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cDNA cloning of run, a Caenorhabditis elegans Runt domain encoding gene
Gene 241 (2000) 255–258 www.elsevier.com/locate/gene
cDNA cloning of run, a Caenorhabditis elegans Runt domain encoding gene Suk-Chul Bae a, *, Junho Lee b a Department of Biochemistry, School of Medicine, Chungbuk National University, Chungju 361-763, South Korea b Department of Biology, Yonsei University, Seoul 120-749, South Korea Received 19 July 1999; received in revised form 11 October 1999; accepted 4 November 1999 Received by A.J. van Wijnen
Abstract PEBP2/CBF is a heterodimeric transcription factor composed of a and b subunits. The essential roles of mammalian PEBP2a genes, PEBP2aA/CBFA1 and PEBP2aB/CBFA2 in osteogenesis and hematopoiesis have been well documented. The PEBP2a proteins contain a 128 amino acid (aa) region which is highly homologous to Drosophila melanogaster runt and lozenge. The evolutionarily conserved region has been named the Runt domain. In this study, we isolated a cDNA encoding the Caenorhabditis elegans homolog of mammalian PEBP2a. The cDNA encodes a 301-aa protein with a highly conserved Runt domain. In addition, a IWRPF five aa motif is present at the C-terminal end. © 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: AML1; CBF; Lozenge; PEBP2; Transcription factor
1. Introduction Recent work has identified a new family of transcriptional regulatory proteins termed PEBP2/CBF (Polyomavirus Enhancer-core Binding Protein 2/Core Binding Factor), whose members play important roles in hematopoiesis and osteogenesis in mouse and human. PEBP2/CBF is a sequence-specific DNA binding protein. Each member of the PEBP2/CBF family of transcription factors is composed of two subunits, a and b (Ogawa et al., 1993a, 1993b; Wang et al., 1993; Bae et al., 1994; Bae and Ito, 1999). The a subunit binds to DNA and the b subunit increases the binding affinity of the a protein for DNA. Three closely related mammalian PEBP2a genes, termed PEBP2aA/CBFA1, PEBP2aB/ CBFA2 and PEBP2aC/CBFA3 have been reported previously (Ogawa et al., 1993b; Bae et al., 1993, 1995; Abbreviations: aa, amino acid(s); AML1, acute myeloid leukemia 1 protein; AML1, gene encoding AML1; CBF, core binding factor; CBFA, a subunit of CBF; CBFA, gene encoding human CBFA; cbfa, gene encoding mouse CBFA; cDNA, DNA complementary to mRNA; nt, nucleotide(s); PEBP2, polyomavirus enhancer binding protein 2; PEBP2, gene encoding human PEBP2; Pebp2, gene encoding mouse PEBP2; PEBP2aA (aB, aC, b), aA (aB, aC, b) subunit of PEBP2. * Corresponding author. Tel.: +82-431-261-2842; fax: +82-431-274-8705. E-mail address: [email protected] (S.-C. Bae)
Levanon et al., 1994). PEBP2aB, also called AML1, has been identified at the breakpoint of chromosome translocations responsible for human leukemia (Miyoshi et al., 1991; Speck and Stacy, 1995; Nucifora and Rowley, 1995). Targeted disruption of the Pebp2aB/ Aml1 gene resulted in lack of definitive hematopoiesis of fetal liver (Okuda et al., 1996; Wang et al., 1996). Analyses of the expression and function of Xaml, the Xenopus homolog of PEBP2aBB/AML1, also suggested that PEBP2aB is required for the development of all blood lineages and that PEBP2aB may function in the early formation of hematopoietic stem cells ( Tracey et al., 1998). More recently, it has been demonstrated that Pebp2aA/Cbfa1, another Pebp2a gene, plays an essential role in osteogenesis. Mice with a homozygous mutation in Pebp2aA died just after birth due to breathing inability