WO2000049048A1

WO2000049048A1 - Ndae1 - a new bicarbonate transporter

Info

Publication number: WO2000049048A1
Application number: PCT/US2000/004028
Authority: WO
Inventors: Michael F. Romero; Alison K. Dillon; Christopher M. Sciortino; Lamara D. Shrode; Caroline R. Sussman
Original assignee: Case Western Reserve University
Priority date: 1999-02-18
Filing date: 2000-02-17
Publication date: 2000-08-24

Abstract

This invention generally relates to novel gene encoding NDAE1 as well as variant amino acid sequences of NDAE1 and novel methods for the screening of compounds that are agonistic or antagonistic to pHi, Na+ or anion channel/transporter activity.

Description

NDAEl - A NEW BICARBONATE TRANSPORTER

This invention was made in part with government support under grants GM-39255 and HL-07415from the NIH and from an NIH supported grant (IRg-91-022-05-IRg) from the Ireland Cancer Center. The government has certain rights in the invention.

FIELD OF THE INVENTION

This invention generally relates to a novel gene designated the Na⁺ driven anion exchanger gene (ndael) and associated peptide and derivatives. Additionally, this inven- tion relates to cells and organisms that are made deficient in expression of this gene or made to express additional copies of this gene. Furthermore, the invention contemplates drug screens for compounds that are agonistic or antagonistic to NDAEl activity. Further still, screens for NDAEl intra- and interspecific homologs as well as NDAEl associated binding molecules are contemplated.

BACKGROUND

Most cellular events are pH sensitive (Busa WB, et al. "Metabolic regulation via intracellular pH" Am J Physiol 246.R409-438, 1984). For example, the rate-limiting enzyme in glycolysis, phosphofructokinase (Trivedi B, and WH Danforth "Effect of pH on the kinetics of frog muscle phosphofructokinase" J Biol Chem 241 41 10-4112, 1966), and a critical ribosomal protein, S6 (Pouyssegur J, et al. "Cytoplasmic pH, a key determinant of growth factor- induced DNA synthesis in quiescent fibroblasts" FEBS Lett 190:115-119, 1985), move from being almost fully active to fully inactive with a pH shift only ~0.1. A sufficiently alkaline pHj (intracellular pH) is required for proliferation in response to several growth factors (Ganz MB, et al. "Effects of mitogens and other agents on rat mesangial cell proliferation, pH, and Ca2+" Am J Physiol 259:F269-278, 1990; Pouyssegur J, et al. "Growth factor activation of an amiloride-sensitive Na+/H+ exchange system in quiescent fibroblasts: coupling to ribosomal protein S6 phosphorylation" Proc Natl Acad Sci U S A 79:3935-3939, 1982; Pouyssegur J, et al. "A specific mutation abolishing Na+/H+ antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH"

Proc Natl Acad Sci U S A 81 :4833-4837, 1984). The list of ion channels with substantial pH sensitivity is ever growing. With so many key processes being pHj sensitive, organisms and cells have evolved acid-base transporters, localized in the plasma membrane, to regulate pH,. Likewise, acid-base transporters are under control of hormones, growth factors or other signals, such as cell volume (see, for example, Akiba T, et al. "Parallel adaptation of the rabbit renal cortical sodium/proton antiporter and sodium/bicarbonate cotransporter in metabolic acidosis and alkalosis" J Clin Invest 80:308-315, 1987; Bierman

AJ, et al. "Bicarbonate determines cytoplasmic pH and suppresses mitogen-induced alkalinization in fibroblastic cells" J Biol Chem 263:15253-15256, 1988; Boron WF. Control of intracellular pH. In: The Kidney: Physiology and Pathophysiology (2nd ed.), edited by Seldin DW, and Giebisch, G. New York: Raven Press, 1992, p. 219-63). The most efficacious of these acid-base transporters carry HCO^~ ₃.

Ion transporters are known to function in the normal physiology of kidneys, nervous tissue, muscle, the cardiovascular system, etc. For example, various transporters control the pH, of myocardium cells. This is of primary importance for the pathological state of ischemia/reperfusion of the myocardium. The ability to control ion transport in myocardial cells could prevent the "runaway-train" scenario and, thus, be a preventative therapy for myocardial infarction and could be routinely used in any procedure requiring stoppage of the heart to prevent damage.

Additionally, regulation of intra- and extra-cellular ion activities (e.g. , H⁺, CI^", Na⁺) is key to normal function of the central nervous system (CNS), digestive tract, respiratory tract, and urinary system. Normal cell function is a balance between inward and outward movement of these ions, often varying in response to intracellular pH. This is especially true in the central nervous system, digestive tract, respiratory tract, and urinary system. The understanding of the physiology of normal physiological function of these organ systems will lead to the development of treatments for diseases, the etiology of which is a malfunc- tion of a regulatory component of the ion transport system. Several ion-transporter protein cDNAs have been cloned in the last few years however, a complete picture of ion transport can not be had with the still limited knowledge of ion transport systems.

As is evident from the above, what is needed are new reagents and methods useful in developing an understanding of ion transport and for the development of new screens for drugs that may be useful in the therapeutic intervention of anion exchange proteins. SUMMARY OF THE INVENTION

The present invention relates to a novel gene sequences (SEQ ID NO: 1 through SEQ ID NO: 7) that encode tissue and species homologs of a Na⁺ driven anion exchanger gene (ndael) as well as the encoded amino acid sequences (SEQ ID NO: 8 through SEQ ID NO: 14). Additionally, the present invention relates to antibodies generated to portions of the amino acid sequences of SEQ ID NO:8 through SEQ ID NO: 14, designated 89035, 89036, 89025, 89033, 89030, 89031 , CWR55, CWR56, CWR57, CWR58.

The present invention generally comprises novel, substantially purified oligonucleotide sequences that encode for the newly discovered gene, ndael. Although the present invention is not limited by any particular mechanism, the expression product of this gene is believed to function as a regulator of intracellular pH (pH,). This gene, and its translation products, is a physiologically unique member of the bicarbonate transport superfamily (BTS) (Romero MF, et al "Expression cloning and characterization of a renal electrogenic Na+/HCO3- cotransporter" Nature 387:409-413, 1997). The exogenous expression of this gene in Xenopus oocytes has been shown to mediate the transport of CI^", Na⁺, H⁺ and

HCO^" ₃, but does not require HCO^" ₃. Transport is blocked by the stilbene DIDS (diisothiocyanatostilbene-2'2-disulfonic acid, an inhibitor of anion transport) and may not be strictly electroneutral. Data suggest this Na⁺ driven anion exchanger (NDAEl) is likely responsible for the Na⁺ dependent Cl-HCO₃ exchange activity characterized in neurons, kidney and fibroblasts. Importantly, since NDAEl does not require HCO^" ₃, its activity could be mistaken for that of Na⁺-H⁺ exchangers (NHEs) when CI^" dependence is not evaluated. In Drospophila disruption of this gene is lethal. This gene and derivative gene products will allow for methods and tools which can be used to regulate the numbers of NDAEl channels on the plasma membrane of cells and, thus, provide novel reagents and methods for the detection of compounds that are agonistic or antagonistic to NDAEl channel function. Additionally, the reagents and methods of the present invention may be used for the diagnoses and treatment of various disease states having an etiology that includes defective channel function or defective pH regulation.

The present invention generally relates to compositions and methods of identifying and testing NDAEl channel pathway agonists and antagonists. The present invention is not limited by the method of the employed screen. In one embodiment, the present invention contemplates screening suspected compounds in a system utilizing transfected cell lines, Xenopus oocytes or microorganisms. In another embodiment, the present invention contemplates using transgeneic organisms or gene "knock out" organisms. In one embodiment, the cells or microorganisms may be transfected transiently. In another embodiment, the cells or microorganisms may be stably transfected. In yet another embodiment, translation products of the invention may be used in a cell-free assay system. For example, pro- viding i) NDAEl, a portion thereof or a NDAEl consensus sequence, and ii) a compound suspected of modulating NDAEl channel activity; a) mixing said NDAEl or portion or a NDAEl consensus sequence with said compound suspected of modulating NDAEl binding activity; b) detecting binding by, for example, Western blot. Any compounds found to bind NDAEl may then be screened in physiological assays to determine any NDAEl chan- nel modulatory effect. In another example, NDAEl cRNA may be translated in vitro in a rabbit reticulocyte in the presence of ³⁵S-methionine. After addition of a test compound, antisera to NDAEl may be used to immunoprecipitate NDAEl . Immune complexes may then be analyzed by SDS-PAGE and fluorography.

Furthermore, in yet another embodiment, antibodies generated to the translation products of the invention may be used in immunoprecipitation assays. In still another embodiment cell based assays incorporating transfected cells (e.g. transiently or stability transfected cells) may be used to screen for NDAEl channel agonists and antagonists. And in still another embodiment, transgenic animals may be generated with the transgene contained in a vector containing an inducible, tissue specific promoter or a restrictive promoter such as a metallothione promoter. Likewise, in another embodiment, "knock out" animals may be made through homologous recombination, thereby producing an organism deficient in gene function. Compounds may be screened in transgeneic or knock out organisms by exposing the organism to the compound or by microinjection techniques. In this regard, the present invention contemplates a Drosophila model system. The present invention also relates to the anti-sense sequence of SEQ ID NO:l through SEQ ID NO: 7, as well as the anti-sense sequence of the transcription product of SEQ ID NO:l through SEQ ID NO: 7. In one embodiment, said sequences are transfected into cells to inhibit the expression of the endogenous ndael gene.

The invention also relates to methods to identify other binding partners of the NDAEl or a NDAEl consensus sequence gene product. The present invention is not limited to the methods employed to identify NDAEl or a NDAEl consensus sequence binding partners. In one embodiment, antibodies generated to translation products of the invention may be used in immunoprecipitation experiments to isolate novel NDAEl binding partners or natural mutations thereof. In another embodiment, the invention may be used to generate fusion proteins (e.g. NDAEl -GST fusion proteins) that could also be used to isolate novel NDAEl binding partners or natural mutations thereof. In yet another embodiment, screens may be conducted using the yeast two-hybrid system using NDAEl or a NDAEl consensus sequence as the bait. In yet another embodiment, screens may be conducted using affinity chromatography using NDAEl or a NDAEl consensus sequence as the ligand.

The invention also relates to the production of derivatives of the ndael gene such as, but not limited to, mutated gene sequences (and portions thereof), transcription products (and portions thereof), expression constructs, transfected cells and transgenic animals generated from the nucleotide sequences (and portions thereof). The present invention also contemplates antibodies (both polyclonal and monoclonal) to the gene product or nucleic acid aptamers, including the products of mutated genes or a NDAEl consensus sequence. Mutated sequences may include non-naturally occurring nucleotides or amino acids. The present invention contemplates using oligonucleotide probes that are at least partly complementary to a portion of the ndael gene sequence or a ndael consensus sequence to detect the presence of the ndael DNA or RNA. Such probes are preferably between approximately 10 and 50 bases and more preferably between approximately 50 and 100 bases. On the other hand, the present invention also contemplates probes that are at least partly complementary to less conserved regions or even unique regions (e.g. a portion of the gene having a sequence unique to the ndael gene).

In addition, the present invention contemplates a diagnostic wherein, for example, a sample of the DNA of the ndael gene sequence or a ndael consensus sequence is determined (e.g. by sequencing) to identify suspected mutations. In such a method, the present invention contemplates isolating the gene from a mixture of DNA. Such isolation can be done using one or more of the probes described above. For example, the present invention contemplates utilizing oligonucleotides that are complementary to the gene as primers in PCR (see U.S. Patent Nos. 4,683,195, 4,683,202 and 4,965,188, all of which are hereby incorporated by reference). Such primers (for example, see Figure 26) can be complemen- tary to internal regions of the gene. More preferably, primers can be designed that will hybridize to each end of the gene so that the entire gene can be amplified and analyzed (e.g. for mutations). The present invention also relates to the identification of new homo logs of NDAEl or mutations thereof. The present invention is not limited to a particular method to identify NDAEl homologs. The present invention contemplates screening for homo logs using a variety of molecular procedures. In one embodiment, screens are conducted using North- era and Southern blotting. In another embodiment, screens are conducted using DNA chip arrays composed of ndael DNA sequences for binding complementary sequences. In yet another embodiment, homologous gene sequences may be isolated from DNA libraries via PCR amplification. The invention contemplates methods for screening for intra- and interspecific homologs of NDAEl, one method comprising (for example): a) providing in any order: i) extracts from cell suspected of containing said homolog, ii) antibodies reactive to

NDAEl or a NDAEl consensus sequence and specific for at least a portion of the peptide of NDAEl or a NDAEl consensus sequence; and b) mixing said antibody with said extract under conditions such that said homolog is detected. The present invention further contemplates a method to screen for homologs of NDAEl comprising: a) extracts from cells suspected of containing said homolog; b) contacting the extract with anti-NDAEl antibody; c) detecting said homolog by techniques known to those practiced in the art, for example Western blotting. Polynucleotides containing the NDAEl gene or a NDAEl consensus sequence may also be fused in frame to a marker sequence which allows for purification of the NDAEl protein, such as the maltose binding protein, which binds to amylose resin, or glutathione, which binds glutathione-S-transferase-coupled resin. Polynucleotides encoding

NDAEl protein, NDAEl peptide fragments or a NDAEl consensus sequence may also be fused in frame to a marker sequence, such as c-myc or eGFP, which encodes an eptitope tag that allows for monitoring the intracellular location of NDAEl using commercially available antibodies. In yet another embodiment, the present invention contemplates the generation functional homologs of the ndael gene sequence and the encoded NDAEl protein via directed molecular evolution (see U.S. Patent No. 5,811,238, incorporated herein by reference). Said homologs generated by directed molecular evolution function to a lesser, a greater or to the same extent as the native gene and protein.

