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Effect of mutations in the upstream promoter on the transcription of human 5S rRNA genes
Biochimica et Biophysica Acta 91565 (2001) 169^173
www.bba-direct.com
Promoter paper
E¡ect of mutations in the upstream promoter on the transcription of human 5S rRNA genes Charlotte Hallenberg 1 , Sune Frederiksen * Department of Medical Biochemistry and Genetics, Biochemistry Laboratory B, Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark Received 23 January 2001; received in revised form 5 June 2001; accepted 11 June 2001
Abstract The human 5S rRNA gene has a 12-mer external promoter, the D box, localized about 30 bp upstream the coding sequence. By site directed mutagenesis 58 different D box promoter mutants were made. While some mutations in the D box allowed full transcription, other mutations decreased the transcriptional activity to 20^50% compared to the bona fide gene, showing the importance of this external promoter in transcription initiation. A number of maxi 5S rRNA genes were constructed from bona fide genes and D box mutated clones. Transfection of HeLa cells with maxi 5S rRNA genes showed that the D box is also important for 5S rRNA gene expression in vivo. Evidence from different eukaryotic cells suggests that expression of 5S rRNA genes is regulated by external promoters in addition to the internal control region. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : 5S rDNA ; 5S rRNA gene; Promoter; RNA polymerase III; Transcription
1. Introduction RNA polymerase III transcribes a number of small RNA encoding genes. Based on di¡erent promoters these genes have been divided into three types. Type I includes only the 5S rRNA genes and the internal promoter consists of an A box, a C box and an intermediate element. Type II includes tRNA, adenovirus VA RNA and others and the internal promoter consists of an A box and a B box. Type III includes U6, 7KS and others and they have no internal promoter but a promoter in the 5P-£anking region (reviews [1,2]). The isolation of 5S rRNA genes from hamster [3], human [4,5], mouse [6], rat [7], and Macaca fascicularis cells [8] has revealed a conserved 12mer sequence in the 5P-£anking region in a position around 330. This 12-mer sequence was designated the D box. The D box is located in positions 332 to 321 in human and in the crab eating macaque, in positions 333 to 322 in mouse and rat, and in positions 336 to 325 in hamster. The importance of the D box for transcription of * Corresponding author. Fax: +45-35-32-77-32. E-mail address : [email protected] (S. Frederiksen). 1 Present address: Department of Forensic Genetics, Institute of Forensic Medicine, University of Copenhagen, Frederik V's Vej 11, DK2100 Copenhagen Ò, Denmark.
mammalian 5S rRNA genes in vitro has been demonstrated by deletion mutants [5,6] and from a genomic rat 5S rDNA clone missing most of the D box [7]. The present paper describes further studies on the importance of the D box for the transcriptional activity of the 5S rRNA genes in vitro and in vivo. The sequence in the D box was changed by site directed mutagenesis. In order to separate the product transcribed from the mutated clones from the cellular 5S rRNA synthesized during the in vivo experiments a number of di¡erent maxi 5S rRNA genes were constructed. 2. D box mutants The D box is located 21^32 bp upstream the coding sequence in the human gene [5]. Point mutations were introduced into the D box by the use of the Altered Sites II in vitro mutagenesis system (Promega). A cloned 5S rDNA fragment (pHU5S3.1) containing the human 5S rRNA gene, the 273 bp upstream sequence and the 237 bp downstream sequence [5] was cloned into the pAlter1 vector and used as template. Primers used for mutagenesis of the D box (shown in bold) were: Mut1: 3P-CGCCCCGACCCGNNANCCCCGTCGGTCCG-5P, Mut2: 3P-CCCGACCCGAGAANCNNGTCGGTCCGCGGA-5P, and
0167-4781 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 1 ) 0 0 2 6 4 - 0
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rRNA synthesized during growth. For that purpose a number of maxi 5S rRNA genes containing a 10 bp insert were constructed. Since the 5S rRNA genes contain an internal promoter as well as a transcription termination signal which overlaps the coding sequence it was necessary to construct an insert that did not in£uence these regulatory mechanisms. A number of 5S rDNA clones and D box mutated clones were selected based on the transcription e¤ciency in vitro. A 10 bp insert was introduced immediately upstream from the termination site of the 5S rRNA gene by use of PCR and tailed primers as de-
Fig. 1. E¡ect of single mutations in the D box on the transcriptional activity in vitro. Site directed mutagenesis was performed with the 640 bp human 5S rDNA pHU5S3.1 and transcription was assayed in a HeLa S-100 extract [5]. The incorporation of 32 P-UTP into RNA was measured with a PhosphorImager. Each D box mutated clone was tested in two or three separate experiments and the transcription calculated in percent of the control gene.
