Vol. 10, No. 7

MOLECULAR AND CELLULAR BIOLOGY, JUlY 1990, p. 3663-3674

0270-7306/90/073663-12$02.00/0

Copyright © 1990, American Society for Microbiology

Activated T Cells Express a Novel Gene on Chromosome 8 That Is Closely Related to the Murine Ecotropic Retroviral Receptor CAROL L. MAcLEOD,1* KIM FINLEY,1 DONALD KAKUDA,1 CHRISTINE A. KOZAK,2 AND MILES F. WILKINSON3

Cancer Biology Program, Department of Medicine and Cancer Center, University of California, San Diego, La Jolla, California 920931; Department of Microbiology and Immunology, Oregon Health Sciences University, Portland, Oregon 972013; and Laboratory of Molecular Microbiology, National Institute ofAllergy and Infectious Diseases, Bethesda, Maryland 208922 Received 22 January 1990/Accepted 19 April 1990

A novel cDNA done (20.5) which is differentially expressed between two closely related T-lymphoma cell clones was isolated by subtraction-enriched differential screening. SL12.4 cells, from which the cDNA was isolated, have characteristics of thymocytes at an intermediate stage in development. A sister cell clone derived from the same tumor, SL12.3, does not express this mRNA, has a distinct phenotype, and expresses fewer genes required for mature T-cell function. The cDNA sequence predicts a highly hydrophobic protein (-49.5 kilodaltons) which contains seven putative membrane spanning domains. The gene was expressed on concanavalin A-activated T lymphocytes and was designated Tea (T-cell early activation gene). The Tea gene mapped to chromosome 8 and appeared to be conserved among mammalian and avian species. The Tea gene is distinct from, but bears extensive amino acid and DNA sequence similarity with, the murine ecotropic retroviral receptor which is encoded by the Rec-l gene. Neither gene product displayed significant homology with other known transmembrane-spanning proteins. Thus, the Tea and Rec-l genes establish a new family encoding multiple membrane-spanning proteins.

A number of genes have been identified which are first expressed in developing thymocytes (reviewed in reference 14). Several encode proteins which are required for the thymocytes to mature into T cells capable of functioning in the immune system, for example, (i) the T-cell receptor for antigen (TCR) and the associated CD3 proteins which are required for antigen recognition and signaling; (ii) CD24 (the interleukin-2 [IL-2] receptor) which must be expressed for the cells to respond to the cytokine IL-2; (iii) gene products important for signal transduction after antigen recognition, such as CD3, CD4, CD8, and CD45; (iv) gene products involved in T-cell homing to peripheral target organs; and (v) genes of unknown function involved in T-cell activation (7, 14, 20, 33, 44). There is remarkable heterogeneity in thymocyte subsets which express different combinations of expressed genes (reviewed in references 1, 14, and 44). Thus, it is likely that many genes that encode products which function in T-cell development, homing, or immune responsiveness remain to be identified. In an effort to isolate from T cells novel cDNA clones which identify new functions, we have chosen to use two closely related T-lymphoma cell clones on the basis of the following reasoning. (i) Numerically infrequent, transient progenitor cells are frequently the target of transformation to malignancy (reviewed in references 16, 19, and 20), and many of their characteristics are preserved in the tumor cells (16, 20, 41). (ii) The heterogeneity of lymphoma cells and cell lines often results from differences in the extent of maturation reached by individual cells present in the population (16, 20). Thus, T-lymphoma cell heterogeneity can be exploited to obtain, from a single individual, closely related cloned cell lines which differ in a limited number of characteristics. These cell lines provide an *

opportunity to work with pure populations of cells with defined and stable phenotypes. Toward this end, the SL12 T-lymphoma model system was developed (23, 34a-37). Two cell clones derived from a single SL12 T-lymphoma cell line were chosen for the isolation of novel, differentially expressed genes because of their known differences in gene expression and their different capacities to cause tumors in syngeneic host animals (23, 34a-37, 47, 51; see Table 1 for a summary of phenotypes). SL12.3 cells express very few of the genes required for T-cell function, and they are highly tumorigenic in syngeneic animals (36, 37). In contrast, the cells of a sister clone, SL12.4, express mRNAs for all the components of the TCR-CD3 complex except TCR-a and in several respects are similar to thymocytes at an intermediate stage in development (36, 51). SL12.4 cells are much less tumorigenic than SL12.3 cells (37). We sought to isolate novel cDNA clones from SL12.4 cells in a effort to identify genes which might be associated with their different capacity to cause tumors in syngeneic mice or those genes which function in T-cell development. In an approach similar to that used by Filmus et al. (13), we used a combination of subtraction hybridization-enriched probes (24, 34a, 49) and classical differential screening (45) to obtain cDNA clones representing genes which were preferentially expressed in the SL12.4 T-cell clone and undetectably expressed in a sister cell clone, SL12.3 (34a). One cDNA clone, 20.5, identified transcripts found in only a limited number of tissues. Tea transcripts were induced in splenocytes activated with the T-cell mitogen concanavalin A (ConA). Unlike other known genes expressed in activated T cells, the Tea gene appears to encode a protein which traverses the membrane multiple times, whereas the large number of known integral membrane proteins which are induced in T-cell activation are single-membrane-spanning proteins (9).

Corresponding author. 3663

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MAcLEOD ET AL.

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MATERIALS AND METHODS Cells. The isolation and culture requirements of the SL12.3 and SL12.4 cloned cell lines have been previously described (35, 37). The phenotypes of the two cell lines SL12.3 and SL12.4 are listed in Table 1 and have been described in detail elsewhere (23, 35-37; MacLeod et al., in press). Cell lines from the following sources were used in the expression studies: embryonal carcinomas F9 and PCC4 (4), pituitary tumor ATt2O (6), thymic epithelial TEPI (3), mammary epithelial (10), BALB/c 3T3 (ATCC CCL 92), and murine embryo fibroblasts (MEF) prepared according to the method of Freshney (15). The cells were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum-glutamine-penicillin-streptomycin. Cells that were used to prepare RNA were harvested during exponential growth from cultures containing 5 x 105 to 8 x 105 cells per ml. Splenocytes obtained from BALB/c mice were seeded at 3 x 106 cells per ml in RPMI 1640 supplemented as described above and stimulated with 10 ,ug of ConA per ml for 0, 6, 24, or 48 h before the harvest of RNA. cDNA library construction and screening. Cytoplasmic poly(A)+ mRNA from SL12.4 cells was used as a template to prepare double-stranded cDNA by a modification of the Gubler and Hoffman method (18); EcoRI linkers were added to the double-stranded DNA, which was previously methylated. Dephosphorylated K gtlO arms (Stratagene, La Jolla, Calif.) were ligated to the cDNA and packaged into bacteriophage A with Stratagene packaging extract according to the instructions of the supplier. Subtraction hybridization was performed essentially as originally described by Timberlake (49) and modified by Hedrick et al. (24). Single-stranded cDNA was prepared from 10 ,g of poly(A)+ SL12.4 RNA with 250 ,uCi of [32P]dCTP (Amersham Corp., Arlington Heights, Ill.) in the presence of 100 ,ug of actinomycin A per ml (24) and hybridized to a Rot of 1,260 (moles of nucleotide per liter x seconds) with 25 ,ug of poly(A)+ RNA from SL12.3 cells in a volume of 8 ,ul at 68°C for 18 h. After hybridization, the single-stranded cDNA was collected by chromatography through a hydroxyapatite column (24, 32). From 1 jig of starting SL12.4 cDNA, approximately 120 ng (12% of the input cDNA containing 3 x 107 cpm) was recovered and used to probe two 150-mm nitrocellulose filters containing 20,000 A gtlO plaques per filter. The first of two duplicate filter lifts from the SL12.4 library was probed with total cDNA from SL12.3 mRNA, and the second filter lift was probed with the SL12.4 subtraction-enriched cDNA prepared as described above. The plaque-purified K phage clone was first identified as SL12.4 specific by two screenings with separately prepared subtracted probes; subsequently, Northern (RNA) analysis was used to confirm that the clone hybridized to mRNA only from SL12.4 cells and not from SL12.3 cells (34a). The cDNA insert was removed from K DNA by digestion with the restriction enzymes HindIII and BglII and subcloned into the plasmid vector pT7/T3 (Bethesda Research Laboratories, Inc., Gaithersburg, Md.). The inserts could not be excised from the A phage with EcoRI because the EcoRI sites were damaged as previously described by Kuziel and Tucker (31). Northern blot analysis. Total cellular RNA was isolated from SL12.4, SL12.3, and fresh tissue preparations from BALB/c mice by the guanidinium isothiocyanate method (34a), modified as described elsewhere (51). Cytoplasmic or nuclear RNA was prepared as previously described (51). For Northern analysis, 10 ,ug of RNA was electrophoresed in 1% formaldehyde agarose gels and transferred to nitrocellulose

