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Isolation of cDNA and Genomic Clones of a Human Ras-Related GTP-Binding Protein Gene and Its Chromosomal Localization to the Long Arm of Chromosome 7, 7q36
SHORT COMMUNICATION Isolation of cDNA and Genomic Clones of a Human Ras-Related GTP-Binding Protein Gene and Its Chromosomal Localization to the Long Arm of Chromosome 7, 7q36 NOBUHISA MIZUKI,*,† MINORU KIMURA,† SHIGEAKI OHNO,* SHOJI MIYATA,† MEGUMI SATO,† HITOSHI ANDO,† MAMI ISHIHARA,* KAORI GOTO,* SATOSHI WATANABE,† MASAAKI YAMAZAKI,‡ AYAKO ONO,‡ SUSUMU TAGUCHI,‡ KATSUZUMI OKUMURA,§ MASAHIRO NOGAMI,§ HIROSHI TAGUCHI,§ ASAKO ANDO,† AND HIDETOSHI INOKO†,1 *Department of Ophthalmology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236, Japan; †Department of Genetic Information, Division of Molecular Life Science, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-11, Japan; ‡Bioscience Laboratory, Fujiya Company, 228 Soya, Hadano, Kanagawa 257, Japan; and §Biochemistry Laboratory, Mie University School of Bioresource, 1515 Kamihamacho, Tsu, Mie 514, Japan Received November 9, 1995; accepted February 1, 1996
A Ras-related GTP-binding protein cDNA has been isolated from a human skin fibroblast cDNA library using a genomic subclone derived from a YAC clone as a probe. The polypeptide, consisting of 184 amino acids deduced from nucleotide sequences, contains five repeats of the Ras-related GTP-binding region and is highly homologous to the rat RHEB (Ras homologue enriched in brain) gene, which encodes a Ras-related growth factor- and synaptic activity-regulated protein, with 98.9% amino acid identity. Therefore, it is suggested to be a human homologue of the rat RHEB protein, and we have designated it human RHEB. Using fluorescence in situ hybridization, we concluded that this human RHEB gene was localized to band q36 on chromosome 7. Considering the chromosomal localization as well as the potential function of this protein, it will be very important to investigate whether it may play a role in the etiopathogenesis of holoprosencephaly type 3 or hereditary sacral agenesis, in which the disease susceptible locus is linked to the microsatellite marker, D7S22, in this chromosomal region, 7q36. q 1996 Academic Press, Inc.
A YAC clone, Y109, was isolated from the YAC library constructed from the B-cell line, CGM1 (A3, B8, Cw-, DR3, DQ2, DR52 and A29, B14, Cw-, DR7, DQ2, DR53), using HLA class I-specific primers (3) and was found to be a chimeric clone made up of the fragments from chromosomes 6 and 7. Y109 with the insert of 600 kb was partially digested by restriction enzyme EcoRI Sequence data from this article have been deposited with the DDBJ, EMBL, and GenBank Data Libraries under Accession No. D78132. 1 To whom correspondence should be addressed. Fax: 81-463-948884.
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and subcloned into the pWE15 cosmid vector. By screening the obtained cosmid clone library using the human Alu-repeat sequence as a probe, 230 cosmid clones containing human genomic DNAs in their inserts were isolated. These cosmid clones were completely digested by EcoRI followed by Southern hybridization using these EcoRI fragments as probes and aligned into a contig (Fig. 1). To assign the chromosomal location of the cosmid contig, fluorescence in situ hybridization (FISH) was performed using representative cosmid clones as probes. Chromosome spreads were obtained from phytohemagglutinin-stimulated blood lymphocytes of a healthy donor after thymidine synchronization and bromodeoxyuridine incorporation by the method of Takahashi et al. (8). Genomic DNA fragments were labeled with biotin-16–dUTP (Boehringer Mannheim) by nick-translation. In situ hybridization was carried out in the presence of COT-1 DNA (GIBCO BRL, Gaithersburg, MD) as a competitor, and hybridized probe was detected with FITC-conjugated avidin (Boehringer Mannheim). Figure 2 shows the location of the cosmid pM118, which includes the Ras-related GTP-binding protein gene described below. The FISH image was processed by a photometrics cooled CCD camera system. Y109 was found to be a chimeric YAC clone, in which about 240 kb of the insert is derived from the chromosome 6 region (6p21) and the remaining 360 kb insert is from the chromosome 7 region (7q36). The chimeric boundary region was determined by hybridization of human chromosome panel blots containing human and hamster somatic cell hybrid DNAs digested with the restriction enzyme EcoRI (BIOS Corp.) (data not shown); the cosmid contig covering the chromosome 7 region is shown in Fig. 1. None of the STS (sequencetagged site) markers such as EN2, CHRM2, D7S427,