Mut3 : 3P-GCCCGCCCCGACCNNAGNACCCCGTCGGTCC-5P. Mutated clones were identi¢ed after sequencing with the Ampli Cycle sequencing kit from Applied Biosystems and 33 P-labeled primers. A total of 58 clones containing one, two or three mutations in the D box were made by site directed mutagenesis. Each D box mutated clone was tested in two or three separate experiments and the transcription calculated in percent of the control gene. The e¡ect of single mutations on the transcriptional e¤ciency is shown in Fig. 1 and the e¡ect of two or three mutations in Fig. 2. Replacing one of the three Ts with A has no or only little e¡ect on the transcriptional activity (80^100% of the control). Replacing a T with a C or G dramatically reduces the e¡ect of the D box on the transcription (20^50% of the control) and replacing a G or a C with an A or a T had none or a moderate e¡ect (80^100% of the control). Two or three substitutions were not necessarily as detrimental as one. 3. Transcriptional e¤ciency of maxi genes In order to study the in vivo transcriptional e¤ciency of the promoter mutants, the product transcribed from the mutated clones must be distinguished from the cellular 5S
Fig. 2. E¡ect of two or three mutations in the D box on the transcriptional activity in vitro. Assay conditions as described in Fig. 1. * indicates a deletion.
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Fig. 3. The transcriptional activity of di¡erent 5S rRNA maxi genes in vitro (¢rst row) and in vivo (second row). The transcription was measured in a HeLa S-100 extract [5] and after transient transfection in HeLa cells. The arrows indicate the coding region including the 10 bp insert between bp 117 and 118. The D box is shown as a box. Mut maxi gene clones are human clones with the mutations in the D box shown under the box. The results are averages of two or three separate experiments and calculated as percent of the transcription of the control genes. The pHU5S maxi gene clones were derived from human 5S rDNA [5] (EMBL accession No. X71793-71803). The maxi genes were transcribed with the same e¤ciency as the parent clones without the 10 bp insert.
scribed by Stemmer and Morris [9]. The 46-mer primers (primers 1 and 2) used to amplify the gene and the plasmid were : primer 1: 3P-GCGGACCCTTATGGCCCACGACATCCG CATGATCACAGAAGCTGGT-5P, primer 2: 5P-TGGACGAAGACTAACTAGTG CTTTTTCTTTGGCTTTTTGCTGTTTC-3P. The tails are shown in italics and the BbsI sites are underlined. Primer 1 anneals to the transcribed region of the 5S rRNA gene whereas primer 2 anneals to the termination site and downstream region of the human gene. By digestion of the PCR product with BbsI followed by ligation, clones containing the 10 bp insert were obtained. The insert contains a site for the restriction endonuclease SpeI and the presence of this site was used to select clones containing the correct insert. Finally the maxi 5S rRNA genes were sequenced by cycle sequencing. The 10-mer sequence CGTACTAGTG was inserted between nucleotides 117 and 118 in the 3P-end of the coding sequence. The di¡erent maxi 5S rRNA gene clones constructed are shown in Fig. 3. The transcriptional activities of the di¡erent maxi genes were investigated in a HeLa cell S-100 extract as described previously [5] and the maxi gene products had the expected size when analyzed on polyacrylamide gels. The transcriptional e¤ciency in vitro of the di¡erent maxi genes is shown in Fig. 3 (¢rst row) and this e¤ciency was in all cases the same as that observed for the parent 5S rRNA gene without the 10 bp insert. 4. Gene expression in vivo HeLa cells were transfected with di¡erent maxi 5S rRNA genes and co-transfected with pSV2CAT in order to monitor the transfection e¤ciency. The cells were maintained in RPMI 1640 medium but transferred into DMEM medium 2^4 h before transfection [10] by the calcium phosphate method [11]. HeLa cells in T75 Falcon £asks
