- Email: [email protected]
DNA sequence flanking the protein coding regions of the rat Hprt gene
Mutation Research Genomics 382 Ž1998. 79–80
Short communication
DNA sequence flanking the protein coding regions of the rat Hprt gene Tao Chen a
a,b,)
, Roberta A. Mittelstaedt a , Robert H. Heflich
a
DiÕision of Genetic and ReproductiÕe Toxicology, HFT-120, National Center for Toxicological Research, 3900 NCTR Rd., Jefferson, AR 72079, USA b Interdisciplinary Toxicology Program, UniÕersity of Arkansas for Medical Sciences, Little Rock, AR 72205, USA Received 27 August 1997; accepted 6 October 1997
Keywords: Gene mutation; Chemical mutagenesis; Multiplex PCR; Exon; Intron; Promoter region
Hypoxanthine-guanine phosphoribosyltransferase Ž Hprt . is a constitutively expressed housekeeping gene that participates in the purine salvage pathway. Its properties have made it a widely used reporter locus for detecting mammalian somatic cell mutation w1x. As a continuation of our interest in characterizing 6-thioguanine-resistant rat lymphocyte mutants w2x, we have determined approximately 6 kb of DNA sequence of the rat Hprt gene, including the portions of introns 1, 2, 3, 4, 5 and 6 that immediately flank protein coding sequences, the complete sequence of introns 7 and 8, and sequences from the 5X- and 3X-untranslated regions of the gene ŽGenBank Accession nos. AF001278 – AF001282, AF009655AF009656, U06049, U31249, U48799.. The sequence was obtained from a rat genomic l clone containing exons 2 and 3 Žkindly provided by Dr. Barbara Hoebee, Bilthoven, The Netherlands., two rat genomic P1 clones obtained from Genome Sys-
) Corresponding author. Tel.: q1 870 5437399; Fax: q1 870 5437393; E-mail: [email protected]
tems ŽSt. Louis, MO. that contained the entire gene, and from polymerase chain reaction ŽPCR. products generated using a rat PromoterFinder e kit ŽClontech Laboratories, Palo Alto, CA.. The protein-coding regions of the sequence are identical to those previously reported for rat Hprt cDNA w3,4x, and the introns are positioned as in the mouse, hamster, and human genes w5–7x. The structures of the intronrexon boundaries are consistent with those of other mammalian genes and contain the ‘‘invariant’’ splice donor and acceptor site dinucleotides w8x. As is the case with other housekeeping genes w1x, the promoter region of the rat Hprt gene is GC-rich Ž68% of the 250 bp immediately upstream of the initiation codon is GC. and lacks TATA and CAAT boxes. The sequence of the rat promoter region is very similar to that of the mouse ŽFig. 1., but contains five, rather than three copies of a GGGCGG GC-box, which is the core of the consensus Sp1 transcriptional activator binding site. The hamster and human genes each contain four nonoverlapping copies of this sequence w6,7x. Upstream of the GC-boxes, the promoter region also contains a CCC sequence ŽFig. 1. in the same general location
1383-5734r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 2 6 Ž 9 7 . 0 0 0 1 4 - 9
80
T. Chen et al.r Mutation Research Genomics 382 (1998) 79–80
screens all nine exons of the rat Hprt gene for deletion mutation w12x.
References
Fig. 1. Comparison of the promoter regions of the Hprt genes from rat and mouse Žsequence from GenBank accession nos. AF001278 and K01507, respectively.. The major transcription start site for mouse w5x is indicated Žw.; the initiation codons Žlast three bases. are italicized. Numbering is from the first base of the initiation codon for the rat sequence only. Potential Sp1 transcriptional activator binding sites are shown in brackets. A potential AP2 binding site is underlined, and a potential unknown initiator binding site is double underlined.