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and D7S483, which were suggested to be localized near the 7q36 region, have been detected so far on the cosmid contig by the PCR technique. These cosmid clones were digested with rare-cutter enzymes such as NotI, BssHII, EagI, SacII, and MluI to construct a more accurate and detailed physical map of the contig and also to search for CpG islands, which are known to be frequently associated with the 5* upstream region of housekeeping and also of some tissue-specific genes. We have recognized five NotI cutting sites strongly associated with CpG islands in the chromosome 7 region of this contig (Fig. 1). A pM118 cosmid clone derived from the 7q36 region was found to contain a typical CpG island in the 3.7kb EcoRI fragment. To pursue the possibility that this CpG island is linked to an expressed gene, a human skin fibroblast cDNA library constructed using the lgt10 phage vector and oligo(dT)-primed cDNAs (Clontech Lab. Inc.) was screened using total EcoRI-digested pM118 cosmid DNA as a probe in the presence of human DNA as a competitor. One cDNA clone with the insert of 1.0 kb, termed pSC118 cDNA, was isolated. The pSC118 cDNA was found to be a housekeeping gene expressed ubiquitously with a transcript of 1.1 kb in every tissue examined, including skin, heart, brain, placenta, liver, skeletal muscle, kidney, and pancreas analyzed by Northern blot using the pSC118 cDNA clone as a probe (Fig. 3). FISH analysis using the pM118 cosmid clone as a probe implies that this gene is a single locus localized to the chromosome 7q36 region (Fig. 2). By Southern blot analysis, the pSC118 cDNA clone was revealed to hybridize with 5.6-, 3.3-, 12-, 2.8-, 5.5-, and 7.0kb EcoRI fragments shared by several overlapping cosmid clones (Fig. 1), indicating that the gene encoded by the pSC118 cDNA clone spans at least 61 kb at 7q36 with an exon– intron structure. Furthermore, the 5* upstream region of the pSC118 cDNA hybridized with the 2.8-, 5.5-, and 7.0-kb EcoRI fragments, defining the orientation of this gene. This indicates that the CpG island in the 7.0-kb fragment shared by the cosmid clones pM130, pM119, pM87, pM104, pM94, pM57, pM81, pM221, pM258, M102, and pM93, but not in pM118, was associated with the pSC118 cDNA defined gene. Since we have not performed any cDNA library screening using the other CpG islands that exist in this contig as probes, several housekeeping or tissue-specific genes may be located in this region.
The pSC118 cDNA insert was cloned into the pBluescript II KS(/) vector (Stratagene) and subjected to double-stranded DNA sequencing by the dideoxy chain termination method (7) using the Applied Biosystems 373A automatic DNA sequencer (Applied Biosystems Japan Inc.) with dye primers. The ends of the cDNA clone were initially sequenced using oligonucleotide primers derived from the flanking sequences of the pBluescript II KS(/) vector, 021M13, and RP1 primers (Applied Biosystems Japan Inc.). For further sequencing, the primer walking strategy was adopted, using several primers designed from the pSC118 nucleotide sequences obtained in the dideoxy chain termination reaction. The pSC118 cDNA insert was completely sequenced on both strands. As a result, the nucleotide length of the pSC118 insert was 975 bp, but an initiation codon ATG (methionine) could not be found in a putative open reading frame. Therefore, to obtain the fulllength coding sequence of the pSC118 cDNA defined transcript covering the 5* upstream region of the pSC118 cDNA sequence, a unique primer designed from the 5* end sequence of the pSC118 insert (R115: 5*-TGGTTGGATCGTAGGAGTCC-3 *) along with one of the primers derived from the sequences flanking the cloning site of the lgt10 