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Cloning and developmental expression of Xenopus cDNAs encoding the Enhancer of split groucho and related proteins
Gene 195 (1997) 41–48
Cloning and developmental expression of Xenopus cDNAs encoding the Enhancer of split groucho and related proteins Barun K. Choudhury a, Jaebong Kim b, Hsiang-Fu Kung b, Steven S.-L. Li a,c,* a Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA b Laboratory of Biochemical Physiology, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA c Institute of Life Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan 80424, ROC Received 29 October 1996; accepted 1 February 1997; Received by A. Nakazawa
Abstract The two full-length cDNAs encoding ESG1 (Enhancer of split groucho) and related AES (Amino Enhancer of split) proteins of 767 and 197 amino acids, respectively, were cloned and sequenced from the African frog Xenopus laevis. The amino acid sequence of Xenopus ESG1 protein had 61% identity to the full-length Drosophila groucho. Xenopus AES protein exhibited 91%, 58% and 48% identity to the mouse AES, amino-terminal regions of Xenopus ESG1 and Drosophila groucho, respectively. Northern blot analysis showed that widespread RNA expression of ESG1 of 2.8 kb, ESG2 of 3.6 kb and AES of 2.2 kb transcripts were seen in adult tissues, whereas ESG1 and AES transcripts of 2.8 kb and 2.2 kb, respectively, were ubiquitously expressed in the developing embryos. The overall structural relationships of ESG and AES proteins among human, mouse, rat, Xenopus and Drosophila were analysed. © 1997 Elsevier Science B.V. Keywords: Nucleotide sequence; Protein; Gene expression; Tissues; Phylogenetic tree; Amphibian
1. Introduction In the fruitfly, Drosophila melanogaster, neurogenic loci, namely, Enhancer of split, Notch, Delta, mastermind, big brain and neuralized, are required for the proper segregation of neural and epidermal progenitor cells during formation of both the central and peripheral nervous systems (Artavanis-Tsakonas et al., 1991; Cabrera, 1992; Campos-Ortega and Jan, 1991; Ghysen et al., 1993). Loss-of-functions mutations in these genes cause most ventral ectodermal cells to become neuroblasts with little or no formation of epidermis (Lehman * Corresponding author. Address c. Tel. +886 7 5253616; Fax: +886 7 5253696. Abbreviations: aa, amino acid(s); AES, Amino Enhancer of split; cdc2K, cdc2 kinase site; bHLH, basic helix–loop–helix; CKII, casein kinase II site; ESG, Enhancer of split groucho; G-protein, guanine nucleotide binding protein; kb, kilobase(s); NLS, nuclear localization sequence; nt, nucleotide(s); ORF, open reading frame; TLE, transducin-like Enhancer of split; UTR, untranslated region(s); WD-40 repeat, the repeat of approximately 40 aa demarcated by Trp-Asp ( WD). 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 1 50 - 9
et al., 1983). The Enhancer of split gene complex contains at least 13 transcription units. Molecular studies have shown that most of these transcription units encode proteins with the basic helix–loop–helix (bHLH ) motif characteristic of certain transcription factors (Delidakis and Artavanis-Tsakonas, 1992; Knust et al., 1992; Schrons et al., 1992). Another structurally unrelated transcript, the m9/10, was originally identified by a viable mutant, groucho, which has specific head bristle duplications. The groucho gene encodes a nuclear protein of 719 aa, including the repeat of approximately 40 aa demarcated by Trp-Asp ( WD-40 repeat) present in a guanine nucleotide binding protein (G-protein) b-subunit (Hartley et al., 1988; Fong et al., 1986). To further our understanding of the mechanisms regulating vertebrate neurogenic genes during neural development, we have reported the nucleotide and deduced aa sequences of mouse ESG cDNA encoding the mammalian homolog of Drosophila groucho protein (Miyasaka et al., 1993). In addition, we have cloned human and mouse cDNAs encoding AES proteins, a novel member of this gene family which exhibits strong similarity to the N-terminal domain of Drosophila
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groucho, mammalian ESG and TLE proteins, but lacks WD-40 repeats (Miyasaka et al., 1993). The embryogenesis of the African frog Xenopus laevis has been extensively studied and it is an excellent model for studying vertebrate development. To begin the studies on amphibian homologs of these groucho and related genes, this study describes the molecular cloning, nucleotide and deduced aa sequences, tissue-specific and developmental expression of Xenopus ESG and AES genes. Data are presented showing that at least two different ESG genes are present in the Xenopus genome. Structural relationships among the groucho and related proteins from human, mouse, rat, and Xenopus and Drosophila were also analysed.
2. Experimental and discussion
Xenopus ESG2. These 3∞-UTR sequence results indicate that Xenopus ESG1 and ESG2 are encoded by two different genes. Xenopus AES cDNA clone 825 was isolated from the same ovary cDNA library, and the nt sequence of the insert DNA was completely determined. The sequence of 2088 bp from the Xenopus AES cDNA insert was found to contain an ORF of 591 bp encoding a predicted protein comprised of 197 aa, 151 bp 5∞-UTR, 1328 bp 3∞-UTR and a poly(A) tail ( Figs. 1A and 2B). This 3∞-UTR appeared to be relatively long and only a single putative polyadenylation signal AATAAA was found at 18 bp 5∞ to the poly(A)-tail. The deduced sequence of 197 aa from Xenopus AES cDNA exhibits 91%, 58% and 48% identity to the mouse AES (Miyasaka et al., 1993), Xenopus ESG1 and Drosophila groucho (Hartley et al., 1988) proteins, respectively.
