DEVELOPMENTAL DYNAMICS 210:11–18 (1997) Sequence and Developmental Expression of AmphiTob, an Amphioxus Homolog of Vertebrate Tob in the PC3/BTG1/Tob Family of Tumor Suppressor Genes NICHOLAS D. HOLLAND,1* SHI-CUI ZHANG,2 MATTHEW CLARK,3 GEORGIA PANOPOULOU,3 HANS LEHRACH,3 AND LINDA Z. HOLLAND1 1Marine Biology Research Division, Scripps Institution of Oceanography, La Jolla, California 2Institute of Oceanology, Academia Sinica, Qingdao, People’s Republic of China 3Max-Planck-Institut für Molekulare Genetik, Berlin (Dahlem), Germany ABSTRACT Tob is a member of the PC3/ BTG1/Tob family of vertebrate tumor suppressor genes; its expression is known to inhibit proliferation of cells in vitro, but its possible roles during normal development have not been investigated previously. The present study concerns the structure and developmental expression of AmphiTob in an invertebrate chordate, amphioxus. This is the first investigation of any Tob gene during embryological development. The 311 amino acid AmphiTob protein is similar to vertebrate Tob but lacks the C-terminal PQ-rich domain of the latter. In early embryos of amphioxus, in situ hybridization first reveals AmphiTob expression in the hypoblast at the gastrula stage on the likely dorsal side of the embryo. During subsequent development, expression is seen in several tissues of the ectoderm, mesoderm, and endoderm. The most striking expression domains are in the developing somitic musculature and dorsal nerve cord. In the medial wall of each somite, AmphiTob is expressed strongly by cells destined to differentiate into the axial trunk muscles; this pattern persists until late in the larval stage, evidently because undifferentiated cells are continually becoming myogenic as the muscles grow. Nerve cord cells conspicuously transcribe AmphiTob from the late neurula until the early larval stage: Expression occurs in a few cells scattered along the nerve cord and in a group of cells located in the cerebral vesicle (in a region presumably homologous to the vertebrate diencephalic forebrain). During development, an intense and transitory transcription of AmphiTob may be an early event in cells exiting the cell cycle in preparation for differentiation. Dev. Dyn. 1997;210:11–18. r 1997 Wiley-Liss, Inc. genes and growth-constraining genes (protooncogenes and tumor suppressor genes, respectively). Such genes, as their names indicate, were originally discovered in mutated states that caused the hyperactivity of the former and the impaired activity of the latter. Tumor suppressor genes often have their most conspicuous effects when they are completely lost from a genome, a fact that has made them more difficult to study experimentally than growth-promoting genes. Even so, several methodological approaches have now revealed nearly a score of tumor suppressor genes in vertebrates (Weinberg, 1991). Compared with vertebrate tumor suppressor genes, those of invertebrates have been less studied. Although many of the tumor suppressor genes so far discovered in embryonic and adult Drosophila (Watson et al., 1994) lack firmly established links with known tumor suppressor genes of vertebrates, the following clear homologies have recently come to light: patched (Johnson et al., 1996), merlin/NF2 (McCartney and Fehon, 1996), and adenomatous popyposis coli (Hayashi et al., 1997). The present paper concerns a gene in the invertebrate chordate, amphioxus, that has a clear homolog with a vertebrate tumor suppressor gene, Tob. In the vertebrates, Tob genes (Matsuda et al., 1996) are closely related to PC3 genes (Kujubu et al., 1987; Lim et al., 1987; Fletcher et al., 1991) and BTG1 genes (Roualt et al., 1992, 1993). All three are rapidly expressed in response to a variety of stimuli and have been implicated in the suppression of cell proliferation (Rayburn et al., 1995; Matsuda et al., 1996; Montagnoli et al., 1996). Structurally, vertebrate PC3, BTG1, and Tob are sufficiently similar to comprise a distinct gene family. The proteins encoded by rat PC3 and human BTG1 are relatively small (158 and 171 amino acids, respectively), whereas the protein encoded by human Tob (345 amino acids) is about