- Email: [email protected]
The human RGL (RalGDS-like) gene: cloning, expression analysis and genomic organization
Biochimica et Biophysica Acta 1491 (2000) 285^288 www.elsevier.com/locate/bba
Short sequence-paper
The human RGL (RalGDS-like) gene: cloning, expression analysis and genomic organization Raman Sood a; *, Izabela Makalowska b , John D. Carpten a , Christiane M. Robbins a , Dietrich A. Stephan a , Timothy D. Connors c , Sharon D. Morgenbesser c , Kui Su c , Heather W. Pinkett a , Christopher L. Graham a , Matthew I. Quesenberry a , Andreas D. Baxevanis b , Katherine W. Klinger c , Je¡rey M. Trent a , Tom I. Bonner d a
Cancer Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Building 36, Room 3D05, 9000 Rockville Pike, Bethesda, MD 20892, USA b Genome Technology Branch, National Human Genome Research Institute, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA c Genzyme Genetics Corporation, Framingham, MA, USA d Laboratory of Genetics, National Institute of Mental Health, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA Received 9 November 1999; accepted 31 January 2000
Abstract Ral GDP dissociation stimulator (RalGDS) and its family members RGL, RLF and RGL2 are involved in Ras and Ral signaling pathways as downstream effector proteins. Here we report the precise localization and cloning of two forms of human RGL gene differing at the amino terminus. Transcript A, cloned from liver cDNA libraries has the same amino terminus as the mouse RGL, whereas transcript B cloned from brain has a substitution of 45 amino acids for the first nine amino acids. At the genomic level, exon 1 of transcript A is replaced by two alternative exons (1B1 and 1B2) in transcript B. Both forms share exons 2 through 18. The human RGL protein shares 94% amino acid identity with the mouse protein. Northern blot analysis shows that human RGL is expressed in a wide variety of tissues with strong expression being seen in the heart, brain, kidney, spleen and testis. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: RGL; Prostate cancer; Gene structure; Expression
Ras plays a critical role in signal transduction for many cellular processes, including cell proliferation, di¡erentiation and transformation [1]. Mutations in Ras are associated with several human cancers [2]. The biological activities of Ras are mediated by interactions of the Ras protein with downstream sig-
* Corresponding author. Fax: +1-301-435-5465; E-mail: [email protected]
naling proteins referred to as Ras e¡ectors, each of which is believed to initiate a cascade of signal transduction reactions. Ras cycles between an active GTPbound form, in which it binds e¡ector molecules, and an inactive GDP-bound form. The ratio of these two forms is largely controlled by GTP binding induced by guanine nucleotide dissociation stimulators (GDSs) and GTP hydrolysis induced by GTPase-activating proteins. An increase in the GTP-bound active form of Ras leads to continuous signaling to its
0167-4781 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 0 ) 0 0 0 3 1 - 2
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e¡ector proteins, thereby causing the malignant transformation of cells. The mouse RGL gene was cloned as a Ras interacting protein using a yeast two-hybrid system [3]. RGL shares 50% amino acid identity with Ral guanine nucleotide dissociation stimulator (RalGDS), a GDP/GTP exchange protein for Ral p24 [4]. The Cterminal domains of RGL and RalGDS contain a Ras-interacting domain that interacts with the GTP-bound form of Ras through its e¡ector loop [5]. Both proteins have been shown to function as Ras e¡ector proteins [3,6,7]. RGL also contains a CDC25-like domain, which is shared among several GDP/GTP exchange proteins. RGL is part of a Rasmediated signaling pathway, distinct from the Raf pathway, which mediates Ras signals to stimulate cfos gene expression and activate Ral [8]. As part of the physical and transcript mapping of the 1q25 region aimed towards identi¢cation of the HPC1 gene causing susceptibility to prostate cancer [9], we cloned the human homologue of the RGL gene. Here we present two alternative forms of human RGL cDNA di¡ering at the amino terminus suggesting alternative splicing and promoter usage in di¡erent tissues. We have also characterized the genomic organization of these two forms and their relative orientation with respect to the Bac contig. To identify candidate genes for HPC1, we mapped ESTs from the human gene map (http:// www.ncbi.nlm.nih.gov/genemap/) for the region of interest to our YAC/BAC map [10]. These ESTs were then extended into full-length transcripts by various cDNA extension technologies. STS-Z40837 mapping to the D1S2640^D1S461 interval was localized to BAC clone b110O11. Initially 1.7 kb of sequence was generated using Unigene cluster Hs.79219 and sequencing of IMAGE clone 360212 (GenBank accession AA013091). Oligonucleotides designed from this sequence were used to identify cDNA clones from pooled brain, fetal brain and liver cDNA libraries using the Gene-Trapper cDNA selection system (Life Technologies). Thirty-four clones with insert sizes ranging from 1.6 to 3.5 kb were identi¢ed to be part of the RGL transcript by sequence alignment using Sequencher (Genecodes). 