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membranes (38, 45). Equal loading and transfer of RNA per lane was assessed by acridine orange staining (45) and by hybridization with murine actin, cyclophyllin (48), and CHO-A (21) cDNA. For the quantitation of spleen cell induction of 20.5 transcripts, Northern blots were hybridized with random-primed (Amersham), 32P-labeled cDNA inserts in the presence of 10o dextran sulfate and 50% formamide for 12 to 18 h at 42°C (45) and washed stepwise as described previously (36, 45) with a final 30-min wash in 0.1 x SSPE (45)-0.1% sodium dodecyl sulfate at 42°C. The blots were exposed to film, stripped, and probed with actin cDNA probes. After exposure of the film within the linear range, the autoradiograms were assessed by densitometry with a laser scanner. A ratio of 20.5 transcripts to actin transcripts was made, and the specific induction of 20.5 mRNA was calculated from that ratio. Southern blot analysis. Total cellular DNA was isolated from SL12.4 cells, murine and hamster livers, and somatic cell hybrids as described elsewhere (45). DNA from chicken and human livers was obtained commercially from Clonetec, Palo Alto, Calif. The DNA was digested with the restriction enzymes noted in the figure legends according to the instructions of the supplier. Digested DNA (10 ,ug) was applied to each lane of a 0.8% agarose gel, electrophoresed in Tris acetate buffer for at least 48 h, blotted onto Nytran supports essentially as described previously (38), hybridized, and washed as described for Northern blot analysis. The blots containing DNA from other species were washed at a lower stringency, and the final wash was carried out at room temperature with 2x SSPE. DNA sequence analysis. From a restriction endonuclease map, fragments were subcloned into pT7T3, and the plasmid was purified on cesium chloride and directly sequenced by double-stranded dideoxy-sequencing methods with Sequenase reagents (U.S. Biochemical Corp., Cleveland, Ohio). Part of the sequence was determined with primers to the host plasmid, and other specific oligonucleotide primers (17mers) were prepared to the cDNA in the University of California San Diego (UCSD) Cancer Center Core Molecular Biology Facility. Both DNA strands were sequenced in their entirety, and all sequences were determined in at least two reactions performed in duplicate. Microgenie (Beckman Instruments, Inc., Fullerton, Calif.) computer programs were used to assemble the overlapping sequence information and perform the initial analysis of the DNA sequence. Further sequence analysis of the DNA and predicted protein was performed by using IntelliGenetics programs (PC GENE). Genetic mapping. The production and characterization of Chinese hamster X mouse somatic cell hybrids has been described previously (25). NFS/N strain mice were obtained from the Division of Natural Resources, National Institutes of Health, Bethesda, Md. Mus musculus musculus mice were obtained from a laboratory colony derived from mice originally trapped in Skive, Denmark, and maintained by M. Potter (National Cancer Institute contract NO1-CB2-5584) at Hazelton Laboratories, Rockville, Md. Hybrid NFSIN x M. musculus musculus females were backcrossed with M. musculus musculus males to produce the experimental animals. DNA from mouse livers was digested with Sacl and BamHI, electrophoresed in 0.4% agarose gels for 48 h, transferred to Hybond N+ membranes (Amersham), and hybridized with the 32P-labeled 20.5 cDNA or a 438-base-pair (bp) probe representing the DNA polymerase B gene which is present on chromosome 8 (O. W. MacBride, C. A. Kozak, and S. H. Wilson, Cytogenet. Cell Genet., in press). Kidney samples

ISOLATION OF NOVEL MURINE cDNA CLONE

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from the same mice were typed for inheritance of the markers Gr-J and Es-i by histochemical staining after electrophoresis on starch gels (22). The most likely gene order was determined from the recombinant inbred mouse experiments by minimizing the number of crossovers among all of the genes (5, 17). RESULTS Isolation and DNA sequence of the 20.5 cDNA clone. A 40,000-member SL12.4 cDNA library in X gtlO (34a) was screened with an SL12.4 cDNA probe subtracted against SL12.3 mRNA (24, 49) and, simultaneously, on duplicate filters with total SL12.3 cDNA (13, 49) as described in Materials and Methods. The characterization of one of several cDNAs isolated by this screening method is the subject of this report. On the basis of the expression characteristics presented below, the cDNA clone has been designated Tea (T-cell early-activation gene). The insert from this clone was sequenced by double-stranded methods on both strands, and the sequence is shown in Fig. 1. The cDNA insert was 2,397 bp in length and contained a single long open reading frame beginning at bp 409 and extending to bp 1769. The cDNA did not contain a polyadenylation signal sequence or a poly(A) tract. The 20.5 cDNA sequence predicts a multiple-membranespanning protein. Figure 1 shows the 453-amino-acid sequence of the predicted protein which has a molecular mass of 49.57 kilodaltons (unmodified). The cDNA sequence may contain the entire coding region since the predicted Nterminal methionine codon is surrounded by a Kozak consensus sequence (GXC AUG G, where X is any nucleotide), which is an optimal translation start site (29), and the predicted 5' and 3' untranslated region contains multiple stop codons. The physical properties of the predicted protein were analyzed by using Microgenie and PC GENE programs. Three potential N-glycosylation sites are represented by stars in Fig. 1. The predicted protein has nine highly hydrophobic regions (underlined in Fig. 1); seven of these have characteristics of transmembrane-spanning domains, on the basis of an analysis with the IntelliGenetics software programs SOAP, HELIXMEM, NOVOTNY, and RAOARGOS. The Tea gene is differentially expressed in T-lymphoma cells and activated T-lymphoid cells from normal spleen. The expression of transcripts recognized by the 20.5 cDNA clone was assessed. The 20.5 cDNA probe recognized two transcripts of approximately 4.5 and 8.5 kilobases (kb) which are present in SL12.4 cells but not in SL12.3 T-lymphoma cells (Fig. 2A). The subcellular location of the two transcripts was examined to determine whether they were both mature transcripts found in the cytoplasm or whether the larger RNA was a nuclear precursor. Both transcripts appeared to be fully processed RNAs since they were found in the cytoplasm (Fig. 2B). The origin of the two transcripts is not yet known; they could arise from alternate initiation of transcription, alternate splicing of the transcript, or utilization of alternate polyadenylation signals (50). Both of the mature cytoplasmic transcripts (8.5 and 4.5 kb) are larger than the cDNA clone (2.4 kb). Thus, our cDNA is not full length, although it appears to contain the entire coding region (see preceding paragraph). A 3' probe (nucleotides 2005 to 2394) detected both mature transcripts, suggesting that the long 3' region is not an intron sequence (data not shown). Both transcripts were also detected with a probe made to a 5' region (nucleotides 1 to 380) of the cDNA clone,