twice the size of the other two genes, resembling them most closely in its N-terminal Key words: cephalochordata; myogenesis; neurogenesis INTRODUCTION During the life history of an animal, both normal development and adult homeostasis are ensured by a balance between the activities of growth-promoting r 1997 WILEY-LISS, INC. Grant sponsor: NSF; Grant number: IBN 96-309938; Grant sponsor: Natural Science Foundation of China; Grant sponsor: K.C. Wong Education Foundation (Hong Kong). *Correspondence to: Nicholas D. Holland, Marine Biology Research Division, Scripps Institution of Oceanography, La Jolla, CA 920930202. E-mail: firstname.lastname@example.org Received April 1997; Accepted 19 May 1997 12 HOLLAND ET AL. half. The proteins encoded by all of these genes have an abundance of nonpolar amino acids at the N-terminal end. It was originally thought that this hydrophobic domain was involved in secretion of the gene product (Bradbury et al., 1991), but subsequent work has shown that the proteins function intracellularly (Roualt et al., 1992; Iacopetti et al., 1994; Varnum et al., 1994; Matsuda et al., 1996). Genes of the PC3/BTG1/Tob family have been reported so far only from higher vertebrates, and the occurrence of homologous genes in lower vertebrates and invertebrates is an open question. The present paper reports the discovery of a Tob homolog from the cephalochordate, amphioxus. In addition, because the developmental transcription patterns have not yet been described for any Tob gene—even for the vertebrates—we describe the expression of AmphiTob during the embryonic and larval stages of amphioxus. MATERIALS AND METHODS Obtaining DNA and Library Screening Ripe adults of the Florida amphioxus, Branchiostoma floridae, were collected in Tampa Bay, Florida. The eggs were fertilized, and the embryos and larvae were cultured at 23°C (Holland and Holland, 1993). A cDNA library was constructed from 26-hr larval mRNA in the pSPORT 1 vector (GIBCO-BRL, Gaithersburg, MD), and genomic DNA for Southern blots was extracted from a sample of 20 adults (L.Z. Holland et al., 1996). When probing the cDNA library (60,000 clones) at moderately low stringency (N.D. Holland et al., 1996) with a 148-bp stretch of the paired box of the amphioxus Pax-6 gene, we incidentally obtained three strongly hybridizing clones (29F16, 6A22, and 60K21) that we called AmphiTob due to their homology with the human Tob gene (Matsuda et al., 1996). The two longest clones, 29F16 and 6A22, were sequenced in their entirety. When aligning sequences, Clustal V was used to introduce gaps. Amino acid substitutions with Dayhoff scores of 9 or higher were considered to be conserved (Doolittle, 1987). Southern Blot Analysis Southern blots of genomic DNA were prepared according to L.Z. Holland et al. (1996). To determine the number of amphioxus genes related to AmphiTob and the gene copy number, blots were hybridized with an ApaI-EcoRI fragment (1,232 bp; delimited by arrows in Fig. 1), which comprises the C-terminal two-thirds of the coding region plus the upstream half of the 38 untranslated region. Low-stringency hybridization was in 6 3 standard saline citrate (SSC), 0.2% sodium dodecyl sulfate (SDS), 10 3 Denhardt’s, 1 mM EDTA overnight at 60°C. Washes were at 55°C in 2 3 SSC, 0.1% SDS. In Situ Hybridization The developmental expression of AmphiTob was studied by in situ hybridizations of developing amphioxus fixed at frequent intervals. A schedule of normal development at 23°C has been published previously (N.D. Holland et al., 1996). The ApaI-EcoRI fragment mentioned in the preceding paragraph was subcloned into pBluescript SK1 (Stratagene, La Jolla, CA) and was used as a template for an antisense riboprobe approximately 1,000 bp long. This riboprobe was designed to hybridize with AmphiTob mRNA but not with that of related genes (i.e., possible amphioxus homologs of PC3 or BTG1). Methods of fixation, riboprobe synthesis, and in situ hybridization were according to L.Z. Holland et al. (1996). RESULTS Sequence Analysis Figure 1 shows cDNA of AmphiTob clone 29F16 from B. floridae (superscripts indicate differences in clone 6A22 relative to clone 29F16). Both