RETINOIC ACID INDUCIBLE DNA SEQUENCES FROM LUNG CELLS

Abstract: Nucleic acid sequences and corresponding polypeptides that are expressed by lung tissue, the expression of which is induced by retinoic acid or inhibited by retinoic acid, are provided. These polypeptide sequences, and the human counterparts, can be used to promote cell differentiation and/or proliferation, especially of alveolar tissue. Also, these cDNAs can be used to identify other retinoic acid inducible sequences.

Description: TTT E OF THE INVENTION RETINOIC ACID INDUCIBLE DNA SEQUENCES FROM LUNG CELLS FIELD OF THE INVENTION The invention relates to the identification of nucleic acid sequences and corresponding polypeptides, the expression of which are induced or inhibited by retinoic acid. These nucleic acid sequences and polypeptides are useful for inducing cell differentiation and/or proliferation and especially for inducing alveolar formation. This work was partially funded by the United States government through the National Institutes of Health Grants HL 20366 and 37666. BACKGROUND OF THE INVENTION Pulmonary emphysema is a common disease in which the destruction of the lung's gas-exchange structures (alveoli) leads to inadequate oxygenation, disability and, frequently, death. Lung transplantation has previously provided the only effective means of remediation. Alveoli are formed by the developmentally regulated subdivision of saccules that constitute the gas-exchange region of the immature lung. The molecular signals responsible for the formation of septa and their spacing are poorly understood. However, in rats and other mammals, retinoids may play a key regulatory role. Fibroblasts rich in vitamin A (retinol) storage granules occupy a large fraction of the alveolar wall when septa are being formed. During the same period, the concentration of cellular-retinol binding protein I, cellular retinoic acid-binding Protein I, and nuclear retinoic acid receptor-τ mRNA peak in the lung. Treatment with dexamethasone, a glucocorticosteroid hormone, prevents septation in a seemingly irrevocable fashion, and diminishes the expression in the lung of cellular retinol-binding protein and retinoic acid receptor- β mRNA. The use of all-trans retinoic acid for treatment of diseases relating to growth and tissue maintenance has previously been known. Retinoic acid receptors belong to a family of nuclear receptors that includes receptors for steroids, thyroid hormone, and calcitriol. It has previously been disclosed that retinoic acid prevents inhibition of alveoli in rats arising from exposure to dexamethasone. (Am. J. Physiol. 270: L305-L310 (1996)). However, there is no teaching therein regarding the use of retinoic acid for treatment of emphysema. U.S. Patent 3,171,781 teaches the use of vitamin A and guaiacol for the treatment of contagious "air sac disease" in fowl. However, there is no teaching therein regarding the administration of retinoic acid for the treatment of emphysema. U.S. Patent 4,606,920 teaches the use of vitamins A and C for the treatment of inflammatory changes in the bronchial mucosa. However, there is no teaching therein regarding the treatment of diseases involving alveoli, and especially alveolar development. U.S. Patent 5,534,261, which is incorporated herein in its entirety as though fully copied herein, teaches the use of retinoic acid to prevent formation of adhesion between organ surfaces in body cavities, especially in the peritoneal cavity. However, no information relating to the use of retinoic acid for the treatment of emphysema is contained therein. U.S. Patent 5,556,611, which is incorporated herein in its entirety as though fully copied herein, teaches the use of retinoic acid and esters thereof in the form of aerosols for treatment of mucosal diseases. Diseases purported to be treatable by use of such aerosols include bronchial carcinoma, acute and chronic bronchitis, acute and chronic functional disturbances due to impairment of the trachealbronchial epithelium following inhalation of dusts and gases, bronchopulmonary dysplasia of newborns and carthagena syndrome. However, there is no mention therein that retinoic acid, its esters or analogues thereof can be administered for treatment of emphysema, which arises from destruction of the lung alveoli. Recently, Drs. Donald and Gloria Massaro showed that retinoic acid treatment abrogates elastase-induced emphysema in adult rats and that treatment of newborn rats with retinoic acid increases alveolar number 50% without lung enlargement. Furthermore, they have shown that treatment with retinoic acid prevents the glucocorticoid-induced inhibition of alveolus formation. (Am. J. Physiol. 