3666

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TATGTCCGTGTGCGTACATGTATGTCTGCGATGTGAGTGTTCAATGTTGTCCGTTATTAG TCTGTGACATAATTCCAGCATGGTAATTGGTGGCATATACTGCACACACTAGTAAACAGT ATATTGCTGAATAGAGATGTATTCTGTATATGTCCTAGGTGGCTGGGGAAATAGTGGTGG TTTCTTTATTAGGTATATGACCATCAGTTTGGACATACTGAAATGCCATCCCCTGTCAGG

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FIG. 1. DNA and predicted protein sequence of clone 20.5 cDNA. The DNA sequence was determined by sequencing both strands from subclones and across junction points in the intact insert with primers specific for the pT7T3 plasmid (Bethesda Research Laboratories) and the 20.5 insert. Microgenie (Beckman) programs were used to prepare the predicted amino acid sequence, identify the potential N-linked glycosylation sites (*), and identify the highly hydrophobic transmembrane-spanning domains (underlined). The sequence has been deposited with GenBank (accession no. M32485).

indicating that the cDNA is not a cloning artifact which joined two different transcripts (data not shown). In addition to the two mature RNAs there were several larger, much less abundant transcripts present in the nuclear and total cell RNA preparations that may be unspliced or partially spliced nuclear precursors (Fig. 2B). Several murine cell lines of embryonic (F9 and PCC4), mammary epithelial (MME), and neuronal (ATt2O) origin were examined for the expression of Tea RNA. Only PCC4 had barely detectable Tea RNA; the other cell lines did not express detectable Tea transcripts. In contrast, cell lines of thymic epithelial (TEPI) and fibroblast (BALB/c 3T3, MEF) origin contained reasonable amounts of Tea mRNA, although at much lower levels than those expressed in SL12.4 T-lymphoma cells (Fig. 2C). To determine whether normal tissues and cells of the lymphoid lineage express the Tea gene, Northern blots prepared from murine tissue mRNA were examined. Cells from thymus, quiescent spleen, gut-associated lymphoid tissue, and bone marrow lacked detectable expression of Tea mRNA (Fig. 2D). However, Tea transcripts were induced in normal spleen cells activated with the T-cell mitogen ConA (Fig. 2D). ConA was used to mimic the activation of splenic T cells which normally occurs in a cell clone-specific manner upon the appropriate presentation of foreign antigen (9). Liver was the only nonlymphoid tissue tested which expressed moderate amounts of Tea mRNA. Tea transcripts were undetectable in intestine, stomach, ovary (Fig. 2D), brain, heart, lung, kidney, pancreas, or testes (data not shown). Thus, it is apparent that Tea gene expression is limited to a few cell types, such as activated spleen, thymic epithelial, T-lymphoma, and liver cells.

To further investigate the induction of Tea mRNA, we prepared RNA 6, 24, and 48 h after ConA was added to splenocytes (Fig. 3). The Tea transcripts are not detectable in quiescent spleen lymphocytes but become detectable within 6 h, peaking at about 24 h. Both the 8.5- and 4.5-kb transcripts present in SL12.4 cells were induced during activation. Two additional transcripts were also detectable in the activated spleen cells, one of about 9 kb and one of about 3.8 kb. Neither of these represent cross hybridization to the murine ecotropic retroviral receptor (ERR) (see following paragraph). The RNA from spleen cells was derived from total cellular RNA; thus, those additional transcripts could be nuclear products which did not get transported to the cytoplasm (Fig. 2). The total amount of RNA was equivalent in each lane (10 ,ug), as assessed by acridine orange staining. However, the relative ratio of rRNA to mRNA changed during T-cell activation because the amount of actin, CHO-A, and cyclophyllin transcripts per 10 p,g of total RNA increased (Fig. 3; data not shown). However, there was clearly a specific induction of Tea gene expression relative to these control RNAs during T-cell activation since the induction was greater than 16-fold (at 24 h) even when normalized to the amount of actin mRNA (see Materials and Methods). Similarity of the 20.5 DNA and amino acid sequence with the murine ERR. Homology searches with the Bionet data base revealed no significant sequence similarity between 20.5 cDNA and other DNA sequences previously reported (11). However, the sequence was compared with that in a recent report (2) of the murine ERR cDNA clone and found to have extensive sequence identity. The gene which encodes the ERR has been designated Rec-J (28). Figure 4

ISOLATION OF NOVEL MURINE cDNA CLONE

VOL. 10, 1990

3667

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FIG. 2. Tea gene expression. Purified inserts containing the entire coding region were labeled with 32P by random priming and used to probe Northern blots in which each lane contained 10 p.g of total cellular RNA (or in the case of panel B, cytoplasmic and nuclear RNA) from SL12.4 or SL12.3 cells, the indicated cell lines, or tissues from normal BALB/c mice. Autoradiograms of Northern blots are shown. (A) Comparison of Tea mRNA expression in SL12.3 and SL12.4 cell lines, (B) Tea mRNA expression in total cellular, cytoplasmic, and nuclear RNA from SL12.4 cells; (C) Tea mRNA expression in a series of murine cell lines described in the text, (D) RNA from the indicated normal tissues from BALB/c mice (GALT, gut-associated lymphoid tissue). The transcripts are 8.5 and 4.5 kb, as estimated by their relative migration against Bethesda Research Laboratories markers and 18 and 28S endogenous rRNA. Equivalent loading and transfer of RNA in all lanes was assessed by acridine orange staining and by hybridization with 32P-cyclophyllin (Cyc) (38) or 32P-CHO-A- (14) labeled cDNA.

shows a comparison of the 20.5 cDNA sequence with the ERR cDNA sequence. The sketch of the two cDNAs (Fig. 4) shows that the ERR cDNA is longer at the 5' end, while the 20.5 cDNA sequence is much longer at the 3' end; the two cDNAs are of similar overall length. The predicted coding region is depicted in Fig. 4 by the cross-hatched portion of each cDNA clone. The DNA alignment reveals an overall

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FIG. 3. Induction of Tea gene expression during T-cell activation. Northern analysis of RNA from quiescent and activated spleen cells harvested at the indicated times following activation with the T-cell mitogen, ConA. RNA from SL12.4 cells was included for comparison. The lower panel shows the same blot stripped and subsequently probed with murine actin to assess the amount of RNA loaded in each lane. Although 10 ,ug of RNA was loaded in each lane and appeared evenly loaded by acridine orange staining (data not shown), the amount of actin mRNA increased during T-cell activation (see text).