caused by a complete lack of ossification ( Komori et al., 1997; Otto et al., 1997). The PEBP2aA gene is also responsible for the human disease, cleidocranial dysplasia (CCD), an autosomal dominant disorder (Lee et al., 1997; Mundlos et al., 1997). Each of the three PEBP2a/CBFA gene products is highly homologous to Drosophila runt ( Kania et al., 1990) and lozenge (Daga et al., 1996) within a 128 amino acid region which harbors two different activities, the ability to bind DNA and the ability to interact with the b subunit. The
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evolutionarily conserved 128 amino acid region has been called the Runt domain ( Kagoshima et al., 1993). Runt was initially characterized because of its role as one of the pair-rule genes during segmentation (Gergen and Wieschaus, 1985) and was subsequently found to have roles in two other developmental processes, fly-sex determination and neurogenesis (Duffy and Gergen, 1991; Duffy et al., 1991). Lozenge is involved in prepatterning photoreceptor precursors in the developing fruit-fly eye (Daga et al., 1996). Since the Runt domain is evolutionarily conserved, and as proteins containing this domain play important biological roles in the fruit fly, mouse and human, we examined whether nematode also contains Runt domain encoding genes. Amino acid sequence homology searches in the Caenorhabditis elegans database revealed that C. elegans contains a Runt domain-encoding gene in its genome. In this study, we describe the isolation of cDNA clones encoding the C. elegans Runt domain, by screening a cDNA library. We propose that this gene be referred to as run (Runt homologous gene). Sequence analysis revealed that run cDNA encodes a highly conserved Runt domain and an IWRPF motif.
2. Materials and methods A C. elegans mixed-stage cDNA library in the UniZAP XR vector (Stratagene) was screened by plaque hybridization (Sambrook et al., 1989). C. elegans cosmid clone B0414 was obtained from the C. elegans genome project. The probe DNA was prepared by digesting B0414 cosmid clone with BglII and RsrII restriction enzymes followed by agarose gel electrophoresis and gel elution. The probe contains the region from nt 12116 to 13699 of B0414 and covers part of the Runt domain. Double positive clones were isolated and the pBluescript phagemids were excised from the Uni-ZAP XT vector in vivo according to the manufacturer’s manual (Stratagene). The nucleotide sequences of the phagemids containing the cDNA insert were analyzed by an automatic sequencer (ABI prism 377). The complete sequence was verified from both directions.
3. Results and discussion 3.1. Isolation of the C. elegans homolog of mammalian PEBP2a/CBFA cDNA To obtain cDNAs encoding the C. elegans homolog of mammalian PEBP2a gene, a cDNA library derived from the mRNAs of mixed developmental stages of C. elegans was screened using the segment of the C. elegans cosmid B0414 which encodes the Runt domain. From 1×106 phage plaques, two positive clones were isolated.
Fig. 1. Amino acid sequence and genomic structure of run. (A) Predicted amino acid sequence from the open reading frame of run cDNA. The Runt domain is underlined. (B) The genomic structure of run. Boxes represent exons and filled boxes represent coding regions. Comparison of C. elegans cosmid clone B0414 and the cDNA sequences (the GenBank accession No. of the cDNA sequence is AF153275) determined nucleotide numbers of the exon–intron junctions. The nt numbers of only the 5∞-ends of the exons are indicated.