were transfected with equimolar amounts of the di¡erent maxi 5S rRNA genes (6 pmoles, 12^15 Wg plasmid DNA). Cells were in the logarithmic growth phase (1.1^3.2U106 cells per £ask) and total RNA synthesis in the di¡erent £asks was compared by the incorporation of 5-3 H-uridine. The transfection e¤ciency was monitored by co-transfection with 15 Wg pSV2CAT. The transfection was measured from the amount of CAT mRNA by means of RT-PCR in the same assay as the maxi gene 5S rRNA product was determined. Primer sequences: CAT-142: CTGCCGACATGGAAG, CAT-248: TGGACAACTTCTTCG [12]. The results obtained with RT-PCR were veri¢ed by measurement of the chloramphenicol acetyltransferase activity [13]. RNA was extracted with 0.01 M potassium phosphate bu¡er (pH 7.2) and phenol at 0³C and precipitated with 1/ 10 vol. saline (0.14 M NaCl, 1 mM MgCl2 , in 0.05 M NaAc pH 5.1) and 2.5 vols. 96% ethanol. The pellet that contained no or very little nuclear DNA [14] was dissolved in PCR bu¡er (20 mM Tris^HCl (pH 8.4), 50 mM KCl) with 10 mM MgCl2 and digested with 75 units DNase I (FPLC-pure, Pharmacia) at 37³C for 30 min to get rid of plasmid DNA. The reaction mixture was extracted with phenol and RNA precipitated with ethanol. The puri¢ed RNA (80^200 Wg) was dissolved in water and the absence of plasmid DNA was con¢rmed by PCR using 5S rRNA maxi gene speci¢c primers (Vivo-1 and Vivo-2) before the RNA preparation was used for RT-PCR. The amount of maxi gene products was measured at ¢ve di¡erent concentrations of total RNA in the range of 0.2^ 1.8 ng per RT-PCR reaction mixture. The RT step was performed in a 20 Wl reaction mixture. The mixture contained 17 mM Tris^HCl (pH 8.4), 42.5 mM KCl, 4.25 mM MgCl2 , 0.1 mM dNTP, 15 pmoles 32 P-5P-end labeled Vivo2 primer, 15 pmoles 32 P-5P-end labeled CAT-248 primer, 0.2^1.8 ng RNA and 8.8 units MULV reverse transcriptase. The reaction was carried out as follows : 37³C for
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30 min, 90³C for 5 min, 4³C. The reaction mixtures were diluted to 50 Wl with PCR bu¡er (20 mM Tris^HCl (pH 8.4), 50 mM KCl) and added 15 pmoles Vivo-1 primer, 15 pmoles CAT-142 primer and 1.7 units Taq DNA polymerase. The PCR reaction was carried out with the following program: 95³C for 15 s, 51³C for 30 s, 72³C for 30 s, (25 cycles), 72³C for 5 min, 4³C. Primer sequences: Vivo-1: GTCTACGGCCATACC, Vivo-2: AAAGCACTAGTACGCC. The transcription of the pHU5S3.2 maxi gene in transfected HeLa cells was used as control in all assays and set to 100%. This clone is transcribed with the same e¤ciency in vitro as two other clones, pHU5S3.0 maxi and pHU5S3.1 maxi with longer 5P-£anking sequences (Fig. 3, ¢rst row). In transfection experiments neither of these clones with longer 5P-£anking regions showed improved transcriptional e¤ciency. The results of the in vivo transcription assays are shown in Fig. 3 (second row). The results are obtained as averages from two or three separate transfection experiments. The pHU5S3.1 Acc maxi clone without 5P-£anking sequence was transcribed with an e¤ciency of 18% that is comparable to the 0^10% observed in vitro. The Mut 23 maxi clone contained one mutation in the D box (T to A substitution) that did not in£uence transcription in vitro. In the transfection experiments with the Mut 23 maxi clone the transcription was measured to 106%. The Mut 1 clone (T to G substitution) was transcribed with 59% e¤ciency and the Mut 26 maxi clone (T to C substitution) with 50% e¤ciency. This is comparable to the e¤ciency observed in vitro which was 40% and 25%, respectively. Changes in the composition of the D box had a similar e¡ect on transcription in vitro and in vivo showing the importance of the external promoter on the regulation of 5S rRNA gene expression.