DNA sequence identity between the overlapping regions of 20.5 cDNA (bp 1 to 2047) and ERR cDNA (bp 400 to 2425) of 59%; one region of 20.5 cDNA (bp 1011 to 1088) is 80% identical. The 5' noncoding region of the 20.5 cDNA sequence (bp 1 to 410) has 68% sequence identity with the overlapping 5' coding region of the ERR cDNA sequence, suggesting that the two genes were derived from a common sequence through a gene duplication event. Although the two sequences are highly related, they did not cross hybridize on Northern blots in the conditions used in our experiments (data not shown). The predicted Tea protein has structural similarity to other proteins with multiple membrane-spanning domains, such as the transducin proteins, ion channels, ion pumps, and sugar transport (8, 30, 34, 39, 46, 52). However, neither has significant sequence similarity (11) with these gene families nor any significant similarity with other known protein sequences, with the notable exception of the ERR predicted protein (2). An alignment of the Tea predicted protein with the ERR protein shows two regions of extensive amino acid sequence similarity (Fig. 5). Region 1, defined in Fig. 5 by parentheses, had 81% sequence identity and 91% similarity over 193 amino acids. Region 2 had 62% sequence identity and 75% similarity over a length of 60 amino acids. Conservative amino acid differences (11) are indicated in Fig. 5 by two dots, and amino acid identities are shown by long dashes between the rows. In contrast to the Tea predicted gene product, the ERR protein is larger and it has 13 to 14 predicted transmembranespanning regions (2). Furthermore, it has a shorter hydrophiic carboxy terminus and an amino terminus of similar length. The predicted carboxy and amino termini of the proteins show the most sequence divergence. Similar to ERR and other multiple membrane-spanning proteins, the Tea gene product contains no signal sequence (2, 34). The comparison shows extensive regions of amino acid similarity, yet the two proteins are clearly distinct gene products. Figure 6 shows a sketch of the predicted protein structure

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GCGTGTCTTTCCTCATCGCTGCCCTGGCCTCGGTTATGGCCGGCCTTTGCTATGCTGATTTGCGGCCCGAGTACCCAAGACTGGATCTGCGTATCTATACACTTACGTCACGGTCGGAG ATCTCCT TCT TGATTGCTGC.TCTCGCCTCCGTGCTGCCGCGCCCTGTGCTACGGCGAGT TTGGTGCCCGCTGTCCCCAACACGGGCTCAGCCTACCTCTACAGCTACGTGACGGTGGGGG

1#0

121 AGCTGTGGGCCTTCATCACTGGCTGGATCTCATCCTGTCATATGTCATAGGTACGTCCAGTGTCGCAAGAGCATGGAGTGGCACCTTTGACGAACTTCTTATACAGA TGGCCCAGT

i l llti1l11 1111111 111tl 11111Il 1i lilt111ti1I1111111i1l111l11111tit111i11l1 i 11111 1111111111111111

S 1J AGCTTTGGCCTT*CATCACTGGCTCGGACCTGAT TCTCTCCTACATCAT CGGTACTYCAAGCGTGGCAAGAGCCTGGAGTGCGACTTTCGACGAGCTGATAGGAGCCTCGGAGAGT 241

TTTTCAAAACGTACTTCAAAATGAAT

TACACTGGT CTGGCAGAGTATCCAGACTTCTTTGCCGTGTGCCTTGTATTACTCCTGGCAGGTCTTTTATCTTTTGAGTACGAGTCTG

358 CTTGGGTGAA TTTTTTACAGCTATTATATCCTGGTCCTTCTCTTTGTCATTGGCTGGGTTTGTGAAAGGATGtGCCTGGAGATCAGTGAAGAGTTTCTCTA

I

11111 111111111

ill ii lli 11111

liltl 111 1111111111111111111

111111 11111 1 ill

111111

758 CCATGGtCAACAAAATTT CACCTGTATCAATGTCCtGGTCtTGTGCTTCATCGTGGTGTCCGGGTTCGTGAAAGGCTCCATTAAAAACTGGCAGCTCA CG GAG

AAAAATT

47n TATCAGCAAGTGCTAGAGAACCACCTTCTGAGAACGGCAACAACATCTACGGGGCTGGCGGCTTTATGCCCTATGGCT TTACAGGGACGTTGGCTGTGCTGTCAACGTGCT TTTAtGCCT

ii11I

869 TCTC C

11111 11111 I1 l1111 ill1

111111l 11111i11111111l111111 111

111l111111111111111111111t

TACGGTGAGGGAGGGTTTATGCCCTTTGGATTCTCTGGTGTCCTGTCAGGGGCAGCGACCTGCTTTTATGCCT

tG TA ACM CAACGACACA AACGTGA AA

597 tTTGTGGCTTTGACTGCAT TGCAACAACCGGTGAAGAGTTCGGAATCCACAAAGGCGATCCCCATCGGAATAGTGACGCtCTTACTTGTCTGCtTTTATGGCT TACT TTGGGCTTTCTG

I illllliilitilli1i ii 11 1111111 ilili

11111ii111111tIll 1 11111ill111111 11 1 1111111111111111lt111111

974 tCGTGCtCCttGACCtGCATCGCCACCACAGGGGAAGMATCAAGAACCCCCAGAAGGCCAT TtCCTGtGGGCATCGTGCGTCCCTCCTCAT TTCCTTCATAGCGTAC T TGCGTGTCtCG 717 CAGCTTTAACGCTTATGATGCCCTTACTACCTCCTGGATGAGAAATCCACTCCCAGTCCGCGTTGtAGTATGTCAGATGGGGCCCCGCCCAAATACGTTGTCGCACAGGCCTCCCTtCTCG

ii1i11i111111l11lti1li1111 111111

1 11111111

lilt 11 lI

11111

lIltlil liii11

11111 1111111

109 CCGCTCtCACGCTCATGATGCCT TACT TCTGCCTGGCATCGtACAGCCCGCTGCCCTGGTGCCT tCAAGCACCAGGGCCtGGGAAGAAGCTAAGTACGCAGTGGCCAT TGCTCTtCTCTGCG

837 CCTTATCAACAAGtCT tCTTGGATCCAttTTTTCCCAATGCCTCGTGTATCTATGCTATGCGGCAGGATGGGT TGCT TTTCAAATTCtCAGCTCAAATCAAT TCCAAACGAAGACACCAG

ll II11111

tIl11111 tit11111

tl tltI11111 11111111lii lil 1111111111111111111

i111111

I liii1111111

1214 CACT TTCCACCAGCTCtCTAGGCTCCAtGT tTCCCATGCCCCGAGt tATCTATGCCAGCTGCtMAAGTGGACTACtGT t tAAttttTGGCCCAAAATCAACAATAGGACCAAAACACCCG

957T AATTCCTACTTTGCACTCCGGGTGCAGTGGCAGCTGTGATGGCCT*TCTTTTTGtACCTGAAGGCCCTCGTGGACATGATGTCTATTGGCACCCTCATGGCCTACTCTtCGGTCCCGCCT

