprises an expression vector and a nucleic acid sequence encoding one or more M-MIER genes or partial sequences thereof, wherein upon administration into a mammal free from cancer, said mammal is protected from developing cancer; and (ii) a pharmaceutically acceptable carrier, buffer, solvent, or diluent, comprising:

(i) the DNA vaccine, either lyophilized or in solution;

(ii) contained in a container, such as a syringe, pipette, eye dropper, vial, nasal spray, or inhaler; and (iii) instructions for use.

56. A method of using a DNA vaccine to protect a mammal against cancer, comprising administering to said mammal an effective amount ofthe DNA vaccine of claim 44, wherein said DNA vaccine comprises an expression vector and a nucleic acid encoding one or more nm-MIER genes or partial sequences thereof, wherein upon administration into a mammal free from cancer, said mammal is protected from developing cancer.

57. The method of claim 56, comprising the additional step of administering single or multipl booster vaccinations to the mammal.

58. An antisense oligonucleotide complementaiy to nm-MIER er-1 , nm-MIER S3, nm-MIER S14, nm-MIER S16, nm-MIER S17, nm-MIER S24 or nm-MIER S30 or partial sequences thereof .

59. The antisense oligonucleotide of claim 58 that is DNA.

60. The antisense oligonucleotide of claim 58 that is RNA.

61. A method of inhibiting the expression of nm-MIER genes comprising contacting tissues o cells which express nm-MIER genes with the antisense oligonucleotide of claim 46.

62. The method of claim 61 wherein said expression of nm-MEER genes is abnormal expression.

63. A method of inhibiting hypeφroliferation of human cells comprising contacting hypeφroliferating human cells with the antisense oligonucleotide of claim 46.