and in the same sequence context ŽAAAATTCCCA. as a CCC site believed to function as an AP2 binding element in the human gene w9x. This sequence motif is not present in the mouse Hprt gene w10x ŽFig. 1.. Downstream of the GC-boxes, the promoter region possesses a GAGGCGGCG sequence ŽFig. 1. that has been identified by DNA footprinting of the human and mouse genes as a potential binding site for an unknown initiation factor w9,10x. There were no GGAACGG sequences in the 674 bp upstream of the initiation codon; GGAACGG is the core target sequence for a possible negative regulatory element in the human HPRT gene w11x. The 3X untranslated region of the rat Hprt gene ŽAF001282. has polyŽA. recognition sequences ŽAATAAA. 434 and 487 bp downstream of the TAA stop signal. Besides its value in interspecies comparisons of the Hprt gene, the sequence described in this report should be useful to investigators employing the rat Hprt gene for mutagenesis studies. We have designed PCR primers from this sequence for screening exons 2, 3 and 8 for point mutations by denaturing gradient gel electrophoresis w2x. The sequence has also been used in developing a multiplex PCR that
w1x J.T. Stout, C.T. Caskey, HPRT: gene structure, expression, and mutation, Annu. Rev. Genet. 19 Ž1985. 127–148. w2x R.H. Heflich, R.A. Mittelstaedt, M.G. Manjanatha, L.E. Lyn-Cook, A. Aidoo, DNA sequence analysis of hprt mutations in lymphocytes from Sprague-Dawley rats treated with 7,12-dimethylbenzwaxanthracene, Environ. Mol. Mutagen. 28 Ž1996. 5–12. w3x T.A. Chiaverotti, N. Battula, R.J. Monnat Jr., Rat hypoxanthine phosphoribosyltransferase cDNA cloning and sequence analysis, Adv. Exp. Med. Biol. 309B Ž1991. 117–120. w4x J.G. Jansen, H. Vrieling, A.A. van Zeeland, G.R. Mohn, The gene encoding hypoxanthine-guanine phosphoribosyltransferase as target for mutational analysis: PCR cloning and sequencing of the cDNA from the rat, Mutation Res. 266 Ž1992. 105–116. w5x D.W. Melton, D.S. Konecki, J. Brennand, C.T. Caskey, Structure, expression, and mutation of the hypoxanthine phosphoribosyltransferase gene, Proc. Natl. Acad. Sci. USA 81 Ž1984. 2147–2151. w6x B.J. Rossiter, J.C. Fuscoe, D.M. Muzny, M. Fox, C.T. Caskey, The Chinese hamster HPRT gene: restriction map, sequence analysis, and multiplex PCR deletion screen, Genomics 9 Ž1991. 247–256. w7x W. Ansorge, C.T. Caskey, H. Erfle, J. Zimmermann, C. Schwager, J. Stegemann, A. Civitello, P. Rice, H. Voss, A. Edwards, Automated DNA sequencing of the human HPRT locus, Genomics 6 Ž1990. 593–608. w8x P. Senapathy, M.B. Shapiro, N.L. Harris, Splice junctions, branch point sites, and exons: sequence statistics, identification, and applications to genome project, Methods Enzymol. 183 Ž1990. 252–278. w9x I.K. Hornstra, T.P. Yang, Multiple in vivo footprints are specific to the active allele of the X-linked human hypoxanX thine phosphoribosyltransferase gene 5 region: implications for X chromosome inactivation, Mol. Cell. Biol. 12 Ž1992. 5345–5354. w10x M.D. Litt, I.K. Hornstra, T.P. Yang, In vivo footprinting and high-resolution methylation analysis of the mouse hypoxanX thine phosphoribosyltransferase gene 5 region on the active and inactive X chromosomes, Mol. Cell. Biol. 16 Ž1996. 6190–6199. w11x D.E. Rincon-Limas, F. Amaya-Manzanares, M.L. Nino´ ˜ Rosales, Y. Yu, T.P. Yang, P.I. Patel, Ubiquitous and neuronal DNA-binding proteins interact with a negative regulatory element of the human hypoxanthine phosphoribosyltransferase gene, Mol. Cell. Biol. 15 Ž1995. 6561–6571. w12x T. Chen, R.A. Mittelstaedt, R.H. Heflich, Multiplex PCR for the analysis of deletion mutation in the rat lymphocyte hprt assay, Environ. Mol. Mutagen. 29 ŽSuppl. 28. Ž1997. 9.