11ii11111111111111

Itt11111111111111111111111Iill111111 itlttlt 111111111111I1tiIi tltl 111111 111111111iii

1334 TAATCGCCACTGTGACCTCAGCGCCCAtTCTGCTGCTGCATGGCCt TCCTCT t GAACtCGAAGGACCTCCTGACCTCATGtCCAtT*GGCACTCtCCtCCCTTACtCtTTTGGCCTGCtCCT

1077 GTGTGCTTATTCTCAGGTAC

1454

CAACCTGGCTTGTGTTACGAGCAGCCCAAATACACCCCTGAGAAAGAAACTCTGGAATCATGTACCAATGCGACttTGAAGAGCGAGTCCC

I I 11111I I I I 11 11 III111 I III11 1111 III III 11111 1111 I I I IIIII GGTTGTTGGTCTTACGGTACCAGCCAGAACAACCTAATCTGGTATACCAGATGGCCAGAACCACCGAGGAGCTAGATCGAGTAG AtCA GAATGAGCTGGTCAGTG

1188 AG

GTCACCATGCTGCAAGGACAGGGTTTCAGCCTACGAACCCTCTTCAGCCCCTCTGCCCT

i1l1l

lul l 11 11

It11

1 1111

ll it1

11 1111 1

CCAGTGAATCAC

GCCCACACGACAGTCGGCTtCCCTTGTGAGCTTTCTGGGGGATTCCTGGCTT

ll

til 1111lllllli11ti ltt t111

l1571 AGACAGCCTTTT tACCGGTAGCCGAGAAGTTTTCTCtGSAAtCCATCCTCTCACCCAAGAACGTGGAGCCCtCCAATTCtCAGGGCtAATTGTGAACATTTCAGCCGCCTCCTAGtCCG 1302 TCCTCATCCtGGCCT TGAGTATTCtAACCACGTATGGCGTCCAGGCCAttGCCAGACTGGAAGCCTGGAGCCTGCTCTCTt CCGCCCTGTTCCTTGTCCTCtGCGCTGCCGCTCATTCTGA

liil 11111 1 111ttIt 1111li111 1111itl lti

l Ilt

I1 Il111111

li

1691 CtCttATCATCACCGTGTGCAtTCTGGCCGtGCT TGGAAGAGAGCCCCTGGCCGAAGGGACACTGTGGGCAGtttTCTTGAATGACAGGGtCAGCCTCtCTCTGCATGCTGGtGACAGGCA

1422 CCATTTGGAGGCAGCCACAGAATCAGCtAAAGGCCCttCATGGTCCCGT TCTTACCGCTTTtGCCGGCCT *CAGCATCCTGGTCAACAT TTACTTGCATGGTCCAGtt^AGTGCGUCAC 1811 TCAtCtGGAGACAGCCtG^AGACAAGACCAAGCTCTCAT tTAAGGtACCCT*tSTGCCCCGtACT TCCTGTCT TGAGCAtCCTCtGTGACAtCtATCTCAtGTCAGCCTGGACCAGGC

1542 CTTGGATCAGATtCAGCATCTGGATGGCGCTTGGCTtTCTGATCTATTTCGCCTATGGCATTAGACACAGCTTGG AGGGTAC CCCAG GGACGMAGGACCATCAGGATGCCTTTt CCTATTGTATGGGATtCCGCACAGTGAGACGTCCCCTGCtGCGCTGGCCAGCAAACTCCTGACAGCAACT 1931 CGtGGGCtCCGGt t GCAGtGTGGAtGCtCAtAGGTTTCACCtCATt

1659 CAGAAAACATCAATGTAGCAACAGAAGAAAAGTCCGtCATGCAAGCAAATGAC CATCACCAAAGAAACCTCAGCTTACCTTTCATACTTCATGAAAACACMAGTCTGTTCATGCTGG

I Il 11111 i111llllllltlllIlIllIl 1111111l 111 111l11 1 i11 11li11 ACC CTCACAATCTCTCCACTCATGCCTCAGGATCAGCTCACA

2051 TGGACCAGTGCAAATGACGTGCAGCCCCACCCACCAGGGTGACAGCGGTTGACGGGTGCCCGTAGAAGCCTGGG

1778 CCCTCGGTCTTACCACGCATACCTTAACAATGAGTACACTGTGGCCGG ATGCCACCATCGTGCTGGCCTGTCGTGGGTCTGCTGTGGACATGGCTTGCCTAACTTGTACTTCCTCCTC CC 2167 CCCCCCTGTCGACCACTGGTCTtCTGCCAGCTCGTGAGATCCTGGTCATTTCTGGACAGTCCCTtGtTTACTCATCTCCCTCTCAGAGCAGCCCTTCTCCTTC

1 896 CAGCAG^CTTCTCTTCAGATGGTGGATTCTGTTCTGAGGAACC^^tGCCTGAGAGCACTCCTCAGCTATATGTATCCCCCAAACATATGTCCGTGTGCGTACATGTATGCTGCCGAtTGT

2286 CGGCCGG GCGCTTC GCTGCTGCGGCCCCAG CAGAAGGGA GGCC 2016 AGTGTTCAATGTTGTCCGTTATTAG CTGTGA

11l Iii

1l

l l

CCCTTCTCCTC TCACTTGGGAAGCAGGCCTCCCTCCCTCCCTGGGACCACCCTGGCATCGCCCATGTG

Ccatettccagcatggtoattggtggcatatagtgc c cactogt acogtatattgctgatagaoatgtottcttsttatgtcc

2397 CACACTCCA GATGGC TAGTGAGCCTCTCCEND 2425 Hatches 1212. Mismtches 789, Uruatched 74

taggtggetggggaactagtggtgt t tctt tat toggtatatgoccatcogtt tggacatactgaeatgccatcccctgtcaggatgtt taesgtggtcotggtggg§"g9ate ggaatggcat tgtc tateat tgtaetgcatatatcct tctcctact tgctaagacagcogt t tcttoaacggccanagagotgt t tctttcctctgtatgacoagatg"ggta

tctgtggctggagatggccaatcc 2397

FIG. 4. Alignment of 20.5 and ERR cDNA sequences. (A) The regions of the ERR and 20.5 cDNA sequences included in the alignment analysis are indicated by the vertical lines. The open reading frames (ORF) for each cDNA are indicated. (B) The entire cDNA sequence of 20.5 is shown on the top line of each pair of lines; the cDNA sequence of ERR (bp 400 to 2425) lacks the first 400 bp and is shown on the bottom of each pair. The vertical lines mark the positions of sequence identity. The comparison was made by using Microgenie software, with gaps generated to allow alignment of the most highly similar sequences. Identity is reported only for the regions of cDNA which are clearly overlapping. Note that the 20.5 cDNA is longer at the 3' end and the ERR cDNA is longer at the 5' end. 3668

VOL. 10, 1990

ISOLATION OF NOVEL MURINE cDNA CLONE

3669

A. Carboxy Terminus

Amino Terminus

-

Tea Protein

Rec-1 Protein

Region of Aligrnent

B. 204

Amino Terminus (Extracettutar) A G F V K G N V A N W K M V II : I : I I I I I I V V S G f V K G S I K N W