5P-RACE on brain and liver cDNA templates (Clontech) was used to identify the remainder of the tran-
script with high GC RACE techniques being used for the 5P-most portions. The liver RACE products gave the same sequence as two recently submitted partial human RGL sequences (GenBank accessions AL080117 and AB023176 = KIAA0959) but extending slightly farther 5P (GenBank accession AF186779). AL080117 and several of our cDNA clones terminate at a stretch of 14 As at base 4357, whereas KIAA0959 and other clones terminate at the more distal authentic poly(A) site at base 4726. A consensus polyadenylation signal (AATAAA) was found at nucleotide position 4678^4683, 43 nucleotides upstream of the poly(A) tail. There is no in-frame upstream terminator sequence but divergence between the human and mouse cDNA sequences immediately upstream of the ¢rst ATG suggests it to be the most likely initiation codon. Human liver RGL thus has an open reading frame of 2307 bp, coding for a protein of 768 amino acids. In contrast, the brain cDNA has an alternative 5P end (GenBank accession AF186780), resulting from transcription initiation at an alternative ¢rst exon. This alternative transcript substitutes 44 amino
Fig. 1. Alignment of human and mouse RGL at the amino acid level. Amino acids that di¡er between human and mouse are shown in the mouse sequence, a dashed line represents the identical residues. Amino acid numbering is given on the left side. Arrows indicate the residues critical to Ras^RGL interaction within the Ras-interacting domain. The boxes denote domains identi¢ed using protein families database (pfam). From left to right: (1) RasGEFN domain (Pfam accession PF00618), (2) RasGEF domain (Pfam accession PF00617) and (3) RA domain (Pfam accession PF00788).
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acids for the ¢rst nine amino acids of the liver transcript resulting in a protein of 803 amino acids. Comparison of the coding sequences of human and mouse RGL genes shows 89% nucleotide and 94% amino acid identity. Both proteins contain three highly conserved domains identi¢ed by matches from protein families database (http://www.sanger.ac.uk/ Software/Pfam/index.shtml) (Fig. 1). Most of the divergence is seen in the inter-domain regions. Of two amino acids critical to the interaction of mouse RGL with Ras [11], Lys685 is conserved in human RGL while Glu689 becomes Asp, a conservative change also observed in other GDS family members. Overall, only three amino acids are di¡erent between Rasinteracting domains of the human and the mouse RGL proteins. Northern blot analysis showed expression of human RGL in all tissues examined (Fig. 2). A major band of V5 kb is seen in most tissues with a slightly larger band (V5.3 kb) being evident in brain. This is consistent with the di¡erent cDNA form isolated from brain cDNA library. Taking into account the poly(A) tails, these mRNA sizes are V0.2 kb larger than the largest composite cDNAs from liver and brain, 4.735 and 5.1 kb, respectively. This result
287
Fig. 2. Northern blot analysis of human RGL gene. Multipletissue Northern blots (Clontech) were hybridized with a 3.5-kb probe corresponding to bases 941^4367 of transcript A. Molecular weight standards are indicated on the left. The blots were washed at high stringency using 0.1% SDS and 0.1USSC at 65³C.
leaves open the possibilities that either there is one more exon upstream which is common to all transcripts or that we have essentially the complete mRNA and two very di¡erent transcription start sites. Our Northern data combined with RT-PCR ELISA screening of KIAA0959 [12] shows that human RGL is expressed in a wide variety of tissues, suggesting its general importance in the Ras signaling pathway. It is not yet clear whether the altered amino terminus of the brain protein alters the function of RGL in the brain or simply re£ects usage of a
Table 1 Exon^intron organization of the human RGL gene Exon
Exon size (bp)
Splice donor site
... Splice acceptor site
Accession No.