to be the case in the protist Acanthamoeba castellanii [15], the fungus Neurospora crassa [16,17], the yeast Saccharomyces cerevisiae [18], the fruit £y Drosophila melanogaster [19], the silkworm Bombyx mori [20,21], the loach Misgurnus fossilis [22], the nematode Caenorhabditis elegans [23] and the toad Xenopus laevis [24]. This evidence together with the evidence presented in the present paper suggests that the de¢nition of type I genes transcribed by RNA polymerase III ought to be revised. The upstream promoter sequences in lower eukaryotes are more AT-rich than the GC-rich D box found conserved in higher eukaryotes [3^8]. Many substitutions of nucleotides in the D box with As or Ts still allow full or almost full transcriptional activity as demonstrated in the present paper (Figs. 1 and 2). The evolutionary history of the D box may therefore have run in parallel with that of the coding sequence of the 5S rRNA genes. Acknowledgements We thank Rita Jensen and Irene JÖrgensen for excellent technical assistance. The work was supported by the Novo î ge Haensch FoundaNordisk Foundation, Gerda and A tion and the Danish Natural Science Research Council.
References [1] [2] [3] [4] [5] [6] [7]
5. External promoters in 5S rRNA genes
[8]
A conserved 12-mer sequence is found in the upstream region of di¡erent mammalian 5S rRNA genes [3^8]. Deletion mutants made from 5S rDNA isolated from human cells [5] and mouse cells [6] suggested that the 12-mer sequence designated the D box had an e¡ect on the transcription of 5S rRNA genes in vitro. A more detailed study of the 12-mer sequence was undertaken in the present paper. The results show that point mutations affect the transcription both in vitro and in vivo and thereby demonstrate the importance of the D box for transcription of the 5S rRNA genes. In the original de¢nition of type I genes transcribed by RNA polymerase III, gene expression was regulated only by internal promoters (reviews [1,2]). The in£uence of upstream sequences on the transcription of 5S rRNA genes has, however, been demonstrated for a number of eukaryotic genes. By means of 5P deletion mutants this was found
[9] [10]
[11] [12] [13]
[14] [15] [16] [17] [18] [19]
I.M. Willis, Eur. J. Biochem. 212 (1993) 1^11. M.R. Paule, R.J. White, Nucleic Acids Res. 6 (2000) 1283^1298. R.P. Hart, W.R. Folk, J. Biol. Chem. 257 (1982) 11706^11711. R.D. Little, D.C. Braaten, Genomics 4 (1989) 376^383. J. Nederby Nielsen, C. Hallenberg, S. Frederiksen, P.D. SÖrensen, B. Lomholt, Nucleic Acids Res. 21 (1993) 3631^3636. C. Hallenberg, J. Nederby Nielsen, S. Frederiksen, Gene 142 (1994) 291^295. S. Frederiksen, H. Cao, B. Lomholt, G. Levan, C. Hallenberg, Cytogenet. Cell Genet. 76 (1997) 101^106. L. Ri¡-Jensen, S. Frederiksen, Biochim. Biophys. Acta 1492 (2000) 537^542. W.P.C. Stemmer, S.K. Morris, BioTechniques 13 (1992) 215^220. K. Docherty, A.R. Clark, in: Hanes, Higgens (Eds.), Gene Transcription. A Practical Approach, IRL Press^Oxford University Press, Oxford, 1993, pp. 64^77. F. Graham, A. van der Eb, Virology 52 (1973) 456^457. C. Gorman, in: D.M. Glover (Ed.), DNA cloning ^ A Practical Approach, Vol. II, IRL Press, Oxford, 1986, pp. 143^190. J.D. Watson, M. Gilman, J. Witkowsky, M. Zoller, Recombinant DNA, 2nd edn., Scienti¢c American Books, W.H. Freeman and Co., New York, 1992, p. 155. P. Hellung-Larsen, S. Frederiksen, Biochim. Biophys. Acta 262 (1972) 290^307. M.G. Zwick, M.A. Imboden, M.R. Paule, Nucleic Acids Res. 19 (1991) 1681^1686. E.U. Selker, E. Morycka-Wroblewska, N.J. Stevens, R.L. Metzenberg, Mol. Gen. Genet. 205 (1986) 189^192. B.M. Tyler, J. Mol. Biol. 196 (1987) 801^811. Y. Lee, W.M. Wong, D. Guyer, A.M. Erkine, R.N.J. Nazar, Mol. Biol. 269 (1997) 676^683. S.J. Sharp, A.D. Garcia, Mol. Cell. Biol. 8 (1988) 1266^1274.