40

G

1

Region 1 A G G

I

Transf

II

Y

G

F

P

I1

1: 11: 1

F

236

G

E

G

G

F

N

P

F

G

F

80

0

K

A

I

P

I

G

I

V

T

I I I 1 1:

1

1

1:

276

120

L

K A

I

P

A

II

V I

P

V G

F

E

Y

T

G

1

E

T E

S G

1

A

T

G

M

A

Y

F

:

1

1

1

I

I

Y

A

N

A

E

D

G

L

L

F

C

L

I

I

I

I

I

I

I

Y

A

A

E

D

I

I

356

I

G

L

L

F

200

F

D

L

K

A

L

V

D

1: 1

1

1 1 1

396

F

E

L

K

D

L

V

D

L

237

0

P

K

Y

T

P

E

K

E

315

A

1 T

E

L

D

R

P

T

R

0

S

I

Transmembrane 4 G T L M A Y

V

D

a

A

S

W S L A L

L A

L

T

L

W

A

V

F

V

355

F

L

P

F

L

P

A

F

S

I

395

A

Y

G

P

V

H

I

I I

R

I

I

Y

I

W.

I

G

H

S

S

V

G

F

D

C

I

A T

T

G E

S A A

L

T

L

M M P Y

Y

L

K

I

I

F

N NR

L

T

L

L

K T

C

A

L

S

T

P

V

I

A

T

I

I

T

K

T P

I

N

I

Y

I

I

I

L

I II:II I IIJ

A Y

S

L

V A A

S

A

G

I I

C N

NV

O

V

L

V

L

Q

V

T

N

L

I

S O

Transnen rane 5 L V G F L A

I

L

I

F

I I

I

G

F

L

I

L

G

L

S

L R T

II1:::

S L K S

L

T

T

Y

G 1

P

N

a

V A

S

A

D

T

W

I I

D

G

Q

K

1II 1I 11

W R

0

P

E

S

I

F

S A

I

W N

:

R

I I

T WV RF AV

K

T

K

u

V

I

WNIL

L

S

:

I I

I

G

I

0

F

L I

1

R E A L F

H V P

: 1

L

L

0 A

F

FT

1

1

Y

F

K V p

Transmembrane 7 A L G F L I

I

I

I

L V Y

F

L

f

I

I

L

A

F

L

I

L

G

I

I

F

I

V

G

A

till1

N

L

T

I

P

V A V

V

V

0

I

L

R

K

I

I

I

E

V C

0

V

L C Y E

::

R

R

A A V

I I T Region 2 U

P

VN

L

T

D

P V A E S

P

I

1

A A

E

ASLAAGOAKKTPDSNLDOCKU

P

S

I I S P

P

L

I

L

0

I D

L

1: 11 NN O

Y

II

E GNPRDEEDDDEDAFSENINVATEEKSVNQANDHHORNLSLPFILHEKTSEC

ITYI

R

P

I I

E V K N P

P SG

I

L

N

L

C

E

V

Y a

C V L

S

F

I

I

G A

I

L

I

S

A

Transmeudrane 6 F L V LC A A

V

1:

T V T R

S

I

F

S V L

A

G

I:

I II

Transsmembrane 3 L S S G A V A

L

A

S E

Iy

E V R N

P

P

L

I

S

G

F

:

F

S

1::

I

I

l

V

V

T

V

S

I

L V S

V N

G

L

E

I

L

I

N

I jy

V K

L D E K

C L

S

I

S

G T

I I

F

I I

I

L

L

I

s c T N A T L K S E S

A

L

I

E

E

V

I

L

L

P

G E

I

G

R

V

T

I

I

L

F

A T

I

S

S G

G

A

1 I I I

F

E

G

S

E

555

I

I

Y A

G S L

N SK T

L

V

A

I

F

N

K

A Q

K

K

515

595

K

P

P

C

I

S

I

I

N

I

T

I

T

F D

S

I

N

I

G P A K Y V V AA G S L C A L

F

E

I

A

W

160

475

V G

S

N D

R

AY

A K Y A V AI

L

f

P

V

I

E

A

Y

P

L M N P Y

C F

G W E

S

S C N N

E

T

I

0

P

F

R

L

L

H

S

K N

V

S A

G V S A A

L

K

276

S A

S

F

E

I

I

A

A

T

K N

V

u

T

F F

P

R

C

AT C

L

A

L

I

316

436

F

I

I

I

L

Transnmembrane 2 S L L V C F

11~~I

E

S

membrane 1 T L A G A A

V L

S

I

::

I

I I

I

Y

F

453

623

FIG. 5. Alignment of Tea predicted protein sequence with the murine ERR sequence. (A) Regions of the two predicted protein products which were compared. The Tea protein extends from amino acids 1 to 404, and the ERR protein extends from amino acids 204 to 603. (B) Alignment of the two predicted proteins shows the amino acid sequence predicted by the 20.5 cDNA on top and that predicted by the ERR cDNA on the bottom. Parentheses delineate the borders of two regions of extensive amino acid identity; Region 1 is 81.3% identical over 192 amino acids, and Region 2 shows 51.9% identity over 79 amino acids.

MOL. CELL. BIOL.

MAcLEOD ET AL.

3670

A.

B.

Tea Protein

IIIllIIIIIIIIINhIIIIIIIIII l U IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIllllgll Rec-1 Protein Region of Alignment

C. 50

40

30 20

I0 0 -10

-20 -30

-40 -50

50

40 30 20 10

0

-10

-20

-30 -40 -50

FIG. 6. Physical properties of the predicted protein products of the Tea and Rec-l genes. (A) Model for possible structure of the Tea predicted protein. Boldface lines indicate the two regions of extensive similarity with the ERR protein. PC GENE software programs from IntelliGenetics (SOAP, HELIXMEM, NOVOTNY, and RAOARGOS) were used to examine the physical properties of the predicted protein to prepare the model shown. (B) Regions of the two predicted proteins which were analyzed for the hydrophobicity comparison (amino acids 1 to 403 for the Tea protein and amino acids 204 to 603 for the ERR protein). (C) Hydrorhobicity plots of the Tea predicted protein (top) and the ERR predicted protein (bottom). Numbers on the x axis refer to the amino acid -sitions included in the analysis as explained in panel B. The y axis gives the hydropathicity values from the SOAP program (11).

ISOLATION OF NOVEL MURINE cDNA CLONE

VOL. 10, 1990

A.

B.