vector (lgt105: 5*-AGCAAGTTCAGCCTGGTTAAGT-3 *; lgt103: 5*-TTATGAGTATTTCTTCCAGGG-3*) were synthesized and subjected to polymerase chain reaction to generate an amplified fragment from the skin fibroblast cDNA library constructed by l gt10. In this way, the 5* end 19-bp nucleotide sequence including the 5*untranslated region and an initiation codon ATG lacking in the pSC118 sequence was determined by the direct sequencing of the obtained PCR product. The full-length coding nucleotide and deduced amino acid sequences of the pSC118 defined gene are shown in Fig. 4. The sequence has a single long open reading frame encoding 184 amino acid residues, followed by an in-frame termination codon and the 3*untranslated region of 412 bp with a poly(A)/ addition signal (AATAAA) 21 nucleotides from the 3* terminal poly(A) tail. Comparison of these sequences with the protein and nucleotide sequence databases (GenBank, EMBL) indicated that the pSC118 cDNA has complete identity to an expressed sequence tag corresponding to a Ras-related GTP-binding protein (Accession No. Z29677) isolated from human keratinocytes, except for one nucleotide substitution (G r
FIG. 1. Genomic restriction map in the chromosome 7 region defined by the overlapping cosmid clones derived from YAC clone Y109. EcoRI sites are shown as short vertical lines without any alphabetical characters. However, the order of the EcoRI fragments is not definitely correct in the regions where enough overlapping cosmid clones have not been obtained. Vertical lines with alphabetical characters indicate recognition sites of the following restriction enzymes. N, NotI site; B, BssHII site; E, EagI site; S, SacII site; M, MluI site. Both open and solid triangles (n, m) show the cosmid fragments hybridized to the pSC118 cDNA. Open triangles in particular show the cosmid fragments hybridized to the PCR product amplified by the lgt105 and the R115 primers, which contain the 5*-untranslated region of the pSC118 defined transcript, defining the orientation of this gene.
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FIG. 2. Chromosomal localization of the Ras-related GTP-binding protein gene by FISH and idiogram of human chromosome 7 showing the map location of the pM118 defined locus at 7q36. Human prometaphase chromosomes counterstained with propidium iodide for Rbanding and FITC signals of the pM118 probe are shown by arrows. See text for details.
T) resulting in one amino acid change from glycine to tryptophan at position 118. This one-base mismatch may represent a limited polymorphism at this site in the Ras-related GTP-binding protein gene. Since this position is located in one of the Ras canonical boxes that are involved in GTP binding (Fig. 4) (1),
FIG. 3. Expression of pSC118 mRNA in human tissues by Northern blot analysis. A multiple tissue Northern blot membrane (Clontech Lab. Inc.) was used, containing 2 mg poly(A)/ RNA per lane from disease-free human tissues as indicated in the figure. Hybridization was performed using the 32P-labeled pSC118 cDNA as a probe according to the manufacturer’s manual.
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the polymorphism may have some biological significance. The nucleotide sequence of the pSC118 cDNA is 89.9% identical to that of the rat RHEB (Ras homologue
FIG. 4. The nucleotide sequence of the pSC118 cDNA and its predicted amino acid sequence. The nucleotide numbers to the right are from an arbitrary position in the putative 5*-untranslated region. The first 19-bp nucleotide sequence, on the 5* side of the vertical bar, was determined by the direct sequencing of the PCR product amplified by the lgt105 and R115 primers, and the remaining 975bp sequence, on the 3* side of the vertical bar, was determined by sequencing the pSC118 cDNA clone isolated from a skin fibroblast cDNA library (see text for details). The deduced amino acid sequence is shown under the nucleotide sequence; (*) indicates a stop codon. The poly(A)/ addition signal in the 3 *-untranslated region is underlined. G1– G5 indicate Ras canonical boxes that are involved in GTP binding (1). CAAX represents a signal for posttranslational farnesylation and targeting to specific membrane (4).