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C. Hallenberg, S. Frederiksen / Biochimica et Biophysica Acta 91565 (2001) 169^173 [20] D.G. Morton, K.U. Sprague, Proc. Natl. Acad. Sci. USA 81 (1984) 5519^5522. [21] T.P.L. Smith, L.S. Young, L.B. Bender, K.U. Sprague, Nucleic Acids Res. 23 (1995) 1244^1251. [22] P. Felgenhauer, J. Sedman, N. Shostak, M. Timofeeva, A. Lind, A. Bayev, Gene 90 (1990) 243^248.
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[23] D.W. Nelson, R.M. Linning, P.J. Davison, B.M. Honda, Gene 218 (1998) 9^16. [24] S.-L. Oei, T. Pieler, J. Biol. Chem. 265 (1990) 7485^7491.
BBAEXP 91565 3-8-01
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Promoter paper
E¡ect of mutations in the upstream promoter on the transcription of human 5S rRNA genes Charlotte Hallenberg 1 , Sune Frederiksen * Department of Medical Biochemistry and Genetics, Biochemistry Laboratory B, Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark Received 23 January 2001; received in revised form 5 June 2001; accepted 11 June 2001
Abstract The human 5S rRNA gene has a 12-mer external promoter, the D box, localized about 30 bp upstream the coding sequence. By site directed mutagenesis 58 different D box promoter mutants were made. While some mutations in the D box allowed full transcription, other mutations decreased the transcriptional activity to 20^50% compared to the bona fide gene, showing the importance of this external promoter in transcription initiation. A number of maxi 5S rRNA genes were constructed from bona fide genes and D box mutated clones. Transfection of HeLa cells with maxi 5S rRNA genes showed that the D box is also important for 5S rRNA gene expression in vivo. Evidence from different eukaryotic cells suggests that expression of 5S rRNA genes is regulated by external promoters in addition to the internal control region. ß 2001 Elsevier Science B.V. All rights reserved. Keywords : 5S rDNA ; 5S rRNA gene; Promoter; RNA polymerase III; Transcription
1. Introduction RNA polymerase III transcribes a number of small RNA encoding genes. Based on di¡erent promoters these genes have been divided into three types. Type I includes only the 5S rRNA genes and the internal promoter consists of an A box, a C box and an intermediate element. Type II includes tRNA, adenovirus VA RNA and others and the internal promoter consists of an A box and a B box. Type III includes U6, 7KS and others and they have no internal promoter but a promoter in the 5P-£anking region (reviews [1,2]). The isolation of 5S rRNA genes from hamster [3], human [4,5], mouse [6], rat [7], and Macaca fascicularis cells [8] has revealed a conserved 12mer sequence in the 5P-£anking region in a position around 330. This 12-mer sequence was designated the D box. The D box is located in positions 332 to 321 in human and in the crab eating macaque, in positions 333 to 322 in mouse and rat, and in positions 336 to 325 in hamster. The importance of the D box for transcription of * Corresponding author. Fax: +45-35-32-77-32. E-mail address : [email protected] (S. Frederiksen). 1 Present address: Department of Forensic Genetics, Institute of Forensic Medicine, University of Copenhagen, Frederik V's Vej 11, DK2100 Copenhagen Ò, Denmark.