TABLE 2. Analysis of concordance between specific mouse chromosomes and the presence of the Tea gene in a series of mouse-hamster somatic cell hybrids

ell CL

C X

E1

3671

Backcross Mice

X

w

UI

IX2

No. of hybrids (DNA hybridization per chromosome)

Mouse chromosome ++b

9.0-

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X

6.6-

4.4-

2.32.0-

-

FIG. 7. Southern analysis of DNA from different species and analysis of recombinant inbred DNA to position the Tea gene on chromosome 8. The 32P-labeled cDNA probe for both panels was a nearly full-length Ball fragment of the 20.5 cDNA clone. (A) Autoradiogram showing the hybridization pattern of PstI-digested DNA (from humans, hamsters, mice, and chickens) probed with the BanI fragment as described in Materials and Methods. (B) Autoradiogram of liver DNA derived from interspecies backcross mice as fully described in Materials and Methods. The figure shows the pattern of hybridization obtained from several backcross animals as indicated and from the NFS/N parental DNA digested with SacI. Sizes in kilobases are indicated to the left of each panel.

prepared by using PC GENE programs to examine physical properties as noted in the figure legend. The sketched model highlights the regions of extensive similarity with the ERR protein. Since ERR encodes a cell surface protein, we compared the hydrophobicity profiles in the regions of overlap (amino acids 1 to 401 of the Tea predicted protein and amino acids 204 to 600 of the ERR protein). The line sketch of Fig. 6B shows the region of the two proteins that was compared in the hydrophobicity plots. The comparison covers a region of 401 contiguous amino acids and shows a remarkable similarity. Thus, it is likely that the product of the Tea gene is also a cell surface protein. The Tea gene appears conserved in higher vertebrates. To determine whether the Tea gene sequences are conserved in evolution, DNA from humans, hamsters, mice, and chickens was tested for cross hybridization with the 20.5 cDNA probe. When the full-length cDNA clone was used to probe the Southern blot, detectable hybridization occurred with all the genomic DNAs tested (Fig. 7). To test whether any of the DNA fragments detected were derived from the Rec-i gene, a probe to the 3' region of the 20.5 cDNA clone (bp 2005 to 2394, which is highly divergent from the ERR cDNA sequence) was found to hybridize to DNA fragments of the same size (data not shown). Thus, the Tea gene itself has been conserved since the sequence which is most divergent from Rec-J hybridized to DNA from other species. These data suggest that the two genes diverged prior to mammalian radiation. It is likely that the Tea gene encodes a protein distinct from ERR which is found in other species. The

5 7 3 4 2 6 5 6 4 0 0 3 5 1 4 3 6 6 5 5

9 5 6 3 11 6 4 13 10 11 13 4 7 10 0 9 4 6 7 8

% Discordancya

+/_

_/+

2 0 2 2 5 1 2 Vc 3 7 7 2 2 5 0 2 0 1 2 2

4 7 3 3 2 6 8 1c 4 2 0 3 7 2 8 4 7 4 5 6

30.0 36.8 35.7 25.0 35.0 36.8 52.6 9.5 33.3 45.0 35.0 41.7 42.9 38.9 66.7 33.3 41.2 29.4 36.8 38.1

Derived from the sum of the +/- and -/+ observations. b Symbols at the left of the slash indicate the presence (+) or absence (-) of the mouse Tea restriction fragment as related to (at the right of the slash) the presence (+) or absence (-) of the particular mouse chromosome indicated, as detected by hybridization with the 20.5 cDNA probe. c Not karyotyped, but typed for other markers. Thus, it is possible that the +/- hybrid cell contains fragments of chromosome 8 or that a small percentage of the cells contain the chromosome. The -/+ exception may contain a portion of chromosome 8 but lack the region containing the Tea gene. a

divergence of the amino acid sequence at the carboxy and amino termini suggest that the two proteins may have distinct functions. Mapping of the Tea gene to chromosome 8. Since the ERR gene product functions as a viral receptor, it was of interest to determine whether the Tea gene mapped to a chromosome known to encode one of the other known retroviral receptors (27). Several different somatic cell hybrids formed between Chinese hamster and mouse cells each retain a limited number of different mouse chromosomes (25). These hybrids were used to map the Tea gene (25). Southern analysis of DNAs digested with PstI from hybrid cells demonstrated that 7 of 21 hybrids contained mouse-specific DNA fragments. A comparison of the known mouse chromosome content with the positive hybridization to mouse-specific DNA fragments indicated that the best correlation is with mouse chromosome 8 (Table 2). To confirm and extend this observation, Tea was positioned on chromosome 8 by analysis of an interspecies backcross (5, 27, 28). DNA digested with SstI showed that NFS/N mice produced cross-reactive bands of 10.0, 6.4, and 5.5 kb. M. musculus musculus DNA produced 10-, 7.4-, and 5.5-kb fragments. Figure 7B shows the pattern of hybridization of the 20.5 cDNA probe in backcrosses of NFS/N x M. musculus musculus Fl mice with M. musculus musculus. The segregation pattern of this restriction fragment-length polymorphism with other markers on chromosome 8 demonstrated that this gene is linked to Gr-J and Es-i with the gene order centromere-Polb-GrI-Tea-Es-i (Tables 3 and 4). The location of the Tea gene on chromosome 8 provides

3672

MOL. CELL. BIOL.

MAcLEOD ET AL.

TABLE 3. Segregation of the Tea hybridizing fragment with alleles of Es-i in 57 progeny of an interspecies backcross

TABLE 4. Recombination

Polb, Gr-i, ahd

Inheritancea of NFS/N allele

Mice

Gr-i

Tea

Es-i

+

+

+

+

22

-

-

-

-

13

+

+

+

_

-

5

_

-

+

9

+

+

Parent

Single

recombinants

_

a

+,

Inherited the

No. of mc m]ce

Polb

allele; -,

-

-

0

+

+

2

+

-

-

-

1

_

+

+

+

5

Locus pair

No. of

Polb, Gr-i Gr-i, Tea Tea, Es_lb Polb, Tea Gr-i, Es-i Polb, Es-I

6 2 14

cM

+

SEa

10.5 ± 4.1 3.5 ± 2.4 24.5 ± 5.7

8

14.0 ± 4.6

16

28.0 ± 5.9 38.6 (Not sigrificant)

22

a Distances in centimorgans (cM) and standard error for each locus pair were calculated according to the method of Green (21) from the number of recombinants per sample size of 57. b An additional 46 mice were typed for Tea and Es-i, for a total number of recombinants of 24 in 103 backcross mice (23.3 cM ± 4.2).

did not inherit the allele.

proof that the gene is distinct from the Rec-i gene which is localized to chromosome 5 (28). It also eliminates the possibility that Tea is the mink cell focus retroviral receptor, which has been localized to chromosome 1 (27). It is possible that the Tea gene product encodes the amphotropic retroviral receptor which is present on chromosome 8. However, little information is yet available on the tissue specificity of amphotropic retroviral infection, and further study is required to test the possibility that the product of the Tea gene functions as a receptor for this virus. DISCUSSION Seventy genes or gene products are known to increase in expression when T cells activate in response to a combination of antigen and self-histocompatibility molecules on the surfaces of antigen-presenting cells (reviewed in reference 9). Polyclonal activators such as lectins, calcium ionophores, or antibodies to the T-cell receptor for antigen can mimic the response induced by antigen (reviewed in reference 9). Some of these activation genes are involved in the tranisition between Go and G1 of the cell cycle. Some encode cytokines and their receptors, nuclear regulatory proteins, and still others are involved in the transport of ions and nutrients into the cells to prepare them for growth. At least 26 T-cell activation gene products have been localized to the cell membrane (9). To our knowledge, 20.5 is the first example of a cloned gene or cDNA that has the potential to encode a multiple transmembrane-spanning protein which is induced during T-cell activation (9). The process of splenic T-cell activation initiated by ConA or antigen begins within minutes of contact with lectin or antigen presentation and continues over a period of about 7 to 14 days. Changes in gene expression occur throughout the activation period. Crabtree (9) has categorized these changes in gene expression by analogy with viral gene activations (immediate, early, late, and very late). By the criteria of Crabtree, Tea is an early gene because Tea mRNA is virtually undetectable in normal quiescent T cells, increases to detectable levels within 6 h, and peaks at about 24 h. The function of the Tea gene is not yet known; it could function to transduce signals or transport small molecules which are signal transducers, or it could function as a receptor for an unidentified ligand. The rather long carboxy terminus of the putative Tea protein might function as a signal transducer, although no evidence to support that speculation exists. Since SL12.3 T-lymphoma cells and numerous other T- and B-tumor cell lines did not express this gene, Tea gene