The invention also contemplates novel compositions such as the ndael gene sequence (or portion thereof) or a ndael consensus sequence inserted into a transfection vector. The invention is not limited to a particular transfection vector. Many commercial vectors are available. Additionally, viral vectors and novel vectors may be made and utilized. The present invention also contemplates a composition comprising said transfection vector transfected into primary cells, a cell line, a microorganism (e.g. paramecium) or embryonic cells (e.g. Xenopus oocytes). The invention is not limited to a particular cell line, cell type or to any particular species from which the cells are derived. The present invention is not limited to a particular transfection method. Many transfection methods are envisioned by the present invention including electroporation, lipofectamine methods, CaCl₂ methods (see, generally, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., and Current Protocols in Molecular Biology (1996) John Wiley and Sons, Inc., N.Y., ) and particle bombardment (Boileau, A.J., et al, "Transformation of Paramecium tetraurelia by electroporation or particle bombardment" J Euk Microbiol 46:56-65, 1999), which is incorporated herein by reference), all of which are known in the art. The present invention contemplates the use of cDNA or cRNA for transfections. Additionally, the transfection of the ndael or a ndael consensus sequence protein is also contemplated by the present invention. In embodiments where more than one vector or sequence is transfected, the vec- tors or sequences may be transfected either simultaneously or sequentially. The present invention is not limited by the number of different expression vectors or sequences that may be transfected simultaneously. Another contemplated composition comprises the ndael gene sequence in an appropriate vector used to make a transgenic animal or microorganism. Such ndael gene sequences may be mutated by methods know in the art such that they are loss of function (lof), gain of function (gof) or change of function (cof) mutants. Additionally, they may be combined with other gene sequences (the secondary gene sequence) for the purposes of producing a fusion product. The invention is not limited to any specific secondary gene sequence. The secondary gene sequence may be used to permit, for example, the isolation of the gene, the isolation of transcription product or the isolation of translation product (e.g., with a His tag). Likewise, said secondary sequence may serve as a marker for identifying or visualizing the vector, the translated RNA or the transcribed protein.

Furthermore, the present invention also contemplates using the above-named sequences and derived products in screening assays. The invention is not limited to any par- ticular screening method. In one embodiment, the invention contemplates drug screens for compounds that are agonistic or antagonistic for NDAEl function. In one embodiment cells (e.g. mammalian, Xenopus oocytes or paramecium) are transfected with vectors con- taining a ndael gene, a complementary DNA (cDNA), a complementary RNA (cRNA) or a ndael consensus sequence. In another embodiment cells are made defective in ndael gene expression through homologous recombination (i.e., genetic recombination involving exchange of homologous loci useful in the generation of null alleles (knockouts) in transgenic animals) (See generally, te Riele, H, et al, "Consecutive inactivation of both alleles of the pim-1 protooncogene by homologous recombination in embryonic stem cells" Nature 348:649-651, 1990). In one embodiment, the expression vectors are under the control of tissue specific promoters (e.g. the metallothione promoter). Cells can be exposed to the compound suspected of altering NDAEl function. The culture can then be exposed to metal ions to activate transcription of the ndael gene and inhibition or enhancement of

NDAEl channel activity can measured by techniques known to those practiced in the art. The invention is not limited to any particular measurement technique. Various methods are envisioned. For example, NDAEl channel activity could be measured by the using the conventional two micro electrode voltage-clamp technique or by using micro-pH electrodes. In another embodiment, the transfection and use of paramecium in said screening assay would allow for the large-scale screening of compounds since chemoattractant methods may be used to quantitate the effect of the suspected compound on NDAEl channel activity.

In one embodiment, the present invention contemplates a composition comprising isolated and purified DNA having an oligonucleotide sequence of SEQ ID NO:l through

SEQ ID NO: 7 (or portion thereof, e.g. a ndael consensus sequence). The present invention further contemplates a composition comprising RNA transcribed from such DNA as well as a composition comprising protein translated from transcribed RNA. The protein (or portion thereof) can be used as an antigen and the present invention specifically con- templates an antibody produced from the protein or portion of the protein.

The present invention contemplates that the isolated and purified DNA (i.e. having an oligonucleotide sequence of SEQ ID NO:l through SEQ ID NO: 7) can be used to make transgenic organisms. For example, the present invention contemplates both transgenic animals comprising such DNA sequences as well as transgenic microorganisms (e.g. paramecium) comprising such DNA sequences. Such transgeneic animals and microorganisms will typically be made using such DNA sequences in operable combination with promoters and enhancers in a transfection vector. The present invention also contemplates such vectors and expression constructs comprising such DNA sequences.

A variety of screening methods are contemplated. In one embodiment, the present invention contemplates a method to detect NDAEl channel agonists and antagonists, com- prising: a) providing i) one or more compounds suspected of modulating NDAEl channel activity, ii) a first mammalian, Xenopus oocytes or paramecium cell line comprising the ndael gene; b) contacting a portion of said cells from said transfected cell line with said one or more compounds under conditions such that said compound can enter said cells, so as to create treated portions and untreated portions of cells; and c) comparing the amount NDAEl channel activity in said treated portion of cells as compared to said untreated portion of cells.

In another embodiment, the present invention contemplates a method to detect NDAEl channel agonists and antagonists, comprising: a) providing i) one or more compounds suspected of modulating NDAEl channel activity, ii) a first mammalian, Xenopus oocytes or paramecium cell line comprising the ndael gene; b) contacting a portion of said cells from said transfected cell line with said one or more compounds under conditions such that said compound can enter said cells, so as to create treated portions and untreated portions of cells; and c) comparing the amount NDAEl channel activity in said treated portion of cells as compared to said untreated portion of cells. The present invention contemplates transgenic animals and microorganisms that express increased levels of NDAEl or have the expression of the ndael gene diminished or inhibited (i.e. gene knock-out animals and microorganisms). Such animals and microorganisms can be made by methods known to those practiced in the art.

DESCRIPTION OF THE FIGURES

Figure 1. Panel A shows a multiple sequence alignment of NDAEl (Gen Bank accession number AF047468), human muscle NBC3 (Sodium Bicarbonate Co-transporter) (Gen Bank accession number AF047033), human retinal NBC2 (AB012130), human pancreas NBC (Gen Bank accession number AF011390), and rat cardiac AE3 (Gen Bank ac- cession number A42497). In the multiple sequence alignment, identical amino acids across all 5 sequences are highlighted black and in reverse type. Similar functional groups across all 5 sequences are in grey highlight and black type. Examples of these sequences are ITFGGLL (SEQ ID NO:84) and FLYMGV (SEQ ID NO:85). Similar amino acids are defined by 6 groups: DN, EQ, ST, KR, FYW, and LIVM. NDAEl predicted transmem- brane spans (TMs) (i.e., 12 hydrophobic regions in Figure 2B) are indicated by brackets and a numbered line over the sequence. Panel B shows suspected physiological functions of the peptide sequences in panel A. Figure 2 shows, A; hydropathy plot of NDAEl. Predicted transmembrane regions

(TMs) are numbered 1-12. The bar between TM 5 and 6 indicates the location of the predicted extracellular loop with N-glycosylation sites. B; dendrogram illustrating % divergence of sequences in panel A. The NDAEl protein is 43-47% and 32-33% identical to the NBCs and the AEs, respectively. C; putative membrane model of NDAEl protein. Twelve TMs are predicted from the primary amino acid sequence using a Kyte-Doolittle algorithm (window size, 18 aa) and by comparison of similar NBC and AE areas.

Figure 3 shows NDAEl expression in Drosophila. Ndael localized to region 54A on Drosophila polytene chromosome 2R by in situ hybridization (not shown). A; southern blot illustrating RT-PCR of Drosophila tissues and rkNBC. NDAEl -gene specific primers were used to amplify -750 bp fragments from RT reactions of Drosophila stage and tissue poly(A)" RNA, with NDAEl and rkNBC included as positive and negative PCR controls, respectively. The male and female lanes are thorax without heads. The "control" lane is unrelated DNA and the "water" lane contained no template. Products are obvious in Drosophila embryos and tissues. Southern blotting showed that all of the ethidium bromide stained NDAEl bands hybridized authentic, biotin-labeled NDAEl probes. B-C; in situ hybridization of NDAEl RNA in Drosophila embryos. Drosophila embryos were fixed and probed for NDAEl mRNA using a single-stranded, digoxigenin-labeled antisense NDAEl RNA probe. In panel B (stage 6 embryo), staining was detectable in the cephalic furrow, gut primordium (arrow), all mesoderm, and some ectoderm. In panel C (stage 16-17 em- bryo), there was strong staining in a subset of CNS cells and possibly some peripheral nervous system. Sense RNA probes revealed no staining (not shown).

Figure 4 shows a putative NDAEl transport model-schematic illustrating ion movements through NDAEl . Panels A & B illustrate the non-HCO^"3 modes of transport while panels C & D illustrate our understanding of the HCO^"3-mode transport model. This mod- el does not imply paired binding or that the exact transport pathway via NDAEl is known.

Panel A illustrates the direction of NDAEl transport, indicated as "forward," at steady-state in the normal oocyte Ringer, ND96. Our model indicates that in comparison to controls NDAEl oocytes at steady-state should (i) have a higher pH„ (ii) have a higher αNa,, and (iii) have a lower αCl,. This "forward" transport is observed experimentally with addition of bath HCO^~ ₃ or removal of bath CI^". Panel B shows the direction of the transported ions for bath removal, "reverse" transport, of either Na⁺ or HCO^" ₃. This "reverse" transport should (i) decrease pH„ (ii) decrease αNa^ and (iii) increase αCl,. NDAEl does not require

HCO ₃ to function (see Figure 5 and text). Although the present invention is not limited to any particular mechanism, incorporated into the transport models are current interpretations of experimental data for NDAEl expressed in Xenopus oocytes. Specifically, Li⁺, but not K⁺ or choline⁺, can substitute for Na⁺ in the presence or absence of HCO^'3. Br^" and NO₃ ^" (and presumably halides) can substitute for CI^" in the presence or absence of HCO 3. Additionally, NO₃ ^" also competes with the HCO^"3 site on NDAEl.

Figure 5 shows physiology of NDAEl expressed in Xenopus oocytes. Oocytes were injected with 50 nL of water or cRNA in water. Panel A, C and E are water- injected control oocytes. Panels B, D and F through I are injected with 35 ng / oocyte of NDAEl cRNA. All solutions are pH 7.5, and all HCO^" ₃ solutions are 1.5% CO₂ / 10 mM

HCO^" ₃. Each panel shows the response of an oocyte to CO₂/HCO^" ₃ addition, removal of Na⁺, removal of Na⁺ and Cf, and replacement of Na⁺ in the absence of CI^". Panel A; pH, of water injected (control) oocyte. Both Na⁺ and CI^" are removed ± CO₂/HCO^" ₃. Panel B; pH, of a NDAEl -injected oocyte. Similar experiment to panel A with a NDAEl -expressing oocyte. Starting pH,'s for NDAEl -oocytes are -0.3 pH units higher than controls as expected for a HCO^" ₃ influx transporter, i.e., an acid extruder. Panel C; αCl, of a water injected oocyte. Note that αCl, is minimally altered by bath solution manipulations. Panel D; αCl, of a NDAEl -injected oocyte. Non-CO₂/HCO-₃ solutions are bubbled with 100% O₂, illustrating that NDAEl does not require HCO ₃ to function. Starting αCl,'s are -10 mM less than control oocyte indicating basal CI^" extrusion from the NDAEl -oocytes. Panel E; αNa, of a water injected oocyte. The αNa, is unaltered by any of the bath solution manipulations. Panel F; αNa, of a NDAEl -injected oocyte. The steady-state αNa, is elevated in comparison to the control oocyte. Panels G-I illustrate DIDS inhibition of ion transport via NDAEl. Panel G; DIDS inhibition of NDAEl -mediated pH, changes. The oocyte was exposed twice to CO₂/HCON first without DIDS (not shown) and second with 200 μM DIDS. Exposure to DIDS appears to completely block NDAEl activity, resulting in a response similar to control oocytes. Panel H; DIDS inhibition of NDAEl -mediated αCl, changes, second pulse shown. Panel I; using a double CO₂/HCO^" ₃ protocol as in Panel G; DIDS also blocks the αNa_; changes. The hatched bar at the bottom right corner represents 10 minutes for that experiment.

Figure 6 shows NDAEl stimulated currents in Xenopus oocytes. Panels A and C are water-controls; Panels B, D, and E are oocytes expressing NDAEl . Panels A-D are voltage clamp experiments where voltage was held at -60 mV while the indicated solutions superfuse the oocyte. Panels C-D are I-V responses acquired in the indicated solutions at 20 mV intervals from -160 to +60 mV from the holding potential (-60 mV). Panel E; inhibitor sensitivity of NDAEl associated voltage changes (undamped): 50 μM niflumic acid (NA), 50 μM diphenylamine carboxylic acid (DPC), and 200 μM DIDS. All of the anion inhibitors tested blocked CI^" dependent transmembrane potentials (V_m) changes in NDAEl oocytes. NA and DPC also block NDAEl -currents as well as NDAEl ion fluxes. NA and DPC were dissolved in dimethyl sulfoxide (DMSO) and diluted 1000-fold (0.1% DMSO) for experiments. DMSO alone does not affect the recorded currents (see figure). Since the number of NDAEl transporters in the oocyte membrane is unknown, it is unclear what percentage of NDAEl -mediated transport is accounted for by these currents. Nevertheless, as indicated in the text, the J(ion) for CI^", HCO ₃, and Na⁺ is at least 100-fold greater than the J(current).

Figure 7 shows PCR products inserting restriction enzyme sites 5' and 3' of the last 95 amino acids of NDAEl for insertion onto a GST-fusion protein vector.

Figure 8 shows PCR products inserting restriction enzyme sites 5' and 3' of the first 100 amino acids of NDAEl for insertion into a GST-fusion protein vector.

Figure 9 shows GST-CTERM95 and GST-NTERM100 fusion protein production in DH5α upon the incubation with 0.4 mM IPTG for 2 h. Figure 10 shows a fusion protein isolation from DH5α bacteria transformed with pGEX-4T-l alone or with the pGEX-4T-l/CTERM95. Isolation of fusion protein using glutathione beads and reduced glutathione, with reduced glutathione removed with differential centrifugation.

Figure 11 shows a fusion protein isolation from DH5α bacteria transformed with the pGEX-4T-l/NTERM100. Fusion protein was isolated using glutathione beads and reduced glutathione, with reduced glutathione removed with differential centrifugation.