1A 1B1 1B2 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
s 205 s 367 164 111 209 78 185 125 216 104 85 90 87 33 122 90 187 255 115 s 615
CAAGCTAAAATGgtaacgagagct AACCTGAACTCGgtatcaaatcta AAGACAGAGGAGgtaagatgactc AGATGGCTAGGGgtgagtaaagct ACTGCTGGACAGgtaagaatgtaa GGTGATCAGGAAgtgagtctccct TGGAAACTGACAgtgagtacttgt TACATGGATGCAgtatgtcttctc AACATCGCTCATgtaattgtcttt TGCCGTCCCAAGgtaagttcccaa CTACTGATGAAGgtgaggctctta CAGAAGGACATGgtatgtctggcc GACTACATCGAGgtgagttccatg ND AGAGGAGGAGAGgtgggatcacct GAGACTCAGCCTgtgagtgtcccc CCTCAAAAAAAGgtatatactcaa AAGAGCATCATGgtgaggagcaag CGGAGGACAAAGgtgggcatcctt ND, contains stop codon
... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... p
AF186781 AF186782 AF192520 AF186783 AF186784 AF186785 AF186786 AF186787 AF186787 AF186788 AF186789 AF186790 AF186791 AF186792 AF186793 AF186794 AF186795 AF186796 AF186797 AF186798
cgtccctggcagAGCTCGATTCAG tcttccacacagATCCGTAATGCC cgtccctggcagAGCTCGATTCAG tttattatacagGTTGAAGGGGAC ttatctctacagGTATGGAAACCT tgtctttctcagTGCAATCGCTTC atatgtcaacagATGGGCTTCCCA cttttgttccagCAACTCTTCAAG ctccccatgcagGAATGTAGACTC cccttttcatagGGACCGAATGCT tctgtttctcagGAAGGAACCTCA gatgctcttcagGGTGTGATGCAG ND tgtctgctttagGAATTTGAAGTG tctctttctcagCTATGCCCTGTC gtttttttccagACTGTTTCTAGG ttactcttccagCTCTCTGAGTCC ttgtctttgcagTTGACGAGCCAG ND 3PUTR, might be the last exon
ND: not determined.
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brain speci¢c promoter that coincidentally uses a different initiation codon. Exon-traps from two overlapping BAC clones, b96P08 and b110O11 [10], de¢ned 10 exons with the 3P exons occurring only in b110O11, thus providing its transcription orientation with respect to the Bac contig. Primers derived from the cDNA were used to sequence the BAC DNA to identify 8 additional exons from the coding region of the liver cDNA. The alternative 5P end of the brain cDNA was mapped to two BAC clones centromeric to b96P08 and exons 1B1 and 1B2 were de¢ned by sequencing these clones. Table 1 lists the position of exons and adjacent intronic sequences. The intron located between exons 6 and 7 is 910 bp in size. The size of the other introns was not determined. All introns have splice donor and acceptor sites that conform to the general GT-AG consensus motif (Table 1). The putative translation initiation codon of the liver transcript (transcript A) is located in exon 1A and the stop codon is in exon 18. The initiation codon of the brain transcript (transcript B) is in exon 1B2 that lies 5P of exon 1A. The marker stsG30928, also mapped to the HPC1 candidate region, is found in the adjoining intron sequence of exon 1B1. Evaluation of RGL as a candidate gene for prostate cancer by SSCP analysis of cDNA from patients from families showing linkage to 1q25 region markers failed to identify any disease-associated mutations in the coding region of transcript A. Southern blot analysis also failed to detect any alterations in the gene. Because of the many biological activities of Ras, the identity of its e¡ectors and the elucidation of the various signaling pathways leading to cell growth and di¡erentiation have been subject of intense research over the past decade. Identi¢cation of the human RGL gene with alternative forms in di¡erent tissues, its expression analysis and genomic organization should prove useful in continuation of such research. References