expression is not required for cell growth. However, our studies do not exclude the possibility that normal T cells require Tea gene expression to undergo the normal cell proliferation which accompanies T-cell activation. The striking homology between the Tea and ERR proteins suggests that the Tea gene product might function as a murine retroviral receptor since at least four classes of murine retroviral receptors have been genetically defined (26a-28), and only one has been molecularly cloned (2). The localization of the Tea gene to chromosome 8 eliminates the possibility that Tea encodes the mink cell focus virus receptor and raises the possibility that it encodes the receptor for the lOAl amphotropic virus, which is closely related to mink cell focus viruses and uses an unmapped surface receptor on mouse cells (40). Alternatively, the novel gene could encode the receptor for other amphotropic retroviruses which has been localized to chromosome 8. In contrast to the Rec-i gene (encoding ERR), which is ubiquitously expressed in mouse tissues (2; J. Cunningham, personal communication), the Tea gene has a much more limited tissue distribution. If the Tea gene encodes a protein which functions as a retroviral receptor, limited tissue distribution could provide specificity for the retroviruses which are restricted to the lymphoid lineage (42). There is a precedent for receptormediated restriction in the human immunodeficiency virus, which uses the CD4 protein as its primary receptor. However, cell-specific receptors are unlikely to be entirely responsible for tissue specificity. The tissue tropism exhibited by retroviruses is likely to result from a complex series of factors, such as the tissue specificity of long terminal repeats, variations in gp7O env proteins, cellular factors, and the expression of appropriate cell surface receptors (26a). When the ERR binding site for virus is identified, it will be possible to determine whether the site is in a region of high similarity with the Tea protein. Viral binding and infection studies are required to determine whether Tea protein functions as a viral receptor (43). In spite of the high degree of similarity to the ERR gene product, there are substantial differences in the physical properties of the Tea and ERR predicted proteins. They map to different chromosomes, and their tissue expression patterns are distinct. Furthermore, the ERR cDNA probe detects sequences of distinct sizes from those detected by the 20.5 cDNA probe. Since the natural cellular functions of both these genes are unknown, further study is required to determine any functional similarities that might exist between the two proteins. An analysis of the conserved and nonconserved amino acids may provide insight into similar or disparate functions of the ERR protein and Tea gene product. In particular, the identification of this new gene

VOL. 10, 1990

family and the regions of DNA sequence which are highly conserved between the two molecules will now permit searches for new members by the approach employed to find members of the steroid receptor-thyroid receptor gene family (12). ACKNOWLEDGMENTS We thank Russell Doolittle (UCSD) for his help in identifying the homology of ERR with 20.5 cDNA; Robert Tukey (UCSD) for his generous help and advice in assessing the nucleic acid and predicted protein sequence of the 20.5 cDNA clone, for critical reading of the manuscript, and for hamster DNA; Don Geske of the UCSD Cancer Center Core Molecular Biology Facility for preparing the oligonucleotide primers; D. Kabet and E. Barklis (Oregon Health Sciences University), J. Cunningham (Harvard Medical School, Cambridge, Mass.), and Alice Barrieux (UCSD) for helpful discussion; Nissi Varki (UCSD) and Lian Qian (Oregon Health Services University) for providing normal murine tissue for RNA preparation; and Charlene Adamson and Lory Walls for technical assistance. This work was conducted, in part, by the Clayton Foundation for Research, California Division, and supported by Public Health Service grants CA37778 and CA42495 to C.L.M. and GM39586 to M.F.W. from the National Institutes of Health. C.L.M. is a Clayton Foundation Investigator. M.F.W. was supported during the earlier phase of this work by Public Health Service postdoctoral fellowship HL07107 from the National Institutes of Health, and K.F. and D.K. were supported, in part, by Public Health Service grant CA37778 and predoctoral fellowships HL07212 and GM07752, respectively, from the National Institutes of Health. LITERATURE CITED 1. Adkins, B., C. Mueller, C. Okada, I. Weissman, and G. Spangrude. 1987. Early events in T cell maturation. Annu. Rev. Immunol. 5:325-365. 2. Albritton, L. M., L. Tseng, D. Scadden, and J. M. Cunningham. 1989. A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection. Cell 57:659-666. 3. Beardsley, T., M. Pierschbacher, G. Wetzel, and E. Hays. 1983. Induction of T cell maturation by a cloned line of thymic epithelium (TEPI). Proc. Natl. Acad. Sci. USA 80:6005-6009. 4. Bernstine, E., M. Hooper, S. Grandchamp, and B. Ephrussi. 1973. Alkaline phosphatase activity in mouse teratoma. Proc. Natl. Acad. Sci. USA 70:3899-3903. 5. Bishop, D. T. 1985. The information content of phase-known matings for ordering genetic loci. Genet. Epidemiol. 2:349-361. 6. Buonassisi, V., G. Sato, and A. Cohen. 1962. Hormone-producing cultures of adrenal and pituitary tumor origin. Proc. Natl. Acad. Sci. USA 48:1184-1192. 7. Burd, P., G. Freeman, S. Wilson, M. Berman, R. DeDruyff, P. Billings, and M. Dorf. 1987. Cloning and characterization of a novel T cell activation gene. J. Immunol. 139:3126-3131. 8. Christie, M. J., J. P. Adelman, J. Douglas, and R. A. North. 1989. Expression of a cloned rat brain potassium channel in Xenopus oocytes. Science 244:221-224. 9. Crabtree, G. R. 1989. Contingent genetic regulatory events in T-lymphocyte activation. Science 243:355-361. 10. Damsky, C., D. Knudsen, R. Dorio, and C. Buck. 1981. Manipulation of cell-cell and cell-substratum interactions in mouse mammary tumor epithelial cells using broad spectrum antisera. J. Cell Biol. 89:173-184. 11. Doolittle, R. 1986. Of URFS and ORFS: a primer on how to analyze derived amino acid sequences. University Science Books, Mill Valley, Calif. 12. Evans, R. M. 1988. The steroid and thyroid hormone receptor superfamily. Science 240:889-894. 13. Filmus, J., J. Church, and R. N. Buick. 1988. Isolation of a cDNA corresponding to a developmentally regulated transcript in rat intestine. Mol. Cell. Biol. 8:4243-4249. 14. Fowlkes, B. J., and D. M. Pardoll. 1989. Molecular and cellular events of T cell development. Adv. Immunol. 44:207-264. 15. Freshney, R. E. 1983. Culture of animal cells, p. 99-110. Alan R.

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Activated T cells express a novel gene on chromosome 8 that is closely related to the murine ecotropic retroviral receptor.

A novel cDNA clone (20.5) which is differentially expressed between two closely related T-lymphoma cell clones was isolated by subtraction-enriched di...
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