2.1. Cloning and sequence analysis of Xenopus ESG and AES cDNAs
2.2. Tissue-specific and developmental expression of ESG and AES mRNAs
A Xenopus clone 701 was isolated from an oocyte cDNA library (Burglin et al., 1987) using mouse ESG cDNA as a probe (Miyasaka et al., 1993) and found to contain the partial ESG cDNA encoding the C-terminal region of 298 aa (S.S.-L. Li, unpublished results). This Xenopus ESG cDNA and N-terminal region of mouse ESG cDNA were used as probes to isolate the ESG1 clone containing a full-length cDNA of 2.7 kb from the Xenopus ovary cDNA library. The Xenopus ESG1 cDNA contains an insert of 2668 bp with an open reading frame (ORF ) of 2301 bp encoding a predicted protein comprising 767 aa (Figs. 1A and 2A). The nt sequence includes a 193 bp 5∞-UTR (untranslated region) based on the use of the first start codon in the ORF. The 151 bp 3∞-UTR contains a poly(A) tail. In addition, a putative polyadenylation signal AATAAA was present at 13 bp 5∞ to the poly(A)-tail. The deduced aa sequence of this Xenopus ESG1 protein exhibits 61% and 71% identity to the Drosophila groucho and mouse ESG proteins, respectively (Hartley et al., 1988; Miyasaka et al., 1993). A Xenopus liver cDNA library was screened using the 2.0 kb coding region of the ESG1 cDNA. A total of 22 clones was purified after three repeated screenings. Only one clone seemed to have a 1.7 kb insert of the ESG2 cDNA, while the other 21 clones appeared to contain Xenopus ESG1 cDNA. This Xenopus ESG2 cDNA contains a partial 597 bp ORF and 3∞-UTR of 1101 bp. The ORF nt sequence identity between ESG1 and ESG2 is 80%, and the deduced 198 aa sequence from this highly conserved WD-40 domain of Xenopus ESG2 shows 14 aa substitutions (7.1% differences) to the corresponding aa numbers 570–767 of the Xenopus ESG1 protein. Moreover, the 3∞-UTR sequence of Xenopus ESG1 cDNA is as much as 64% different from that of
Northern blot analyses with the 1701 bp ESG1 coding region probe ( Figs. 1A, 3A, ESG1), including the variable and conserved WD-40 sequence, revealed that the ESG mRNA migrated as two transcripts of 3.6 kb and 2.8 kb in all adult tissues examined, except in muscle where the 2.8 kb species was apparently undetectable ( Fig. 3A). A 653 bp ESG2-specific probe was prepared from the 3∞-UTR sequence of the 1.7 kb clone isolated from the Xenopus liver library ( Fig. 1A). Only the 3.6 kb band was detected from the same Northern blot with this ESG2 probe ( Fig. 3A, ESG2). These results of Northern blot analysis as well as the sequence analyses strongly suggest that Xenopus ESG1 and ESG2 are indeed two different genes and are not alleles of the same gene. The ESG1 cDNA was presumably derived from the 2.8 kb transcript and it is abundantly expressed in brain, lung, testis and ovary in comparison with liver, heart, kidney and spleen, whereas the 3.6 kb ESG2 transcript is abundantly expressed in spleen and ovary. Differences in mRNA level among these adult tissues suggest that Xenopus ESG mRNAs are subjected to tissue-specific regulation. The Northern blot analysis of AES mRNA from adult tissues using a 593 bp AES coding probe showed that AES transcript of 2.2 kb is widespread and predominantly present in brain, testis and ovary ( Fig. 3A, AES). Xenopus ESG1 and AES transcripts of 2.8 kb and 2.2 kb, respectively, were ubiquitously expressed in the developing embryos. These two transcripts were also expressed in both the unfertilized and fertilized eggs, suggesting that the ESG1 and AES genes are expressed maternally. Similarly, Drosophila groucho protein was found to be deposited maternally in the eggs and showed a ubiquitous distribution throughout embryonic development (Delidakis et al., 1991). Unlike adult tissues,
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Fig. 1. (A) Xenopus ESG and AES cDNA clones. (B) The relationship between AES and groucho/ESG/TLE proteins and structural domains. The ORF sequence is shown by boxes. The 5∞- and 3∞-UTR are indicated by single solid lines. Solid bars under the different clones show the regions used as the gene-specific probes in Northern blot studies. The hatched area indicates the similarity between ESG and AES and designated as AES domain. The central variable region contains NLS, CKII and cdc2K sites, and is denoted as NLS/CKII/cdc2K domain. The C-terminal region of ESG protein containing four WD-40 repeats (dotted area) is indicated as WD-40 domain.