clones are about 2.4 kb long and code for a 311 amino acid protein with the same amino acid sequence; thus, despite some nucleotide differences, the two clones evidently represent polymorphic forms of the same gene. The 38 untranslated region includes three AT-rich sequences with ATTTA motifs (starting at nucleotides 1,356, 1,745, and 2,390) that characterize genes transcribing rapidly degradable mRNA (Shaw and Kamen, 1986). Because there is no polyadenylation signal (AATAAA) near the 38 end of the nucleotide sequence, the 38 untranslated region is presumably incomplete. Figure 2A shows that AmphiTob and human Tob (Matsuda et al., 1996) share 51% identical amino acids (75% identities plus conservative amino acid substitutions). A comparison of hydrophobicity plots of each protein (data not shown) reveals that the 16 N-terminal amino acids of both comprise a very similar nonpolar domain. Like vertebrate Tob, AmphiTob is rich in PEST residues (stretches rich in proline, glutamic acid, serine, and threonine), which characterize relatively unstable proteins (Rogers et al., 1986). The C-terminal half of Tob includes a PQ-rich domain that is not present in the AmphiTob protein. Figure 2B compares the entire amino acid sequences of AmphiTob, rat PC3 (Bradbury et al., 1991), and human BTG1 (Roualt et al., 1992). For AmphiTob vs. PC3, the amino acid identities are 42%, and the identities plus conservative amino acid substitutions are 66%. For AmphiTob vs. BTG1, the amino acid identities are 37%, and the identities plus conservative amino acid substitutions are 65%. Southern Blot Analysis The Southern blot was probed with a stretch of AmphiTob selected to avoid extensive sequence similarities to vertebrate PC3 and BTG1 genes. At low stringency, a single hybridization band resulted from digestion by 7 of 15 restriction enzymes (Fig. 3). This result strongly suggests that AmphiTob is the only Tob gene in the amphioxus genome and is present in a single copy. Developmental Expression of AmphiTob Wholemount in situ hybridization during the cleavage and blastula stages of amphioxus reveals no detect- Fig. 1. AmphiTob clone 29F16 from cDNA of 26-hr larvae of the Florida amphioxus, Branchiostoma floridae: nucleotide and deduced amino acid sequences (GenBank accession number U95824). The in-frame stop codon preceding the presumed translational start site is underlined. The two superscript arrows delimit the ApaI-EcoRI fragment used for the Southern blot analysis and for antisense riboprobe synthesis. The superscript numbers locate the following differences in clone 6A22 relative to clone 29F16 (no amino acid substitutions resulted from nucleotide differences in the coding region): 1, 58 start of clone; 2, C; 3, A; 4, T; 5, A; 6, G; 7, C; 8, C; 9, G; 10, A; 11, C; 12, A; 13, C; 14, C; 15, G; 16, T; 17, delete ACA; 18, insert A; 19, A; 20, insert T; 21, T; 22, delete TG; 23, 38 end extended by eight additional As. 14 HOLLAND ET AL. Fig. 2. A: Amino acids encoded by AmphiTob compared with amino acids encoded by human Tob (Matsuda et al., 1996). B: Amino acids encoded by AmphiTob compared with those encoded by rat PC3 (Bradbury et al., 1991) and human BTG1 (Roualt et al., 1992). Identical amino acids and conserved amino acid substitutions are indicated by lines and dots, respectively. able expression of AmphiTob. Expression is first seen at the late gastrula (Fig. 4A), chiefly in the hypoblast on the probable dorsal side of the embryo (as indicated by subsequent expression patterns). In the early neurula stage, AmphiTob is expressed in cells that comprise the anterior wall of the foregut endoderm and in the STRUCTURE AND EXPRESSION OF AMPHIOXUS Tob Fig. 3. Genomic southern blot analysis of DNA pooled from 20 amphioxus adults. Numbers at the top of lanes refer to digestion in the following restriction enzymes: 1, BstE II; 2, BstX I; 3, Eco0109 I; 4, EcoR V; 5, Hind III; 6, Pst I; 7, EcoR I. The blot was probed at low stringency with a 1,232-bp stretch of AmphiTob. Size markers are at left. paraxial mesoderm (Fig. 4B), in which the presomitic grooves are forming (Fig. 4C). By the stage of the hatching neurula (Fig. 4D,E), expression is