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enriched in brain) gene, which encodes a growth factorand synaptic action-regulated protein (9). There is only a difference of two amino acids between the pSC118 and the rat RHEB (rRHEB) encoded proteins, and the amino acid identity amounts to as high as 98.9% between them, indicating that the pSC118 cDNA defined gene, ubiquitously expressed, is a human homologue of the rRHEB gene. Therefore, this gene was designated human RHEB (hRHEB). The tryptophan at position 118 and the methionine at position 170 of the hRHEB protein are substituted for glycine and isoleucine, respectively, in the rRHEB protein. The hRHEB gene product possesses five GTP-binding regions (G1–G5), like the rRHEB gene product (Fig. 4). These GTP-binding regions in RHEB are highly conserved and homologous to numerous members of the Ras family involving yeast Ras1, human Rap2, and Human H-Ras-1 with 42.7, 37.1, and 35.8% amino acid identity, respectively. Like H-Ras, Rap2, and RHEB, the carboxy-terminal sequence of RHEB encodes a CAAX box (Fig. 4), indicating that the protein undergoes posttranscriptional farnesylation and is targeted to specific membrane (4). Although rRHEB is a housekeeping gene, its mRNA is expressed at a comparatively high level in the hippocampus and the cerebral cortex and at a relatively high level in Embryonic Day 19 cortical plates (9). The rRHEB mRNA is rapidly induced by growth factors and receptor-dependent synaptic activity, suggesting that rRHEB may play an important role in long-term activity-dependent neuronal responses (9). Since the hRHEB gene is highly homologous to this rRHEB gene and is a novel member of the Ras family of small GTPbinding proteins, it is possible that the hRHEB protein is also involved in neuronal activity, response, and/or development in the central nervous system. Since the brain mRNA investigated here in the Northern blot was extracted from total brain and not from specific tissues such as hippocampus or cerebral cortex, the hRHEB expression level in brain might be relatively lower than that in the other tissues examined (Fig. 3). In this respect it is notable that the pathogenic genes of holoprosencephaly type-3 (HPE3) and hereditary sacral agenesis were reported to be contiguous and localized to the same region of chromosome 7, 7q36 (2, 5, 6). A mutation in the hRHEB gene, causing a conformation change in its protein, could affect the neural activity, response, and/or development in the central nervous system, i. e., through impairment of GTP-binding activity, leading to the development of holoprosencephaly type-3 or sacral agenesis. It will be very important to detect genetic mutations or alterations in this gene, including the coding as well as the noncoding regions, and also to compare the expression level of the hRHEB
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locus between patients and healthy controls. We have also isolated mouse RHEB genomic clones (data not shown) and have been constructing a knockout mouse by inactivating the RHEB gene using a gene targeting technique to analyze the function of this gene. In conclusion, the gene product specified by the pSC118 cDNA is suggested to be a human homologue of the rRHEB protein, a Ras-related growth factor- and synaptic activity-regulated protein, and to map to the human chromosome 7q36 region. Based on its chromosomal localization and the predicted function, the hRHEB gene specified by pSC118 is considered to be a strong candidate gene responsible for the pathogenesis of holoprosencephaly-3 or hereditary sacral agenesis. ACKNOWLEDGMENT We thank Dr. Stephan Bech for his careful review of the manuscript and critical comments.
REFERENCES 1. Bourne, H. R., Sanders, D. A., and McCormick, F. (1991). The GTPase superfamily: Conserved structure and molecular mechanism. Nature 349: 117– 127. 2. Gurrieri, F., Trask, B. J., van den Engh, G., Krauss, C. M., Schinzel, A., Pettenati, M. J., Schindler, D., Dietz-Band, J., Vergnaud, G., Scherer, S. W., Tsui, L.-C., and Muenke, M. (1993). Physical mapping of the holoprosencephaly critical region on chromosome 7q36. Nature Genet. 3: 247 –251. 3. Imai, T., and Olson, M. V. (1990). Second-generation approach to the construction of yeast artificial-chromosome libraries. Genomics 8: 297–303. 4. Kinsella, B. T., Erdman, R. A., and Maltese, W. A. (1991). Posttranslational modification of Ha-ras p21 by farnesyl versus geranylgeranyl isoprenoids is determined by the COOH-terminal amino acid. Proc. Natl. Acad. Sci. USA 88: 8934 – 8938. 5. Lynch, S. A., Bond, P. M., Copp, A. J., Kirwan, W. O., Nour, S., Balling, R., Mariman, E., Burn, J., and Strachan, T. (1995). A gene for autosomal dominant sacral agenesis maps to the holoprosencephaly region at 7q36. Nature Genet. 11: 93 –95. 6. Muenke, M., Gurrieri, F., Bay, C., Yi, D. H., Collins, A. L., Johnson, V. P., Hennekam, R. C. M., Schaefer, G. B., Weik, L., Lubinsky, M. S., Daack-Hirsch, S., Moore, C. A., Dobyns, W. B., Murray, J. C., and Price, R. A. (1994). Linkage of a human brain malformation, familial holoprosencephaly, to chromosome 7 and evidence for genetic heterogeneity. Proc. Natl. Acad. Sci. USA 91: 8102 –8106. 7. Sanger, F., Nicklen, S., and Coulson, A. R. (1977). DNA sequencing with chain-termination inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463 – 5467. 8. Takahashi, E., Yamauchi, M., Tsuji, H., Hitomi, A., Meuth, M., and Hori, T. (1991). Chromosome mapping of the human cytidine-5*-triphosphate synthetase (CTPS) gene to band 1p34.1 – p34.3 by fluorescence in situ hybridization. Hum. Genet. 88: 119– 121. 9. Yamagata, K., Sanders, L. K., Kaufmann, W. E., Yee, W., Barnes, C. A., Nathans, D., and Worley, P. F. (1994). RHEB, a growth factor- and synaptic activity-regulated gene, encodes a novel Ras-related protein. J. Biol. Chem. 269: 16333 –16339.