ESG1 probe detected only 2.8 kb transcript in all embryonic stages, and the 2.8 kb transcript appeared to be decreased after stage 9, fine cell blastula ( Fig. 3B). Sequential hybridization of the same blot with an ESG2-specific probe detected none of the 3.6 kb and 2.8 kb transcripts, suggesting that the ESG2 gene is not expressed at the early developmental stages (data not shown). 2.3. Amino acid sequence comparisons among groucho and related proteins The aa sequences of Xenopus ESG1, ESG2 and AES proteins determined in this investigation were aligned with those of Drosophila groucho (Hartley et al., 1988) and related proteins from human, mouse and rat reported previously (Stifani et al., 1992; Miyasaka et al., 1993; Mallo et al., 1993; Schmidt and Sladek, 1993; Scala et al., 1994). The AES proteins from mammals and Xenopus are homologous with the N-terminal domain of Drosophila groucho and vertebrate ESG/TLE proteins, and there is 23% identity at the 221 positions of all 12 species compared (Fig. 4). The C-terminal domain, containing WD-40 repeats of the Drosophila groucho and vertebrate ESG/TLE proteins, exhibits 77%
identity among all of the proteins at the 298 positions compared. The central highly variable domain of these proteins exhibits only 8% identity at the 297 positions. The aa sequence of Xenopus ESG1 protein contains insertions of seven and nine aa at position numbers 15–21 and numbers 134–142, respectively, in comparison with those of other groucho and ESG/TLE proteins. The rat R-esp2 protein contains a deletion of 25 aa at positions 109–142. It is of interest that these corresponding residues of mouse AES protein are encoded by an exon of mouse AES gene (Mallo et al., 1994), indicating that the deletion of rat R-esp2 protein may be due to alternative splicing. The central regions of Drosophila groucho and human TLE proteins were reported to contain nuclear localization sequence (NLS ), casein kinase II (CKII ) and cdc2 kinase (cdc2K ) sites, and these proteins were shown to be present in the nucleus ( Hartley et al., 1988; Stifani et al., 1992). Xenopus ESG1 protein appears to have similar sequences at the NLS, CKII and cdc2K sites and, thus, it may also be present and function in the nucleus. The conserved C-terminal domain of the groucho, TLE and ESG proteins from Drosophila, human, mouse, rat and Xenopus contains at least four WD-40 repeats, and these repeats were also found in an expanding group of unrelated proteins,
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Fig. 2. The nucleotide and deduced aa sequences of (A) Xenopus ESG1 and (B) AES cDNAs. The nt and deduced aa sequences of the Xenopus ESG1 and AES cDNAs are presented. The polyadenylation signal of AATAAA is indicated in bold, and the poly(A)-tail is given. The number of the first aa begins at Met, and is shown using the one-letter code, and the last nt on each row is given on the left and the right, respectively. Methods: The cDNA inserts from the cloned Uni-ZAP XR phages were excised into pBluescript SK(−) phagemid (Stratagene, La Jolla, CA, USA). Sequencing was carried out on both strands with dideoxy nucleotide technology (Sequenase kit, US Biochemicals, Cleveland, OH, USA), using a combination of synthetic oligo primers. The nt and aa sequences have been deposited in the GenBank database under accession nos U18775 and U18776.
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Fig. 3. Northern blot analysis of AES and ESG transcripts. (A) Xenopus adult tissues. Total RNAs from adult tissues: liver, muscle, brain, heart, kidney, lung, spleen, testis and ovary. (B) Xenopus embryos at different developmental stages: unfertilized egg; fertilized egg; stage 6, late morula (~1000 cells); stage 9, fine cell blastula (~6000 cells); stage 11, mid gastrula (~30 000 cells); stage 20, late neurula; stage 24, early tailbud; stage 32, late tailbud. Northern blots were sequentially hybridized with the radiolabelled AES, ESG1- and ESG2-specific cDNA probes (Fig. 1A). The bottom panel of Fig. 3A shows the ethidium bromide-stained 18S and 28S RNAs, whereas the bottom panel of Fig. 3B shows hybridization to a genomic clone containing the genes for Xenopus histones H3 and H4. Methods: Total RNAs from adult tissues were extracted using the TRIzol Reagent (Life Technologies, Gaithersburg, MD, USA). Xenopus eggs were fertilized in vitro and maintained until they had developed to the appropriate stage ( Wallace et al., 1973). Embryos were staged according to Nieuwkoop and Faber (1956). Total nucleic acid from oocytes and embryos was extracted as described previously (Rollins et al., 1993). Either 10 mg of total RNAs or two embryo equivalents of nucleic acid from each specific stage were separated on a 1.2% agarose/2.2 M formaldehyde gel.
including the yeast proteins encoded by CDC4 (a cell cycle gene), TUP1 (a mediator of glucose repression), SLF2 (suppressor gene for floculation), AAR1 (a1–a2 repression and cell control ) and AER2 (control of hemeregulated and catabolite-repressed gene). The TUP1, SLF2, AAR1 and AER2 genes were cloned on the basis of different phenotypes, but were found to be identical ( Yochem and Byers, 1987; Fujita et al., 1990; Williams and Trumbly, 1990; Mukai et al., 1991; Zhang et al., 1991). The overall structural relationships among these groucho and related proteins from human, mouse, rat, Xenopus and Drosophila were analysed and the dendrogram is illustrated in Fig. 5. The evolutionary tree (not presented ) constructed using the UPGMA method
(Swofford and Olsen, 1990) is essentially identical to the dendrogram (Fig. 5). The four AES proteins from human, mouse, rat and Xenopus are clustered into a group, and the groucho, ESG and TLE proteins are clustered into a separate group. In human, four different TLE proteins were found (Hartley et al., 1988). Mouse ESG protein appears to be most similar to human TLE3 protein, and rat R-esp2 and Xenopus ESG2 are similar to human TLE4 as well as to TLE1 protein. Human TLE2 protein seem to be positioned between Drosophila groucho and Xenopus ESG1 proteins. The AES proteins of 197 aa from human, mouse and rat and Xenopus do not contain the middle variable region, including NLS, CKII and cdc2K sites, or the conserved C-terminal WD-40 repeat sequence present
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(2)
(3)
(4) Fig. 5. Structural relationship of groucho and related proteins. The aa sequences of ESG1, ESG2 and AES from Xenopus laevis were determined in this investigation (Genbank accession nos U18775, U63516 and U18776, respectively). The names of the organisms and the Genbank accession numbers of the published groucho and related proteins are as follows: human, Homo sapiens, AES/hesp ( X73358/U04241), TLE1 (M99435), TLE2 (M99436), TLE3 (M99438), TLE4 (M99439); mouse, Mus musculus, AES/Grg ( X73361/L12140), ESG ( X73360); rat, Rattus norvegicus, R-esp1 (L14462), R-esp2 (L14463); and fruitfly, Drosophila melanogaster (M20571). The structural relationship was constructed using the pileup program of the Wisconsin GCG package based on the method of Feng and Doolittle (1987).