detectable in the foregut endoderm, somitic mesoderm, and posterior mesoderm. In the late neurula (Fig. 4F–H), mesodermal expression is strongest in the somites (in the medial wall, where the myoblasts are beginning to differentiate) but is becoming less conspicuous in the posterior mesoderm and the foregut endoderm; in addition, expression is now conspicuous in a group of cells located in the dorsal and ventral wall of the cerebral vesicle near the anterior end of the nerve cord and in individual cells scattered more posteriorly in the nerve cord. In the 24-hr embryo (Fig. 4I,J), the transcription patterns are much the same as at the preceding stage, although an additional expression domain is now detectable in the anterior ectoderm. In the 30-hr larva (one-gill slit stage), expression is present but is weaker in the somites and is no longer detectable in the posterior mesoderm. Expression remains strong in the anterior ectoderm and in cells of the cerebral vesicle of the dorsal nerve cord. In addition, transcripts are now present in scattered cells in the ventral wall of the midgut and hindgut (Fig. 4K). In the subsequent larval stages, AmphiTob transcription diminishes in most tissues to levels undetectable by in situ hybridization. The exception is transcription by some cells of the somitic musculature (Fig. 4L), at least through the 14-day larva (six-gill slit stage). The expressing cells are presumably exiting the cell cycle and are about to differentiate into muscle cells of the axial trunk musculature. DISCUSSION Vertebrates and cephalochordates have been evolving independently for half a billion years. Thus, sequence comparisons of Tob genes (and their protein products) between amphioxus and vertebrates can indicate which regions have diverged and which have tended to be conserved during evolution. The C-terminal half of the vertebrate Tob protein includes a PQ- 15 rich sequence that is 26 amino acids long in human Tob (Matsuda et al., 1996) and 43 amino acids long in murine Tob (Yoshida et al., 1996). On the basis of the PQ-rich domain, Matsuda et al. (1996) tentatively proposed that Tob might act as a transcription factor. If Tob proteins do include such a function, then it is a feature peculiar to vertebrates, because AmphiTob lacks any equivalent of the PQ-rich domain (either through loss of an ancestral PQ-rich region in the cephalochordate line or through de novo acquisition in the vertebrate line). For Tob proteins in general, sequence data by themselves give no clear insights into potential functions. Although the hydrophobic N-terminal domain is not a signal peptide involved in secretion of Tob proteins (Roualt et al., 1992; Iacopetti et al., 1994; Varnum et al., 1994; Matsuda et al., 1996), its function is uncertain; for BTG1 proteins, which have a similar domain, Roualt et al. (1992) suggested that the nonpolar domain might anchor the protein to a cell membrane. AmphiTob, like human and murine Tob, includes several PEST residues, which are characteristic of proteins with relatively short half-lives (Rogers et al., 1986). The intracellular half-life has not been measured directly for any Tob protein, but the half-life of PC3 protein, which has comparable PEST residues, is less than 15 min (Varnum et al., 1994). Like the Tob protein, the mRNA coding for it is probably also short-lived due to the presence of ATTTA motifs (Shaw and Kamen, 1986) in the 38 untranslated regions of vertebrate Tob (Yoshida et al., 1996) and AmphiTob. Although transfection of Tob expression plasmids into cultured cells represses their growth (Matsuda et al., 1996), little is yet known about the detailed relations of Tob to other components of the growthregulating machinery. Matsuda et al. (1996) demonstrated that Tob can interact directly with at least one member of the epidermal growth factor receptor family (p185, which is encoded by Her2 5 erbB2/neu), resulting in the negative regulation of the antiproliferative pathway mediated by Tob; this interaction involves binding of Tob to the cytoplasmic domain of p185, which is a transmembrane receptor tyrosine kinase, although it is not yet known whether Tob is phosphorylated