Figure 12 shows affinity purified 89025 peptide antibody recognizing the GST- CTERM95 fusion protein but not GST alone. Figure 13 shows immunopositive staining of NDAEl in rat kidney cortex. A and

E, 15 μM sections stained with anύ-Drosophila NDAEl antiserum (cND25, i.e., 89025) visualized with Cy3; C, sections incubated with secondary antibodies only and photographed with the same or longer exposure than in A; B, D and F are phase images corresponding to A, C and E, respectively. Scale bar is 50 μM for A-D and 25 μM for E-

F. Glomeruli are indicated by "g".

Figure 14 shows immunopositive staining of NDAEl in rat kidney medulla. A and

E, 15μM sections stained with anύ-Drosophila NDAEl antiserum (cND25, i.e., 89025) visualized with Cy3; C, sections incubated with secondary antibodies only and photographed with the same or longer exposure than in A; B, D and F are phase images corresponding to A, C and E, respectively. Scale bar is 50 μM for A-D and 25 μM for E-

F. Collecting ducts are indicated by "CD".

Figure 15 shows a Western blot against Drosophila whole protein lysate using serum (1 :1500 dilution) from rabbit 89025, which was injected with the C-terminal peptide of NDAEl.

Figure 16 shows a Western blot from a single gel showing competition of antibody for the C-terminal peptide and NDAEl in Drosophila whole cell lysate. Either 10 or 30 μl of lysate were loaded onto the gel, as indicated. The blot was probed with an antibody dilution of 1 :1000 in the absence or presence of 120 μg of C-terminal peptide, as indicated. (Note: differences in indicated molecular weights of NDAEl between the Western blots and gels of the instant application are due to glycosylation differences of the peptides).

Figure 17 shows that Cocalico antibodies (Cocalico Biologicals, Inc Reamstown, PA) recognize GST-CTERM95 fusion protein. Purified GST-CTERM95 fusion proteins were run on a 7.5%o gel, transferred, and then probed with serum from rabbits CWR56 and CWR55 which were immunized with GST-CTERM95 fusion protein. Both antibodies recognize the fusion protein at 35 kDa. In addition, higher molecular weight products were observed.

Figure 18 shows the transfection NDAEl -EGFP into COS-7 cells.

Figure 19 shows the recognition of NDAEl by anti-NDAEl antibody CWR57. Figure 20 shows the recognition of NDAEl by anti-NDAEl antisera 89025.

Figure 21 shows the immunoprecipitation of NDAEl by anti-NDAEl antibodies CWR55, 89025 and 89035. The Immunoprecipitates were recognized by Western blot using anti-NDAEl antibody CWR57 and anti-GFP. Figure 22 shows PCR product using primers that remove the stop codon and insert restriction enzyme sites 5' and 3' of full length Drosophila ndael for insertion into pMH, an HA tag mammalian expression vector.

Figure 23 shows an alignment of nucleotide SEQ ID NOS:l - 7. Figure 24 shows an alignment of amino acid SEQ ID NOS: 8 - 14.

Figure 25 shows the amino acid sequences of SEQ ID NOS: 18 and 19.

Figure 26 shows the nucleotide seqeunces (SEQ ID NOS:20 - 83) of various primers used herein.

Figure 27, panel A and B show NDAEl does not require HCO₃ ^". Panel A is in the absence of CO₂ / HCO₃ ^". NDAEl -mediated transport can use Na⁺ or Li⁺ at the cation site and CI^", Br^" or NO₃ ^" at the "anion site". Interestingly, neither K⁺ nor choline⁺ can be substituted at the "cation site". Panel B is in the presence of CO₂ / HCO₃ ^". NO₃ ^" elicits a decrease in intracellular pH rather than the increase observed with gluconate replacement of CI^". These data indicate that NO₃ ^" competes for both the "anion binding site": the "HCO₃ ^" site" as well as the "CI^" site". Panels C and D show NO₃ ^" is transported by

NDAEl (see Example 13).

DEFINITIONS

To facilitate understanding of the invention, a number of terms are defined below. "Nucleic acid sequence", "nucleotide sequence" and "polynucleotide sequence" as used herein refer to an oligonucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.

As used herein, the terms "oligonucleotides" and "oligomers" refer to a nucleic acid sequence of at least about 10 nucleotides and as many as about 100 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20-25 nucleotides, which can be used as a probe or amplimer.

The term "nucleotide sequence of interest" refers to any nucleotide sequence, the manipulation of which may be deemed desirable for any reason, by one of ordinary skill in the art. Such nucleotide sequences include, but are not limited to, coding sequences of structural genes (e.g., reporter genes, selection marker genes, oncogenes, drug resistance genes, growth factors, etc.), and of non-coding regulatory sequences do not encode an mRNA or protein product (e.g., promoter sequence, enhancer sequence, polyadenylation sequence, termination sequence, etc.).

"Amino acid sequence", "polypeptide sequence", "peptide sequence" and "peptide" are used interchangeably herein to refer to a sequence of amino acids. A "variant" of a first nucleotide sequence is defined as a nucleotide sequence which differs from the referenced, parent or wild type nucleotide sequence e.g., by having one or more deletions, insertions, or substitutions that may be detected using hybridization assays or using DNA sequencing. Included within this definition is the detection of alterations to the genomic sequence of the first nucleotide sequence. For example, hybridization assays may be used to detect alterations in (1) the pattern of restriction enzyme fragments capable of hybridizing to a genomic sequence of the first nucleotide sequence (i.e., RFLP analysis), (2) the inability of a selected portion of the first nucleotide sequence to hybridize to a sample of genomic DNA which contains the first nucleotide sequence (e.g., using allele- specific oligonucleotide probes), (3) improper or unexpected hybridization, such as hybrid- ization to a locus other than the normal chromosomal locus for the first nucleotide sequence (e.g., using fluorescent in situ hybridization (FISH) to metaphase chromosomes spreads, etc.). One example of a variant is a mutated wild type sequence.

The term "portion" when used in reference to a nucleotide sequence refers to fragments of that nucleotide sequence. The fragments may range in size from 5 nucleotide residues to the entire nucleotide sequence minus one nucleic acid residue.

An oligonucleotide sequence which is a "homolog" of a first nucleotide sequence is defined herein as an oligonucleotide sequence which exhibits greater than or equal to 50% identity, and more preferably greater than or equal to 70% identity, to the first nucleotide sequence when sequences having a length of 10 bp or larger are compared. A "derivative" is a modification of a natural nucleotide or amino acid sequence the incorporates synthetic mutations. Synthetic mutations may include non-naturally occurring nucleotides or amino acids. Likewise, synthetic mutations may include mutations of a sequence generated by non-natural methods (e.g. random mutagenesis, directed peptide evolution, chimeric proteins or fusion proteins, etc.) that include either naturally occurring or non-naturally occurring nucleotides or amino acids. Additionally, "derivative" refers to downstream products of a nucleotide or amino acid sequence (e.g. antibodies).

A "chimeric" or "fusion" nucleotide sequence or peptide is a nucleotide sequence or peptide that includes sequences from one or more natural nucleotide sequences or peptides, in an operable condition. The chimeric or fusion nucleotide sequence or peptide may or may not have a increased, decreased or changed function.

DNA molecules are said to have "5' ends" and "3' ends" because mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one mono- nucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotide is referred to as the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is referred to as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of another mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5' and 3' ends. In either a linear or circular DNA molecule, discrete elements are referred to as being "upstream" or 5' of the "downstream" or 3' elements. This terminology reflects that transcription proceeds in a 5' to 3' direction along the DNA strand. The promoter and enhancer elements which direct transcription of a linked gene are generally located 5' or upstream of the coding region. However, enhancer elements can exert their effect even when located 3 ' of the promoter element and the coding region. Transcription termination and polyadenylation signals are located 3' or downstream of the coding region.

The term "recombinant DNA molecule" as used herein refers to a DNA molecule which is comprised of segments of DNA joined together by means of molecular biological techniques.

The term "recombinant protein" or "recombinant polypeptide" as used herein refers to a protein molecule which is expressed using a recombinant DNA molecule.

As used herein, the terms "vector" and "vehicle" are used interchangeably in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another. The term "expression vector" or "expression cassette" as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

The terms "in operable combination", "in operable order" and "operably linked" as used herein refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The terms also refer to the linkage of amino acid sequences in such a manner so that a functional protein is produced.

The term "transfection" as used herein refers to the introduction of foreign DNA into cells. Transfection may be accomplished by a variety of means known to the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, biolistics (i.e., particle bombardment) and the like. As used herein, the terms "complementary" or "complementarity" are used in reference to "polynucleotides" and "oligonucleotides" (which are interchangeable terms that refer to a sequence of nucleotides) related by the base-pairing rules. For example, the sequence "5'-CAGT-3\" is complementary to the sequence "5'-ACTG-3\" Complementarity can be "partial" or "total". "Partial" complementarity is where one or more nucleic acid bases is not matched according to the base pairing rules. "Total" or

"complete" complementarity between nucleic acids is where each and every nucleic acid base is matched with another base under the base pairing rules. The degree of complementarity between nucleic acid strands may have significant effects on the efficiency and strength of hybridization between nucleic acid strands. This may be of particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids.

The terms "homology" and "homologous" as used herein in reference to nucleotide sequences refer to a degree of complementarity with other nucleotide sequences. There may be partial homology or complete homology (i.e., identity). A nucleotide sequence which is partially complementary, i.e., "substantially homologous", to a nucleic acid sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid sequence. The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence to a target sequence under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions re- quire that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.

Low stringency conditions comprise conditions equivalent to binding or hybridization at 68°C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH₂PO₄ ^«H₂O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1 % SDS, 5X Denhardt's reagent (50X Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA (Fraction V; Sigma)) and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 2. OX SSPE, 0.1% SDS at room temperature when a probe of about 100 to about 1000 nucleotides in length is employed.

It is well known in the art that numerous equivalent conditions may be employed to comprise low stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g. , the presence or absence of formamide, dextran sulfate, polyethylene glycol), as well as components of the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed condi- tions. In addition, conditions which promote hybridization under conditions of high stringency (e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.) are well known in the art. High stringency conditions, when used in reference to nucleic acid hybridization, comprise conditions equivalent to binding or hybridization at 68°C in a solution consisting of 5X SSPE, 1 % SDS, 5X Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 0.1X SSPE and 0.1 % SDS at 68°C when a probe of about 100 to about 1000 nucleotides in length is employed.

When used in reference to a double-stranded nucleic acid sequence such as a cDNA or genomic clone, the term "substantially homologous" refers to any probe which can hy- bridize to either or both strands of the double- stranded nucleic acid sequence under conditions of low stringency as described above.

When used in reference to a single-stranded nucleic acid sequence, the term "substantially homologous" refers to any probe which can hybridize (i.e., it is the complement of ) the single- stranded nucleic acid sequence under conditions of low stringency as described above.

As used herein, the term "hybridization" is used in reference to the pairing of complementary nucleic acids using any process by which a strand of nucleic acid joins with a complementary strand through base pairing to form a hybridization complex. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the T_m of the formed hybrid, and the G:C ratio within the nucleic acids. As used herein the term "hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bounds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. A hybridization complex may be formed in solution (e.g., C₀t or Rgt analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized to a solid support (e.g., a nylon membrane or a nitrocellulose filter as employed in Southern and Northern blotting, dot blotting or a glass slide as employed in in situ hybridization, including FISH (fluorescent in situ hybridization)). As used herein, the term "T_m" is used in reference to the "melting temperature." The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. The equation for calculating the T_m of nucleic acids is well known in the art. As indicated by standard references, a simple estimate of the T_m value may be calculated by the equation: T_m = 81.5 + 0.41(% G + C), when a nucleic acid is in aqueous solution at 1 M NaCl (see e.g., Anderson and

Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985)). Other references include more sophisticated computations which take structural as well as sequence characteristics into account for the calculation of T_m.

As used herein the term "stringency" is used in reference to the conditions of tem- perature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. "Stringency" typically occurs in a range from about T_m°C to about 20°C to 25°C below T_m. As will be understood by those of skill in the art, a stringent hybridization can be used to identify or detect identical poly- nucleotide sequences or to identify or detect similar or related polynucleotide sequences. Under "stringent conditions" the nucleotide sequence of SEQ ID NO:l through SEQ ID NO: 7, or portions thereof, will hybridize to its exact complement and closely related sequences. As used herein, the term "amplifiable nucleic acid" is used in reference to nucleic acids which may be amplified by any amplification method. It is contemplated that "amplifiable nucleic acid" will usually comprise "sample template."

The term "heterologous nucleic acid sequence" or "heterologous DNA" are used interchangeably to refer to a nucleotide sequence which is ligated to a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Heterologous DNA is not endogenous to the cell into which it is introduced, but has been obtained from another cell. Generally, although not necessarily, such heterologous DNA encodes RNA and proteins that are not normally produced by the cell into which it is expressed. Examples of heterologous DNA include reporter genes, transcriptional and translational regulatory sequences, selectable marker proteins (e.g., proteins which confer drug resistance), etc.

As used herein, the term "sample template" refers to nucleic acid originating from a sample which is analyzed for the presence of a target sequence of interest. In contrast, "background template" is used in reference to nucleic acid other than sample template which may or may not be present in a sample. Background template is most often inadvertent. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample. "Amplification" is defined as the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction technologies well known in the art (Dieffenbach CW and GS Dveksler (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, NY). As used herein, the term "polymerase chain reaction" ("PCR") refers to the method of K.B. Mullis U.S. Patent Nos. 4,683,195 and 4,683,202, hereby incorporated by reference, which describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. The length of the amplified segment of the desired target sequence is determined by the relative positions of two oligonucleotide primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the "polymerase chain reaction" (hereinafter "PCR"). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be "PCR amplified."

With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of ³²P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.

The terms "reverse transcription polymerase chain reaction" and "RT-PCR" refer to a method for reverse transcription of an RNA sequence to generate a mixture of cDNA sequences, followed by increasing the concentration of a desired segment of the transcribed cDNA sequences in the mixture without cloning or purification. Typically, RNA is reverse transcribed using a single primer (e.g., an oligo-dT primer) prior to PCR amplification of the desired segment of the transcribed DNA using two primers. As used herein, the term "primer" refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and of an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.

As used herein, the term "probe" refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, which is capable of hybridizing to another oligonucleotide of interest. A probe may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene sequences. It is contemplated that any probe used in the present invention will be labeled with any "reporter molecule", so that it is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.

As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes, each of which cut double- or single-stranded DNA at or near a specific nucleotide sequence.