(5)
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the ESG1 and ESG2 are amphibian homologs of the Drosophila groucho gene. Widespread RNA expression of ESG1 of 2.8 kb, ESG2 of 3.6 kb and AES of 2.2 kb transcripts was seen in adult tissues. Differences in mRNA level among these adult tissues suggest that Xenopus ESG mRNAs are subjected to tissue-specific regulation. In the adult tissues, the ESG2-specific non-coding region probe only detected the 3.6 kb transcript, which may originate from a different ESG gene, showing that at least two different ESG genes are present in the Xenopus genome. ESG1 and AES transcripts of 2.8 kb and 2.2 kb, respectively, were ubiquitously expressed during early development. These two transcripts were also expressed in both the unfertilized and fertilized eggs, suggesting that the ESG1 and AES genes are expressed maternally. The ESG2-specific probe detected none of the 3.6 kb or 2.8 kb transcripts in oocytes and developing embryos, suggesting that the ESG2 gene is not expressed during the early developmental stages.
Acknowledgement in the G-protein b-subunit ( Fong et al., 1986). As noted previously (Miyasaka et al., 1993), the AES proteins and the N-terminal domain of groucho, ESG and TLE proteins contain potential leucine zipper, which may provide a basis for the direct interaction between these different types of protein. It may be noted that the rat R-esp1 protein (AES homolog) was also shown to be present in the nucleus (Schmidt and Sladek, 1993). As to the function of AES proteins, it is tempting to suggest that these AES proteins may modulate the function of the groucho, ESG and TLE proteins. The strong evolutionary conservation of this protein, across the species boundary, indicates that it plays an important role in cellular function and/or embryonic development. Finally, it would be of interest to determine whether the AES gene is present in the Drosophila genome. 2.4. Conclusions
(1) The nt sequences of Xenopus ESG1, ESG2 and AES cDNAs have been cloned and determined. Comparison of the deduced aa sequences suggests that
We thanks Dr Matthew T. Andrews for the generous gifts of the Xenopus embryo RNA samples and histones H3-H4 probe, and Dr Masood A. Qureshi for isolation of Xenopus clone 701. This investigation was supported in part by grants NSC85-2732-B-110-002 and NSC862313-B-110-002 from the National Science Council of Taiwan, ROC.
References Artavanis-Tsakonas, S., Delidakis, C., Fehon, R.G., 1991. The Notch locus and the cell biology of neuroblast segregration. Annu. Rev. Cell. Biol. 7, 427–452. Burglin, T.R., Mattaj, I.W., Newmeyer, D.D., Zeller, R., De Robbertis, E.M., 1987. Cloning of nucleoplasmin from Xenopus laevis oocytes and analysis of its developmental expression. Genes Dev. 1, 97–107. Cabrera, C.V., 1992. The generation of cell diversity during early neurogenesis in Drosophila. Development 115, 893–901. Campos-Ortega, J.A., Jan, Y.N., 1991. Genetic and molecular bases of neurogenesis in Drosophila melanogaster. Annu. Rev. Neurosci. 14, 399–420. Delidakis, C., Artavanis-Tsakonas, S., 1992. The Enhancer of split
Fig. 4. Amino acid sequence comparison of Xenopus ESG and AES proteins with Drosophila groucho and related proteins from human, mouse and rat. The aa sequences were aligned using the Wisconsin GCG package. Identical residues among all sequences are shown in bold, and gaps are indicated by hyphens. The NLS, CKII and cdc2K sites are indicated, and the WD sequences demarcating WD-40 repeats are underlined. The translation stop codon is denoted by a star. The N-terminal region exhibits 23% identity (50/221 positions) among all 12 sequences. The middle region, containing NLS, CKII and cdc2K sites, is variable with several gaps and only 8% identity (39/297 positions) and the C-terminal WD-40 region possesses 77% identity (228/398 positions) among eight sequences.
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(E(spl)) locus of Drosophila encodes seven independent helix–loop–helix proteins. Proc. Natl Acad. Sci. USA 89, 8731–8735. Delidakis, C., Preiss, A., Hartley, D.A., Artavanis-Tsakonas, S., 1991. Two genetically and molecularly distinct functions involved in early neurogenesis reside within the Enhancer of split locus of Drosophila melanogaster. Genetics 129, 803–823. Feng, D.F., Doolittle, R.F., 1987. Progressive sequence alignment as a prerequisite to correct phylogenetic trees. J. Mol. Evol. 25, 351–360. Fong, H.K.W., Hurley, J.B., Hopkins, R.S., Miake-Lye, R., Johnson, M.S., Doolittle, R.F., Simon, M.I., 1986. Repetitive segmental structure of the transducin beta subunit: homology with the CDC4 gene and identification of related mRNAs. Proc. Natl Acad. Sci. USA 83, 2162–2166. Fujita, A., Matsumoto, S., Kuhara, S., Misumi, Y., Kobayashi, H., 1990. Cloning of the yeast SFL2 gene: its disruption results in pleiotropic phenotypes characteristic for tup1 mutants. Gene 89, 93–99. Ghysen, A., Dambly-Chaudiere, C., Jan, L.Y., Jan, Y.N., 1993. Cell interactions and gene interactions in peripheral neurogenesis. Genes Dev. 7, 723–733. Hartley, D.A., Preiss, A., Artavanis-Tsakonas, S., 1988. A deduced gene product from the Drosophila neurogenic locus Enhancer of split, shows homology to the mammalian G protein b subunit. Cell 55, 785–795. Knust, E., Schrons, H., Grawe, F., Campos-Ortega, J.A., 1992. Seven genes of Enhancer of split complex of Drosophila melanogaster encode helix–loop–helix proteins. Genetics 132, 505–518. Lehman, R., Jimenez, F., Dietrich, U., Campos-Ortega, J.A., 1983. On the phenotype and development of mutants of early neurogenesis in Drosophila melanogaster. Whilhelm Roux’s Arch. Dev. Biol. 192, 62–74. Mallo, M., Franco del Amo, F., Gridley, T., 1993. Cloning and developmental expression of Grg, a mouse gene related to the groucho transcript of the Drosophila Enhancer of split complex. Mech. Dev. 42, 67–76. Mallo, M., Steingrimsson, E., Copeland, N.G., Jenkins, N.A., Gridley, T., 1994. Genomic organization, alternative polyadenylation, and chromosomal localization of Grg, a mouse gene related to the groucho transcript of the Drosophila Enhancer of split complex. Genomics 21, 194–201. Miyasaka, H., Choudhury, B.K., Hou, E.W., Li, S.S.-L., 1993. Molecular cloning and expression of mouse and human cDNAs encoding