270 pp. L305-L310 (1996)). However, to the best of the inventor's knowledge, there is no information relating to nucleic acid sequences and polypeptides expressed selectively by lung tissue in the presence or absence of retinoic acid. Such nucleic acid sequences and polypeptides would be potentially significant in promoting alveolar development and treatment of disorders relating thereto. OBJECTS OF THE INVENTION Therefore, it is an object of the invention to provide novel nucleic acid sequences that encode proteins that are induced or inhibited by retinoic acid or a fragment thereof. More specifically, it is an object of the invention to provide cDNAs or fragments thereof that encode a polypeptide the expression of which is induced or inhibited in pulmonary cells in the presence or absence of retinoic acid. It is another object of the invention to use the novel nucleic acid sequences provided herewith as a hybridization probe to identify related nucleic acid sequences that encode proteins or polypeptides, the expression of which is induced or inhibited by retinoic acid. It is still another object of the invention to utilize the corresponding proteins as potential targets to screen compound libraries to identify compounds that antagonize or agonize the expression of retinoic acid inducible nucleic acid sequences. It is another object of the invention to utilize the novel polypeptides or fragments encoded by said novel nucleic acid sequences to treat pulmonary disorders, e.g. emphysema and immature lung development in premature infants. It is another specific object of the invention to provide pharmaceutical compositions comprising the novel proteins and fragments encoded by said nucleic acid sequences. BRIEF DESCRIPTION OF THE FIGURES Figures 1 A through 1C contain a Table that identifies the different clones, size, homologous sequence, and hypothesized function, identified by differential display. Figures 2 through 36 contain the nucleic acid sequences of clones isolated by differential display. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the identification of a novel nucleic acid sequences, the expression of which in rat lung tissue is induced or inhibited by retinoic acid. As discussed supra, it has been reported that retinoic acid induces the expression of genes involved in the proliferation and differentiation of different mammalian cells and tissues. This apparently occurs because of the presence of retinoic acid receptors in mammalian cells, which comprises a family of nuclear receptors that interact with various ligands such as retinoids, steroids, thyroid hormone and calcitriol, and which ligand interaction are believed to trigger key cellular events that result in cell proliferation, differentiation and apoptosis. Therefore, the identification of proteins, the expression of which is induced or inhibited by retinoic acid, is useful as such proteins may play an active role in modulation of cell proliferation, differentiation and/or apoptosis. Related to the present invention, it was recently reported by Massaro et al (Id.), that retinoic acid, esters and analogs, thereof may be used to treat pulmonary disorders and, in particular, emphysema. More specifically, it has been discovered that retinoic acid and related compounds induce the formation of alveoli and reduce lung volume. This has been shown in a rat model of emphysema (elastase-induced emphysema). Specifically, it was found that treatment with retinoic acid in adult and newborn rats with retinoic acid increased alveoli number about 50%. Based thereon, it was hypothesized by the present inventor that retinoic acid may induce or suppress the expression of specific proteins or nucleic acid sequences that promote or inhibit alveolar formation. In order to test such hypothesis, experiments were conducted comparing gene expression in rat lungs which were treated or not treated with retinoic acid. After such treatment, it was determined by differential expression whether any mRNA transcripts were induced or inhibited as a result of retinoic acid treatment or absence of treatment. The results of this experiment demonstrated that a specific mRNA transcript which corresponds to the cDNA sequence set forth below was induced