Restriction enzyme analysis showed that two clones contained 1.5 kb- and 0.9 kb-long inserts with overlapping restriction patterns. The nucleotide sequences of longer cDNAs were completely determined. The total number of nucleotides was 1512. An oligo-dA stretch of 20 residues was located at the 3∞ end. This stretch is likely to represent the poly(A) tail of the mRNA, since there was a polyadenylation signal, AATAAA, 12 nucleotides upstream of the oligo-dA stretch and the genomic DNA did not have any A-rich stretch around this region. There was a long open reading frame starting at nt 388 with the capacity to encode a protein of 301 aa. The ATG at nt 388 is likely to be the start codon, since this ATG is the only initiation codon in the open reading frame which allows translation of the Runt domain. This gene was named run. Fig. 1A shows the aa sequence of RUN. The 1512-nt sequence of run cDNA was deposited in GenBank under the accession No. AF153275. 3.2. Genomic structure Nucleotide sequence comparison of run cDNA with the C. elegans genome sequence revealed the run gene to be composed of 11 exons. There is an exceptionally long intron (7.2 kb) between exons 3 and 4. The putative
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fourth exon, containing the most probable translation initiation codon, also encodes the first part of the Runt domain. The remaining Runt domain is encoded by exon 5, 6, 7 and 8. The physical map of the run gene is depicted in Fig. 1B. 3.3. Amino acid sequence homology The amino acid sequence homology within the Runt domain of Runt family members including C. elegans RUN is shown in Fig. 2A. It is interesting to note that, while the homology between RUN (nematode)/runt (fruit fly) and RUN/Lozenge (fruit fly) are 52 and 53%, respectively, the homology between the two fruit-fly runt domains is only 69%. Furthermore, the homologies
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between RUN and three PEBP2a (human) homologs are about 50%, which is not so different from the homology between nematode and fruit fly. One possible explanation of these results would be that diversification of the Runt domain occurred at a very early stage of evolution. The apparent evolutionary relationships of nine members of the Runt family are shown in Fig 2B. It is worthwhile noting that the N-terminal part of the RUN Runt domain is much less homologous to other Runt domains than the C-terminal part. It has been proposed that the N-terminal part is important for redox regulation as well as heterodimerization with the b subunit. The major function of the b subunit seems be protection of the cysteine residues in the N-terminal region from oxidation ( Kagoshima et al., 1996). The Runt domain of nematode RUN, however, is only weakly homologous in its N-terminal region and does not contain redox sensitive cysteine residues (Fig. 2A). This observation and our failure to find a PEBP2bCBFB homologous gene in the C. elegans genome database suggest that RUN may be functional in the absence of the b protein. It has been observed that the C-terminal-most five-aa sequence, VWRPY, is 100% conserved from sea urchin to human and that this sequence is shared by all members of the full-size Runt domain family identified so far. RUN also contains a very similar aa sequence, IWRPF, although it is not identical, at its corresponding region (Fig. 1A). The conservation of this five-aa sequence at the C-terminal end of the protein might imply that the VWRPY/IWRPF motif functions as a part of the Runt domain. Examination of the expression pattern of the run gene, and identification and characterization of null mutations in development, will elucidate the biological functions of run gene in the nematode.
4. Conclusions
Fig. 2. The amino acid sequence alignment of the Runt domain. (A) The sequence information of sea urchin (SpRunt-1), fruit fly (runt, lozenge), frog ( Xaml1), chicken (runtB) and human (PEBP2aA/CBFA1, PEBP2aB/CBFA2/AML1, PEBP2aC/CBFA3) were taken from Coffman et al. (1996), Kania et al. (1990), Daga et al. (1996), Tracey et al. (1998), Castagnola et al. (1996), Ogawa et al. (1993b), Bae et al. (1993, 1995) and Levanon et al. (1994). The Runt domains of the mouse and human proteins are identical. Identical amino acids between more than four species are indicated by shadowing. Asterisks indicate the putative redox sensitive cysteine residues. (B) A dendrogram showing sequence similarities among known Runt domain proteins. Amino acid sequences of Runt domains were aligned and evolutionary relationship was calculated using CLUSTAL method (Higgins and Sharp, 1988). The numbers represent relative relationships between branch-points.
(1) We isolated the C. elegans homolog (run) of PEBP2a/CBFA which is known to play important functions in hematopoiesis and osteogenesis in mouse and human. (2) The run gene is composed of 11 exons and a 7.2 kb long intron is located between exons 3 and 4.