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A Ras-related GTP-binding protein cDNA has been isolated from a human skin fibroblast cDNA library using a genomic subclone derived from a YAC clone as a probe. The polypeptide, consisting of 184 amino acids deduced from nucleotide sequences, contains five repeats of the Ras-related GTP-binding region and is highly homologous to the rat RHEB (Ras homologue enriched in brain) gene, which encodes a Ras-related growth factor- and synaptic activity-regulated protein, with 98.9% amino acid identity. Therefore, it is suggested to be a human homologue of the rat RHEB protein, and we have designated it human RHEB. Using fluorescence in situ hybridization, we concluded that this human RHEB gene was localized to band q36 on chromosome 7. Considering the chromosomal localization as well as the potential function of this protein, it will be very important to investigate whether it may play a role in the etiopathogenesis of holoprosencephaly type 3 or hereditary sacral agenesis, in which the disease susceptible locus is linked to the microsatellite marker, D7S22, in this chromosomal region, 7q36. q 1996 Academic Press, Inc.
A YAC clone, Y109, was isolated from the YAC library constructed from the B-cell line, CGM1 (A3, B8, Cw-, DR3, DQ2, DR52 and A29, B14, Cw-, DR7, DQ2, DR53), using HLA class I-specific primers (3) and was found to be a chimeric clone made up of the fragments from chromosomes 6 and 7. Y109 with the insert of 600 kb was partially digested by restriction enzyme EcoRI Sequence data from this article have been deposited with the DDBJ, EMBL, and GenBank Data Libraries under Accession No. D78132. 1 To whom correspondence should be addressed. Fax: 81-463-948884.
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and subcloned into the pWE15 cosmid vector. By screening the obtained cosmid clone library using the human Alu-repeat sequence as a probe, 230 cosmid clones containing human genomic DNAs in their inserts were isolated. These cosmid clones were completely digested by EcoRI followed by Southern hybridization using these EcoRI fragments as probes and aligned into a contig (Fig. 1). To assign the chromosomal location of the cosmid contig, fluorescence in situ hybridization (FISH) was performed using representative cosmid clones as probes. Chromosome spreads were obtained from phytohemagglutinin-stimulated blood lymphocytes of a healthy donor after thymidine synchronization and bromodeoxyuridine incorporation by the method of Takahashi et al. (8). Genomic DNA fragments were labeled with biotin-16–dUTP (Boehringer Mannheim) by nick-translation. In situ hybridization was carried out in the presence of COT-1 DNA (GIBCO BRL, Gaithersburg, MD) as a competitor, and hybridized probe was detected with FITC-conjugated avidin (Boehringer Mannheim). Figure 2 shows the location of the cosmid pM118, which includes the Ras-related GTP-binding protein gene described below. The FISH image was processed by a photometrics cooled CCD camera system. Y109 was found to be a chimeric YAC clone, in which about 240 kb of the insert is derived from the chromosome 6 region (6p21) and the remaining 360 kb insert is from the chromosome 7 region (7q36). The chimeric boundary region was determined by hybridization of human chromosome panel blots containing human and hamster somatic cell hybrid DNAs digested with the restriction enzyme EcoRI (BIOS Corp.) (data not shown); the cosmid contig covering the chromosome 7 region is shown in Fig. 1. None of the STS (sequencetagged site) markers such as EN2, CHRM2, D7S427,
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and D7S483, which were suggested to be localized near the 7q36 region, have been detected so far on the cosmid contig by the PCR technique. These cosmid clones were digested with rare-cutter enzymes such as NotI, BssHII, EagI, SacII, and MluI to construct a more accurate and detailed physical map of the contig and also to search for CpG islands, which are known to be frequently associated with the 5* upstream region of housekeeping and also of some tissue-specific genes. We have recognized five NotI cutting sites strongly associated with CpG islands in the chromosome 7 region of this contig (Fig. 1). A pM118 cosmid clone derived from the 7q36 region was found to contain a typical CpG island in the 3.7kb EcoRI fragment. To pursue the possibility that this CpG island is linked to an expressed gene, a human skin fibroblast cDNA library constructed using the lgt10 phage vector and oligo(dT)-primed cDNAs (Clontech Lab. Inc.) was screened using total EcoRI-digested pM118 cosmid DNA as a probe in the presence of human DNA as a