as a result of retinoic acid treatment: 1. TTTTTTTTAT AGAAGTGATG TTTGAATAGT TTTTGTCAGG GCAATGTCAG 51 CCCAATAGTT TTATTGGGCT CATTCTTCTA AGAAATAGTT ATGAATTTGA 101 GTCTCTGACT TGTATCATTC TGCCCAGGGA AATTCTATAG TACTGAAGTC 151 TTGGAGGCTG CCCAGCAACT CCACTGGGCA TTGCCATGTG CCTAGATATA 201 ATACCTCCAT GGCAGATTTC TGACTAGGCT GCCAGAGACT GTTAATTGCT 251 TCGTAGCTCA TGTATTCTGA ATACTTGCAC AATTAGTACC ACCAGGGCAT 301 TACTAAGGCT TACCATTGTG TTTTCCAGAT TTTTCAGTAG AATAATTGTC 351 CAGCCTAAGA TACACTTCAG CTTGTTTGCC CTATAGTGGT TATGTTGACT 401 GAAGGACAAC TATACTGAAA CTCAGAGAGC AGAGCCTGTA GTATTCTTGG 451 CAAGCAAATG GTGACATCCA GTTAGTACAT CTCTGTAATC TTACAATAAA 501 GATTCTTACT TGCCTTGAAA AAAAAAAA in a pharmaceutically acceptable carrier. Also, the results of this differential display experiment identified the cloned cDNA sequences identified in Figures 1 through 36, and Tables 1 through 3 infra. Based on sequence homology, it appears that many of these sequences may be involved in cell differentiation and/or proliferation. In particular, many of these sequences exhibit homology to embryonic gene sequences and reproductive tissue gene sequences such as rat embryonic and ovary tissue, and rat placental and mammary tissue. Also, several of these sequences exhibit sequence homology to mitochondrial, nebulette, histone genes, and G proteins and therefore may be significant in protein synthesis, cell proliferation or development. The lead sequence, C72Rilb, exhibits homology to alpha-2-macroglobulin (A2M) gene which is on human chromosome 12 at map position 12pl2-13. This gene is related to a form of emphysema. In this same gene locus are SGC31674, alpha-2-macroglobulin recursor, 12pl2.3-13.3, SCC34867, which is homologous to cellular retinol-binding protein I, WI- 15438, which is proliferating-cell nucleolar antigen pi20, and SGC32489, ubitiquin carboxyl- terminal hydrolase. It would appear that this sequence corresponds to a 3' fragment of a gene expressed by lung tissue under retinoic acid inducible conditions that is involved in alveolar development. The fact that this sequence co-localizes to the A2M gene suggests that it may be involved in emphysema. Therefore, this sequence will be used as a hybridization probe to identify the full length gene from a cDNA or genomic library. Also, upon identification of the full length sequence, this sequence or fragment thereof will be used to identify the human counterpart sequence. The specific clones identified by differential display are set forth in Tables 1-3 on the following pages. TABLE 1 DD Clone With Homologous Unigene Sequence: TABLE 2 DD Clone With Homologous ESTs TABLE 3: DD Clone With No Homologous Sequence in GenBank and dbEST: Based on the effect of retinoic acid on alveolar formation, and the fact that the subject nucleic acid sequences are transcribed or not transcribed upon retinoic acid induction, it is anticipated that the corresponding polypeptides should encode molecules that are responsible for the effects of retinoic acid on lung tissue. Moreover, given the fact that retinoic acid and related retinoids play an active role in differentiation and/or proliferation of other cells and tissues, these proteins may have broad activity, i.e. they may promote the differentiation and/or proliferation of other tissues also. Therefore, the present invention involves the identification of polypeptides expressed by lung tissue, the expression of which is inhibited or induced by retinoic acid, and especially the amino acid sequence identified above or a fragment thereof for promoting cell differentiation and/or proliferation. More preferably, the present invention is directed toward the use of such polypeptides or fragments thereof for treating diseases wherein promotion of alveolar function is therapeutically beneficial. Examples of such diseases and conditions include in particular emphysema, and immature lung development, especially in premature infants. As noted, the subject cDNAs were isolated from rat lung tissue. It is anticipated that there should exist human counterparts to the subject cDNA having a related sequence and function. This is a reasonable conclusion based on the fact that retinoic acid receptors are present on mammalian cells of different species including humans. Therefore, the present invention further embraces related nucleic acid sequences, and in particular the corresponding human nucleic acid sequences. The corresponding human sequences can be identified from a cDNA library