Acknowledgements We are very grateful to all our colleagues and collaborators who have made this work possible. We also thank Dr. Alan Coulson for the kind gift of B0414 cosmid clone. A Genetic Engineering Research Grant (1998-019-D00047) from the Ministry of Education, Korea supported this study.
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References Bae, S.-C., Ito, Y., 1999. Regulation mechanisms for the heterodimeric transcription factor PEBP2/CBF. Histol. Histopathol. 14, 1213–1221. Bae, S.-C., Ogawa, E., Maruyama, M., Oka, H., Satake, M., Shigesada, K., Jenkins, N.A., Gilbert, D.J., Copeland, N.G., Ito, Y., 1994. PEBP2aB/ mouse AML1 consists of multiple isoforms that possesses differential transactivation potentials. Mol. Cell. Biol. 14, 3242–3252. Bae, S.-C., Takahasi, E., Zhang, Y.W., Ogawa, E., Shigesada, K., Namba, Y., Satake, M., Ito, Y., 1995. Cloning, mapping and expression of PEBP2aC, a third gene encoding the mammalian Runt domain. Gene 159, 245–248. Bae, S.-C., Yamaguchi-Iwai, Y., Ogawa, E., Maruyama, M., Inuzuka, M., Kagoshima, H., Satake, M., Ito, Y., 1993. Isolation of PEBP2aB cDNA representing the mouse homologue of human acute myeloid leukemia gene AML1. Oncogene 8, 809–814. Castagnola, P., Gennari, M., Gaggero, A., Rossi, F., Daga, A., Corsetti, M.T., Calabi, F., Cancedda, R., 1996. Expression of runtB is modulated during chondrocyte differentiation. Exp. Cell. Res. 223, 215–226. Coffman, J.A., Kirchhamer, C.V., Harrington, M.G., Davidson, E.H., 1996. SpRunt-1, a new member of the runt domain family of transcription factors, is a positive regulator of the aboral ectodermspecific CyIIIA gene in sea urchin embryos. Dev. Biol. 174, 43–54. Daga, A., Karlovich, C.A., Dumstrei, K., Banerjee, J., 1996. Patterning of cells in the Drosophila eye by Lozenge, which shares homologous domain with AML1. Genes Dev. 10, 1194–1205. Duffy, J.B., Gergen, J.P., 1991. The Drosophila segmentation gene runt acts as a position-specific numerator element necessary for the uniform expression of the sex determining gene, Sex-lethal. Genes Dev. 5, 2176–2187. Duffy, J.B., Kania, M.A., Gergen, J.P., 1991. Expression and function of the Drosophila gene runt in early stages of neural development. Development 113, 1223–1230. Gergen, J.P., Wieschaus, E., 1985. The localized requirement for a gene affecting segmentation in Drosophila: analysis of larvae mosaic for runt. Dev. Biol. 109, 321–335. Higgins, D.G., Sharp, P.M., 1988. CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene 73, 237–244. Kagoshima, H., Akamatsu, Y., Ito, Y., Shigesada, K., 1996. Functional dissection of the alpha and beta subunits of transcription factor PEBP2 and the redox susceptibility of its DNA binding activity. J. Biol. Chem. 271, 3074–3082. Kagoshima, H., Satake, M., Miyoshi, H., Ohki, M., Pepling, M., Gergen, P., Shigesada, K., Ito, Y., 1993. The Runt domain identifies a new family of heterodimeric transcriptional regulators. Trends Genet. 9, 338–341. Kania, M.A., Bonne, A.S., Duffy, J.B., Gergen, J.P., 1990. The Drosophila segmentation gene runt encodes a novel nuclear regulatory protein that is also expressed in the developing nervous system. Genes Dev. 4, 1701–1713. Komori, T., Yagi, H., Nomura, S., Yamaguchi, A., Sasaki, K., Deguchi, K., Shimizu, Y., Bronson, R.T., Gao, Y.-H., Inada, M.,