[2] J.L. Bos, Ras oncogenes in human cancer: a review, Cancer Res. 49 (1989) 4682^4689. [3] A. Kikuchi, S.D. Demo, Z.-H. Ye, Y.-W. Chen, L.T. Williams, RalGDS family members interact with the e¡ector loop of ras p21, Mol. Cell. Biol. 14 (1994) 7483^7491. [4] C.F. Albright, B.W. Giddings, J. Liu, M. Vito, R.A. Weinberg, Characterization of a guanine nucleotide dissociation stimulator for a ras-related GTPase, EMBO J. 12 (1993) 339^347. [5] S. Koyama, Y.-W. Chen, M. Ikeda, A.J. Muslin, L.T. Williams, A. Kikuchi, Ras-interacting domain of RGL blocks Ras-dependent signal transduction in Xenopus oocytes, FEBS Lett. 380 (1996) 113^117. [6] F. Hofer, S. Fields, C. Schneider, G.S. Martin, Activated Ras interacts with the Ral guanine nucleotide dissociation stimulator, Proc. Natl. Acad. Sci. USA 91 (1994) 11089^ 11093. [7] M.J. Miller, S. Prigent, E. Kupperman, L. Rioux, S.-H. Park, J.R. Feramisco, M.A. White, J.L. Rutkowski, J.L. Meinkoth, RalGDS functions in Ras- and cAMP-mediated growth stimulation, J. Biol. Chem. 272 (1997) 5600^5605. [8] H. Murai, M. Ikeda, S. Kishida, O. Ishida, M. OkazakiKishida, Y. Matsuura, A. Kikuchi, Characterization of Ral GDP dissociation stimulator-like (RGL) activities to regulate c-fos promoter and the GDP/GTP exchange of Ral, J. Biol. Chem. 272 (1997) 10483^10490. [9] J.R. Smith, D. Freije, J.D. Carpten, H. Gronberg, J. Xu, S.D. Isaacs, M.J. Brownstein, G.S. Bova, H. Guo, P. Bujnovsky, D.R. Nusskern, J.-E. Damber, A. Bergh, M. Emanuelsson, O.P. Kallioniemi, J. Walker-Daniels, J.E. BaileyWilson, T.H. Beaty, D.A. Meyers, P.A. Walsh, F.S. Collins, J.M. Trent, W.B. Isaacs, Major susceptibility locus for prostate cancer on chromosome 1 suggested by a genome-wide search, Science 274 (1996) 1371^1374. [10] J.D. Carpten, I. Makalowska, C.M. Robbins, N. Scott, R. Sood, T.D. Connors, T.I. Bonner, J.R. Smith, M.U. Faruque, D.A. Stephan, H. Pinkett, S.D. Morgenbesser, K. Su, C. Graham, S.G. Gregory, H. Williams, L. McDonald, A.D. Baxevanis, K.W. Klinger, G.M. Landes and J.M. Trent, A 6-Mb high-resolution physical and transcription map encompassing the hereditary prostate cancer 1 (HPC1) region, Genomics, in press. [11] M. Shirouzu, K. Hashimoto, A. Kikuchi, S. Yokoyama, Double-mutant analysis of the interaction of Ras with the Ras-binding domain of RGL, Biochemistry 38 (1999) 5103^ 5110. [12] T. Nagase, K.-I. Ishikawa, M. Suyama, R. Kikuno, M. Hirosawa, N. Miyajima, A. Tanaka, H. Kotani, N. Nomura, O. Ohara, Prediction of the coding sequences of unidenti¢ed human genes. XIII. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro, DNA Res. 6 (1999) 63^70.
[1] J.L. Bos, Ras-like GTPases: mini-review, Biochim. Biophys. Acta 1333 (1997) M19^M31.