Short communication
DNA sequence flanking the protein coding regions of the rat Hprt gene Tao Chen a
a,b,)
, Roberta A. Mittelstaedt a , Robert H. Heflich
a
DiÕision of Genetic and ReproductiÕe Toxicology, HFT-120, National Center for Toxicological Research, 3900 NCTR Rd., Jefferson, AR 72079, USA b Interdisciplinary Toxicology Program, UniÕersity of Arkansas for Medical Sciences, Little Rock, AR 72205, USA Received 27 August 1997; accepted 6 October 1997
Keywords: Gene mutation; Chemical mutagenesis; Multiplex PCR; Exon; Intron; Promoter region
Hypoxanthine-guanine phosphoribosyltransferase Ž Hprt . is a constitutively expressed housekeeping gene that participates in the purine salvage pathway. Its properties have made it a widely used reporter locus for detecting mammalian somatic cell mutation w1x. As a continuation of our interest in characterizing 6-thioguanine-resistant rat lymphocyte mutants w2x, we have determined approximately 6 kb of DNA sequence of the rat Hprt gene, including the portions of introns 1, 2, 3, 4, 5 and 6 that immediately flank protein coding sequences, the complete sequence of introns 7 and 8, and sequences from the 5X- and 3X-untranslated regions of the gene ŽGenBank Accession nos. AF001278 – AF001282, AF009655AF009656, U06049, U31249, U48799.. The sequence was obtained from a rat genomic l clone containing exons 2 and 3 Žkindly provided by Dr. Barbara Hoebee, Bilthoven, The Netherlands., two rat genomic P1 clones obtained from Genome Sys-
) Corresponding author. Tel.: q1 870 5437399; Fax: q1 870 5437393; E-mail: [email protected]
tems ŽSt. Louis, MO. that contained the entire gene, and from polymerase chain reaction ŽPCR. products generated using a rat PromoterFinder e kit ŽClontech Laboratories, Palo Alto, CA.. The protein-coding regions of the sequence are identical to those previously reported for rat Hprt cDNA w3,4x, and the introns are positioned as in the mouse, hamster, and human genes w5–7x. The structures of the intronrexon boundaries are consistent with those of other mammalian genes and contain the ‘‘invariant’’ splice donor and acceptor site dinucleotides w8x. As is the case with other housekeeping genes w1x, the promoter region of the rat Hprt gene is GC-rich Ž68% of the 250 bp immediately upstream of the initiation codon is GC. and lacks TATA and CAAT boxes. The sequence of the rat promoter region is very similar to that of the mouse ŽFig. 1., but contains five, rather than three copies of a GGGCGG GC-box, which is the core of the consensus Sp1 transcriptional activator binding site. The hamster and human genes each contain four nonoverlapping copies of this sequence w6,7x. Upstream of the GC-boxes, the promoter region also contains a CCC sequence ŽFig. 1. in the same general location
1383-5734r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 2 6 Ž 9 7 . 0 0 0 1 4 - 9
80
T. Chen et al.r Mutation Research Genomics 382 (1998) 79–80
screens all nine exons of the rat Hprt gene for deletion mutation w12x.
References
Fig. 1. Comparison of the promoter regions of the Hprt genes from rat and mouse Žsequence from GenBank accession nos. AF001278 and K01507, respectively.. The major transcription start site for mouse w5x is indicated Žw.; the initiation codons Žlast three bases. are italicized. Numbering is from the first base of the initiation codon for the rat sequence only. Potential Sp1 transcriptional activator binding sites are shown in brackets. A potential AP2 binding site is underlined, and a potential unknown initiator binding site is double underlined.