As used herein, the term "an oligonucleotide having a nucleotide sequence encoding a gene" means a nucleic acid sequence comprising the coding region of a gene, i.e. the nucleic acid sequence which encodes a gene product. The coding region may be present in either a cDNA, genomic DNA or RNA form. When present in a DNA form, the oligonucleotide may be single- stranded (i.e., the sense strand) or double-stranded. Suitable control elements such as enhancers, promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and/or correct processing of the primary RNA transcript. Alter- natively, the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers, splice junctions, intervening sequences, polyadenylation signals, etc. or a combination of both endogenous and exogenous control elements. Transcriptional control signals in eukaryotes comprise "enhancer" elements. Enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription (Maniatis, T. et al, (1987) Science 236:1237). Enhancer elements have been isolated from a variety of eukaryotic sources including genes in plant, yeast, insect and mammalian cells and viruses. The selection of a particular enhancer depends on what cell type is to be used to express the protein of interest.

The presence of "splicing signals" on an expression vector often results in higher levels of expression of the recombinant transcript. Splicing signals mediate the removal of introns from the primary RNA transcript and consist of a splice donor and acceptor site (Sambrook, J. et al, (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York, pp. 16.7-16.8). A commonly used splice donor and acceptor site is the splice junction from the 16S RNA of SV40.

Efficient expression of recombinant DNA sequences in eukaryotic cells requires expression of signals directing the efficient termination and polyadenylation of the resulting transcript. Transcription termination signals are generally found downstream of the polyadenylation signal and are a few hundred nucleotides in length. The term "poly A site" or "poly A sequence" as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a poly A tail are unstable and are rapidly degraded. The poly A signal utilized in an expression vector may be "heterologous" or "endogenous." An endogenous poly A signal is one that is found naturally at the 3 ' end of the coding region of a given gene in the genome. A heterologous poly A signal is one which is isolated from one gene and placed 3' of another gene.

The term "promoter", "promoter element" or "promoter sequence" as used herein, refers to a DNA sequence which when placed at the 5' end of (i.e., precedes) an oligonucleotide sequence is capable of controlling the transcription of the oligonucleotide sequence into mRNA. A promoter is typically located 5' (i.e., upstream) of an oligonucleotide sequence whose transcription into mRNA it controls, and provides a site for specific binding by RNA polymerase and for initiation of transcription. The term "promoter activity" when made in reference to a nucleic acid sequence refers to the ability of the nucleic acid sequence to initiate transcription of an oligonucleotide sequence into mRNA.

The term "tissue specific" as it applies to a promoter refers to a promoter that is capable of directing selective expression of an oligonucleotide sequence to a specific type of tissue in the relative absence of expression of the same oligonucleotide in a different type of tissue. Tissue specificity of a promoter may be evaluated by, for example, operably linking a reporter gene to the promoter sequence to generate a reporter construct, introducing the reporter construct into the genome of an animal such that the reporter construct is integrated into every tissue of the resulting transgenic animal, and detecting the expres- sion of the reporter gene (e.g., detecting mRNA, protein, or the activity of a protein encoded by the reporter gene) in different tissues of the transgenic animal. Selectivity need not be absolute. The detection of a greater level of expression of the reporter gene in one or more tissues relative to the level of expression of the reporter gene in other tissues shows that the promoter is specific for the tissues in which greater levels of expression are detected.

The term "cell type specific" as applied to a promoter refers to a promoter which is capable of directing selective expression of an oligonucleotide sequence in a specific type of cell in the relative absence of expression of the same oligonucleotide sequence in a different type of cell within the same tissue. The term "cell type specific" when applied to a promoter also means a promoter capable of promoting selective expression of an oligonucleotide in a region within a single tissue. Again, selectivity need not be absolute. Cell type specificity of a promoter may be assessed using methods well known in the art, e.g., immunohistochemical staining as described herein. Briefly, tissue sections are embedded in paraffin, and paraffin sections are reacted with a primary antibody which is specific for the polypeptide product encoded by the oligonucleotide sequence whose expression is controlled by the promoter. As an alternative to paraffin sectioning, samples may be cryosectioned. For example, sections may be frozen prior to and during sectioning thus avoiding potential interference by residual paraffin. A labeled (e.g., peroxidase conjugated) secondary antibody which is specific for the primary antibody is allowed to bind to the sectioned tissue and specific binding detected (e.g., with avidin/biotin) by microscopy.

The terms "selective expression", "selectively express" and grammatical equivalents thereof refer to a comparison of relative levels of expression in two or more regions of interest. For example, "selective expression" when used in connection with tissues refers to a substantially greater level of expression of a gene of interest in a particular tissue, or to a substantially greater number of cells which express the gene within that tissue, as compared, respectively, to the level of expression of, and the number of cells expressing, the same gene in another tissue (i.e., selectivity need not be absolute). Selective expression does not require, although it may include, expression of a gene of interest in a particular tissue and a total absence of expression of the same gene in another tissue. Similarly, "selective expression" as used herein in reference to cell types refers to a substantially greater level of expression of, or a substantially greater number of cells which express, a gene of interest in a particular cell type, when compared, respectively, to the expression levels of the gene and to the number of cells expressing the gene in another cell type.

The term "contiguous" when used in reference to two or more nucleotide sequences means the nucleotide sequences are ligated in tandem either in the absence of intervening sequences, or in the presence of intervening sequences which do not comprise one or more control elements.

The term "transfection" or "transfected" refers to the introduction of foreign DNA or RNA into a cell. As used herein, the terms "nucleic acid molecule encoding", "nucleotide encoding",

"DNA sequence encoding" and "DNA encoding" refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence. As used herein, the term "antisense" is used in reference to RNA sequences which are complementary to a specific RNA sequence (e.g., mRNA). Antisense RNA may be produced by any method, including synthesis by splicing the gene(s) of interest in a reverse orientation to a viral promoter which permits the synthesis of a coding strand. Once introduced into a cell, this transcribed strand combines with natural mRNA produced by the cell to form duplexes. These duplexes then block either the further transcription of the mRNA or its translation. In this manner, mutant phenotypes may be generated or production of gene products may be reduced or inhibited. The term "antisense strand" is used in reference to a nucleic acid strand that is complementary to the "sense" strand. The designation (-) (i.e., "negative") is sometimes used in reference to the antisense strand, with the desig- nation (+) sometimes used in reference to the sense (i.e., "positive") strand.

The term "Southern blot" refers to the analysis of DNA on agarose or acrylamide gels to fractionate the DNA according to size, followed by transfer and immobilization of the DNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized DNA is then probed with a labeled oligo-deoxyribonucleotide probe or DNA probe to detect DNA species complementary to the probe used. The DNA may be cleaved with restriction enzymes prior to electrophoresis. Following electrophoresis, the DNA may be partially depurinated and denatured prior to or during transfer to the solid support. Southern blots are a standard tool of molecular biologists (J. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, pp 9.31-9.58). The term "Northern blot" as used herein refers to the analysis of RNA by electrophoresis of RNA on agarose gels to fractionate the RNA according to size followed by transfer of the RNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized RNA is then probed with a labeled oligo-deoxyribonucleotide probe or DNA probe to detect RNA species complementary to the probe used. Northern blots are a standard tool of molecular biologists (J. Sambrook, J. et al. (1989) supra, pp 7.39-7.52).

The term "reverse Northern blot" as used herein refers to the analysis of DNA by electrophoresis of DNA on agarose gels to fractionate the DNA on the basis of size followed by transfer of the fractionated DNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized DNA is then probed with a labeled oligo- ribonucleotide probe or RNA probe to detect DNA species complementary to the oligo- ribonucleotide probe used. The term "isolated" when used in relation to a nucleic acid, as in "an isolated oligonucleotide" refers to a nucleic acid sequence that is separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is nucleic acid present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids are nucleic acids such as DNA and RNA which are found in the state they exist in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes; RNA sequences, such as a specific mRNA sequence encoding a specific protein, are found in the cell as a mixture with numerous other mRNAs which encode a multitude of proteins. However, isolated nucleic acid encoding a polypeptide of interest includes, by way of example, such nucleic acid in cells ordinarily expressing the polypeptide of interest where the nucleic acid is in a chromosomal or extrachromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature. The isolated nucleic acid or oligonucleotide may be present in single- stranded or double-stranded form. Isolated nucleic acid can be readily identified (if de- sired) by a variety of techniques (e.g., hybridization, dot blotting, etc.). When an isolated nucleic acid or oligonucleotide is to be utilized to express a protein, the oligonucleotide will contain at a minimum the sense or coding strand (i.e., the oligonucleotide may be single- stranded). Alternatively, it may contain both the sense and anti-sense strands (i.e., the oligonucleotide may be double- stranded). As used herein, the term "purified" or "to purify" refers to the removal of one or more (undesired) components from a sample. For example, where recombinant polypeptides are expressed in bacterial host cells, the polypeptides are purified by the re- moval of host cell proteins thereby increasing the percent of recombinant polypeptides in the sample.

As used herein, the term "substantially purified" refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free and more preferably 90% free from other components with which they are naturally associated. An "isolated polynucleotide" is, therefore, a substantially purified polynucleotide.

As used herein the term "coding region" when used in reference to a structural gene refers to the nucleotide sequences which encode the amino acids found in the nascent poly- peptide as a result of translation of a mRNA molecule. The coding region is bounded, in eukaryotes, on the 5 ' side by the nucleotide triplet "ATG" which encodes the initiator me- thionine and on the 3' side by one of the three triplets which specify stop codons (i.e., TAA, TAG, TGA).

As used herein, the term "structural gene" or "structural nucleotide sequence" refers to a DNA sequence coding for RNA or a protein which does not control the expression of other genes. In contrast, a "regulatory gene" or "regulatory sequence" is a structural gene which encodes products (e.g., transcription factors) which control the expression of other genes.

As used herein, the term "regulatory element" refers to a genetic element which controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements include splicing signals, polyadenylation signals, termination signals, etc.

As used herein, the term "peptide transcription factor binding site" or "transcription factor binding site" refers to a nucleotide sequence which binds protein transcription factors and, thereby, controls some aspect of the expression of nucleic acid sequences. For example, Sp-1 and API (activator protein 1) binding sites are examples of peptide transcription factor binding sites.

As used herein, the term "gene" means the deoxyribonucleotide sequences compris- ing the coding region of a structural gene. A "gene" may also include non-translated sequences located adjacent to the coding region on both the 5' and 3' ends such that the gene corresponds to the length of the full-length mRNA. The sequences which are located 5' of the coding region and which are present on the mRNA are referred to as 5' non- translated sequences. The sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene which are transcribed into heterogenous nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5 ' and 3 ' end of the sequences which are present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5 ' or 3 ' to the non-translated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers which control or influence the transcription of the gene. The 3' flanking region may contain sequences which direct the termination of transcription, post- transcriptional cleavage and polyadenylation.

A "non-human animal" refers to any animal which is not a human and includes vertebrates such as rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc. Preferred non-human animals are selected from the order Rodentia. "Non-human animal" additionally refers to amphibians (e.g. Xenopus), reptiles, insects (e.g. Drosophila) and other non-mammalian animal species. A "transgenic animal" as used herein refers to an animal that includes a transgene which is inserted into a cell and which becomes integrated into the genome either of somatic and/or germ line cells of the. A "transgene" means a DNA sequence which is partly or entirely heterologous (i.e., not present in nature) to the animal in which it is found, or which is homologous to an endogenous sequence (i.e., a sequence that is found in the animal in nature) and is inserted into the animal's genome at a location which differs from that of the naturally occurring sequence. Transgenic animals which include one or more transgenes are within the scope of this invention. Additionally, a "transgenic animal" as used herein refers to an animal that has had one or more genes "knocked out" (made non- functional or made to function a reduced amount) by the process of homologous recombination, or by similar processes.

The term "compound" refers to any chemical entity, pharmaceutical, drug, and the like that can be used to treat or prevent a disease, illness, sickness, or disorder of bodily function. Compounds comprise both known and potential therapeutic compounds. A compound can be determined to be therapeutic by testing using the testing methods of the present invention. A "known therapeutic compound" refers to a therapeutic compound that has been shown (e.g., through animal trials or prior experience with administration to humans) to be effective in such treatment or prevention. A compound is said to be "in a form suitable for administration such that the compound is bio-available in the blood of the animal" when the compound may be administered to an animal by any desired route (e.g. , oral, intravenous, subcutaneous, intrathecal, intraperitoneal, intramuscular, etc.) and the compound or its active metabolites appears in the blood of the animal in an active form. As used herein "agonist" refers to molecules or compounds which mimic or augment the action of a "native" or "natural" compound. Agonists may be homologous to these natural compounds in respect to conformation, charge or other characteristics

As used herein "antagonist" refers to molecules or compounds which inhibit the action of a "native" or "natural" compound. Antagonists may or may not be homologous to these natural compounds in respect to conformation, charge or other characteristics.

"Patient" shall be defined as a human or other animal, such as a guinea pig or mouse and the like, that may be in need of alleviation or amelioration from a recognized medical condition.

"Proliferation" refers to the ability of cells to divide into two cells repeatably there- by resulting in a total increase of cells in the population. Said population may be in an organism or in a culture apparatus.

A "cell-targeting mechanism" refers to a process, procedure or reagent that allows a compound or reagent (e.g. an antisense mRNA strand) to locate to a cell or cells in an organism. The targeting need not be absolute. The targeting need not be specific for a particular type of cell. A example of a targeting mechanism is a peptide (that is bound to, for example, an antisense mRNA strand) that may be recognized by a cell surface receptor so that, if in proximity to the cell surface receptor, the peptide will be bound by the cell surface receptor, and, thereby, be localized or targeted to the cell displaying the cell surface receptor, thereby also targeting the bound antisense mRNA to the cell.

A "transformed cell" is a cell or cell line that has acquired the ability to grow in cell culture for many multiple generations, the ability to grow in soft agar and the ability to not have cell growth inhibited by cell-to-cell contact. In this regard, transformation refers to the introduction of foreign genetic material into a cell or organism. Transformation may be accomplished by any method known which permits the successful introduction of nucleic acids into cells and which results in the expression of the introduced nucleic acid. "Transformation" includes but is not limited to such methods as transfection, microinjection, electroporation, and lipofection (liposome-mediated gene transfer). Transformation may be accomplished through use of any expression vector. For example, the use of baculovirus to introduce foreign nucleic acid into insect cells is contemplated. The term "transformation" also includes methods such as P-element mediated germline transformation of whole insects. Additionally, transformation refers to cells that have been trans- formed naturally, usually through genetic mutation.

As used herein "exogenous" means that the gene encoding the protein is not normally expressed in the cell. Additionally, "exogenous" refers to a gene transfected into a cell to augment the normal (i.e. natural) level of expression of that gene.