BBAEXP 91371 29-3-00
Short sequence-paper
The human RGL (RalGDS-like) gene: cloning, expression analysis and genomic organization Raman Sood a; *, Izabela Makalowska b , John D. Carpten a , Christiane M. Robbins a , Dietrich A. Stephan a , Timothy D. Connors c , Sharon D. Morgenbesser c , Kui Su c , Heather W. Pinkett a , Christopher L. Graham a , Matthew I. Quesenberry a , Andreas D. Baxevanis b , Katherine W. Klinger c , Je¡rey M. Trent a , Tom I. Bonner d a
Cancer Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Building 36, Room 3D05, 9000 Rockville Pike, Bethesda, MD 20892, USA b Genome Technology Branch, National Human Genome Research Institute, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA c Genzyme Genetics Corporation, Framingham, MA, USA d Laboratory of Genetics, National Institute of Mental Health, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA Received 9 November 1999; accepted 31 January 2000
Abstract Ral GDP dissociation stimulator (RalGDS) and its family members RGL, RLF and RGL2 are involved in Ras and Ral signaling pathways as downstream effector proteins. Here we report the precise localization and cloning of two forms of human RGL gene differing at the amino terminus. Transcript A, cloned from liver cDNA libraries has the same amino terminus as the mouse RGL, whereas transcript B cloned from brain has a substitution of 45 amino acids for the first nine amino acids. At the genomic level, exon 1 of transcript A is replaced by two alternative exons (1B1 and 1B2) in transcript B. Both forms share exons 2 through 18. The human RGL protein shares 94% amino acid identity with the mouse protein. Northern blot analysis shows that human RGL is expressed in a wide variety of tissues with strong expression being seen in the heart, brain, kidney, spleen and testis. ß 2000 Elsevier Science B.V. All rights reserved. Keywords: RGL; Prostate cancer; Gene structure; Expression
Ras plays a critical role in signal transduction for many cellular processes, including cell proliferation, di¡erentiation and transformation [1]. Mutations in Ras are associated with several human cancers [2]. The biological activities of Ras are mediated by interactions of the Ras protein with downstream sig-
* Corresponding author. Fax: +1-301-435-5465; E-mail: [email protected]
naling proteins referred to as Ras e¡ectors, each of which is believed to initiate a cascade of signal transduction reactions. Ras cycles between an active GTPbound form, in which it binds e¡ector molecules, and an inactive GDP-bound form. The ratio of these two forms is largely controlled by GTP binding induced by guanine nucleotide dissociation stimulators (GDSs) and GTP hydrolysis induced by GTPase-activating proteins. An increase in the GTP-bound active form of Ras leads to continuous signaling to its
0167-4781 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 0 ) 0 0 0 3 1 - 2
BBAEXP 91371 29-3-00
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R. Sood et al. / Biochimica et Biophysica Acta 1491 (2000) 285^288
e¡ector proteins, thereby causing the malignant transformation of cells. The mouse RGL gene was cloned as a Ras interacting protein using a yeast two-hybrid system [3]. RGL shares 50% amino acid identity with Ral guanine nucleotide dissociation stimulator (RalGDS), a GDP/GTP exchange protein for Ral p24 [4]. The Cterminal domains of RGL and RalGDS contain a Ras-interacting domain that interacts with the GTP-bound form of Ras through its e¡ector loop [5]. Both proteins have been shown to function as Ras e¡ector proteins [3,6,7]. RGL also contains a CDC25-like domain, which is shared among several GDP/GTP exchange proteins. RGL is part of a Rasmediated signaling pathway, distinct from the Raf pathway, which mediates Ras signals to stimulate cfos gene expression and activate Ral [8]. As part of the physical and transcript mapping of the 1q25 region aimed towards identi¢cation of the HPC1 gene causing susceptibility to prostate cancer [9], we cloned the human homologue of the RGL gene. Here we present two alternative forms of human RGL cDNA di¡ering at the amino terminus suggesting alternative splicing and promoter usage in di¡erent tissues. We have also characterized the genomic organization of these two forms and their relative orientation with respect to the Bac contig. To identify candidate genes for HPC1, we mapped ESTs from the human gene map (http:// www.ncbi.nlm.nih.gov/genemap/) for the region of interest to our YAC/BAC map [10]. These ESTs were then extended into full-length transcripts by various cDNA extension technologies. STS-Z40837 mapping to the D1S2640^D1S461 interval was localized to BAC clone b110O11. Initially 1.7 kb of sequence was generated using Unigene cluster Hs.79219 and sequencing of IMAGE clone 360212 (GenBank accession AA013091). Oligonucleotides designed from this sequence were used to identify cDNA clones from pooled brain, fetal brain and liver cDNA libraries using the Gene-Trapper cDNA selection system (Life Technologies). Thirty-four clones with insert sizes ranging from 1.6 to 3.5 kb were identi¢ed to be part of the RGL transcript by sequence alignment using Sequencher (Genecodes). 5P-RACE on brain and liver cDNA templates (Clontech) was used to identify the remainder of the tran-