and in the same sequence context ŽAAAATTCCCA. as a CCC site believed to function as an AP2 binding element in the human gene w9x. This sequence motif is not present in the mouse Hprt gene w10x ŽFig. 1.. Downstream of the GC-boxes, the promoter region possesses a GAGGCGGCG sequence ŽFig. 1. that has been identified by DNA footprinting of the human and mouse genes as a potential binding site for an unknown initiation factor w9,10x. There were no GGAACGG sequences in the 674 bp upstream of the initiation codon; GGAACGG is the core target sequence for a possible negative regulatory element in the human HPRT gene w11x. The 3X untranslated region of the rat Hprt gene ŽAF001282. has polyŽA. recognition sequences ŽAATAAA. 434 and 487 bp downstream of the TAA stop signal. Besides its value in interspecies comparisons of the Hprt gene, the sequence described in this report should be useful to investigators employing the rat Hprt gene for mutagenesis studies. We have designed PCR primers from this sequence for screening exons 2, 3 and 8 for point mutations by denaturing gradient gel electrophoresis w2x. The sequence has also been used in developing a multiplex PCR that
w1x J.T. Stout, C.T. Caskey, HPRT: gene structure, expression, and mutation, Annu. Rev. Genet. 19 Ž1985. 127–148. w2x R.H. Heflich, R.A. Mittelstaedt, M.G. Manjanatha, L.E. Lyn-Cook, A. Aidoo, DNA sequence analysis of hprt mutations in lymphocytes from Sprague-Dawley rats treated with 7,12-dimethylbenzwaxanthracene, Environ. Mol. Mutagen. 28 Ž1996. 5–12. w3x T.A. Chiaverotti, N. Battula, R.J. Monnat Jr., Rat hypoxanthine phosphoribosyltransferase cDNA cloning and sequence analysis, Adv. Exp. Med. Biol. 309B Ž1991. 117–120. w4x J.G. Jansen, H. Vrieling, A.A. van Zeeland, G.R. Mohn, The gene encoding hypoxanthine-guanine phosphoribosyltransferase as target for mutational analysis: PCR cloning and sequencing of the cDNA from the rat, Mutation Res. 266 Ž1992. 105–116. w5x D.W. Melton, D.S. Konecki, J. Brennand, C.T. Caskey, Structure, expression, and mutation of the hypoxanthine phosphoribosyltransferase gene, Proc. Natl. Acad. Sci. USA 81 Ž1984. 2147–2151. w6x B.J. Rossiter, J.C. Fuscoe, D.M. Muzny, M. Fox, C.T. Caskey, The Chinese hamster HPRT gene: restriction map, sequence analysis, and multiplex PCR deletion screen, Genomics 9 Ž1991. 247–256. w7x W. Ansorge, C.T. Caskey, H. Erfle, J. Zimmermann, C. Schwager, J. Stegemann, A. Civitello, P. Rice, H. Voss, A. Edwards, Automated DNA sequencing of the human HPRT locus, Genomics 6 Ž1990. 593–608. w8x P. Senapathy, M.B. Shapiro, N.L. Harris, Splice junctions, branch point sites, and exons: sequence statistics, identification, and applications to genome project, Methods Enzymol. 183 Ž1990. 252–278. w9x I.K. Hornstra, T.P. Yang, Multiple in vivo footprints are specific to the active allele of the X-linked human hypoxanX thine phosphoribosyltransferase gene 5 region: implications for X chromosome inactivation, Mol. Cell. Biol. 12 Ž1992. 5345–5354. w10x M.D. Litt, I.K. Hornstra, T.P. Yang, In vivo footprinting and high-resolution methylation analysis of the mouse hypoxanX thine phosphoribosyltransferase gene 5 region on the active and inactive X chromosomes, Mol. Cell. Biol. 16 Ž1996. 6190–6199. w11x D.E. Rincon-Limas, F. Amaya-Manzanares, M.L. Nino´ ˜ Rosales, Y. Yu, T.P. Yang, P.I. Patel, Ubiquitous and neuronal DNA-binding proteins interact with a negative regulatory element of the human hypoxanthine phosphoribosyltransferase gene, Mol. Cell. Biol. 15 Ž1995. 6561–6571. w12x T. Chen, R.A. Mittelstaedt, R.H. Heflich, Multiplex PCR for the analysis of deletion mutation in the rat lymphocyte hprt assay, Environ. Mol. Mutagen. 29 ŽSuppl. 28. Ž1997. 9.