"Gain of function" (gof) shall be defined as all modifications to an oligonucleotide that, when that oligonucleotide is transfected into a host organism and translated into a peptide, that peptide will function with increased efficiency as compared to the wild type peptide when the gene or gene product is induced to function whether that induction be continuous or non-continuous. It may, in effect, function as an augmenter of the natural gene if the natural gene is present and functional in the host into which the gof oligonucle- otide was transfected, or it may add that function to the host if the natural gene is not present or is non-functional.

"Loss of function" (lof) shall be defined as all modifications to an oligonucleotide that, when that oligonucleotide is transfected into a host organism and translated into a peptide, that peptide will function with decreased efficiency as compared to the wild type peptide when the gene or gene product is induced to function whether that induction be continuous or non-continuous. It may, in effect, function as a diminisher of natural gene function if the natural gene is present and functional in the host into which the lof oligonu- cleotide was transfected, or may negatively interfere with processes in the host if the natural gene is not present or is non-functional.

"Change of function" (cof) shall be defined as all modifications to an oligonucleotide that, when that oligonucleotide is transfected into a host organism and translated into a peptide, that peptide will function in a different manner as compared to the wild type peptide when the gene or gene product is induced to function whether that induction be continuous or non-continuous. For example, a mutation that would cause NDAEl to transport different ions that it would normally would be a change of function mutation.

"Consensus sequence" shall be defined as a sequence of amino acids or nucleotides that contain identical amino acids or nucleotides or functionally equivalent amino acids or nucleotides for at least 25 % of the sequence. The identical or functionally equivalent amino acids or nucleotides need not be contiguous.

The terms "channel" and "transporter" shall be interchangeable when referring to NDAEl nucleotide or peptide.

GENERAL DESCRIPTION OF THE INVENTION

The present invention relates to gene sequences for a novel, physiologically unique cation-coupled Cl-HCO₃ exchanger. The present invention also relates to derivatives of the gene sequence, e.g. transcribed RNA, translated protein, antibodies generated from the translated protein, expression vectors and transgenic animals incorporating the gene sequence and assays to identify compounds or agents that are antagonistic or agonistic to NDAEl function as well as assays to identify homologs and binding partners of NDAEl. Those practiced in the art will recognize that these derivatives and assays may also utilize and relate to portions of the gene sequence. For example, a portion of the gene may be translated to produce a truncated protein. In another example, the truncated protein may be used to generate antibodies to the specific epitope encoded in the protein without interference from other epitopes from the complete protein.

The NDAEl gene encodes a channel protein located in the cell membrane of various cell types. Although the present invention is not limited to any particular mechanism, channel proteins, like NDAEl, are critical in the regulation of intracellular ion concentrations. Aberrant behavior in ion channel or transporter proteins may lead to any number of neurological or muscular diseases. Additionally, the regulation of ion concentrations is critical to ensure the proper functioning of the excretory system. Kidneys are necessary for ionic and fluid homeostasis.

The present invention is not limited to any particular mechanism. However, current research supports the idea that cells regulate their pH, using a classic pump-leak mechanism (Boron WF. Control of intracellular pH. In: The Kidney: Physiology and Pathophysiology

(2nd ed.), edited by Seldin DW, and Giebisch, G. New York: Raven Press, 1992, p. 219- 63.). The "pumps" are the acid extruders, active transporters that tend to increase pH,. The "leaks" are acid loaders, passive mechanisms that tend to decrease pH,. Acid extruders include the vacuolar-type H⁺ pump, which is a primary active transporter, as well as the Na-H exchanger and the Na⁺ driven Cl-HCO₃ exchanger (NDAEl), both of which are secondary active transporters, i.e., they use the Na" gradient to move acid equivalents. In kidney, the first two are present at the apical membrane of the proximal tubule, while the latter is basolateral. Acid loaders include the Cl-HCO₃ exchanger (AE1-3) and the 1 :3 electrogenic Na/HCO₃ cotransporter (NBC). NBC is expressed at the proximal-tubule basolateral membrane. Pathways for the passive leak of H⁺ or HCO^" ₃ are also acid loaders.

This includes the GABA-activated CI^" channel (Kaila K, and J Voipio, "Postsynaptic fall in intracellular pH induced by GABA-activated bicarbonate conductance" Nature 330:163-5, 1987), a key player in the CNS, permitting passive HCO^" ₃ efflux, and thus a substantial intracellular acid load. The acid extruders ( pH,) are the Na-H exchanger and the Na⁺-driven Cl-HCO₃ exchanger. Typical of most cells, in physiologic solutions (/. e. , bicarbonate containing) this Na⁺-driven Cl-HCO₃ exchanger mediates about twice the acid flux as the Na-H exchanger (H⁺ out and HCO ₃ in, which is equivalent to 2H⁺ out). These two acid extruders are balanced by a single major acid loader (^pH,), the Cl-HCO₃ exchanger. Thus, the cell invests substantial energy to allow these acid loaders and extruders continuous opposition to each other. Energetically, the Na-K pump ultimately generates the ion gradients that these three transporters dissipate.

The cell expends this seemingly redundant energy because it must stringently control pH,. The key to this pH,-regulatory network is the pH, dependence of the transporters themselves (analogous to voltage dependence for ion channels or phosphorylation status for proteins in signaling cascades): A decrease in pH, simultaneously stimulates the acid extruders and inhibits the acid loaders (Boyarsky G, et al. "pH regulation in single glomer- ular mesangial cells. II. Na+-dependent and -independent Cl(-)-HCO3- exchangers" Am J Physiol 255:C857-869, 1988; Mason MJ, et al. 'Internal pH-sensitive site couples Cl-(-)HCO3- exchange to Na+-H+ antiport in lymphocytes' Am J Physiol 256:C428-433, 1989; Olsnes S, et al. "pH-regulated anion antiport in nucleated mammalian cells" J Cell Biol 102:967- 971, 1986). The result is a rapid return of pH_j toward the initial value. An increase of pH; has the opposite effects on these transporters, i.e., a rapid response causing pHj to decrease toward its initial value.

In acid-transporting epithelial cells, transepithelial acid secretion can be an important by-product of pH, regulation. Thus, in the proximal tubule, the electrogenic Na HCO₃ cotransporter at the basolateral membrane (BLM) is an acid loader that lowers pH_;, stimulating both Na-H exchange and H⁺ pumping at the apical membrane while also stimulating Na⁺ driven Cl-HCO₃ exchange at the basolateral membrane (in cells where both are present). The net effect is efficient pH_j regulation. However, because of the strategic segregation of the acid loaders and extruders on opposite sides of the cell, the proximal tubule also secretes luminal acid and absorbs both NaHCO₃ and NaCl across the BLM. In this regard, it is important to note that inefficient regulation of pH_;, Na⁺ or anion transport can have an adverse impact on cell physiology leading to changes in tissue, organ and organism homeo- stasis, thus leading to disease states.

As many as six HCO^" ₃ transporters have been functionally identified, three acid loaders and three acid extruders. It is believed that the reason for so many is revelaed by the fact the K⁺, Ca⁺², HCO^" ₃, and H⁺ are all vital to cell survival. Thus, these molecules, even when present at high concentrations, need to be strictly regulated to control or maintain ion gradients, or to regulate signaling cascades. Each of the three HCO^~ ₃ -dependent acid extruders, and each of the three acid loaders, functions just a little differently than the other two (Figure IB). They dissipate different ion gradients, put different osmotic loads on the cell, have different effects on V_m (transmembrane potentials), and may also be regulated differently. Variable cytoplasmic domains of the transporter proteins may allow unique cytoskeletal interactions (Ding Y, et al. "The major kidney AE1 isoform does not bind ankyrin (Ankl) in vitro. An essential role for the 79 NH2-terminal amino acid resi- dues of band 3" J Biol Chem 269:32201-32208, 1994), as well as distinct modulation or binding by enzymes / proteins. Nevertheless, when thinking of pH and acid-base transporters, most people focus on H⁺ transporters, particularly Na-H exchangers. Certainly these H⁺ transporters are very important. However, HCO^" ₃ transporters often carry more acid-base equivalents and are more active in a CO₂/HCO^~ ₃ environment. Why, then, do HCO^~ ₃ transporters receive less attention? There are two likely reasons: (i) it is usually difficult to cleanly discern HCO^"" ₃ transporters from one another, and, (ii) HCO^" ₃-containing solutions are demanding to make correctly, tending to quickly lose CO₂ unless special precautions are taken.

Soon after pH-sensitive dyes were first used in mammalian cells, reports of Na-H exchangers abounded. Focus turned away from the Na⁺-driven Cl-HCO₃ exchanger (previ- ously discovered in invertebrate nerve and muscle cells). The Na⁺ driven Cl-HCO₃ exchanger was being referred to as the "invertebrate " pH, regulator. The Na-H exchanger was the "vertebrate " pH, regulator. It was held that vertebrates must have different ways of regulating pH, and ion transport than invertebrates. Surprisingly at the time, nobody had looked for the Na^"-driven Cl-HCO₃ exchanger in a mammalian cell. In 1985, Pouyssegur found substantial Na⁺-driven Cl-HCO₃ exchange activity in fibroblasts (L'Allemain G, et al "Role of a Na+-dependent C1-/HCO3- exchange in regulation of intracellular pH in fibroblasts" J Biol Chem 260: 4877-4883, 1985). Presently, it is appreciated that HCO^~ ₃-dependent acid extruders exist in a wide range of cells and are virtually unrivaled in their ability to transport acid-base equivalents. Despite this rich physiological documentation of HCO ₃ transporters, molecular information has been limited to the Cl-HCO₃ exchangers (AE1-AE3). In fact, as noted, prior to NBC's cloning (Romero MF, et al. "Expression cloning and characterization of a renal electrogenic Na+/HCO3- cotransporter" Nature 387:409-413, 1997), there was no molecular data on any of the five cation-linked HCO^" ₃ transporters. Thus, the molecular information provided by the amphib- ian NBC sequence and modern molecular biology tools have begun an explosive revisiting of HCO^~ ₃ transporter identification, localization, and physiology. Therefore, our discovery of NDAEl will provide novel reagents suited for the elucidation of the mechanisms of pH„ Na⁺ and anion regulation as well as screening for compounds that may modulate NDAEl function. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Generally, the nomenclature used hereafter and the laboratory procedures in cell culture, molecular genetics, and nucleic acid chemistry and hybridization described below are those well known and commonly employed in the art. Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, and microbial culture and transformation (e.g., electroporation, lipofection). Generally enzymatic reactions and purification steps are performed according to the manufacturer's specifications. The techniques and procedures are generally performed according to conventional methods in the art and various general references (see, generally, Sambrook et al. Molecular Cloning: A Laborato- ry Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., and Current Protocols in Molecular Biology (1996) John Wiley and Sons, Inc., N.Y., which are incorporated herein by reference) which are provided throughout this document.

Oligonucleotides can be synthesized on an Applied BioSystems oligonucleotide synthesizer [for details see Sinha et al, Nucleic Acids Res. 12:4539 (1984)], according to specifications provided by the manufacturer. Complementary oligonucleotides are annealed by heating them to 90°C in a solution of 10 mM Tris-HCl buffer (pH 8.0) containing NaCl (200 mM) and then allowing them to cool slowly to room temperature. For binding and turnover assays, duplex DNA is purified from native polyacrylamide (15%> w/v) gels. The band corresponding to double-stranded DNA is excised and soaked overnight in 0.30 M sodium acetate buffer (pH 5.0) containing EDTA (1 mM). After soaking, the supernatant is extracted with phenol/chloroform (1/1 v/v) and precipitated with ethanol. DNA substrates are radiolabeled on their 5' -OH group by treatment with [g-³²P]ATP and T4 polynucleotide kinase. Salts and unincorporated nucleotides are removed by chromatography on Sephadex G columns. Assays for detecting the ability of agents to inhibit or enhance NDAEl channel activity provide for facile high-throughput screening of agent banks (e.g., compound libraries, peptide libraries, and the like) to identify antagonists or agonists. Such NDAEl antagonists and agonists may be further developed as potential therapeutics and diagnostic or prognostic tools for diverse types of neurological and muscular diseases, as well as cardiac arrhythmias, hypertension, hypotension, angina, asthma, diabetes, renal insufficiency, urinary incontinence, acidosis, alkalosis, irritable colon, epilepsy, cerebrovascular ischemia and autoimmune disease. Likewise, the ndael gene, and modifications thereof, may be useful in gene therapy. For example, the incorporation of the ndael gene sequence into cells in context of tissue specific or inducible promoters might be useful in the treatment of hereditary diseases.

1. Screens to identify Agonists of Antagonists of NDAEl Channel / Transporter

Activity

There are several different approaches contemplated by the present invention to look for small molecules that specifically inhibit or enhance NDAEl channel activity. One approach is to transfect expression constructs (vectors) comprising the ndael gene into cells and measure changes in the rate of pH, flux as compared to controls after the cells have been exposed to the compound suspected of modulating NDAEl activity. Cells may be transiently transfected or stably transfected with the construct under control of an inducible or temperature sensitive promoter. Another embodiment is to transfect cRNA for the NDAEl protein. Other embodiments would include translation of the invention and purifi- cation of the peptide. The purified peptide could then be used as a substrate in a cell-free assay, e.g., in screens for compounds that bind to NDAEl. Furthermore, transgenic animals and stably transfected cell lines could be produced allowing for in vivo assays to be conducted.

A. In vitro Assays i. Transfection Assays

Transfection assays allow for a great deal of flexibility in assay development. The wide range of commercially available transfection vectors will permit the expression of the invention in a extensive number of cell types. In one embodiment, cells are transiently transfected with an expression construct comprising, in operable combination, the ndael gene and an inducible promoter allowing for the initiation of translation and transcription when needed. Cells are exposed to the agent suspected of modulating NDAEl activity, NDAEl expression is initiated and changes pH, are measured. Rates of pH, change in cells treated with said compound are compared to rates in cells that are untreated. Rates of pH„ Na" and anion change are quantitated by any of a number of ways reported in the literature and known to those practiced in the art. In another embodiment, stably transfected cells lines are employed. The use of an inducible promoter or temperature sensitive promoter can be utilized in these systems. Screening assays for compounds suspected of modulating NDAEl channel activity are conducted in the same manner as with the transient transfection assays. Using stably transfected cell lines, however, allows for greater consistency between experiments and allows for inter-experimental comparisons.

