Proc. Nat. Acad. Sci. USA Vol. 72, No. 12, pp. 4835-4839, December 1975
Biochemistry
Strand-selective transcription of globin genes in rabbit erythroid cells and chromatin (cDNA/RNA nucleotidyltransferase/DNA-RNA hybridization/mRNA/nuclear RNA)
G. N. WILSON, A. W. STEGGLES, AND A. W. NIENHUIS Molecular Hematology Branch, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland 20014
Communicated by Donald S. Fredrickson, September 19,1975 In order to investigate the symmetry of gloABSTRACT bin gene transcription, complementary RNA (cRNA) was synthesized using rabbit globin complementary DNA (cDNA) as a template for Escherichia coli DNA-dependent RNA polymerase (RNA nucleotidyltransferase). The cRNA hybridized specifically to its own cDNA template but not to sheep cDNA, rabbit globin mRNA, or poly(dT). Hybridization studies with cRNA demonstrated that RNA sequences transcribed from the DNA strand complementary to the globin gene region (anti-strand) were not present in cellular, total nuclear, or fractionated nuclear RNA from rabbit marrow. Such sequences were detected in RNA transcribed from rabbit marrow chromatin by E. coli or sheep liver RNA polymerases, but amounted to less than 50% of the globin mRNA sequences present in the same transcript. The evidence indicates that globin mRNA transcription is predominantly DNA strand specific.
While the selection of one strand of DNA as a template for RNA synthesis is a hallmark of transcription in prokaryotes, the rule of asymmetric transcription in higher organisms has not been established. The most definitive evidence concerns the transcription of the clustered and moderately reiterated ribosomal genes of Xenopus laevis, where hybridization to separate strands of ribosomal DNA (1) and electron microscopic evidence (2) conclusively demonstrate asymmetric transcription of ribosomal RNA precursor. Symmetrical transcription has been documented, however, in HeLa cell mitochondria (3) and during the maturation of animal viruses (4, 5). Of great interest is the symmetry of transcription of single-copy eukaryotic DNA. The fact that eukaryotic mRNA is predominantly single-stranded (6) does not rule out the transient existence of RNA transcribed from the DNA strand complementary to unique gene regions (anti-strand transcript) as an intermediate in processing or as a template for putative reverse transcriptases or RNA replicases (7). With regard to the former possibility, sequences complementary to polysomal RNA have been identified in HeLa cell nuclear RNA (8). Double-stranded RNA has also been detected in nuclear RNA (9, 10) but its renaturation properties favor hairpin configurations of single RNA molecules rather than true symmetrical intermediates (11). Globin genes are present in one or only a few copies in several species (12, 13) and thus are suitable for investigating the symmetry of transcription of single copy genes. In this report, we describe the synthesis of a specific hybridization probe for detection of RNA sequences transcribed from the DNA strand complementary to the globin genes, document the absence of such sequences in rabbit marrow cells, and Abbreviations: cDNA, DNA complementary to globin mRNA; cRNA, RNA complementary to cDNA; cRNAr, RNA complementary to rabbit globin mRNA; Hepes, N-hydroxyethylpiperazineN'-2-ethanesulfonic acid.
4835
examine the symmetry of globin gene transcription in a chromatin-primed cell-free system.
METHODS
Preparation of cDNA. Rabbit and sheep globin mRNA were prepared as described previously (14, 15). Rabbit and sheep cDNAs were synthesized as before (16) but purified by a modification of the method of Harrison et al. (17). The reaction mixtures (1 ml) were centrifuged through Sephadex G-25 in a 10 ml syringe, adjusted to 0.1 M NaOH, 0.9 M NaCI, 0.01 M EDTA, and applied to a 12 ml 5-30% sucrose gradient containing the same buffer. After centrifugation at 40,000 rpm for 18 hr at 20° in the Spinco SW A1 rotor, fractions were collected and monitored for their content of [3H]or [32P]cDNA. Those fractions containing cDNA of greater size than 300 nucleotides by comparison to an Escherichia coli DNA standard were pooled, neutralized with HCI, mixed with S A260 units of yeast tRNA (Miles), and precipitated with two volumes of ethanol at -20°. The precipitate was collected by centrifugation, dissolved in distilled water, refiltered on Sephadex G-25, and lyophilized before use. Preparation of cRNA. Ribonucleic acid complementary to sheep or rabbit cDNA (cRNA) was synthesized essentially as described (18, 19) in a reaction mixture (1 ml) containing 50 4mol of Tris-HCI, pH 7.9, 3 ,umol of MgCI2, 8 pgmol of KCI, 1.2 ,mol of 2-mercaptoethanol, 5% (vol/vol) glycerol, 0.01 ,umol of [3H]UTP (30 Ci/mmol; Schwarz/Mann), 0.2 ,umol of ATP, GTP, and CTP (Schwarz/Mann), 50 ng of [32PlcDNA (1000 cpm/ng) and 100 units of E. coli RNA polymerase (RNA nucleotidyltransferase; Grand Island Biologicals). After incubation for 30 min at 370, 2 ,umol of CaCl2 and 20 ,ug of DNase I (Worthington) were added and the mixture incubated for 10 min at 370. Sodium dodecyl sulfate was added to 0.25% and the mixture was extracted at room temperature with an equal volume of phenol saturated with 0.5 M KC1. The aqueous phase was purified by Sephadex G-50 column chromatography in distilled water and lyophilized. Approximately 250 ng of [3H]cRNA (2000 cpm/ ng) were recovered, containing less than 0.1 ng (100 cpm) of the cDNA template. Sizing of the cRNA's was accomplished by sodium dodecyl sulfate-polyacrylamide electrophoresis (14) or by sucrose gradient centrifugation in 70% formamide
(29).