provided from human lung tissue or cells cultured in the presence of retinoic acid. It is anticipated that the rat cDNA will hybridize to the human cDNA counterpart and therefore will be identified from a cDNA library by hybridization. This can be effected by using the subject cDNAs, or a fragment thereof, of sufficient length, typically at least 10-12 nucleotides in length, as a hybridization probe. Methods for effecting hybridization are well known in the art. Preferably such probes will be at least about 50 nucleotides in length. The present invention therefore embraces cDNAs that specifically hybridize to the cDNA identified supra which have substantially the same sequence. In the present specification, two sequences will: (a) both form a base- pair duplex with the same segment, and (b) the melting temperatures of said two duplexes in a solution of 0.5 x SSPE will differ by less than 10°C. Also, if the hybridizing segments have "substantially the same sequence", at least 70% of the aligned portion of said nucleic acid sequences, without allowing for any gaps, will be identical, and more typically at least 80% of the aligned nucleotides will be identical, without allowing for any gaps. Therefore, the present invention embraces cDNAs that hybridize to the subject cDNAs discovered by differential display, or a fragment thereof, under the above-recited hybridization conditions, wherein said sequences exhibit at least 70% sequence identity to the subject cDNAs in the coding region, and more preferably will exhibit at least 80% sequence identity. Alternatively, related cDNAs can be isolated from a cDNA expression library prepared from human or other species lung tissue which has been induced by retinoic acid using an antibody produced against the rat polypeptide as an immunoaffinity probe. It is anticipated that the rat and human polypeptide will have conserved amino acid sequence and therefore that the anti-rat polypeptide antibody or antiserum will cross-react with the human counterpart. The present invention further embraces the use of the polypeptides encoded by the subject cDNA or its human or other species counterpart as a target to screen compound libraries for molecules that agonize or antagonize retinoic acid induced cell differentiation or proliferation. Essentially this will entail screening compound libraries for compounds that specifically bind to the subject polypeptide or its human or other non-human counterparts. These molecules may be useful for promoting or inhibiting cell differentiation and proliferation, e.g. alveolar development. This may be effected, e.g., by expressing the subject polypeptide in phase display libraries and screening phage that express the polypeptide for those that bind to screened compounds. Such compounds may be preferred to the subject polypeptide as potential therapeutics, e.g., because of solubility , stability and cost concerns. Most significantly, the present invention is directed to the use of the subject polypeptide and related counterparts in other species, in particular human, for promoting alveolar development. This is beneficial for the treatment of any condition involving abnormal or inadequate alveoli formation and development. Examples of such conditions include especially emphysema and immature lung development in premature infants. The subject polypeptide or a fragment thereof which is at least 50 amino acids and more preferably at least 100 amino acids in length is administered by any pharmaceutically acceptable route. These routes include any systemic or parental mode of administration, e.g. intravenous, intraarterial, intramuscular, subcutaneous, oral, intranasal, via inhalation, etc. A preferred means of administration comprises transdermal administration using known transdermal cancer diseases, DMSO and via inhalation. This will increase the delivery of the compound to the desired site, i.e. the lung. In general, the preferred dosage will range from about 0.005 to 10 mg 1 kg body weight, more preferably 0.05 to 0.1 mg 1kg body weight. Also, the subject polypeptide may be delivered to the mucus membranes in the form of cyclodextrin inclusion complexes. Additionally, the invention relates to the identification of other genes that are expressed by lung tissue in response to retinoic acid. In general this will involve differential display techniques wherein RNA is obtained from lung tissue cultured in the presence or absence of retinoic acid, cDNA libraries