In order to test the stimulatory or inhibitory effect of a compound, particularly a pharmacological agent, it is desirable to have a model system comprising a population of cells that have increased numbers of NDAEl channels on their cellular plasma membrane. Such a model system is especially suitable for measuring small changes in pH,. Such model systems are prepared by transfecting into host cells cDNA or RNA molecules operably encoding ND AE 1. Such model systems are especially useful for monitoring the effect of a compound on pH,. After transfection the cells are cultured for a time and under conditions which permit transformation of the host cells, i.e., expression of the transfected operably encoded ndael cDNA or RNA.

The compound (which, depending on the compound, may be dissolved in a suitable carrier) is added to the culture medium of a test population of transformed host cells. Preferably, a plurality of concentrations of the compound are added to a corresponding plurality of test populations. The compound is also added to the culture medium of a con- trol population of cells that have not been transformed, i.e., cRNA or cDNA molecules encoding ndael are not transfected into the cell, or an empty expression vector (i.e., an expression vector without ndael encoding cDNA or cRNA) is transfected into the cell. Thereafter, pH, is measured using conventional techniques, such as, for example, using a microprobe for pH. A difference in intracellular pH in the control population verses the test population is indicative of a stimulatory or inhibitory effect of the compound on the

NDAEl channels formed by transfection of the ndael encoding oligonucleotide into the cells. Such measurements are also used to determine the effective compound dosage. ii. Cell-free Assays

NDAEl protein may be utilized in cell-free assays. For example, a compound suspected of binding NDAEl could be added to a reaction. Modulation of binding activity could be measured by changes in pH,. Such assays would allow for the identification com- pounds that may modulate NDAEl activity in vivo and be adaptable for high-through put screening assays.

B. In Vivo Assays i. Transgenic Animal Assays In one embodiment, transgenic animals are constructed using standard protocols, including homologous recombination (i.e., genetic recombination involving exchange of homologous loci useful in the generation of null alleles (knockouts) in transgenic animals) (See generally, te Riele, H, et al, "Consecutive inactivation of both alleles of the pim-1 protooncogene by homologous recombination in embryonic stem cells" Nature 348:649- 651, 1990). The ndael gene may be placed under the control of a tissue specific promoter or inducible promoter. The generation of transgenic animals will allow for the creation of model systems to investigate the numerous diseases associated with aberrant NDAEl channel activity which may provide the means for determining the physiology of the disease or its treatment. ii. Paramecium Based Assays

In yet another embodiment, paramecium are transfected with the ndael gene by methods known to those in the art (e.g. electroporation or particle bombardment; Boileau, A.J., et al, "Transformation of Paramecium tetraurelia by electroporation or particle bombardment" J Euk Microbiol 46:56-65, 1999), which is incorporated herein by reference). Said transfected paramecium are then exposed to compounds suspected of modulating

NDAEl channel activity. Rates of pH₍ flux in the paramecium may be measured by che- mosensory assays known in the art (Fraga, D., et al, "Introducing antisense oligodeoxynucleotides into Paramecium via electroporation" J Euk Microbiol 45:582-588, 1998) and compared to rates of pH, flux in untreated paramecium. iii. Xenopus Oocyte Based Assays

Tn still yet another embodiment, Xenopus oocytes are transfected by methods known to those in the art (see Experimental section). Said transfected oocytes are then exposed to compounds suspected of modulating NDAEl channel activity. Rates of pH₍ change in treated, transfected oocytes are measured by assays known in the art (e.g. intracellular pH probe) and compared to the pH, of untreated, transfected oocytes. 2. Screens to Identify NDAEl Binding Partners

A. In vitro Assays

There are several different approaches to identifying NDAEl interactive molecules or binding partners. Techniques that may be used are, but not limited to, immunoprecipita- tion of NDAEl with antibodies generated to the translation product of the invention. This would also bring down any associated bound proteins, i.e. proteins in the cell with affinity for the NDAEl polypeptide. Another method is to generate fusion proteins comprising NDAEl connected to a generally recognized pull-down protein such as glutathione S-trans- ferase (GST). Bound proteins can then be eluted and analyzed. Yet another method is to bind NDAEl to a solid support and expose the bound NDAEl to cell extracts suspected of containing an NDAEl interactive molecule or binding partner. i.

Immunoprecipitation

After the generation of antibodies to NDAEl, cells expressing transfected NDAEl are lysed and then incubated with one of the antibodies. Antibodies interact with the bound NDAEl and any associated proteins can then be pulled down with protein- A

Sepharose or protein-G Sepharose beads, using standard techniques. Where yeast binding partners are sought, yeast cells are lysed. ii. Fusion Protein Pull-down

A method similar to immunoprecipitation is to construct fusion proteins of the mu- tant and wild type NDAEl and glutathione S-transferase (GST). The GST-NDAE1 fusion proteins are then incubated with cell extracts and then removed with glutathione Sepharose beads. Any bound, NDAEl -associated proteins are then characterized.

B. In Vivo Assays i. Yeast Two-hybrid System The yeast two-hybrid system identifies the interaction between two proteins by reconstructing active transcription factor dimers (Chien, C.T., et al. "The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest" Proc Natl Acad Sci, USA 88:9578-9582, 1991). The dimers are formed between two fusion proteins, one of which contains a DNA-binding domain (DB) fused to the first pro- tein of interest (DB-X, where X will be NDAEl) and the other, an activation domain (AD) fused to the second protein of interest (AD-Y, where Y will be a candidate NDAEl -bind- ing protein encoded by cDNA from a commercially available library). The DB-X:AD-Y interaction reconstitutes a functional transcription factor that activates chromosomally-inte- grated reporter genes driven by promoters containing the relevant DB binding sites. Large cDNA libraries can be easily screened with the yeast-two hybrid system. Yeast cDNA libraries are commercially available. Standard molecular biological techniques can be employed to isolate and characterize the interacting protein.

3. Screens to Identify NDAEl Homologs

Standard molecular biological techniques can be used along with the reagents of the present invention to identify NDAEl homologs in various species. For example, preferred embodiments may included, but are not limited to, DNA-DNA hybridization techniques (e.g. Southern blots) and DNA-RNA hybridization techniques (e.g. Northern blots). Hybridization may be to key consensus sequences (see Figure 1A). Additional techniques may include, for example, immunoscreening of proteins made from library stocks by anti- bodies generated from the invention. The present invention also contemplates a number of approaches including, but not limited to, immunoprecipitation and affinity purification of cell and tissue extracts and immunoscreening of proteins and glycoproteins translated from DNA and RNA library stocks. Furthermore, hybridization screens of RNA and DNA library stocks could be accomplished using RNA and DNA sequences reverse engineered from isolated NDAEl protein or by using anti-sense DNA or amino RNA sequences.

Additionally, homolog screens may be conducted using degenerative PCR wherein primers incorporate inosine (or other non-naturally occurring nucleotide). Said non-naturally occurring nucleotide may be in the first, second or third codon position.

EXPERIMENTAL

The following examples serve only to illustrate certain aspects of the present invention and in no way are meant to limit the present invention.

Example 1

Cloning. We identified a Drosophila expressed sequence tag (EST, Gen Bank accession number AA567741, deposited by the Berkeley Drosophila Genome Project). We obtained this Drosophila clone (Research Genetics, St. Louis, MO) and sequenced it (Fig- ure 1) (W. M. Keck Biotechnology Resource Laboratory, New Haven, CT). This 3225 base pair clone has an initial Met followed by a 3090 base pair open reading frame (ORF) and a 3' untranslated region (104 bp). We directionally subcloned the cDNA into a Xenopus expression plasmid as previously described (Romero, M. F., et al. "Cloning and func- tional expression of rNBC, an electrogenic Na(+)-HCO3- cotransporter from rat kidney"

Am J Physiol 274:F425-32, 1998). Linearized cDNA was used to make capped cRNA with the SP6 mMessage mMachine kit (Ambion, Austin, TX) as previously described (Romero, M. F., et al "Cloning and functional expression of rNBC, an electrogenic Na(+)- HCO3- cotransporter from rat kidney" Am J Physiol 274:F425-32, 1998). The full cDNA sequence of Drosophila NDAEl (SEQ ID NO:5) is GenBank accession number AF047468.

Example 2

Sequencing. DNA sequencing of our clone revealed a single, long open reading frame (ORF) flanked by 5' and 3' untranslated regions (UTR'S, 426 bp and 104 bp, respectively). The predicted protein is 43% similar to the cloned NBCs and 32% similar to the AEs (Figure 1). Multiple sequence alignments were performed using the Clustal method and the PAM250 residue weight table (DNAstar program, Lasergene, Madison, Wl) with % divergence and % similarity calculated as previously reported (Romero, M. F., et al. "Expression cloning and characterization of a renal electrogenic Na+/HCO3- cotransporter" Nature 387:409-413, 1997) and the alignment shaded and annotated using GeneDoc^® (http://www.cris.com/~ketchup/genedoc.shtml). While the NDAEl hydropathy plot (Figure

2A) is similar to those of the BTS members, it is most similar to the AEs (anion exchangers). A dendrogram of the published BTS sequences (Figure 2B) illustrates that NDAEl forms a new branch of the superfamily. (The dendrogram and "% similarity" calculations ignore poor matches on the ends of either sequence, so that the values are misleadingly high. For example, consider the sequences: CDEF, CDE, DEF, and CEF.

CDEF is calculated to be 100% similar to both CDE and DEF, but only 67% similar to CEF. By inspection, it is clear that the two examples of 100% similarity are over estimates, because 25% of the respective sequences are ignored for the comparison). Our NDAEl topology model (Figure 2C) predicts (i) intracellular NH₂- and COOH-termini, (ii) 12 transmembrane spans (TMs), (iii) a central exofacial loop with putative N-glycosylation sites, and (iv) multiple putative phosphorylation sites. The NDAEl topology is most easily fit by the proposed 12-14 TM models for the AEs (Kopito, R. R., et al. "Primary structure .and transmembrane orientation of the murine anion exchange protein" Nature 316:234-238, 1985; Alper, S. L., et al. "Cloning and characterization of a murine band 3- related cDNA from kidney and from a lymphoid cell line" J Biol Chem 263:17092-17099, 1988; Kopito, R. R. et al. "Regulation of intracellular pH by a neuronal homolog of the erythrocyte anion exchanger" Cell 59:927-937, 1989; Reithmeier, R A. F. "The erythro- cyte anion transporter (band 3)" Curr. Opin. Struct. Biol. 3:515-523, 1993). A large exofacial loop is predicted at the TM 5, 6 junction, containing 2 predicted N-linked glycosylation sites (N600 and N618). For the AEs, the last 2 hydrophobic regions are long enough to span the membrane twice (Reithmeier, R. A. F. "The erythrocyte anion trans- porter (band 3)" Curr. Opin. Struct. Biol. 3:515-523, 1993), consistent with 12 TMs; others have suggested as many as 14 (Reithmeier, R. A. F. "The erythrocyte anion transporter (band 3)" Curr. Opin. Struct. Biol. 3:515-523, 1993; Alper, S. L. "The band 3-related anion exchanger (AE) gene family" Annu Rev Physiol 53:549-564, 1991). Both the NH₂- and COOH- -termini are predicted to be intracellular as in the NBCs and AEs. Putative TMs are indicated by numbered rectangles. Predicted starts and stops of TMs are indicated by amino acid letter and number. A single predicted DIDS-reaction motif is indicated as a diamond.

Example 3

Localization. We determined the location of NDAEl mRNA in Drosophila. To facilitate future genetic analysis of NDAEl in Drosophila, we localized the ndael gene to region 54A on polytene chromosome 2R by in situ hybridization (not shown). Chromosomes were prepared and hybridized by standard methods (Pardue, M. L. "Looking at polytene chromosomes" Methods Cell Biol 44:333-351, (1994). Biotin-labeled probes were generated by random hexamer-priming with Biotin-HighPrime^® (Boehringer-Mannheim) and the entire NDAEl -cDNA (i.e., the EcoRI / Hind III fragment of the pSport 2 construct), according to manufacturer's instructions. Horseradish peroxidase-labeled anti- biotin antibodies were used for detection.

Using Northern analysis of poly(A)⁺ RNA, we were unable to detect NDAEl mRNA in embryos, isolated adult heads or body parts. Northern blotting was performed as follows. We isolated poly(A)⁺ RNA from Drosophila developmental stages and tissues as previously described (Romero, M. F., et al. "Expression cloning and characterization of a renal electrogenic Na+/HCO3- cotransporter" Nature 387:409-413, 1997). We used 2 μg of poly(A)⁺ RNA from these stages for denaturing electrophoresis .and electroblotting. The NDAEl -cDNA was random primed and ³²P-labeled. Hybridization overnight at 60°C in ExpressHyb (Clontech, Palo Alto, CA) followed by low stringency washing (42°C with 2xSSC), did not result in discrete hybridization. However, by RT-PCR we could detect NDAEl mRNA in heads as well as several embryonic stages (Figure 3A). Reverse transcription was performed using Superscript RT kit according to manufacturer directions (Life Technologies, Gaithersburg, MD) with Drosophila poly(A)⁺ RNA. Using Drosophila ndael specific primers, ΕxTaq (Panvera, Madison, Wl), and dNTPs, we performed PCR with 30 cycles of 94°C (30s), 55°C (45s), and 72°C (45s). Products were verified with a 0.65% agarose / TBE gel. The gel was Southern blotted onto Zeta-probe (BioRad) and detected using random primed, digoxigenin- labeled NDAEl cDNA according to manufacture's instructions (Boehringer Mannheim). Detection of digoxigenin-labeled DNA probe was performed using DIG Luminescent Detection (Boehringer Mannheim, Indianapolis, IN), recorded on X-ray film, and digitized using Adobe PhotoShop (Figure 3 A).