competitor. One cDNA clone with the insert of 1.0 kb, termed pSC118 cDNA, was isolated. The pSC118 cDNA was found to be a housekeeping gene expressed ubiquitously with a transcript of 1.1 kb in every tissue examined, including skin, heart, brain, placenta, liver, skeletal muscle, kidney, and pancreas analyzed by Northern blot using the pSC118 cDNA clone as a probe (Fig. 3). FISH analysis using the pM118 cosmid clone as a probe implies that this gene is a single locus localized to the chromosome 7q36 region (Fig. 2). By Southern blot analysis, the pSC118 cDNA clone was revealed to hybridize with 5.6-, 3.3-, 12-, 2.8-, 5.5-, and 7.0kb EcoRI fragments shared by several overlapping cosmid clones (Fig. 1), indicating that the gene encoded by the pSC118 cDNA clone spans at least 61 kb at 7q36 with an exon– intron structure. Furthermore, the 5* upstream region of the pSC118 cDNA hybridized with the 2.8-, 5.5-, and 7.0-kb EcoRI fragments, defining the orientation of this gene. This indicates that the CpG island in the 7.0-kb fragment shared by the cosmid clones pM130, pM119, pM87, pM104, pM94, pM57, pM81, pM221, pM258, M102, and pM93, but not in pM118, was associated with the pSC118 cDNA defined gene. Since we have not performed any cDNA library screening using the other CpG islands that exist in this contig as probes, several housekeeping or tissue-specific genes may be located in this region.
The pSC118 cDNA insert was cloned into the pBluescript II KS(/) vector (Stratagene) and subjected to double-stranded DNA sequencing by the dideoxy chain termination method (7) using the Applied Biosystems 373A automatic DNA sequencer (Applied Biosystems Japan Inc.) with dye primers. The ends of the cDNA clone were initially sequenced using oligonucleotide primers derived from the flanking sequences of the pBluescript II KS(/) vector, 021M13, and RP1 primers (Applied Biosystems Japan Inc.). For further sequencing, the primer walking strategy was adopted, using several primers designed from the pSC118 nucleotide sequences obtained in the dideoxy chain termination reaction. The pSC118 cDNA insert was completely sequenced on both strands. As a result, the nucleotide length of the pSC118 insert was 975 bp, but an initiation codon ATG (methionine) could not be found in a putative open reading frame. Therefore, to obtain the fulllength coding sequence of the pSC118 cDNA defined transcript covering the 5* upstream region of the pSC118 cDNA sequence, a unique primer designed from the 5* end sequence of the pSC118 insert (R115: 5*-TGGTTGGATCGTAGGAGTCC-3 *) along with one of the primers derived from the sequences flanking the cloning site of the lgt10 vector (lgt105: 5*-AGCAAGTTCAGCCTGGTTAAGT-3 *; lgt103: 5*-TTATGAGTATTTCTTCCAGGG-3*) were synthesized and subjected to polymerase chain reaction to generate an amplified fragment from the skin fibroblast cDNA library constructed by l gt10. In this way, the 5* end 19-bp nucleotide sequence including the 5*untranslated region and an initiation codon ATG lacking in the pSC118 sequence was determined by the direct sequencing of the obtained PCR product. The full-length coding nucleotide and deduced amino acid sequences of the pSC118 defined gene are shown in Fig. 4. The sequence has a single long open reading frame encoding 184 amino acid residues, followed by an in-frame termination codon and the 3*untranslated region of 412 bp with a poly(A)/ addition signal (AATAAA) 21 nucleotides from the 3* terminal poly(A) tail. Comparison of these sequences with the protein and nucleotide sequence databases (GenBank, EMBL) indicated that the pSC118 cDNA has complete identity to an expressed sequence tag corresponding to a Ras-related GTP-binding protein (Accession No. Z29677) isolated from human keratinocytes, except for one nucleotide substitution (G r
FIG. 1. Genomic restriction map in the chromosome 7 region defined by the overlapping cosmid clones derived from YAC clone Y109. EcoRI sites are shown as short vertical lines without any alphabetical characters. However, the order of the EcoRI fragments is not definitely correct in the regions where enough overlapping cosmid clones have not been obtained. Vertical lines with alphabetical characters indicate recognition sites of the following restriction enzymes. N, NotI site; B, BssHII site; E, EagI site; S, SacII site; M, MluI site. Both open and solid triangles (n, m) show the cosmid fragments hybridized to the pSC118 cDNA. Open triangles in particular show the cosmid fragments hybridized to the PCR product amplified by the lgt105 and the R115 primers, which contain the 5*-untranslated region of the pSC118 defined transcript, defining the orientation of this gene.