mammalian 5S rRNA genes in vitro has been demonstrated by deletion mutants [5,6] and from a genomic rat 5S rDNA clone missing most of the D box [7]. The present paper describes further studies on the importance of the D box for the transcriptional activity of the 5S rRNA genes in vitro and in vivo. The sequence in the D box was changed by site directed mutagenesis. In order to separate the product transcribed from the mutated clones from the cellular 5S rRNA synthesized during the in vivo experiments a number of di¡erent maxi 5S rRNA genes were constructed. 2. D box mutants The D box is located 21^32 bp upstream the coding sequence in the human gene [5]. Point mutations were introduced into the D box by the use of the Altered Sites II in vitro mutagenesis system (Promega). A cloned 5S rDNA fragment (pHU5S3.1) containing the human 5S rRNA gene, the 273 bp upstream sequence and the 237 bp downstream sequence [5] was cloned into the pAlter1 vector and used as template. Primers used for mutagenesis of the D box (shown in bold) were: Mut1: 3P-CGCCCCGACCCGNNANCCCCGTCGGTCCG-5P, Mut2: 3P-CCCGACCCGAGAANCNNGTCGGTCCGCGGA-5P, and
0167-4781 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 0 1 ) 0 0 2 6 4 - 0
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rRNA synthesized during growth. For that purpose a number of maxi 5S rRNA genes containing a 10 bp insert were constructed. Since the 5S rRNA genes contain an internal promoter as well as a transcription termination signal which overlaps the coding sequence it was necessary to construct an insert that did not in£uence these regulatory mechanisms. A number of 5S rDNA clones and D box mutated clones were selected based on the transcription e¤ciency in vitro. A 10 bp insert was introduced immediately upstream from the termination site of the 5S rRNA gene by use of PCR and tailed primers as de-
Fig. 1. E¡ect of single mutations in the D box on the transcriptional activity in vitro. Site directed mutagenesis was performed with the 640 bp human 5S rDNA pHU5S3.1 and transcription was assayed in a HeLa S-100 extract [5]. The incorporation of 32 P-UTP into RNA was measured with a PhosphorImager. Each D box mutated clone was tested in two or three separate experiments and the transcription calculated in percent of the control gene.
Mut3 : 3P-GCCCGCCCCGACCNNAGNACCCCGTCGGTCC-5P. Mutated clones were identi¢ed after sequencing with the Ampli Cycle sequencing kit from Applied Biosystems and 33 P-labeled primers. A total of 58 clones containing one, two or three mutations in the D box were made by site directed mutagenesis. Each D box mutated clone was tested in two or three separate experiments and the transcription calculated in percent of the control gene. The e¡ect of single mutations on the transcriptional e¤ciency is shown in Fig. 1 and the e¡ect of two or three mutations in Fig. 2. Replacing one of the three Ts with A has no or only little e¡ect on the transcriptional activity (80^100% of the control). Replacing a T with a C or G dramatically reduces the e¡ect of the D box on the transcription (20^50% of the control) and replacing a G or a C with an A or a T had none or a moderate e¡ect (80^100% of the control). Two or three substitutions were not necessarily as detrimental as one. 3. Transcriptional e¤ciency of maxi genes In order to study the in vivo transcriptional e¤ciency of the promoter mutants, the product transcribed from the mutated clones must be distinguished from the cellular 5S
Fig. 2. E¡ect of two or three mutations in the D box on the transcriptional activity in vitro. Assay conditions as described in Fig. 1. * indicates a deletion.
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Fig. 3. The transcriptional activity of di¡erent 5S rRNA maxi genes in vitro (¢rst row) and in vivo (second row). The transcription was measured in a HeLa S-100 extract [5] and after transient transfection in HeLa cells. The arrows indicate the coding region including the 10 bp insert between bp 117 and 118. The D box is shown as a box. Mut maxi gene clones are human clones with the mutations in the D box shown under the box. The results are averages of two or three separate experiments and calculated as percent of the transcription of the control genes. The pHU5S maxi gene clones were derived from human 5S rDNA [5] (EMBL accession No. X71793-71803). The maxi genes were transcribed with the same e¤ciency as the parent clones without the 10 bp insert.