in the process. The downstream targets of Tob proteins are unknown at present. However, it has recently been found that PC3 and BTG1 can bind to a protein-arginine N-methyltransferase, which, in turn, may modulate splicing during the maturation of mRNA, with ultimate effects on the cell cycle (Lin et al., 1996). Thus, one can speculate that Tob might have a comparable target and mode of action. Alternatively, it has been suggested that PC3 might influence the phosphorylation state of the key growth-suppressor protein, pRB, as part of an antiproliferation pathway (Montagnoli et al., 1996). Expression of vertebrate genes of the PC3/BTG1/Tob family has been studied in cells stimulated by administration of exogenous inducers (Kujubu et al., 1987; Lim et al., 1987, 1995; Bradbury et al., 1991; Fletcher et al., 16 HOLLAND ET AL. Fig. 4. Wholemount in situ hybridization showing expression of AmphiTob in developing amphioxus; anterior in all specimens (except A) is toward the left; all scale lines are 50 mm. A: Late gastrula in an optical section midway along animal-vegetal axis; the hypoblast (probably on the dorsal side) is expressing AmphiTob. B: Side view of an early neurula embryo with expression in the foregut wall (arrow) and in the paraxial mesoderm. C: The preceding embryo in dorsal view focused at the level of the paraxial mesoderm; expressing cells are associated with the presomitic grooves (arrowheads). D: Side view of a hatching neurula with expression in the foregut wall (single arrow), paraxial mesoderm, and posterior mesoderm (double arrow). E: Dorsal view of the preceding stage showing expression in the paraxial mesoderm and posterior mesoderm. F: Side view of a late neurula with conspicuous expression near the anterior end of the dorsal nerve cord (arrow) and segmental expression in the somitic mesoderm. G: Late neurula in dorsal view focused at the level of the notochord (n) and somitic myocoels (arrows); AmphiTob is strongly expressed in cells in the myogenic medial wall of each somite. H: The preceding specimen in dorsal view focused at the level of the dorsal nerve cord; expressing cells are clustered near the anterior end (single arrow) and are also scattered more posteriorly along the nerve cord (double arrow). I: Side view of 24-hr embryo with expression in the anterior ectoderm (arrowhead), nerve cord (arrow), and somites. J: Anterior region of the preceding specimen with expression in the anterior ectoderm and cerebral vesicle of the nerve cord. K: Side view of a 30-hr larva with expression not only in the anterior ectoderm and nerve cord but also in the endoderm, especially in a few midgut and hindgut cells (arrowheads). L: Side view of a 2-week larvae showing three segments of the trunk musculature delimited by connective tissue septa (arrowheads); within each segment, some cells (arrow) express AmphiTob. STRUCTURE AND EXPRESSION OF AMPHIOXUS Tob 1991; Roualt et al., 1992; Cmarik et al., 1994), in unstimulated cell cultures (Matsuda et al., 1996), and also in the normal tissues of embryos (Iacopetti et al., 1994), juveniles (Tippets et al., 1988; Lim et al., 1994), and adults (Tippets et al., 1988; Roualt et al., 1992; Lim et al., 1994; Rayburn et al., 1995; Matsuda et al., 1996). In most normal tissues of postnatal vertebrates, transcription of these genes can usually be demonstrated by Northern blot analysis; presumably, much of this expression is at a low level for maintaining tissue homeostasis, with a possible admixture of more intense expression in predifferentiation compartments of renewing cell populations (Rayburn et al., 1995). The present study with wholemount in situ hybridization may lack the sensitivity to register low background levels of transcription. Therefore, our positive reactions identify cells that transcribe AmphiTob conspicuously. By analogy with what is known for PC3 (Iacopetti et al., 1994), it seems likely that this intense expression characterizes cells that are exiting from the cell cycle; however, this function of Tob during normal development has not yet been unequivocally established. There has been little work on expression patterns of vertebrate PC3/BTG1/Tob