Sato, M., Okamoto, R., Kitamura, Y., Yoshiaki, S., Kishimoto, T., 1997. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89, 755–764. Lee, B., Thirunavukkarasu, K., Zhou, L., Pastore, L., Baldini, A., Hecht, J., Geoffroy, V., Ducy, P., Karsenty, G., 1997. Missense mutations abolishing DNA binding of the osteoblast-specific transcription factor OSF2/CBFA1 in cleidocranial dysplasia. Nat. Genet. 15, 303–306. Levanon, D., Negreanu, V., Bernstein, Y., Bar-Am, I., Avivi, L., Groner, Y., 1994. AML1, AML2 and AML3 the human members of the runt domain gene-family: cDNA structure, expression, and chromosomal localization. Genomics 23, 425–432. Miyoshi, H., Shimizu, K., Kozu, T., Maseki, N., Kaneko, Y., Ohki, M., 1991. t(8,21) breakpoints on chromosome 21 in acute myeloid leukemia are clustered within a limited region of a single gene, AML1. Proc. Natl. Acad. Sci. USA 88, 10431–10434. Mundlos, S., Otto, F., Mundlos, C., Mulliken, J.B., Aylsworth, A.S., Albright, S., Lindhout, D., Coel, W.G., Mertelsman, R., Zabel, B.U., Olsen, B.R., 1997. Mutation involving the transcription factor CBFA1 causes cleidocranial dysplasia. Cell 89, 773–779. Nucifora, G., Rowley, J.D., 1995. AML1 and the 8;21 and 3;21 translocation in acute and chronic myeloid leukemia. Blood 86, 1–14. Ogawa, E., Inuzuka, M., Maruyama, M., Satake, M., Naito-fujimoto, M., Ito, Y., Shigesada, K., 1993a. Molecular cloning and characterization of PEBP2b, the heterodimeric partner of a novel Drosophila runt-related DNA binding protein PEBP2a. Virology 194, 314–331. Ogawa, E., Maruyama, M., Kagoshima, H., Inuzuka, M., Satake, M., Shigesada, K., Ito, Y., 1993b. PEBP2/PEA2 represents a family of transcription factors homologous to the products of the Drosophila runt gene and human AML1 gene. Proc. Natl. Acad. Sci. USA 90, 6859–6863. Okuda, T., van Deursen, J., Hiebert, S.W., Grosveld, G., Downing, J.R., 1996. AML1, the target of multiple chromosomal translocations in human leukemia is essential for normal fetal liver hematopoiesis. Cell 84, 321–330. Otto, F., Thornell, A.P., Crompton, T., Owen, M.J., 1997. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome is essential for osteoblast differentiation and bone development. Cell 89, 765–771. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. second ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Speck, N.A., Stacy, T., 1995. A new transcription factor family associated with human leukemias:. Crit. Rev. Eukaryotic Gene Expr. 5, 337–364. Tracey Jr., W.D., Pepling, M.E., Horb, M.E., Thomsen, G.H., Gergen, J.P., 1998. A Xenopus homologue of aml-1 reveals unexpected patterning mechanisms leading to the formation of embryonic blood. Development 125, 1371–1380. Wang, Q., Stacy, T., Binder, M., Matine-Padilla, M., Sharpe, A.H., Speck, N.A., 1993. Disruption of the cbfa2 gene causes necrosis and hemorrhaging in the central nervous system and blocks definitive hematopoiesis. Proc. Natl. Acad. Sci. USA 92, 3444–3449. Wang, S., Wang, Q., Crute, B.E., Melnikova, I.N., Keller, S.R., Speck, N.A., 1993. Cloning and characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding factor. Mol. Cell. Biol. 13, 3324–3339.