AES and ESG proteins with strong homology to Drosophila Enhancer of split groucho protein. Eur. J. Biochem. 216, 343–352. Mukai, Y., Harashima, S., Oshima, Y., 1991. AAR1/TUP1 protein, with a structure similar to that of the beta subunit of G proteins, is required for a1-alpha 2 and alpha 2 repression in cell type control of Saccharomyces cerevisiae. Mol. Cell Biol. 11, 3773–3779. Nieuwkoop, P.D., Faber, J., 1956. In: Normal Table of Xenopus laevis (Daudin). Elsevier–North Holland, Amsterdam. Rollins, M.B., Del-Rio, S., Galey, A.L., Setzer, D.R., Andrews, M.T., 1993. Role of TFIIIA zinc fingers in vivo: analysis of single-finger function in developing Xenopus embryos. Mol. Cell Biol. 13, 4776–4783. Scala, L.A., Tirumalai, P.T., Piparo, K.E., Howells, R.D., 1994. Molecular cloning, sequence analysis and characterization of a human homolog of Drosophila enhancer of split m9/m10. FASEB J. 8, A1419 Schmidt, C.J., Sladek, T.E., 1993. A rat homolog of the Drosophila Enhancer of split ( groucho) locus lacking WD-40 repeats. J. Biol. Chem. 268, 25681–25686. Schrons, H., Knust, E., Campos-Ortega, J.A., 1992. The Enhancer of split complex and adjacent genes in the 96F region of Drosophila melanogaster are required for segregation of neural and epidermal progenitor cells. Genetics 132, 481–503. Stifani, S., Blaumueller, C.M., Redhead, N.J., Hill, R.E., ArtavanisTsakonas, S., 1992. Human homologs of Drosophila Enhancer of split gene product define a novel family of nuclear proteins. Nat. Genet. 2, 119–127. Swofford, D.L., Olsen, G.J., 1990. In: Hillis, D.M., Moritz, C. ( Eds.), Molecular Systemics. Sinauer, Sunderland, MA, pp. 411–501. Wallace, R.A., Jared, D.W., Durmont, J.N., Sega, M.W., 1973. Protein incorporation by isolated amphibian oocytes. II. Optimum incubation conditions. J. Exp. Zool. 184, 321–334. Williams, F.E., Trumbly, R.J., 1990. Characterization of TUP1, a mediator of glucose repression in Saccharomyces cerevisiae. Mol. Cell Biol. 10, 6500–6511. Yochem, J., Byers, B., 1987. Structural comparison of the yeast cell division cycle gene CDC4 and a related pseudogene. J. Mol. Biol. 195, 233–245. Zhang, M., Rosenblum-Vos, L.S., Lowry, C.V., Boakye, K.A., Zitomer, R.S., 1991. A yeast protein with homology to the betasubunit of G proteins is involved in control of heme-regulated and catabolite-repressed genes. Gene 97, 153–161.
Cloning and developmental expression of Xenopus cDNAs encoding the Enhancer of split groucho and related proteins Barun K. Choudhury a, Jaebong Kim b, Hsiang-Fu Kung b, Steven S.-L. Li a,c,* a Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA b Laboratory of Biochemical Physiology, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA c Institute of Life Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan 80424, ROC Received 29 October 1996; accepted 1 February 1997; Received by A. Nakazawa
Abstract The two full-length cDNAs encoding ESG1 (Enhancer of split groucho) and related AES (Amino Enhancer of split) proteins of 767 and 197 amino acids, respectively, were cloned and sequenced from the African frog Xenopus laevis. The amino acid sequence of Xenopus ESG1 protein had 61% identity to the full-length Drosophila groucho. Xenopus AES protein exhibited 91%, 58% and 48% identity to the mouse AES, amino-terminal regions of Xenopus ESG1 and Drosophila groucho, respectively. Northern blot analysis showed that widespread RNA expression of ESG1 of 2.8 kb, ESG2 of 3.6 kb and AES of 2.2 kb transcripts were seen in adult tissues, whereas ESG1 and AES transcripts of 2.8 kb and 2.2 kb, respectively, were ubiquitously expressed in the developing embryos. The overall structural relationships of ESG and AES proteins among human, mouse, rat, Xenopus and Drosophila were analysed. © 1997 Elsevier Science B.V. Keywords: Nucleotide sequence; Protein; Gene expression; Tissues; Phylogenetic tree; Amphibian
1. Introduction In the fruitfly, Drosophila melanogaster, neurogenic loci, namely, Enhancer of split, Notch, Delta, mastermind, big brain and neuralized, are required for the proper segregation of neural and epidermal progenitor cells during formation of both the central and peripheral nervous systems (Artavanis-Tsakonas et al., 1991; Cabrera, 1992; Campos-Ortega and Jan, 1991; Ghysen et al., 1993). Loss-of-functions mutations in these genes cause most ventral ectodermal cells to become neuroblasts with little or no formation of epidermis (Lehman * Corresponding author. Address c. Tel. +886 7 5253616; Fax: +886 7 5253696. Abbreviations: aa, amino acid(s); AES, Amino Enhancer of split; cdc2K, cdc2 kinase site; bHLH, basic helix–loop–helix; CKII, casein kinase II site; ESG, Enhancer of split groucho; G-protein, guanine nucleotide binding protein; kb, kilobase(s); NLS, nuclear localization sequence; nt, nucleotide(s); ORF, open reading frame; TLE, transducin-like Enhancer of split; UTR, untranslated region(s); WD-40 repeat, the repeat of approximately 40 aa demarcated by Trp-Asp ( WD). 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 1 50 - 9