scribed by Stemmer and Morris [9]. The 46-mer primers (primers 1 and 2) used to amplify the gene and the plasmid were : primer 1: 3P-GCGGACCCTTATGGCCCACGACATCCG CATGATCACAGAAGCTGGT-5P, primer 2: 5P-TGGACGAAGACTAACTAGTG CTTTTTCTTTGGCTTTTTGCTGTTTC-3P. The tails are shown in italics and the BbsI sites are underlined. Primer 1 anneals to the transcribed region of the 5S rRNA gene whereas primer 2 anneals to the termination site and downstream region of the human gene. By digestion of the PCR product with BbsI followed by ligation, clones containing the 10 bp insert were obtained. The insert contains a site for the restriction endonuclease SpeI and the presence of this site was used to select clones containing the correct insert. Finally the maxi 5S rRNA genes were sequenced by cycle sequencing. The 10-mer sequence CGTACTAGTG was inserted between nucleotides 117 and 118 in the 3P-end of the coding sequence. The di¡erent maxi 5S rRNA gene clones constructed are shown in Fig. 3. The transcriptional activities of the di¡erent maxi genes were investigated in a HeLa cell S-100 extract as described previously [5] and the maxi gene products had the expected size when analyzed on polyacrylamide gels. The transcriptional e¤ciency in vitro of the di¡erent maxi genes is shown in Fig. 3 (¢rst row) and this e¤ciency was in all cases the same as that observed for the parent 5S rRNA gene without the 10 bp insert. 4. Gene expression in vivo HeLa cells were transfected with di¡erent maxi 5S rRNA genes and co-transfected with pSV2CAT in order to monitor the transfection e¤ciency. The cells were maintained in RPMI 1640 medium but transferred into DMEM medium 2^4 h before transfection [10] by the calcium phosphate method [11]. HeLa cells in T75 Falcon £asks
were transfected with equimolar amounts of the di¡erent maxi 5S rRNA genes (6 pmoles, 12^15 Wg plasmid DNA). Cells were in the logarithmic growth phase (1.1^3.2U106 cells per £ask) and total RNA synthesis in the di¡erent £asks was compared by the incorporation of 5-3 H-uridine. The transfection e¤ciency was monitored by co-transfection with 15 Wg pSV2CAT. The transfection was measured from the amount of CAT mRNA by means of RT-PCR in the same assay as the maxi gene 5S rRNA product was determined. Primer sequences: CAT-142: CTGCCGACATGGAAG, CAT-248: TGGACAACTTCTTCG [12]. The results obtained with RT-PCR were veri¢ed by measurement of the chloramphenicol acetyltransferase activity [13]. RNA was extracted with 0.01 M potassium phosphate bu¡er (pH 7.2) and phenol at 0³C and precipitated with 1/ 10 vol. saline (0.14 M NaCl, 1 mM MgCl2 , in 0.05 M NaAc pH 5.1) and 2.5 vols. 96% ethanol. The pellet that contained no or very little nuclear DNA [14] was dissolved in PCR bu¡er (20 mM Tris^HCl (pH 8.4), 50 mM KCl) with 10 mM MgCl2 and digested with 75 units DNase I (FPLC-pure, Pharmacia) at 37³C for 30 min to get rid of plasmid DNA. The reaction mixture was extracted with phenol and RNA precipitated with ethanol. The puri¢ed RNA (80^200 Wg) was dissolved in water and the absence of plasmid DNA was con¢rmed by PCR using 5S rRNA maxi gene speci¢c primers (Vivo-1 and Vivo-2) before the RNA preparation was used for RT-PCR. The amount of maxi gene products was measured at ¢ve di¡erent concentrations of total RNA in the range of 0.2^ 1.8 ng per RT-PCR reaction mixture. The RT step was performed in a 20 Wl reaction mixture. The mixture contained 17 mM Tris^HCl (pH 8.4), 42.5 mM KCl, 4.25 mM MgCl2 , 0.1 mM dNTP, 15 pmoles 32 P-5P-end labeled Vivo2 primer, 15 pmoles 32 P-5P-end labeled CAT-248 primer, 0.2^1.8 ng RNA and 8.8 units MULV reverse transcriptase. The reaction was carried out as follows : 37³C for