In situ hybridization to NDAEl mRNA in whole-mount Drosophila embryos (Figure 3B, C) illustrates that NDAEl is present during embryogenesis. In situ hybridization was performed as follows. We made whole mounts of 0-24 hr Drosophila embryos. To make antisense cRNA, the complete Drosophila NDAEl was directionally cloned into pSport 2 (Life Technologies) at EcoRI and Sal I. Digoxigenin-labeled antisense NDAEl - cRNA was synthesized using the SP6 promoter and mMessage mMachine (Ambion) as described above and reduced to a mean size of -200 bp by alkaline hydrolysis (Cox, K. H.,et al "Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes" Dev Biol 101 :485-502, 1984). Embryos were permeabilized using proteinase K treatment. Digoxigenin label was visualized using an anti-digoxigenin antibody coupled to alkaline phosphatase (Tautz, D., et al. "A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback" Chromosoma 98:81-85, 1989). Embryo staining was documented on slide-film and subsequently digitized. Hybridization was determined specific: (i) NDAEl staining was evident in discrete cells making DNA hybridization unlikely and (ii) staining with another antisense RNA probe was also discrete, yet present in difference cells than NDAEl. CNS staining is apparent throughout embryogenesis (Figure 3B, C). Staining of the gut primordium and mesoderm is evident in stage 6/7 (Figure 3B). Staining of a specific subset of cells in the CNS is detectable by late embryogenesis (Figure 3C) as is staining of the anal plate (not shown), i.e., the larval absorptive apparatus. Example 4

Physiologic function. To evaluate the physiologic function of NDAEl, we expressed it in Xenopus oocytes. Oocyte experimental solutions were made as follows. The CO₂ / HCOγfree ND96 contained 96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂, and 5 mM HEPES (pH 7.5 and 195-200 mOsm). In CO₂ / HCO^" ₃ -equilibrated solutions, 10 mM NaHCO₃ replaced 10 mM NaCl. In 0-Na⁺ solutions, choline replaced

Na⁺. In 0-C solutions, gluconate replaced CI^". Non-HCO ₃ solutions were bubbled with 100 % O₂ to remove trace CO₂ and HCO^"" ₃. The experiments were performed as follows. 50 nL of water (control) or RNA solution (35 ng of NDAEl-cRNA) was injected into stage V/VI Xenopus oocytes. Voltage electrodes, made from fiber-capillary borosilicate and filled with 3 M KCl, had resistances of 1-10 MΩ (Romero, M. F., et al. "Expression cloning and characterization of a renal electrogenic Na+/HCO3- cotransporter" Nature 387:409-413, 1997). Ion selective electrodes (pH, CI , and Na⁺) were pulled similarly, and silanized with bw-(dimethylamino)-dimethylsilane (Fluka Chemical Corp., Ronkonkoma, NY). pH electrodes tips were filled with hydrogen ionophore 1 cocktail B (Fluka), and back filled with phosphate buffer (pH 7.0). CI^" electrode tips were filled with a CI^" ionophore (Corning, Corning, NY) and backfilled with 0.5 M NaCl; Na⁺ electrode tips were filled with sodium ionophore 1 cocktail B (Fluka), and backfilled with 0.15 M NaCl. Electrodes were connected to a high- impedance electrometer (WPI-FD223 for pHj , αCl_j, or Na_j and V_m experiments) and digitized output data acquired by computer. All ion-selective microelectrodes had slopes of -54 to -57 mV / decade ion concentration (or activity). pH electrodes were calibrated at pH 6.0 and 8.0; CI^" and Na⁺ electrodes were calibrated with 10 and 100 mM NaCl. Selectivity of CI^" was checked using 100 mM NaHCO₃ and for Na⁺ using 100 mM KCl. Na⁺ electrodes were greater than 50-fold selective for Na⁺ and CI^" electrodes were at least 10-fold selective vs. HCO^~ ₃. For voltage- clamp experiments (Warner Inst. Co., Oocyte Clamp), electrodes were filled with 3 M

KCl/agar and 3 M KCl and had resistances of 0.2-0.5 MΩ. Oocytes were clamped at -60 mV and stepped from -160 to +60 mV in 20 mV steps; the resulting I-V traces were fil- tered at 5 kHz (8 pole Bessel filter, Frequency Devices) and sampled at 1 kHz. Data were acquired and analyzed using Pulse and PulseFit (HEKA Instruments, Germany).

Statistical analysis. Values quantitated are indicated as the mean ± s.e.m. Ion activities between control and NDAEl oocytes were shown by a two-tailed t-test to have a significance of p < 0.016 or less.

The results indicate that NDAEl is indeed functionally unique in the BTS. Although the present invention is not limited to any particular mechanism, Figure 4 is a model illustrating ion transport suspected of Na⁺ dependent Cl-HCO₃ exchange activity. The model was tested with oocytes expressing NDAEl . Figure 5 A shows that removal and replacement of bath Na\ CI^", or both, with and without HCO^~ ₃ did not alter intracellular pH (pHj) of a water-injected control cell. However, expression of NDAEl elevated resting pH, by -0.3 pH units (Figure 5B), i.e., control = 7.27+0.03 (n=9) and NDAEl = 7.54+0.03 (n=18). The acidification elicited by CO₂ / HCO^" ₃ (Figure 5 A) was markedly reduced in NDAEl oocytes (Figure 5B) and greatly increased intracellular [HCO^" ₃] (control = 3.1±0.2 mM, n=9; NDAEl = 7.4±0.4 mM, n=16) ([HCO^" ₃] was calculated using the pH; obtained just before CO₂, steady-state pH, in the presence of CO₂ / HCO^" ₃, and the Henderson- Hasselbalch equation.). The higher resting pH_s and elevated [HCO^" ₃] were consistent with NDAEl 's role as an acid extruder, "forward" transport in Figure 4A. Bath Na⁺ removal elicited a robust pH, decrease illustrating that NDAEl is readily reversible (Figure 4B). Subsequent removal of CI^" stopped and slightly reversed the acidification, while readdition of Na⁺ in the sustained absence of CI^" triggered a rapid pH_j recovery. A similar response was completely blocked by 200 μM DIDS (Figure 5G). These pH_j changes were consistent with Na^* and HCO^~ ₃ cotransport in exchange for CI^" and H" as observed in snail neurons (Thomas, R. C. "The role of bicarbonate, chloride and sodium ions in the regulation of intracellular pH in snail neurones" J Physiol (Lond) 273:317-338, 1977) and squid axons

(Russell, J. M., et al. "Role of chloride transport in regulation of intracellular pH" Nature 264:73-74, 1976).

We tested our transport model (Figure 4) by measuring intracellular CI^" activity (αClj). Figure 5C shows that a control oocyte has -31 mM αCl, which only slightly changes with ion replacement ± CO₂/HCO^" ₃ (37.0±1.6 mM, n=9). Figure 5D illustrates that an oocyte expressing the NDAEl transporter has -22 mM αCl; (29.5+2.1 mM, n=6). NDAEl oocytes showed both rapid and robust responses to ion replacement and addition of CO₂/HCO^" ₃ , i.e., changes of 3-8 mM activity Figure 5D). CO₂/HCO^" ₃ supplied to the bath decreases αCl,, and Na⁺ removal reversed this response. With both Na⁺ and CI^" removed αCl, change stopped, but readdition of Na⁺ elicited a large and rapid fall in αCl,. The removal of bath CO₂/HCO^~ ₃ brought αCl, back to resting levels (Figure 5D). These alter- ations of αCl, were also blocked by 200 μM DIDS (Figure 5H). Moreover, the beginning of Figure 4D illustrates that HCO^~ ₃ was not required for ionic movements through the transporter (solutions bubbled with 100% O₂). This physiologic characteristic is reminiscent of the multiple transported anions (e.g., OH , Br^", I^") and HCO^" ₃ stimulated activity of AEs. To discriminate between Na⁺ dependence (gradient) vs. Na⁺ driven (transport), we measured the effect of NDAEl activity on intracellular Na⁺ activity (αNa,) of oocytes. Figure 5E-F are representative traces from control and NDAEl oocyte experiments, respectively, using similar solution protocols as in Figure 5A-D. A control oocyte (Figure 5E) has -2.6 mM αNa, (3.1±0.5 mM, n=10) which did not change with bath ion substitutions. Figure 5F shows that αNa, is increased to -5 mM in NDAEl -expressing oocyte (4.6±0.3 mM, n=10). Na⁺ was transported by NDAEl as evidenced by (i) increased αNa, with the addition of CO₂ / HCO^~ ₃, (ii) reduced αNa, with Na⁺ removal, and (iii) increased αNa, with CI removal. Na⁺ transport via NDAEl was blocked by 200 μM DIDS (Figure 51). Changes of αNa, were always in the opposite direction as αCl, changes indicating a Na⁺ for CI exchange. As shown for both the pH, and αCl, responses, Na⁺ transport was also observed in the complete absence of HCO^" ₃ (not shown). Thus, our data indicate that this Drosophila Na⁺ dependent Cl-HCO₃ exchanger is more appropriately named a Na⁺ driven anion exchanger or NDAEl.

Example 5 Effect of transport inhibitors. We noted that CI^" removal or the addition of HCO ₃ resulted in depolarizations only in NDAEl oocytes (Figure 5B). Therefore, we voltage clamped and used anion transport inhibitors (DIDS, DPC, and niflumic acid) on NDAEl oocytes to evaluate the electrical nature of this transporter (Figure 6). In a voltage clamped oocyte, this depolarization is measured as an inward (negative) current. A com- parison of water- injected control (Figure 6A) and NDAEl oocytes (Figure 6B) illustrates that both CI^" removal and HCO^" ₃ addition elicit current specific to NDAEl expression. The reversal potential of both control (Figure 6C) and NDAEl oocytes (Figure 5D) was about -20 mV. In the absence of CI^", there was also a HCO^" ₃ stimulated current only in NDAEl -oocytes (Figure 6B). This current had a linear voltage dependence (Figure 5D). DIDS, DPC, and niflumic acid block the depolarization (undamped cell) due to CI^" removal (Figure 5E). However, the measured currents in NDAEl oocytes were small compared to the pH_j, αCl_j, and Na_j changes. The voltage deflections and associated currents are either endogenous to the oocyte uncovered by NDAEl activity or more likely a "leak" current through the NDAEl transporter. Present data imply that the NDAEl current represents a leak current rather than NDAEl being "electrogenic": (i) the J(ion) / J(current) ratio for CI^", HCO ₃, and Na⁺ is > 100, and (ii) the pH₍ changes are 2-3 times greater for NDAEl than rkNBC while the transport currents are at least 10-fold smaller (30 nA vs.

300-500 nA, respectively) (Sciortino, C. M., et al. "Cation and voltage dependence of rat kidney, electrogenic Na⁺/HCO₃ ^" cotransporter, rkNBC, expressed in oocytes" Am. J. Physiol. 277:F611-623, 1999). The J(ion) was calculated from the initial rate of ionic change elicited by CI^" removal and the volume to surface area ratio (V/SA) of the oocyte ([diameter/2]/3). Similarly, J(current) was calculated from the current in 0 CI^" at -20mV, i.e., the voltage obtained in undamped oocytes with CI^" removal, and SA/N. The resulting values was divided by the Faraday constant to yield a true flux, J(current). These transporter currents (or voltage changes) would not have been detectable in snail neurons (Thomas, R. C. "The role of bicarbonate, chloride and sodium ions in the regulation of intracellular pH in snail neurones" J Physiol (Lond) 273:317-338, 1977) or squid axons

Expression of ΝDAE1 in Xenopus oocytes shows all the physiologic properties of the Νa⁺ dependent Cl-HCO₃ exchanger: CI^" transport, Na⁺ transport, Na7HCO^~ ₃ cotransport (or Na⁺ - H⁺ exchange), and sensitivity to DIDS. NDAEl does not require

HCO^" ₃ and appears to be a more general anion exchanger. Our data infer that NDAEl exchanges Na⁺ and HCO ₃ (or an anion) for CI and H⁺ (Figure 4). Thus, it is likely that NDAEl is the Na⁺ dependent Cl-HCO₃ exchanger functionally identified in neurons, fibroblasts, mesangial cells, and renal tubule cells. Physiologically, the activity of the Na⁺ dependent Cl-HCO₃ exchanger appears to be regulated. In mesangial cells, agents such as angiotensin II, serotonin, and vasopressin, which act locally as growth factors (Ganz, M. B., et al. "Long-term effects of growth fac- tors on pH and acid-base transport in rat glomerular mesangial cells" Am J Physiol 266:F576-585, 1994), as well as EGF and PDGF, stimulate ion transport activity including Na⁺ dependent Cl-HCO₃ exchange (Ganz, M. B., et al. "Arginine vasopressin enhances pH_j regulation in the presence of HCO3 ^y stimulating three acid-base transport systems" Nature 337:648-651, 1989). Recently, Na⁺ dependent Cl-HCO₃ exchange activity was shown to increase during normal renal development (Ganz, M. B., et al "Development of pH regulatory transport in glomerular mesangial cells" Am J Physiol 274:F550-555, 1998). And, in NIH-3T3 fibroblasts, Kaplan and Boron (Kaplan, D. L., et al. "Long-term expression of c-H-ras stimulates Na-H and Na(+)-dependent CI- HCO3 exchange in NIH-3T3 fibroblasts" J Biol Chem 269:4116-4124, 1994) found that transformation with c-H-ras not only increased the activity of the Na⁺ dependent Cl-HCO₃ exchanger but also shifted activation to more alkaline pH values, effectively removing intracellular pH (pH,) as the transporters control mechanism. Moreover, some studies postulate that mis- or de-regulation of stilbene-sensitive HCO^" ₃ transport (Lee, A. H., et al. "Heterogeneity of intracellular pH and of mechanisms that regulate intracellular pH in populations of cultured cells" Cancer Res

58:1901-1908, 1998) or Na-H exchange (Rotin, D., et al. "Requirement of the Na+/H+ exchanger for tumor growth" Cancer Res 49:205-211, 1989) is involved in neoplasia.

To begin investigating the role of NDAEl in vivo, we searched the Berkeley Drosophila P element insertion site database (BDGP) with the ndael sequence and identified a P element insertion mutation that lies 408 bases 5' of the predicted ndael initiation codon.

We determined that the insertion site of this P element mutation lies within the ndael 5' untranslated sequence by RT PCR, using wild type poly(A)⁺ RNA and primers that flank the site of insertion. While the gene affected by this mutation was previously unknown, it was known to be essential for viability, since the mutation was isolated in a screen for lethal P element induced mutations (Torok, T., et al. "P-lacW insertional mutagenesis on the second chromosome of Drosophila melanogaster: isolation of lethals with different overgrowth phenotypes" Genetics 135:71-80, 1993; Roch, F., et al. "Screening of larval pupal P-element induced lethals on the second chromosome in Drosophila melanogaster: clonal analysis and morphology of imaginal discs" Mol Gen Genet 257:103- 112, 1998). Example 6

Production of fusion proteins. The production of glutathione- S-transferase (GST) fusion proteins of NDAEl would provide us with the necessary reagent required for: (1) in situ protein competition in Xenopus oocytes, (2) binding column protocols and (3) fur- ther antibody production and affinity purifications.