genes during normal development. The one existing study, which concerned PC3 and was focused exclusively on the developing central nervous system (Iacopetti et al., 1994), revealed that PC3 transcription transiently becomes intense in neuronal precursor cells entering growth arrest in preparation for subsequent differentiation. For interpreting the present in situ data for AmphiTob expression in developing amphioxus, it also seems likely that transiently intensified transcription of the gene occurs at the time when cells exit the growth cycle, soon before differentiation begins. In general, it seems likely that, like Tob, many genes that are viewed as tumor suppressors in one context will ultimately be found to play important roles during normal embryology. At the gastrula stage of amphioxus, conspicuous AmphiTob transcription in cells of the dorsal hypoblast is one of the earliest manifestations of dorsoventral polarity in the embryo. During the subsequent neurula stage, many of these cells evidently differentiate into axial trunk muscles along in the median wall of each somite (Holland, 1996). Later in development—even in advanced larval stages—some cells of the trunk musculature continue to express AmphiTob conspicuously, presumably because they are on the verge of differentiating into definitive myocytes to accommodate the continuing growth of the axial muscles. Other obvious domains of AmphiTob expression during amphioxus development include a region in the anterior part of the dorsal nerve cord that is probably homologous to the vertebrate diencephalic forebrain (N.D. Holland et al., 1996). Presumably, the intense transcription of AmphiTob is in neuroblasts exiting the cell cycle in preparation for differentiation. The strong expression of AmphiTob in the wall of the developing midgut and hindgut probably identifies cells that are 17 ceasing to divide prior to differentiation into unicellular endocrine cells synthesizing peptide hormones, such as insulin, secretin, and neurotensin (Thorndyke and Falkmer, 1985). For the vertebrates, it will be interesting to determine whether Tob is expressed during normal development, especially during myogenesis, neurogenesis, and the differentiation of certain endocrine organs. ACKNOWLEDGMENTS We are grateful to John Lawrence and Ray Wilson for laboratory facilities at the University of South Florida (Tampa and St. Petersburg). The Pax-6 probe was generously provided by Sacha Glardon. This work was supported in part by NSF research grant IBN 96309938 to N.D.H. and L.Z.H. and also by grants to S.C.Z. from the Natural Science Foundation of China and the K.C. Wong Education Foundation (Hong Kong). REFERENCES Bradbury A, Possenti R, Shooter EM, Tyrone F. Molecular cloning of PC3, a putatively secreted protein whose mRNA is induced by nerve growth factor and depolarization. Proc. Natl. Acad. Sci. USA 1991;88: 3353–3357. Cmarik JL, Herschman H, Colburn NH. Preferential primaryresponse gene expression in promotion-resistant vs. promotionsensitive JB6 cells. Mol. Carcinogen. 1994;11:115–124. Doolittle R. Of URFS and ORFS. Mill Valley, CA: University Scientific Books, 1987. Fletcher BS, Lim RW, Varnum BC, Kujubu DA, Koski RA, Herschman H. R. Structure and expression of TIS21, a primary response gene induced by growth factors and tumor promoters. J. Biol. Chem. 1991;266:14511–14518. Hayashi S, Rubinfeld B, Souza B, Polakis P, Wieschaus E, Levine AJ. A Drosophila homolog of the tumor suppressor gene adenomatous polyposis coli down-regulates b-catenin but its zygotic expression is not essential for regulation of armadillo. Proc. Natl. Acad. Sci. USA 1997;94:242–247. Holland LZ. Muscle development in amphioxus: Morphology, biochemistry, and molecular biology. Israel J. Zool. 1996;42(Suppl.):235–246. Holland LZ, Holland PWH, Holland ND. Revealing homologies between body parts of distantly related animals by in situ hybridization to developmental genes: Amphioxus vs. vertebrates. In: Ferraris JD, Palumbi SR, eds. Molecular Zoology: Advances, Strategies, and Protocols. New York: Wiley-Liss, Inc., 1996:267–282. Holland ND, Holland LZ. Embryos and larvae of invertebrate deuterostomes. In: Stern CD, Holland PWH, eds. Essential Developmental Biology: A Practical Approach. Oxford: IRL Press, 1993: 21–32. Holland ND, Panganiban T, Henyey E, Holland LZ. Sequence and developmental expression of AmphiDll, an amphioxus Distal-less gene transcribed in the ectoderm, epidermis, and nervous system: Insights into evolution of craniate forebrain and neural crest. Development 1996;122:2911–2920. Iacopetti P, Barsacchi G, Tirone F, Maffei L, Cremisi F. Developmental expression of PC3 gene is correlated with neuronal cell birthday. Mech. Dev. 1994;47:127–137. Johnson RL, Rothman AL, Xie JW, Goodrich LV, Bare JW, Bonifas JM, Quinn AG, Meyers RM, Cox DR, Epstein EH, Scott MP. (1996). Human homolog of patched, a candidate gene for the basal cell nevus syndrome. Science 1996;272:1668–1671. Kujubu DA, Lim RW, Varnum BC, Herschman H. Induction of transiently expressed genes in PC-12 pheochromocytoma cells. Oncogene 1987;1:257–262. Lim IK, Kim NK, Lee MS, Lee SH. Expression of TIS-21 gene during the development of Balb/c mice and the liver regeneration. Korean J. Biochem. 1994;26:169–175. 18 HOLLAND ET AL. Lim IK, Lee MS, Lee SH, Kim NK, Jou I, Seo JS, Park SC. Differential expression of TIS21 and TIS1 genes in the various organs of Balb/c mice, thymic carcinoma tissues and human cancer cell lines. J. Cancer Res. Clin. Oncol. 1995;121:279–284. Lim RW, Varnum BC, Herschman HR. Cloning of tetradecanoyl phorbol ester-induced ‘‘primary response’’ sequences and their expression in density-arrested Swiss 3T3 cells and a TPA nonproliferative variant. Oncogene 1987;1:263–270. Lin WJ, Gary JD, Yang MC, Clarke S, Herschman HR. The mammalian immediate-early TIS21 protein and the leukemia-associated BTG1 protein interact with a protein-arginine N-methyltransferase. J. Biol. Chem. 1996;271:15034–15044. Matsuda S, Kawamura-Tsuzuku J, Oshugi M, Yoshida M, Emi M, Nakamura Y, Onda M, Yoshida Y, Nishiyama A, Yamamoto T. Tob, a novel protein that interacts with p185erbB2, is associated with antiproliferative activity. Oncogene 1996;12:705–713. McCartney BM, Fehon RG. Distinct cellular and subcellular patterns of expression imply distinct functions for the Drosophila homologues of moesin and the neurofibromatosis 2 tumor suppressor, merlin. J. Cell Biol. 1996;133:843–852. Montagnoli A, Guardavaccaro D, Starace G, Tirone F. Overexpression of the nerve growth factor-inducible PC3 immediate early gene is associated with growth inhibition. Cell Growth Diff. 1996;7:1327– 1336. Rayburn DJ, Hamil KG, Tsuruta JK, O’Brien DA, Hall SH. Stagespecific expression of B cell translocation gene 1 in rat testis. Endocrinology 1995;136:5769–5777. Rogers S, Wells R, Rechsteiner M. Amino acid sequences common to rapidly degraded proteins: The PEST hypothesis. Science 1986;234: 364–368. Roualt JP, Rimokh R, Tessa C, Paranhos G, Ffrench M, Duret L, Garoccio M, Germain D, Samarut J, Magaud JP. BTG1, a member of a new family of antiproliferative genes. EMBO J. 1992;11:16631670. Roualt JP, Samarut C, Duret L, Tessa C, Samarut J, Magaud JP. Sequence analysis reveals that the BTG1 antiproliferative gene is conserved throughout evolution in its coding and 38 noncoding regions. Gene 1993;129:303–306. Shaw G, Kamen R. A conserved AU sequence from the 38 untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 1986;46:659–667. Thorndyke MC, Falkmer S. The brain-gut axis in protochordates. In: Foreman RE, Gormban A, Dodd JM, Olsson R, eds. Evolutionary Biology of Primitive Fishes. New York: Plenum Press, 1985:379– 400. Tippets MT, Varnum BC, Lim RW, Herschman HR. Tumor promoterinducible genes are differentially expressed in the developing mouse. Mol. Cell. Biol. 1988;8:4570–4572. Varnum BC, Reddy ST, Koski RA, Herschman HR. Synthesis, degradation, and subcellular localization of proteins encoded by the primary response genes TIS7/PC4 and TIS21/PC3. J. Cell. Physiol. 1994;158: 205–213. Watson KL, Justice RW, Bryant PJ. (1994) Drosophila in cancer research: The first fifty tumor suppressor genes. J. Cell Sci. 1994; 18(Suppl.):19–33. Weinberg RA. Tumor suppressor genes. Science 1991;254:1138–1146. Yoshida Y, Matsuda S, Yamamoto T. Unpublished GenBank Sequence D78382, 1996.