are generated therefrom, and these cDNA libraries screened by differential display in order to identify those cDNAs which are selectively expressed by lung tissue in the presence of retinoic acid, or inhibited in the absence of retinoic acid. Methods for effecting differential display to identify nucleic acids that are expressed under particular conditions are well known in the art. A suitable differential display protocol is provided in the Example infra. Preferably, the present invention will comprise the use of differential display methods to identify nucleic acid sequences that are transcribed by human lung tissue under retinoic acid inducible conditions. It is anticipated that such methods will result in the identification of the human counterpart of the particular rat cDNA sequence described in the examples which follow. Also, the invention embraces the use of the subject polypeptide to make polyclonal antiserum or monoclonal antibodies specific to the subject polypeptide. These antiserum or monoclonal antibodies will be useful immunoprobes to identify related polypeptide, e.g. human polypeptide that are expressed under retinoic acid inducible conditions. Methods for making polyclonal antiserum and monoclonal antibodies are well known in the art. In general such methods involve immunizing an appropriate host against the polypeptide, typically of a different species, to generate antiserum. Monoclonal antibodies can be obtained by the production of hybridomas that secrete a specific immunoglobin against the polypeptide. Typical hosts for producing antiserum include hamsters, mice, guinea pigs, rabbits, monkeys, etc. Also, the present invention embraces the expression by recombinant methods of the cloned sequences disclosed herein and especially the polypeptide encoded by the cDNA below. l TTTΓTTTTAT AGAAGTGATG TTTGAATAGT TTTTGTCAGG GCAATGTCAG 51 CCCAATAGTT TTATTGGGCT CATTCTTCTA AGAAATAGTT ATGAATTTGA 101 GTCTCTGACT TGTATCATTC TGCCCAGGGA AATTCTATAG TACTGAAGTC 151 TTGGAGGCTG CCCAGCAACT CCACTGGGCA TTGCCATGTG CCTAGATATA 201 ATACCTCCAT GGCAGATTTC TGACTAGGCT GCCAGAGACT GTTAATTGCT 251 TCGTAGCTCA TGTATTCTGA ATACTTGCAC AATTAGTACC ACCAGGGCAT 301 TACTAAGGCT TACCATTGTG TTTTCCAGAT TTTTCAGTAG AATAATTGTC 351 CAGCCTAAGA TACACTTCAG CTTGTTTGCC CTATAGTGGT TATGTTGACT 401 GAAGGACAAC TATACTGAAA CTCAGAGAGC AGAGCCTGTA GTATTCTTGG 451 CAAGCAAATG GTGACATCCA GTTAGTACAT CTCTGTAATC TTACAATAAA 501 GATTCTTACT TGCCTTGAAA AAAAAAAA To produce the recombinant protein encoded by the sequence, this sequence can be incorporated into any suitable expression vector system. Typically, eukaryotic cells, and especially mammalian cells, are preferred. For example, the mammalian expression vector pcDNA3, available from Invitrogen, Carlesbad, California can be used. The plasmid containing the cDNA is then transfected into suitable cells, e.g., Chinese Hamster Ovary cells. The cells are cultured for an appropriate time, e.g., about 24 hours, to enable expression of the recombinant protein. The recombinant protein produced thereby is then isolated from the cell lysate or culture media by immunoaffinity column chromatography, dependent upon whether the protein is expressed using a secretion or intracellular expression system. EXAMPLE 1 Differential Display Protocol Used to Isolate Subject Cloned Sequences: RT-DD to identify differentially expressed band: 0.2 μg total cellular RNA was used as a template in a 20 μl reverse transcription reaction with Moloney mouse leukemia virus (MMLV) reverse transcriptase. One-tenth of the RT cDNA product was used for each differential display PCR. Each DD reaction is a 20 μl PCR carried out in a Perking Elmer 2400 for 40 cycles in the presence of Amplitaq DNA polymerase (Perking Elmer, Foster City, CA) and 10 μCi 33 P alpha dATP (New England Nuclear). The PCR products were separated by a 6% sequencing gel. Differential display with HAP-1 and HT U C as primer using the RT product from 4 sets of lung samples, namely Saline-Oil (SO), Saline-Retinoic Acid (SR), Elastase-Oil (EO), and Elastase-Retinoic Acid (ER), respectively , allows isolation of one differentially expressed band, rClRil.is expressed very lightly in the SO group (+), but expressed stronger in the SR group (++), and most strongly in the ER (+++) group. This protocol resulted in the isolation of rClRil (rat T U C-HAP1 primer, induced by by retinoic acid, band 1). As rClRil was the only RA induced band common between the control and elastase group after 