script with high GC RACE techniques being used for the 5P-most portions. The liver RACE products gave the same sequence as two recently submitted partial human RGL sequences (GenBank accessions AL080117 and AB023176 = KIAA0959) but extending slightly farther 5P (GenBank accession AF186779). AL080117 and several of our cDNA clones terminate at a stretch of 14 As at base 4357, whereas KIAA0959 and other clones terminate at the more distal authentic poly(A) site at base 4726. A consensus polyadenylation signal (AATAAA) was found at nucleotide position 4678^4683, 43 nucleotides upstream of the poly(A) tail. There is no in-frame upstream terminator sequence but divergence between the human and mouse cDNA sequences immediately upstream of the ¢rst ATG suggests it to be the most likely initiation codon. Human liver RGL thus has an open reading frame of 2307 bp, coding for a protein of 768 amino acids. In contrast, the brain cDNA has an alternative 5P end (GenBank accession AF186780), resulting from transcription initiation at an alternative ¢rst exon. This alternative transcript substitutes 44 amino
Fig. 1. Alignment of human and mouse RGL at the amino acid level. Amino acids that di¡er between human and mouse are shown in the mouse sequence, a dashed line represents the identical residues. Amino acid numbering is given on the left side. Arrows indicate the residues critical to Ras^RGL interaction within the Ras-interacting domain. The boxes denote domains identi¢ed using protein families database (pfam). From left to right: (1) RasGEFN domain (Pfam accession PF00618), (2) RasGEF domain (Pfam accession PF00617) and (3) RA domain (Pfam accession PF00788).
BBAEXP 91371 29-3-00
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acids for the ¢rst nine amino acids of the liver transcript resulting in a protein of 803 amino acids. Comparison of the coding sequences of human and mouse RGL genes shows 89% nucleotide and 94% amino acid identity. Both proteins contain three highly conserved domains identi¢ed by matches from protein families database (http://www.sanger.ac.uk/ Software/Pfam/index.shtml) (Fig. 1). Most of the divergence is seen in the inter-domain regions. Of two amino acids critical to the interaction of mouse RGL with Ras [11], Lys685 is conserved in human RGL while Glu689 becomes Asp, a conservative change also observed in other GDS family members. Overall, only three amino acids are di¡erent between Rasinteracting domains of the human and the mouse RGL proteins. Northern blot analysis showed expression of human RGL in all tissues examined (Fig. 2). A major band of V5 kb is seen in most tissues with a slightly larger band (V5.3 kb) being evident in brain. This is consistent with the di¡erent cDNA form isolated from brain cDNA library. Taking into account the poly(A) tails, these mRNA sizes are V0.2 kb larger than the largest composite cDNAs from liver and brain, 4.735 and 5.1 kb, respectively. This result
287
Fig. 2. Northern blot analysis of human RGL gene. Multipletissue Northern blots (Clontech) were hybridized with a 3.5-kb probe corresponding to bases 941^4367 of transcript A. Molecular weight standards are indicated on the left. The blots were washed at high stringency using 0.1% SDS and 0.1USSC at 65³C.
leaves open the possibilities that either there is one more exon upstream which is common to all transcripts or that we have essentially the complete mRNA and two very di¡erent transcription start sites. Our Northern data combined with RT-PCR ELISA screening of KIAA0959 [12] shows that human RGL is expressed in a wide variety of tissues, suggesting its general importance in the Ras signaling pathway. It is not yet clear whether the altered amino terminus of the brain protein alters the function of RGL in the brain or simply re£ects usage of a
Table 1 Exon^intron organization of the human RGL gene Exon
Exon size (bp)
Splice donor site
... Splice acceptor site
Accession No.