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FIG. 2. Chromosomal localization of the Ras-related GTP-binding protein gene by FISH and idiogram of human chromosome 7 showing the map location of the pM118 defined locus at 7q36. Human prometaphase chromosomes counterstained with propidium iodide for Rbanding and FITC signals of the pM118 probe are shown by arrows. See text for details.
T) resulting in one amino acid change from glycine to tryptophan at position 118. This one-base mismatch may represent a limited polymorphism at this site in the Ras-related GTP-binding protein gene. Since this position is located in one of the Ras canonical boxes that are involved in GTP binding (Fig. 4) (1),
FIG. 3. Expression of pSC118 mRNA in human tissues by Northern blot analysis. A multiple tissue Northern blot membrane (Clontech Lab. Inc.) was used, containing 2 mg poly(A)/ RNA per lane from disease-free human tissues as indicated in the figure. Hybridization was performed using the 32P-labeled pSC118 cDNA as a probe according to the manufacturer’s manual.
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the polymorphism may have some biological significance. The nucleotide sequence of the pSC118 cDNA is 89.9% identical to that of the rat RHEB (Ras homologue
FIG. 4. The nucleotide sequence of the pSC118 cDNA and its predicted amino acid sequence. The nucleotide numbers to the right are from an arbitrary position in the putative 5*-untranslated region. The first 19-bp nucleotide sequence, on the 5* side of the vertical bar, was determined by the direct sequencing of the PCR product amplified by the lgt105 and R115 primers, and the remaining 975bp sequence, on the 3* side of the vertical bar, was determined by sequencing the pSC118 cDNA clone isolated from a skin fibroblast cDNA library (see text for details). The deduced amino acid sequence is shown under the nucleotide sequence; (*) indicates a stop codon. The poly(A)/ addition signal in the 3 *-untranslated region is underlined. G1– G5 indicate Ras canonical boxes that are involved in GTP binding (1). CAAX represents a signal for posttranslational farnesylation and targeting to specific membrane (4).
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enriched in brain) gene, which encodes a growth factorand synaptic action-regulated protein (9). There is only a difference of two amino acids between the pSC118 and the rat RHEB (rRHEB) encoded proteins, and the amino acid identity amounts to as high as 98.9% between them, indicating that the pSC118 cDNA defined gene, ubiquitously expressed, is a human homologue of the rRHEB gene. Therefore, this gene was designated human RHEB (hRHEB). The tryptophan at position 118 and the methionine at position 170 of the hRHEB protein are substituted for glycine and isoleucine, respectively, in the rRHEB protein. The hRHEB gene product possesses five GTP-binding regions (G1–G5), like the rRHEB gene product (Fig. 4). These GTP-binding regions in RHEB are highly conserved and homologous to numerous members of the Ras family involving yeast Ras1, human Rap2, and Human H-Ras-1 with 42.7, 37.1, and 35.8% amino acid identity, respectively. Like H-Ras, Rap2, and RHEB, the carboxy-terminal sequence of RHEB encodes a CAAX box (Fig. 4), indicating that the protein undergoes posttranscriptional farnesylation and is targeted to specific membrane (4). Although rRHEB is a housekeeping gene, its mRNA is expressed at a comparatively high level in the hippocampus and the cerebral cortex and at a relatively high level in Embryonic Day 19 cortical plates (9). The rRHEB mRNA is rapidly induced by growth factors and receptor-dependent synaptic activity, suggesting that rRHEB may play an important role in long-term activity-dependent neuronal responses (9). Since the hRHEB gene is highly homologous to this rRHEB gene and is a novel member of the Ras family of small GTPbinding proteins, it is possible that the hRHEB protein is also involved in neuronal activity, response, and/or development in the central nervous system. Since the brain