96 displays, it was cloned and sequenced as set forth below. EXAMPLE 2 TA cloning and sequence determination of rClRil: The band corresponding to rClRil was sliced from the polyacrylamide gel and the amplified DNA was recovered by boiling as described in the RNA image protocol. The cDNA recovered was re-amplified in 40 μl reaction by 40 cycles of PCR using the same 5' and 3' primers used previously but without the radioactive dNTP. Amplified cDNAs were ligated into the pCR ® II TA vector (3.9 kb, Invitrogen, San Diego, CA) according to the protocol described in the TA Cloning Kit manual from Invitrogen. Thirty clones were picked and the cloned inserts examined by mapping with Eco RI (cloning site) and Rsa I. These clones are identified in Tables 1 through 3 supra. The insert of selected clones was sequenced using the Sanger dideoxy method with the USB sequencing kit (United States Biochemical, Cleveland, OH), with either Ml 3 forward or reverse primers. The results of this experiment indicated that the dDNA inserts of all 30 clones possessed the same restriction enzyme map, indicating that the rClRil band is homogeneous. rClRil was found to be 160 bp in size with 11 As 17 bp downstream from a classical AATAAA polyadenylation signal motif. AGCTTGATTC CCCTATAGTG GTTATGTTGA CTGAAGGACA ACTATACTGA AACTCAGAGA GCAGAGCCTG TAGTATTCTT GGCAAGCAAA TGGTGACATC CAGTTAGTAC ATCTCTGTAA TCTTACAATA AAGATTCTTA CTTGCCTTGA AAAAAAAAAA It was concluded, based thereon, that rClRil represents the cDNA of the 3' end of a poly (A) mRNA. The fact that the rClRil band was homogeneous suggested that differential display very effectively isolated differentially expressed genes from lung tissue, in particular genes induced or inhibited by retinoic acid. The entire sequence of rClRil was also used to search the GenBank database. The best score was human chromosome 12pl3 (U47924) sequence [reported by Ansari-Lari et al, Genome Res. 6(1966)314] with 60.7% identity in an 89 bp overlap [nucleotide 21-103 in rClRil and 84,000-84,088 in U47924] with one 1 bp gap and one 5 bp gap. The second best score was human V beta T-cell receptor (TCRBV) gene locus (U03115) with 90.0% identity in 20 bp overlap. The 12pl3 human chromosomal fragment contains 6 genes of known function and 2 genes of unknown function (Gene A and Gene B). Gene A has 3 alternative splicing forms. Gene B is distinct. However, Gene A and Gene B may also be one gene with a collection of alternative transcripts. Exon 4 of Gene A3 is spliced into exon 1 of Gene B. Gene A and its variants are present mainly in the brain, with low level in the liver. It is not expressed in the lung. Gene B is present in a very low level in the heart, lung, ovary and skeletal muscle. Gene A shows no similarity to any known protein. Gene A3 has 7 transmembrane domains. Gene B shows a weak similarity (16.5% at the protein level) to the rat synaptonemal complex protein Sc 65. It is hypothesized by the inventor that overlap with ClRil will likely be at the 5 th exon of Gene Al and Gene A2. Sequence comparison result in the following observations: (i) rClRil vs Al - 40.63% starting at nt 1 -end vs nt 630-786, with one 3 bp gap; (ii) rClRil vs A2 - 40.63% starting at nt 13-end vs nt 1633-1777, with one 3 bp gap; (iii) the overlapping regions of Al and A2 with rClRil are the same; (iv) rClRil vs A3 - 38.61%, starting at nt 7-end vs nt 445-589, with one 2 bp gap; and (v) rClRil vs B - 38.75%, starting at nt 7-end vs nt 567-801 , with one 3 bp gap- As noted, the 12pl2-13 chromosomal map position is correlated to emphysema due to alpha-2-macroglobulin (A2M) deficiency. In this chromosomal locus are : SGC31674 alpha 2-macroglobulin precursor, 12pl2.3-13.3 SCG34687 highly homologous to cellular retinol-binding protein I WI- 15438 proliferating-cell nucleolar antigen P 120 SGC32489 ubiquitin carboxyl-terminal hydrolase. Also, rClRil was compared with various rat alpha-2-macroglobulin gene from the GenBank. The results of this comparison are: (i) rClRil vs rat A2M mRNA (M84368) - 38.5%, with one 5 bp gap; (ii) rClRil vs rat A2M mRNA (M84369) - 46.38%, nt 81-150 vs nt 1-68, one 2 bp gap; (iii) rClRil vs rat A2M (M84360) - 34.92%, nt 20-145 vs nt 1-126; (iv) rClRil vs rat A2M (M22667, exon 1 ) - 40.63%, nt 5-end vs nt 1107- 1266, one 5 bp gap; (v) rClRil vs rat A2M (M22668, exon 2) - 41.875%, nt 11-end vs nt 52-210, one 2 bp and one 7 bp gap; (vi) rCiRIL vx rat A2M (M22669, exons 3 and 4) - 35.625%, nt 7-end vs 70- 220, no