1A 1B1 1B2 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
s 205 s 367 164 111 209 78 185 125 216 104 85 90 87 33 122 90 187 255 115 s 615
CAAGCTAAAATGgtaacgagagct AACCTGAACTCGgtatcaaatcta AAGACAGAGGAGgtaagatgactc AGATGGCTAGGGgtgagtaaagct ACTGCTGGACAGgtaagaatgtaa GGTGATCAGGAAgtgagtctccct TGGAAACTGACAgtgagtacttgt TACATGGATGCAgtatgtcttctc AACATCGCTCATgtaattgtcttt TGCCGTCCCAAGgtaagttcccaa CTACTGATGAAGgtgaggctctta CAGAAGGACATGgtatgtctggcc GACTACATCGAGgtgagttccatg ND AGAGGAGGAGAGgtgggatcacct GAGACTCAGCCTgtgagtgtcccc CCTCAAAAAAAGgtatatactcaa AAGAGCATCATGgtgaggagcaag CGGAGGACAAAGgtgggcatcctt ND, contains stop codon
... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... p
AF186781 AF186782 AF192520 AF186783 AF186784 AF186785 AF186786 AF186787 AF186787 AF186788 AF186789 AF186790 AF186791 AF186792 AF186793 AF186794 AF186795 AF186796 AF186797 AF186798
cgtccctggcagAGCTCGATTCAG tcttccacacagATCCGTAATGCC cgtccctggcagAGCTCGATTCAG tttattatacagGTTGAAGGGGAC ttatctctacagGTATGGAAACCT tgtctttctcagTGCAATCGCTTC atatgtcaacagATGGGCTTCCCA cttttgttccagCAACTCTTCAAG ctccccatgcagGAATGTAGACTC cccttttcatagGGACCGAATGCT tctgtttctcagGAAGGAACCTCA gatgctcttcagGGTGTGATGCAG ND tgtctgctttagGAATTTGAAGTG tctctttctcagCTATGCCCTGTC gtttttttccagACTGTTTCTAGG ttactcttccagCTCTCTGAGTCC ttgtctttgcagTTGACGAGCCAG ND 3PUTR, might be the last exon
ND: not determined.
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R. Sood et al. / Biochimica et Biophysica Acta 1491 (2000) 285^288
brain speci¢c promoter that coincidentally uses a different initiation codon. Exon-traps from two overlapping BAC clones, b96P08 and b110O11 [10], de¢ned 10 exons with the 3P exons occurring only in b110O11, thus providing its transcription orientation with respect to the Bac contig. Primers derived from the cDNA were used to sequence the BAC DNA to identify 8 additional exons from the coding region of the liver cDNA. The alternative 5P end of the brain cDNA was mapped to two BAC clones centromeric to b96P08 and exons 1B1 and 1B2 were de¢ned by sequencing these clones. Table 1 lists the position of exons and adjacent intronic sequences. The intron located between exons 6 and 7 is 910 bp in size. The size of the other introns was not determined. All introns have splice donor and acceptor sites that conform to the general GT-AG consensus motif (Table 1). The putative translation initiation codon of the liver transcript (transcript A) is located in exon 1A and the stop codon is in exon 18. The initiation codon of the brain transcript (transcript B) is in exon 1B2 that lies 5P of exon 1A. The marker stsG30928, also mapped to the HPC1 candidate region, is found in the adjoining intron sequence of exon 1B1. Evaluation of RGL as a candidate gene for prostate cancer by SSCP analysis of cDNA from patients from families showing linkage to 1q25 region markers failed to identify any disease-associated mutations in the coding region of transcript A. Southern blot analysis also failed to detect any alterations in the gene. Because of the many biological activities of Ras, the identity of its e¡ectors and the elucidation of the various signaling pathways leading to cell growth and di¡erentiation have been subject of intense research over the past decade. Identi¢cation of the human RGL gene with alternative forms in di¡erent tissues, its expression analysis and genomic organization should prove useful in continuation of such research. References
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