mRNA investigated here in the Northern blot was extracted from total brain and not from specific tissues such as hippocampus or cerebral cortex, the hRHEB expression level in brain might be relatively lower than that in the other tissues examined (Fig. 3). In this respect it is notable that the pathogenic genes of holoprosencephaly type-3 (HPE3) and hereditary sacral agenesis were reported to be contiguous and localized to the same region of chromosome 7, 7q36 (2, 5, 6). A mutation in the hRHEB gene, causing a conformation change in its protein, could affect the neural activity, response, and/or development in the central nervous system, i. e., through impairment of GTP-binding activity, leading to the development of holoprosencephaly type-3 or sacral agenesis. It will be very important to detect genetic mutations or alterations in this gene, including the coding as well as the noncoding regions, and also to compare the expression level of the hRHEB
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locus between patients and healthy controls. We have also isolated mouse RHEB genomic clones (data not shown) and have been constructing a knockout mouse by inactivating the RHEB gene using a gene targeting technique to analyze the function of this gene. In conclusion, the gene product specified by the pSC118 cDNA is suggested to be a human homologue of the rRHEB protein, a Ras-related growth factor- and synaptic activity-regulated protein, and to map to the human chromosome 7q36 region. Based on its chromosomal localization and the predicted function, the hRHEB gene specified by pSC118 is considered to be a strong candidate gene responsible for the pathogenesis of holoprosencephaly-3 or hereditary sacral agenesis. ACKNOWLEDGMENT We thank Dr. Stephan Bech for his careful review of the manuscript and critical comments.
REFERENCES 1. Bourne, H. R., Sanders, D. A., and McCormick, F. (1991). The GTPase superfamily: Conserved structure and molecular mechanism. Nature 349: 117– 127. 2. Gurrieri, F., Trask, B. J., van den Engh, G., Krauss, C. M., Schinzel, A., Pettenati, M. J., Schindler, D., Dietz-Band, J., Vergnaud, G., Scherer, S. W., Tsui, L.-C., and Muenke, M. (1993). Physical mapping of the holoprosencephaly critical region on chromosome 7q36. Nature Genet. 3: 247 –251. 3. Imai, T., and Olson, M. V. (1990). Second-generation approach to the construction of yeast artificial-chromosome libraries. Genomics 8: 297–303. 4. Kinsella, B. T., Erdman, R. A., and Maltese, W. A. (1991). Posttranslational modification of Ha-ras p21 by farnesyl versus geranylgeranyl isoprenoids is determined by the COOH-terminal amino acid. Proc. Natl. Acad. Sci. USA 88: 8934 – 8938. 5. Lynch, S. A., Bond, P. M., Copp, A. J., Kirwan, W. O., Nour, S., Balling, R., Mariman, E., Burn, J., and Strachan, T. (1995). A gene for autosomal dominant sacral agenesis maps to the holoprosencephaly region at 7q36. Nature Genet. 11: 93 –95. 6. Muenke, M., Gurrieri, F., Bay, C., Yi, D. H., Collins, A. L., Johnson, V. P., Hennekam, R. C. M., Schaefer, G. B., Weik, L., Lubinsky, M. S., Daack-Hirsch, S., Moore, C. A., Dobyns, W. B., Murray, J. C., and Price, R. A. (1994). Linkage of a human brain malformation, familial holoprosencephaly, to chromosome 7 and evidence for genetic heterogeneity. Proc. Natl. Acad. Sci. USA 91: 8102 –8106. 7. Sanger, F., Nicklen, S., and Coulson, A. R. (1977). DNA sequencing with chain-termination inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463 – 5467. 8. Takahashi, E., Yamauchi, M., Tsuji, H., Hitomi, A., Meuth, M., and Hori, T. (1991). Chromosome mapping of the human cytidine-5*-triphosphate synthetase (CTPS) gene to band 1p34.1 – p34.3 by fluorescence in situ hybridization. Hum. Genet. 88: 119– 121. 9. Yamagata, K., Sanders, L. K., Kaufmann, W. E., Yee, W., Barnes, C. A., Nathans, D., and Worley, P. F. (1994). RHEB, a growth factor- and synaptic activity-regulated gene, encodes a novel Ras-related protein. J. Biol. Chem. 269: 16333 –16339.
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AP: Genomics