et al., 1983). The Enhancer of split gene complex contains at least 13 transcription units. Molecular studies have shown that most of these transcription units encode proteins with the basic helix–loop–helix (bHLH ) motif characteristic of certain transcription factors (Delidakis and Artavanis-Tsakonas, 1992; Knust et al., 1992; Schrons et al., 1992). Another structurally unrelated transcript, the m9/10, was originally identified by a viable mutant, groucho, which has specific head bristle duplications. The groucho gene encodes a nuclear protein of 719 aa, including the repeat of approximately 40 aa demarcated by Trp-Asp ( WD-40 repeat) present in a guanine nucleotide binding protein (G-protein) b-subunit (Hartley et al., 1988; Fong et al., 1986). To further our understanding of the mechanisms regulating vertebrate neurogenic genes during neural development, we have reported the nucleotide and deduced aa sequences of mouse ESG cDNA encoding the mammalian homolog of Drosophila groucho protein (Miyasaka et al., 1993). In addition, we have cloned human and mouse cDNAs encoding AES proteins, a novel member of this gene family which exhibits strong similarity to the N-terminal domain of Drosophila
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groucho, mammalian ESG and TLE proteins, but lacks WD-40 repeats (Miyasaka et al., 1993). The embryogenesis of the African frog Xenopus laevis has been extensively studied and it is an excellent model for studying vertebrate development. To begin the studies on amphibian homologs of these groucho and related genes, this study describes the molecular cloning, nucleotide and deduced aa sequences, tissue-specific and developmental expression of Xenopus ESG and AES genes. Data are presented showing that at least two different ESG genes are present in the Xenopus genome. Structural relationships among the groucho and related proteins from human, mouse, rat, and Xenopus and Drosophila were also analysed.
2. Experimental and discussion
Xenopus ESG2. These 3∞-UTR sequence results indicate that Xenopus ESG1 and ESG2 are encoded by two different genes. Xenopus AES cDNA clone 825 was isolated from the same ovary cDNA library, and the nt sequence of the insert DNA was completely determined. The sequence of 2088 bp from the Xenopus AES cDNA insert was found to contain an ORF of 591 bp encoding a predicted protein comprised of 197 aa, 151 bp 5∞-UTR, 1328 bp 3∞-UTR and a poly(A) tail ( Figs. 1A and 2B). This 3∞-UTR appeared to be relatively long and only a single putative polyadenylation signal AATAAA was found at 18 bp 5∞ to the poly(A)-tail. The deduced sequence of 197 aa from Xenopus AES cDNA exhibits 91%, 58% and 48% identity to the mouse AES (Miyasaka et al., 1993), Xenopus ESG1 and Drosophila groucho (Hartley et al., 1988) proteins, respectively.
2.1. Cloning and sequence analysis of Xenopus ESG and AES cDNAs
2.2. Tissue-specific and developmental expression of ESG and AES mRNAs
A Xenopus clone 701 was isolated from an oocyte cDNA library (Burglin et al., 1987) using mouse ESG cDNA as a probe (Miyasaka et al., 1993) and found to contain the partial ESG cDNA encoding the C-terminal region of 298 aa (S.S.-L. Li, unpublished results). This Xenopus ESG cDNA and N-terminal region of mouse ESG cDNA were used as probes to isolate the ESG1 clone containing a full-length cDNA of 2.7 kb from the Xenopus ovary cDNA library. The Xenopus ESG1 cDNA contains an insert of 2668 bp with an open reading frame (ORF ) of 2301 bp encoding a predicted protein comprising 767 aa (Figs. 1A and 2A). The nt sequence includes a 193 bp 5∞-UTR (untranslated region) based on the use of the first start codon in the ORF. The 151 bp 3∞-UTR contains a poly(A) tail. In addition, a putative polyadenylation signal AATAAA was present at 13 bp 5∞ to the poly(A)-tail. The deduced aa sequence of this Xenopus ESG1 protein exhibits 61% and 71% identity to the Drosophila groucho and mouse ESG proteins, respectively (Hartley et al., 1988; Miyasaka et al., 1993). A Xenopus liver cDNA library was screened using the 2.0 kb coding region of the ESG1 cDNA. A total of 22 clones was purified after three repeated screenings. Only one clone seemed to have a 1.7 kb insert of the ESG2 cDNA, while the other 21 clones appeared to contain Xenopus ESG1 cDNA. This Xenopus ESG2 cDNA contains a partial 597 bp ORF and 3∞-UTR of 1101 bp. The ORF nt sequence identity between ESG1 and ESG2 is 80%, and the deduced 198 aa sequence from this highly conserved WD-40 domain of Xenopus ESG2 shows 14 aa substitutions (7.1% differences) to the corresponding aa numbers 570–767 of the Xenopus ESG1 protein. Moreover, the 3∞-UTR sequence of Xenopus ESG1 cDNA is as much as 64% different from that of