Polymerase chain reaction (PCR) primers were created to insert a 5' EcoRI restriction enzyme site and a 3' Xhol restriction enzyme site on the last 95 amino acids of NDAEl (CGSTEco_F and CGSTXho_R). These primers were used to PCR a 300 bp fragment from Drosophila NDAEl (drNDAEl) (Figure 7). This fragment was then insert- ed, using the engineered restriction sites, into the pGEX-4T-l GST fusion protein expression vector, resulting in a construct termed pGEX-4T-l/CTERM95 which was used for fusion protein production.

In addition, PCR primers were created to insert a 5 ' EcoRI restriction enzyme site and a 3' Xhol restriction enzyme site on the first 100 amino acids of NDAEl (NGSTEco F and NGSTXho R). These primers were used to PCR an approximately 300 bp fragment from the BAC human clone of NDAEl (Figure 8). This fragment was also inserted into the pGEX 4T-1 vector, to form a construct termed pGEX-4T-l/NTERM100, and was used for fusion protein production.

DH5α bacteria were transformed with either the pGEX4T-l/CTERM95 or pGEX4T-l/NTERM100 constructs, and GST-fusion protein production was induced by the addition of IPTG. Figure 9 shows that incubation with 0.4mM IPTG resulted in the production of the GST-CTERM95 fusion protein with a molecular weight of approximately 35 kDa and of the production of GST-NTERMIOO, with an approximate molecular weight of 39 kDa. Isolation of the GST-fusion proteins was performed by utilizing the ability for GST to bind to glutathione attached to agarose beads. Briefly, after protein induction, bacteria were lysed by snap freeze/thaw, vortexing and sonication while in a PBST solution containing protease inhibitors and 0.1% Triton X-100. Fusion protein was collected by an overnight incubation with glutathione agarose beads at 4°C. Fusion proteins were eluted from the beads by the addition of 40 mM reduced glutathione for 1 hour. This elution step was repeated, the elutions pooled, and reduced glutathione was removed by using a Centricon centrifugal filter device with a molecular weight cutoff of 3,000. Figure 10 shows that GST-CTERM95 can be isolated using glutathione agarose beads, and that the molecular weight of the fusion protein is higher than the 28 kDa of GST alone. Figure 11 shows that GST-NTERMIOO can similarly be isolated. The GST-CTERM95 fusion protein was then used to affinity purify anti-CTP antibody from QCB (Quality Controlled Biologicals, Inc., Hopkinton, MA) rabbit number 89025 (see example 7). Briefly, GST-CTERM95 was blotted onto nitrocellulose, and by using Ponceau S. to detect the region of the blot containing the fusion protein, the nitrocellulose was cut into pieces, blocked with 5% milk and incubated with serum overnight. Antibody to the fusion protein was eluted by a glycine solution pH 2.2. The pH of the solution was immediately returned to pH 7.8 by the addition of Tris. The resulting affinity purified antibody recognizes the GST-CTERM95 fusion protein, but not GST alone (Figure 12). Using these techniqes, NDAEl was also fused to GST as well as GFP (see example 10). Example 7

Production of antibodies. Polyclonal antibodies were generated to NDAEl using techniques known to those in the art. The anύ-Drosphila NDAEl antibody designated cND25 works well in immunohistochemistry (Figures 13 and 14) and Western blots (data not shown). Antibodies were then generated to specific portions of NDAEl. Synthetic peptides of three regions of NDAEl were chosen. The first region was termed N-terminal peptide (NTP) and it consists of the amino acids 20-34 of Drosophila

NDAEl (DDE APKDPRTGGEDF) (SEQ ID NO:15). The second region was termed C-terminal peptide (CTP), which consists of amino acids 1014-1030 (SNANEKEFEAQSSLLKK) (SEQ ID NO: 16). The final region was termed Middle peptide (MP), and consists of amino acids 376-393 (PSQEVRKRPPELPKEEVD) (SEQ ID NO: 17). All of these regions are predicted to be intracellular. These peptides were cross-linked to keyhole limpet hemacyanin (KLH) for immunization of rabbits. After four immunizations, serum from several rabbits (rabbit numbers 89035 and 89036 for NTP; 89030 and 89031 for MP; and 89025 and 89033 for CTP) were tested by the Romero laboratory to determine the presence of specific antibodies raised against the respective region of NDAEl . Figure 15 is a representative Western Blot showing the presence of antibodies to the C-terminal peptide of NDAEl. The predicted molecular weight of NDAEl is 115 kDa. A prominent band was observed at this approximate molecular weight. Detection of the band above 105 kDa was partially competed away if the serum was preincubated with 120 g of the C-terminal peptide (Figure 16). Additionally, we wanted to create antibodies that would recognize larger portions of the protein. Therefore, we sent the GST-CTERM95 (SEQ ID NO: 18) and GST-NTERM100 (SEQ ID NO: 19) fusion proteins to Cocalico Biologicals, Inc (Reamstown, PA) for injection into rabbits (CWR55-56 for GST-CTERM95 and CWR57-58 for GSTNTERM100). Using their standard production protocol and schedule of injections initial test bleeds have been obtained and we have tested them by Western blot (Figure 17).

Example 8

Immunolocalization of NDAEl in Rat Kidney. Glomeruli and tubules in rat kidney cortex were immunopositive for NDAEl (Figure 13). Cryosections of rat kidney were stained with anύ-Drosophila NDAEl antibody (cND25, i.e. 89025) with flurochrome coupled secondary antibodies. Kidneys were fixed with PLP (2%> para formaldehyde, 75 mM lysine, 10 mM NaIO4 in PBS) 4-6 hours and cryoprotected in 30% sucrose overnight before freezing in liquid nitrogen and cryosectioning. Sections were hydrated in PBS, blocked with 10% normal goat serum (NGS) 15 min, incubated with or without primary antibody overnight, incubated in biotin conjugated goat anti-rabbit followed by avidin conjugated to Cy3 each for 1 hour. All antibody and NGS incubations were in PBS containing 10%) normal goat serum and 0.1% Triton X-100. Sections were washed with PBS between all antibody incubations and were mounted with 5% n-propyl galliate in glycerol.

Tubules in rat kidney medulla were immunopositive for NDAEl (Figure 14). Kid- neys were fixed as above. Sections were stained with anύ-Drosophila NDAEl antibody

(cND25, i.e. 89025) and visualized with Cy3. Example 9

Identification of transfected NDAEl and NDAEl fusion proteins with anti- NDAEl. Using lipofectamine technique (GibcoBRL), COS7 cells were transfected with NDAEl -EGFP and plated onto glass coverslips. Coverslips were fixed with 4% paraformaldehyde for 30 min. Coverslips were than washed with 100 mM glycine in phosphate buffered saline (PBS) for 10 min and than permeabilized with 0.1 % Triton-X 100 for 10 min. Next, coverslips were blocked with 10% donkey serum for 2 hr and then exposed to the affinity purified anti-CWR57-NDAEl (NH₂-terminus) primary antibody for 2 hr. Coverslips were then washed with PBS and exposed to the donkey-anti-rabbit conjugated Cy3 secondary antibody for 2 hr. Figure 18 shows that only eGFP positive cells have

NDAEl recombinant protein.

Example 10

Immunoprecipitation of recombinant NDAEl and NDAEl -GFP with anti- NDAEl. Using the lipofectamine technique, COS7 cells were transfected with either EGFP, NDAEl -EGFP or NBC-EGFP. Forty-eight hours later cells were lysed for 10 min on ice with 250 μl of RIPA cell lysis buffer with mammalian protease inhibitors. Cells were then scraped off the dish and lysates were centrifuged to remove nonsoluable material. Supernatants were then diluted with equal amounts of 2X LSB and 30 μl of each sample was loaded onto a 7.5% SDS-acrylamide gel. Proteins were resolved by SDS-PAGE and then transferred to nitrocellulose and blocked for two hours with 10%> milk in Blotto.

The blot was incubated overnight with the primary antibody, rabbit anti-CWR57AP (rbαCWR57AP or CWR57) and then incubated with HRP conjugated anti-rabbit secondary antibody for two hours. Proteins were resolved by ECL (Pierce). The blot was stripped and reprobed with mouse anti-EGFP antibody. Figure 19 shows the recognition of NDAEl -GFP by rbαCWR57 AP antibody. Figure 20 shows the recognition of NDAEl -

GFP by G.89025 antisera.

Figure 21 shows the immunoprecipitation by anti-NDAEl antibodies CWR55, 89025 and 89035 of recombinant NDAE1-EGFP transfected into COS7 cells. Using the lipofectamine technique COS7 cells were transfected with either NDAEl -EGFP or NBC- EGFP (data not shown). Forty-eight hours later cells were lysed for 10 min on ice with

1000 μl of RIPA Western cell lysis buffer with mammalian protease inhibitor cocktail. Cells were then scraped off the dish and lysates were centrifuged to remove nonsoluble material. Supernatants were incubated with either αCWR57AP, 89025 or α89035 for 3 hours at 4°C. Samples were then incubated with Protein A Sepharose beads for 2 hours at 4°C. Beads were washed 5X with RIPA buffer. Protein / antibody complexes were eluded from the beads in 60 μl of 2X LSB (Lamelli sample buffer). Proteins were resolved by SDS-PAGE and then transferred to nitrocellulose and blocked for two hours with 10% milk in Blotto. The blot was incubated overnight with primary antibody rbαCWR57AP and then incubated with HRP conjugated anti-rabbit secondary antibody for two hours. Proteins were resolved by ECL (Pierce). The same blot was then stripped and reprobed with mouse anti-EGFP primary antibody followed by secondary antibody, as above. Example 11

Isolation ofNDAEl homologs. To isolate homologs of NDAEl from membrane preparations, we separate cellular membranes from cell lysates (e.g., cells may be lysed by Western blot lysis buffer with protease inhibitors) by centrifugation, resuspend in lysate buffer (with protease inhibitors), and immunoprecipitate with anti-NDAEl antibodies (e.g., cND25, 89025, 89035, 89033, 89036, 89030, 89031, CRW55AP, CRW56AP, CRW57AP,

CRW58AP or anti-NDAEl -GFP antibody) or with any antibody specific for NDAEl or a portion thereof. Precipitates may then be separated from the precipitating antibodies an a denaturing SDS-PAGE gel. After electrophoresis the proteins may be excised from the gel for characterization. Example 12

Molecular Labeling of NDAEl. To further facilitate us in determining cellular localization of NDAEl, we have labeled NDAEl with hemagglutinin A (HA) protein of human influenza virus. PCR primers were designed to insert a 5' Hindlll restriction enzyme site (NDHindIII_F) and to remove the stop codon and insert a 3' EcoRI restriction enzyme site (NDEcoRlNS_R). These primers were then used to PCR full length NDAEl from Drosophila NDAEl (Figure 22). The PCR product will be inserted into the pMH mammalian expression vector which will place the HA tag on the C-terminal end of NDAEl. Example 13

Nitrate selective electrods were fabricated by pulling filamented borosilicate glass followed by silanization at 200°C. Subsequently, shanks were coated with SylGuard (Corning), and cooled under vacuum. Pipettes were then backfilled with NO₃ ^" backfill solution (200 mM KNO₃ and 200 mM KCl). Negative pressure was then used to draw back the Nitrate lonophore-Cocktail A (Fluka, #72549) to a height of at least 20mm. The slope was determined by calibration on 100, 10 1 and 0.1 mM KNO₃ and was found to be linear in this range and have a slope of at least -55 mV / decade.

It should be clear from the above that the reagents and methods detailed here will allow for the screening of compounds the are agonistic or antagonistic to functioning NDAEl channels.

Claims

1. Purified DNA selected from the group consisting of SEQ ID NOS:l, 2, 3, 4, 5, 6, 7 and 8.

2. RNA transcribed from the DNA of claim 1.

3. Protein translated from the RNA of claim 2.

4. Purified protein selected from the group consisting of SEQ ID NOS:8, 9, 10, 11, 12, 13 and 14.

5. Antibodies produced from the protein of claim 3.

6. Expression constructs comprising the DNA of claim 1.

7. A transgenic animal comprising DNA of claim 1.

8. Antibodies to NDAEl or a portion thereof or a fusion protein thereof, selected from a group consisting of antibodies designated 89035, 89036, 89025, 89033, 89030, 89031, CWR55, CWR56, CWR57 and CWR58.

9. A method, comprising: a) providing i) one or more compounds, ii) a cell line or transfected cells comprising the ndael gene; b) contacting a portion of said cells from said cell line with said one or more compounds under conditions such that said compound can enter said cells, so as to create treated portions and untreated portions of cells; and c) comparing the amount of pH„ Na⁺ or anion channel / transporter activity of said treated cells with the amount of pH„ Na⁺ or anion channel / transporter activity of said untreated cells.

10. A method, comprising: a) providing unmutated cDNA encoding protein, said protein selected from the group consisting of SEQ ID NOS:8, 8, 10, 11, 12, 13 and 14; and, b) treating said cDNA under conditions such that mutated cDNA is produced.

1. The method of Claim 10, wherein said treating comprises: a) deriving mRNA from said cDNA wherein said mRNA is in operational condition and wherein said mRNA comprises a hairpin loop on both the 3' and 5' ends, a ribosome binding site 5' to the coding sequence and a spacer sequence 3' to the coding sequence and wherein said mRNA lacks a stop codon; b) translating said mRNA in an in vitro ribosome based translation system to produce a peptide encoded by said mRNA under conditions that permit the retention of both the mRNA and the translated peptide on the ribo- some, to produce a ribosome/mRNA peptide complex; c) screening for and isolating said ribosome/mRNA/peptide complexes wherein said peptide functions to a greater, lesser or changed degree than the peptide encoded by the original cDNA sequence; d) disassociating said mRNA from said ribosome/mRNA/peptide com- plex; e) reverse transcribing the mRNA into cDNA wherein the polymerase is not capable of error correction thereby incorporating transcription errors into the cDNA, to produce a selection of mutated and nonmutated cDNA sequences; f) transcribing said mutated and nonmutated cDNA sequences into mRNA in operational condition, wherein said mRNA comprises, a hairpin loop on both the 3' and 5' ends, a ribosome binding site 5' to the coding sequence and a spacer sequence 3' to the coding sequence and wherein said mRNA lacks a stop codon; g) continue said steps (a-f) until a peptide is produced that functions to a greater, lesser or changed degree than the peptide encoded by the original cDNA sequence.

11. The oligonucleotide produced by the method of claim 8.

12. The peptide produced by the method of claim 8.