cDNA cloning of run, a Caenorhabditis elegans Runt domain encoding gene Suk-Chul Bae a, *, Junho Lee b a Department of Biochemistry, School of Medicine, Chungbuk National University, Chungju 361-763, South Korea b Department of Biology, Yonsei University, Seoul 120-749, South Korea Received 19 July 1999; received in revised form 11 October 1999; accepted 4 November 1999 Received by A.J. van Wijnen
Abstract PEBP2/CBF is a heterodimeric transcription factor composed of a and b subunits. The essential roles of mammalian PEBP2a genes, PEBP2aA/CBFA1 and PEBP2aB/CBFA2 in osteogenesis and hematopoiesis have been well documented. The PEBP2a proteins contain a 128 amino acid (aa) region which is highly homologous to Drosophila melanogaster runt and lozenge. The evolutionarily conserved region has been named the Runt domain. In this study, we isolated a cDNA encoding the Caenorhabditis elegans homolog of mammalian PEBP2a. The cDNA encodes a 301-aa protein with a highly conserved Runt domain. In addition, a IWRPF five aa motif is present at the C-terminal end. © 2000 Published by Elsevier Science B.V. All rights reserved. Keywords: AML1; CBF; Lozenge; PEBP2; Transcription factor
1. Introduction Recent work has identified a new family of transcriptional regulatory proteins termed PEBP2/CBF (Polyomavirus Enhancer-core Binding Protein 2/Core Binding Factor), whose members play important roles in hematopoiesis and osteogenesis in mouse and human. PEBP2/CBF is a sequence-specific DNA binding protein. Each member of the PEBP2/CBF family of transcription factors is composed of two subunits, a and b (Ogawa et al., 1993a, 1993b; Wang et al., 1993; Bae et al., 1994; Bae and Ito, 1999). The a subunit binds to DNA and the b subunit increases the binding affinity of the a protein for DNA. Three closely related mammalian PEBP2a genes, termed PEBP2aA/CBFA1, PEBP2aB/ CBFA2 and PEBP2aC/CBFA3 have been reported previously (Ogawa et al., 1993b; Bae et al., 1993, 1995; Abbreviations: aa, amino acid(s); AML1, acute myeloid leukemia 1 protein; AML1, gene encoding AML1; CBF, core binding factor; CBFA, a subunit of CBF; CBFA, gene encoding human CBFA; cbfa, gene encoding mouse CBFA; cDNA, DNA complementary to mRNA; nt, nucleotide(s); PEBP2, polyomavirus enhancer binding protein 2; PEBP2, gene encoding human PEBP2; Pebp2, gene encoding mouse PEBP2; PEBP2aA (aB, aC, b), aA (aB, aC, b) subunit of PEBP2. * Corresponding author. Tel.: +82-431-261-2842; fax: +82-431-274-8705. E-mail address: [email protected] (S.-C. Bae)
Levanon et al., 1994). PEBP2aB, also called AML1, has been identified at the breakpoint of chromosome translocations responsible for human leukemia (Miyoshi et al., 1991; Speck and Stacy, 1995; Nucifora and Rowley, 1995). Targeted disruption of the Pebp2aB/ Aml1 gene resulted in lack of definitive hematopoiesis of fetal liver (Okuda et al., 1996; Wang et al., 1996). Analyses of the expression and function of Xaml, the Xenopus homolog of PEBP2aBB/AML1, also suggested that PEBP2aB is required for the development of all blood lineages and that PEBP2aB may function in the early formation of hematopoietic stem cells ( Tracey et al., 1998). More recently, it has been demonstrated that Pebp2aA/Cbfa1, another Pebp2a gene, plays an essential role in osteogenesis. Mice with a homozygous mutation in Pebp2aA died just after birth due to breathing inability caused by a complete lack of ossification ( Komori et al., 1997; Otto et al., 1997). The PEBP2aA gene is also responsible for the human disease, cleidocranial dysplasia (CCD), an autosomal dominant disorder (Lee et al., 1997; Mundlos et al., 1997). Each of the three PEBP2a/CBFA gene products is highly homologous to Drosophila runt ( Kania et al., 1990) and lozenge (Daga et al., 1996) within a 128 amino acid region which harbors two different activities, the ability to bind DNA and the ability to interact with the b subunit. The