Northern blot analyses with the 1701 bp ESG1 coding region probe ( Figs. 1A, 3A, ESG1), including the variable and conserved WD-40 sequence, revealed that the ESG mRNA migrated as two transcripts of 3.6 kb and 2.8 kb in all adult tissues examined, except in muscle where the 2.8 kb species was apparently undetectable ( Fig. 3A). A 653 bp ESG2-specific probe was prepared from the 3∞-UTR sequence of the 1.7 kb clone isolated from the Xenopus liver library ( Fig. 1A). Only the 3.6 kb band was detected from the same Northern blot with this ESG2 probe ( Fig. 3A, ESG2). These results of Northern blot analysis as well as the sequence analyses strongly suggest that Xenopus ESG1 and ESG2 are indeed two different genes and are not alleles of the same gene. The ESG1 cDNA was presumably derived from the 2.8 kb transcript and it is abundantly expressed in brain, lung, testis and ovary in comparison with liver, heart, kidney and spleen, whereas the 3.6 kb ESG2 transcript is abundantly expressed in spleen and ovary. Differences in mRNA level among these adult tissues suggest that Xenopus ESG mRNAs are subjected to tissue-specific regulation. The Northern blot analysis of AES mRNA from adult tissues using a 593 bp AES coding probe showed that AES transcript of 2.2 kb is widespread and predominantly present in brain, testis and ovary ( Fig. 3A, AES). Xenopus ESG1 and AES transcripts of 2.8 kb and 2.2 kb, respectively, were ubiquitously expressed in the developing embryos. These two transcripts were also expressed in both the unfertilized and fertilized eggs, suggesting that the ESG1 and AES genes are expressed maternally. Similarly, Drosophila groucho protein was found to be deposited maternally in the eggs and showed a ubiquitous distribution throughout embryonic development (Delidakis et al., 1991). Unlike adult tissues,
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Fig. 1. (A) Xenopus ESG and AES cDNA clones. (B) The relationship between AES and groucho/ESG/TLE proteins and structural domains. The ORF sequence is shown by boxes. The 5∞- and 3∞-UTR are indicated by single solid lines. Solid bars under the different clones show the regions used as the gene-specific probes in Northern blot studies. The hatched area indicates the similarity between ESG and AES and designated as AES domain. The central variable region contains NLS, CKII and cdc2K sites, and is denoted as NLS/CKII/cdc2K domain. The C-terminal region of ESG protein containing four WD-40 repeats (dotted area) is indicated as WD-40 domain.
ESG1 probe detected only 2.8 kb transcript in all embryonic stages, and the 2.8 kb transcript appeared to be decreased after stage 9, fine cell blastula ( Fig. 3B). Sequential hybridization of the same blot with an ESG2-specific probe detected none of the 3.6 kb and 2.8 kb transcripts, suggesting that the ESG2 gene is not expressed at the early developmental stages (data not shown). 2.3. Amino acid sequence comparisons among groucho and related proteins The aa sequences of Xenopus ESG1, ESG2 and AES proteins determined in this investigation were aligned with those of Drosophila groucho (Hartley et al., 1988) and related proteins from human, mouse and rat reported previously (Stifani et al., 1992; Miyasaka et al., 1993; Mallo et al., 1993; Schmidt and Sladek, 1993; Scala et al., 1994). The AES proteins from mammals and Xenopus are homologous with the N-terminal domain of Drosophila groucho and vertebrate ESG/TLE proteins, and there is 23% identity at the 221 positions of all 12 species compared (Fig. 4). The C-terminal domain, containing WD-40 repeats of the Drosophila groucho and vertebrate ESG/TLE proteins, exhibits 77%
identity among all of the proteins at the 298 positions compared. The central highly variable domain of these proteins exhibits only 8% identity at the 297 positions. The aa sequence of Xenopus ESG1 protein contains insertions of seven and nine aa at position numbers 15–21 and numbers 134–142, respectively, in comparison with those of other groucho and ESG/TLE proteins. The rat R-esp2 protein contains a deletion of 25 aa at positions 109–142. It is of interest that these corresponding residues of mouse AES protein are encoded by an exon of mouse AES gene (Mallo et al., 1994), indicating that the deletion of rat R-esp2 protein may be due to alternative splicing. The central regions of Drosophila groucho and human TLE proteins were reported to contain nuclear localization sequence (NLS ), casein kinase II (CKII ) and cdc2 kinase (cdc2K ) sites, and these proteins were shown to be present in the nucleus ( Hartley et al., 1988; Stifani et al., 1992). Xenopus ESG1 protein appears to have similar sequences at the NLS, CKII and cdc2K sites and, thus, it may also be present and function in the nucleus. The conserved C-terminal domain of the groucho, TLE and ESG proteins from Drosophila, human, mouse, rat and Xenopus contains at least four WD-40 repeats, and these repeats were also found in an expanding group of unrelated proteins,
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Fig. 2. The nucleotide and deduced aa sequences of (A) Xenopus ESG1 and (B) AES cDNAs. The nt and deduced aa sequences of the Xenopus ESG1 and AES cDNAs are presented. The polyadenylation signal of AATAAA is indicated in bold, and the poly(A)-tail is given. The number of the first aa begins at Met, and is shown using the one-letter code, and the last nt on each row is given on the left and the right, respectively. Methods: The cDNA inserts from the cloned Uni-ZAP XR phages were excised into pBluescript SK(−) phagemid (Stratagene, La Jolla, CA, USA). Sequencing was carried out on both strands with dideoxy nucleotide technology (Sequenase kit, US Biochemicals, Cleveland, OH, USA), using a combination of synthetic oligo primers. The nt and aa sequences have been deposited in the GenBank database under accession nos U18775 and U18776.