gap; (vii) rCiRIL vs rat A2M (M22670, exons 5 and 6) - 41.875%, nt 4-end vs 111- 269, no gap. However, the comparisons of rClRil vs M84369, 84370, 22667, 22668, and possibly 22669, may have little significance because rClRil is probably the 3' end of a gene. By contrast, the regions in the A2M represented by these sequences do not correspond to the 3' end of rat A2M because the comparison starts at nucleotide 1 or early exons. Based on these comparisons, it was concluded that: (1) rClRil is not A2M because sequence comparison shows that the two genes are not identical. (2) rClRil is not cellular retinol binding protein 1 because the two genes are most likely not in the same locus. (3) rClRil is likely to be the rat analog of gene A/B in human which is not expressed or expressed at a very low level in the lung without stimulation. (4) Co-localization of rClRil with emphysema due to A2M deficiency suggests that absence of rClRil expression and A2M (due to chromosome deletion) can lead to the development of emphysema. EXAMPLE 3: Construction of Expression Plasmid Based on its potential role in alveoli development, rClRil was expressed by recombinant methods, as follows. The cDNA was obtained from the cloning vector, pBluescript. The cDNA construct was cloned at the Xbal site in the vector upstream of the insert. After addition of a linker containing Xbal, Bgl II and Sstl sites to the 5' end of the insert, the construct was closed again. The construct was then double digested with Bglll and Notl. The recognition sequences of both enzymes were upstream of the translation initiation signal of the cDNA insert. A linker containing Notl, Sail and Bglll was inserted into the construct to close it again. The construct was then double digested with Sail and Hind III, which generated a cDNA with a Sail site on the 5' end and a Hindlll site on the 3' end. The cDNA was then cloned into the multiple cloning site of the mammalian expression vector pcDNA3 predigested with Sail and Hindlll to produce the expression construct. Transfection and Expression Chinese hamster ovary-Kl cells (ATCC CCL61) which were purchased from the American Type Culture Collection, cells were grown in Ham's F21 media supplemented with 10% FBS and 1% antibiotic/antimycotic solution. The cells were cultured at 37 °C in 5% C0 2 /95% air. Culture media was changed twice weekly. (Culture components were purchased from GIBCO/BRL, Gaithersburg, Maryland.) CHO cells were transfected with the expression construct by lipofectin. Cells were seeded in T-75 flasks at 5 X 10 5 per flask. Sixty μg of lipofectin (GIBCO/BRL) were added to 3.5 ml of Opti-MEM containing 0.1% β- mercaptoethanol and 1% non-essential amino acid. Twenty μg of the construct DNA linearized with the enzyme Aatll was dissolved in 1.5 ml Opti-MEM. The DNA/media mixture was filtered through a 0.2 μm, Anotop 10 filter (Alltech Associates, Inc, Deerfield, Illinois) into the Opti-MEM/lipofectin mixture and the solution added to the T-75 flask. After 24 hours, 10 ml of Ham's F12 medium supplemented with 10% FBS was added. After another 24 hours, the medium was removed and 10 ml of Ham's F12 medium supplemented with 10% FBS, 1% antibiotic/antimycotic, 2mM glutamine and 1 mg/ml G418 (Geneticin) were added. The cells were grown in this medium for 10 days. G418 resistant CHO transfectants were tested for expression of the cloned cDNA by nested polymerase chain reaction. Transfectants expressing the cDNA were cloned by limiting dilution. Therapeutic Use The resultant protein can be administered in combination with a pharmaceutically acceptable carrier such as saline to patients having decreased air exchange at the alveolar level. Alternatively, the protein may be administered transdermally with known transdermal carriers such as DMSO. In general, the dosage will be about 0.05 to 0.1 mg/kg body weight. The protein may also be administered to the mucous membranes by means such as inhalation. The protein may also be administered in the form of cyclodextrin inclusion complexes or by transdermal means such as by patch. While the invention has been described with respect to certain specific embodiments, it will be appreciated that many modifications and changes thereof may be made by those skilled in the art without departing from the spirit of the invention. It is intended, therefore, by the appended claims to cover all modifications and changes that fall within the true spirit and scope of the invention.