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evolutionarily conserved 128 amino acid region has been called the Runt domain ( Kagoshima et al., 1993). Runt was initially characterized because of its role as one of the pair-rule genes during segmentation (Gergen and Wieschaus, 1985) and was subsequently found to have roles in two other developmental processes, fly-sex determination and neurogenesis (Duffy and Gergen, 1991; Duffy et al., 1991). Lozenge is involved in prepatterning photoreceptor precursors in the developing fruit-fly eye (Daga et al., 1996). Since the Runt domain is evolutionarily conserved, and as proteins containing this domain play important biological roles in the fruit fly, mouse and human, we examined whether nematode also contains Runt domain encoding genes. Amino acid sequence homology searches in the Caenorhabditis elegans database revealed that C. elegans contains a Runt domain-encoding gene in its genome. In this study, we describe the isolation of cDNA clones encoding the C. elegans Runt domain, by screening a cDNA library. We propose that this gene be referred to as run (Runt homologous gene). Sequence analysis revealed that run cDNA encodes a highly conserved Runt domain and an IWRPF motif.
2. Materials and methods A C. elegans mixed-stage cDNA library in the UniZAP XR vector (Stratagene) was screened by plaque hybridization (Sambrook et al., 1989). C. elegans cosmid clone B0414 was obtained from the C. elegans genome project. The probe DNA was prepared by digesting B0414 cosmid clone with BglII and RsrII restriction enzymes followed by agarose gel electrophoresis and gel elution. The probe contains the region from nt 12116 to 13699 of B0414 and covers part of the Runt domain. Double positive clones were isolated and the pBluescript phagemids were excised from the Uni-ZAP XT vector in vivo according to the manufacturer’s manual (Stratagene). The nucleotide sequences of the phagemids containing the cDNA insert were analyzed by an automatic sequencer (ABI prism 377). The complete sequence was verified from both directions.
3. Results and discussion 3.1. Isolation of the C. elegans homolog of mammalian PEBP2a/CBFA cDNA To obtain cDNAs encoding the C. elegans homolog of mammalian PEBP2a gene, a cDNA library derived from the mRNAs of mixed developmental stages of C. elegans was screened using the segment of the C. elegans cosmid B0414 which encodes the Runt domain. From 1×106 phage plaques, two positive clones were isolated.
Fig. 1. Amino acid sequence and genomic structure of run. (A) Predicted amino acid sequence from the open reading frame of run cDNA. The Runt domain is underlined. (B) The genomic structure of run. Boxes represent exons and filled boxes represent coding regions. Comparison of C. elegans cosmid clone B0414 and the cDNA sequences (the GenBank accession No. of the cDNA sequence is AF153275) determined nucleotide numbers of the exon–intron junctions. The nt numbers of only the 5∞-ends of the exons are indicated.