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Fig. 3. Northern blot analysis of AES and ESG transcripts. (A) Xenopus adult tissues. Total RNAs from adult tissues: liver, muscle, brain, heart, kidney, lung, spleen, testis and ovary. (B) Xenopus embryos at different developmental stages: unfertilized egg; fertilized egg; stage 6, late morula (~1000 cells); stage 9, fine cell blastula (~6000 cells); stage 11, mid gastrula (~30 000 cells); stage 20, late neurula; stage 24, early tailbud; stage 32, late tailbud. Northern blots were sequentially hybridized with the radiolabelled AES, ESG1- and ESG2-specific cDNA probes (Fig. 1A). The bottom panel of Fig. 3A shows the ethidium bromide-stained 18S and 28S RNAs, whereas the bottom panel of Fig. 3B shows hybridization to a genomic clone containing the genes for Xenopus histones H3 and H4. Methods: Total RNAs from adult tissues were extracted using the TRIzol Reagent (Life Technologies, Gaithersburg, MD, USA). Xenopus eggs were fertilized in vitro and maintained until they had developed to the appropriate stage ( Wallace et al., 1973). Embryos were staged according to Nieuwkoop and Faber (1956). Total nucleic acid from oocytes and embryos was extracted as described previously (Rollins et al., 1993). Either 10 mg of total RNAs or two embryo equivalents of nucleic acid from each specific stage were separated on a 1.2% agarose/2.2 M formaldehyde gel.
including the yeast proteins encoded by CDC4 (a cell cycle gene), TUP1 (a mediator of glucose repression), SLF2 (suppressor gene for floculation), AAR1 (a1–a2 repression and cell control ) and AER2 (control of hemeregulated and catabolite-repressed gene). The TUP1, SLF2, AAR1 and AER2 genes were cloned on the basis of different phenotypes, but were found to be identical ( Yochem and Byers, 1987; Fujita et al., 1990; Williams and Trumbly, 1990; Mukai et al., 1991; Zhang et al., 1991). The overall structural relationships among these groucho and related proteins from human, mouse, rat, Xenopus and Drosophila were analysed and the dendrogram is illustrated in Fig. 5. The evolutionary tree (not presented ) constructed using the UPGMA method
(Swofford and Olsen, 1990) is essentially identical to the dendrogram (Fig. 5). The four AES proteins from human, mouse, rat and Xenopus are clustered into a group, and the groucho, ESG and TLE proteins are clustered into a separate group. In human, four different TLE proteins were found (Hartley et al., 1988). Mouse ESG protein appears to be most similar to human TLE3 protein, and rat R-esp2 and Xenopus ESG2 are similar to human TLE4 as well as to TLE1 protein. Human TLE2 protein seem to be positioned between Drosophila groucho and Xenopus ESG1 proteins. The AES proteins of 197 aa from human, mouse and rat and Xenopus do not contain the middle variable region, including NLS, CKII and cdc2K sites, or the conserved C-terminal WD-40 repeat sequence present
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(2)
(3)
(4) Fig. 5. Structural relationship of groucho and related proteins. The aa sequences of ESG1, ESG2 and AES from Xenopus laevis were determined in this investigation (Genbank accession nos U18775, U63516 and U18776, respectively). The names of the organisms and the Genbank accession numbers of the published groucho and related proteins are as follows: human, Homo sapiens, AES/hesp ( X73358/U04241), TLE1 (M99435), TLE2 (M99436), TLE3 (M99438), TLE4 (M99439); mouse, Mus musculus, AES/Grg ( X73361/L12140), ESG ( X73360); rat, Rattus norvegicus, R-esp1 (L14462), R-esp2 (L14463); and fruitfly, Drosophila melanogaster (M20571). The structural relationship was constructed using the pileup program of the Wisconsin GCG package based on the method of Feng and Doolittle (1987).
(5)
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the ESG1 and ESG2 are amphibian homologs of the Drosophila groucho gene. Widespread RNA expression of ESG1 of 2.8 kb, ESG2 of 3.6 kb and AES of 2.2 kb transcripts was seen in adult tissues. Differences in mRNA level among these adult tissues suggest that Xenopus ESG mRNAs are subjected to tissue-specific regulation. In the adult tissues, the ESG2-specific non-coding region probe only detected the 3.6 kb transcript, which may originate from a different ESG gene, showing that at least two different ESG genes are present in the Xenopus genome. ESG1 and AES transcripts of 2.8 kb and 2.2 kb, respectively, were ubiquitously expressed during early development. These two transcripts were also expressed in both the unfertilized and fertilized eggs, suggesting that the ESG1 and AES genes are expressed maternally. The ESG2-specific probe detected none of the 3.6 kb or 2.8 kb transcripts in oocytes and developing embryos, suggesting that the ESG2 gene is not expressed during the early developmental stages.
Acknowledgement in the G-protein b-subunit ( Fong et al., 1986). As noted previously (Miyasaka et al., 1993), the AES proteins and the N-terminal domain of groucho, ESG and TLE proteins contain potential leucine zipper, which may provide a basis for the direct interaction between these different types of protein. It may be noted that the rat R-esp1 protein (AES homolog) was also shown to be present in the nucleus (Schmidt and Sladek, 1993). As to the function of AES proteins, it is tempting to suggest that these AES proteins may modulate the function of the groucho, ESG and TLE proteins. The strong evolutionary conservation of this protein, across the species boundary, indicates that it plays an important role in cellular function and/or embryonic development. Finally, it would be of interest to determine whether the AES gene is present in the Drosophila genome. 2.4. Conclusions
(1) The nt sequences of Xenopus ESG1, ESG2 and AES cDNAs have been cloned and determined. Comparison of the deduced aa sequences suggests that
We thanks Dr Matthew T. Andrews for the generous gifts of the Xenopus embryo RNA samples and histones H3-H4 probe, and Dr Masood A. Qureshi for isolation of Xenopus clone 701. This investigation was supported in part by grants NSC85-2732-B-110-002 and NSC862313-B-110-002 from the National Science Council of Taiwan, ROC.
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