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30 min, 90³C for 5 min, 4³C. The reaction mixtures were diluted to 50 Wl with PCR bu¡er (20 mM Tris^HCl (pH 8.4), 50 mM KCl) and added 15 pmoles Vivo-1 primer, 15 pmoles CAT-142 primer and 1.7 units Taq DNA polymerase. The PCR reaction was carried out with the following program: 95³C for 15 s, 51³C for 30 s, 72³C for 30 s, (25 cycles), 72³C for 5 min, 4³C. Primer sequences: Vivo-1: GTCTACGGCCATACC, Vivo-2: AAAGCACTAGTACGCC. The transcription of the pHU5S3.2 maxi gene in transfected HeLa cells was used as control in all assays and set to 100%. This clone is transcribed with the same e¤ciency in vitro as two other clones, pHU5S3.0 maxi and pHU5S3.1 maxi with longer 5P-£anking sequences (Fig. 3, ¢rst row). In transfection experiments neither of these clones with longer 5P-£anking regions showed improved transcriptional e¤ciency. The results of the in vivo transcription assays are shown in Fig. 3 (second row). The results are obtained as averages from two or three separate transfection experiments. The pHU5S3.1 Acc maxi clone without 5P-£anking sequence was transcribed with an e¤ciency of 18% that is comparable to the 0^10% observed in vitro. The Mut 23 maxi clone contained one mutation in the D box (T to A substitution) that did not in£uence transcription in vitro. In the transfection experiments with the Mut 23 maxi clone the transcription was measured to 106%. The Mut 1 clone (T to G substitution) was transcribed with 59% e¤ciency and the Mut 26 maxi clone (T to C substitution) with 50% e¤ciency. This is comparable to the e¤ciency observed in vitro which was 40% and 25%, respectively. Changes in the composition of the D box had a similar e¡ect on transcription in vitro and in vivo showing the importance of the external promoter on the regulation of 5S rRNA gene expression.
to be the case in the protist Acanthamoeba castellanii [15], the fungus Neurospora crassa [16,17], the yeast Saccharomyces cerevisiae [18], the fruit £y Drosophila melanogaster [19], the silkworm Bombyx mori [20,21], the loach Misgurnus fossilis [22], the nematode Caenorhabditis elegans [23] and the toad Xenopus laevis [24]. This evidence together with the evidence presented in the present paper suggests that the de¢nition of type I genes transcribed by RNA polymerase III ought to be revised. The upstream promoter sequences in lower eukaryotes are more AT-rich than the GC-rich D box found conserved in higher eukaryotes [3^8]. Many substitutions of nucleotides in the D box with As or Ts still allow full or almost full transcriptional activity as demonstrated in the present paper (Figs. 1 and 2). The evolutionary history of the D box may therefore have run in parallel with that of the coding sequence of the 5S rRNA genes. Acknowledgements We thank Rita Jensen and Irene JÖrgensen for excellent technical assistance. The work was supported by the Novo î ge Haensch FoundaNordisk Foundation, Gerda and A tion and the Danish Natural Science Research Council.
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5. External promoters in 5S rRNA genes
[8]
A conserved 12-mer sequence is found in the upstream region of di¡erent mammalian 5S rRNA genes [3^8]. Deletion mutants made from 5S rDNA isolated from human cells [5] and mouse cells [6] suggested that the 12-mer sequence designated the D box had an e¡ect on the transcription of 5S rRNA genes in vitro. A more detailed study of the 12-mer sequence was undertaken in the present paper. The results show that point mutations affect the transcription both in vitro and in vivo and thereby demonstrate the importance of the D box for transcription of the 5S rRNA genes. In the original de¢nition of type I genes transcribed by RNA polymerase III, gene expression was regulated only by internal promoters (reviews [1,2]). The in£uence of upstream sequences on the transcription of 5S rRNA genes has, however, been demonstrated for a number of eukaryotic genes. By means of 5P deletion mutants this was found
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BBAEXP 91565 3-8-01
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