Restriction enzyme analysis showed that two clones contained 1.5 kb- and 0.9 kb-long inserts with overlapping restriction patterns. The nucleotide sequences of longer cDNAs were completely determined. The total number of nucleotides was 1512. An oligo-dA stretch of 20 residues was located at the 3∞ end. This stretch is likely to represent the poly(A) tail of the mRNA, since there was a polyadenylation signal, AATAAA, 12 nucleotides upstream of the oligo-dA stretch and the genomic DNA did not have any A-rich stretch around this region. There was a long open reading frame starting at nt 388 with the capacity to encode a protein of 301 aa. The ATG at nt 388 is likely to be the start codon, since this ATG is the only initiation codon in the open reading frame which allows translation of the Runt domain. This gene was named run. Fig. 1A shows the aa sequence of RUN. The 1512-nt sequence of run cDNA was deposited in GenBank under the accession No. AF153275. 3.2. Genomic structure Nucleotide sequence comparison of run cDNA with the C. elegans genome sequence revealed the run gene to be composed of 11 exons. There is an exceptionally long intron (7.2 kb) between exons 3 and 4. The putative
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fourth exon, containing the most probable translation initiation codon, also encodes the first part of the Runt domain. The remaining Runt domain is encoded by exon 5, 6, 7 and 8. The physical map of the run gene is depicted in Fig. 1B. 3.3. Amino acid sequence homology The amino acid sequence homology within the Runt domain of Runt family members including C. elegans RUN is shown in Fig. 2A. It is interesting to note that, while the homology between RUN (nematode)/runt (fruit fly) and RUN/Lozenge (fruit fly) are 52 and 53%, respectively, the homology between the two fruit-fly runt domains is only 69%. Furthermore, the homologies
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between RUN and three PEBP2a (human) homologs are about 50%, which is not so different from the homology between nematode and fruit fly. One possible explanation of these results would be that diversification of the Runt domain occurred at a very early stage of evolution. The apparent evolutionary relationships of nine members of the Runt family are shown in Fig 2B. It is worthwhile noting that the N-terminal part of the RUN Runt domain is much less homologous to other Runt domains than the C-terminal part. It has been proposed that the N-terminal part is important for redox regulation as well as heterodimerization with the b subunit. The major function of the b subunit seems be protection of the cysteine residues in the N-terminal region from oxidation ( Kagoshima et al., 1996). The Runt domain of nematode RUN, however, is only weakly homologous in its N-terminal region and does not contain redox sensitive cysteine residues (Fig. 2A). This observation and our failure to find a PEBP2bCBFB homologous gene in the C. elegans genome database suggest that RUN may be functional in the absence of the b protein. It has been observed that the C-terminal-most five-aa sequence, VWRPY, is 100% conserved from sea urchin to human and that this sequence is shared by all members of the full-size Runt domain family identified so far. RUN also contains a very similar aa sequence, IWRPF, although it is not identical, at its corresponding region (Fig. 1A). The conservation of this five-aa sequence at the C-terminal end of the protein might imply that the VWRPY/IWRPF motif functions as a part of the Runt domain. Examination of the expression pattern of the run gene, and identification and characterization of null mutations in development, will elucidate the biological functions of run gene in the nematode.
4. Conclusions
Fig. 2. The amino acid sequence alignment of the Runt domain. (A) The sequence information of sea urchin (SpRunt-1), fruit fly (runt, lozenge), frog ( Xaml1), chicken (runtB) and human (PEBP2aA/CBFA1, PEBP2aB/CBFA2/AML1, PEBP2aC/CBFA3) were taken from Coffman et al. (1996), Kania et al. (1990), Daga et al. (1996), Tracey et al. (1998), Castagnola et al. (1996), Ogawa et al. (1993b), Bae et al. (1993, 1995) and Levanon et al. (1994). The Runt domains of the mouse and human proteins are identical. Identical amino acids between more than four species are indicated by shadowing. Asterisks indicate the putative redox sensitive cysteine residues. (B) A dendrogram showing sequence similarities among known Runt domain proteins. Amino acid sequences of Runt domains were aligned and evolutionary relationship was calculated using CLUSTAL method (Higgins and Sharp, 1988). The numbers represent relative relationships between branch-points.
(1) We isolated the C. elegans homolog (run) of PEBP2a/CBFA which is known to play important functions in hematopoiesis and osteogenesis in mouse and human. (2) The run gene is composed of 11 exons and a 7.2 kb long intron is located between exons 3 and 4.
Acknowledgements We are very grateful to all our colleagues and collaborators who have made this work possible. We also thank Dr. Alan Coulson for the kind gift of B0414 cosmid clone. A Genetic Engineering Research Grant (1998-019-D00047) from the Ministry of Education, Korea supported this study.
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