Claims: WHAT IS CLAIMED IS: 1. A protein encoded by a cDNA of the formula: 1. TTTTTTTTAT AGAAGTGATG TTTGAATAGT TTTTGTCAGG GCAATGTCAG 51 CCCAATAGTT TTATTGGGCT CATTCTTCTA AGAAATAGTT ATGAATTTGA 101 GTCTCTGACT TGTATCATTC TGCCCAGGGA AATTCTATAG TACTGAAGTC 151 TTGGAGGCTG CCCAGCAACT CCACTGGGCA TTGCCATGTG CCTAGATATA 201 ATACCTCCAT GGCAGATTTC TGACTAGGCT GCCAGAGACT GTTAATTGCT 251 TCGTAGCTCA TGTATTCTGA ATACTTGCAC AATTAGTACC ACCAGGGCAT 301 TACTAAGGCT TACCATTGTG TTTTCCAGAT TTTTCAGTAG AATAATTGTC 351 CAGCCTAAGA TACACTTCAG CTTGTTTGCC CTATAGTGGT TATGTTGACT 401 GAAGGACAAC TATACTGAAA CTCAGAGAGC AGAGCCTGTA GTATTCTTGG 451 CAAGCAAATG GTGACATCCA GTTAGTACAT CTCTGTAATC TTACAATAAA 501 GATTCTTACT TGCCTTGAAA AAAAAAAA. 2. A nucleic acid sequence selected from the group consisting of (i) the cDNA recited in Claim 1; (ii) a nucleic acid sequence that specifically hybridizes thereto under high stringency conditions which contains a nucleic acid sequence of at least 100 nucleotides that is at least 80% identical to a portion of said cDNA when aligned to said cDNA without allowing for any gaps; and (iii) a nucleic acid sequence that encodes a polypeptide having the same amino acid sequence as the cDN A of (i) or (ii). 3. An antibody or polyclonal antiserum produced against a polypeptide encoded by the nucleic acid sequence of Claim 2. 4. An improved method of identifying a nucleic acid sequence by differential display wherein the improvement comprises screening a cDNA library obtained from lung tissue cultured in the presence and/or absence of retinoic acid to identify a cDNA that is induced or inhibited by retinoic acid and which encodes a polypeptide that promotes alveolar formation. 5. The method of Claim 4 wherein said cDNA libraries are generated against human lung tissue cultured in the presence or absence of retinoic acid. 6. A method of therapy which results in the promotion of alveolar formation which comprises administering a therapeutically effective amount of a polypeptide according to Claim 1 or a fragment thereof of at least 50 amino acids which promotes alveolar formation. 7. The method of Claim 6 which is used to treat emphysema in infants having immature lung development. 8. The method of Claim 6 wherein said polypeptide is administered via inhalation. 9. A method of identifying compounds that affect cell differentiation and/or proliferation which method comprises screening a compound library to identify a compound that specifically binds to a polypeptide encoded by the nucleic acid sequence of Claim 2. 10. The method of Claim 9 wherein said compound binds to the polypeptide encoded by the cDNA having the sequence 1. TTTTTTTTAT AGAAGTGATG TTTGAATAGT TTTTGTCAGG GCAATGTCAG 51 CCCAATAGTT TTATTGGGCT CATTCTTCTA AGAAATAGTT ATGAATTTGA 101 GTCTCTGACT TGTATCATTC TGCCCAGGGA AATTCTATAG TACTGAAGTC 151 TTGGAGGCTG CCCAGCAACT CCACTGGGCA TTGCCATGTG CCTAGATATA 201 ATACCTCCAT GGCAGATTTC TGACTAGGCT GCCAGAGACT GTTAATTGCT 251 TCGTAGCTCA TGTATTCTGA ATACTTGCAC AATTAGTACC ACCAGGGCAT 301 TACTAAGGCT TACCATTGTG TTTTCCAGAT TTTTCAGTAG AATAATTGTC 351 CAGCCTAAGA TACACTTCAG CTTGTTTGCC CTATAGTGGT TATGTTGACT 401 GAAGGACAAC TATACTGAAA CTCAGAGAGC AGAGCCTGTA GTATTCTTGG 451 CAAGCAAATG GTGACATCCA GTTAGTACAT CTCTGTAATC TTACAATAAA 501 GATTCTTACT TGCCTTGAAA AAAAAAAA. 11. A nucleic acid sequence which contains a cDNA selected from the group consisting of: A15Rrl, A15Rr3b, G51Rrla, C72Rrla, C72Rrlb, A51Rilb, A54Rilc, A55Rila/b, A59Rilc, A59Ri2c, C71Rila, C72Rila, C72Rilb, A15Rr3a, C16Rrl/2, C16Rr3/4/5, C42Rrla, A15Ril, G42Ril, A51Rila, G51Rila, A54Rila, A54Rilb, A59Rila, A59Rilb, A59Ri2a, A59Ri2b, C71Rilb, C71Ri2, A15Rr3c, C43Rrlb, G51Rrlb, ClRil, A51Ri2, G51Rilb. 12. A pharmaceutical composition containing a polypeptide encoded by the cDNA of Claim 1 and pharmaceutically acceptable carrier.

Inventors: CHAN WAI-YEE, CHAN, WAI-YEE
Applicants: UNIV GEORGETOWN MED CENTER, GEORGETOWN UNIVERSITY MEDICAL CENTER
Application year: 1999
Main classification: A61K38
IPC codes: A61K38, C07K14, C12Q1, G01N33