Ribonucleic acid complementary to rabbit globin mRNA (cRNAr) was synthesized as previously described (20, 21) in a 1 ml reaction mixture containing 100 ,umol of Tris-HCI, pH 7.9, 3 ,umol of MnCl2, 10 ,mol of KC1, 1.2 ,mol of 2mercaptoethanol, 5% (vol/vol) glycerol, 0.01 ,lmol of [3H]UTP (30 Ci/mmol), 0.2 ,gmol of ATP, GTP, and CTP, 1 ,ug of globin mRNA, and 20 units of Micrococcus lysodeikticus RNA polymerase (Miles). After incubation and extrac-
4836
Proc. Nat. Acad. Sci. USA 72 (1975)
Biochemistry: Wilson et al.
tion of the RNA product as described for cRNA, approximately 20 ng of [3H]cRNAr was recovered as an unresolved mixture with its globin mRNA template. The cRNAr was prepared to provide a test of resistance of RNA duplexes to RNase. Preparation of Rabbit Marrow Cellular and Nuclear RNA. Marrow cells from 1 to 2 kg phenylhydrazine-stimulated rabbits (15) were suspended in NCTC-109 (Grand Island Biologicals), filtered through four layers of cheesecloth (Chickopee Mills), and centrifuged at 1000 X g for 10 min. Ninety-five percent of the cells were erythroid precursors as determined by cytological study. Cellular RNA was extracted from 1 g of washed rabbit marrow cells by the method of Penman (22). Nuclei were prepared from 5 g of cells using the citric acid method described by Knowler et al. (23). Nuclear RNA was extracted and fractionated on sodium dodecyl sulfate for formamide sucrose gradients by the method of Lanyon et al. (24). Preparation and Transcription of Rabbit Marrow Chromatin. Rabbit marrow chromatin was prepared as described previously (16) and transcribed within 2 hr of isolation. The standard transcription reaction (2 ml) contained 30 mM Tris-HCl, pH 7.9, 100 mM KC1, 3 mM MgCl2, 3 mM MnCl2, 1.2 mM 2-mercaptoethanol, 5-15% glycerol, 2 mM ATP, GTP, and CTP, 0.4 mM [32P]UTP (0.1 mCi/mmol), rabbit marrow chromatin containing 500 ,ug of DNA, and either 200 units of E. coli RNA polymerase or 50 units of sheep liver RNA polymerase II prepared as described previously (16). After incubation for 1 hr at 370, the amount of RNA synthesized was estimated by removing aliquots for precipitation with 10% trichloroacetic acid. For control reactions without polymerase, 5-50 Ag of [32P]RNA carrier purified from a standard transcription reaction containing 50 jg of sonicated E. coli DNA (Worthington) and 200 units of E. coli RNA polymerase was added to ensure accurate comparison with the reaction containing enzyme. After centrifugation at 4° for 10 min at 12,000 X g, the procedure of Penman (22) was used to extract RNA from the supernatant with 70-80% yield of acid-insoluble radioactivity. The extracted RNA was purified by Sephadex G-50 chromatography and lyophilized. The purified fraction contained 5075% of the initial acid-insoluble radioactivity (60-100 Mg by 32p cpm, 80-120 Ag by orcinol assay) and less than 0.2 Mg of DNA. Hybridization Assays. The procedure was similar to that described by Harrison et al. (25). Reactions (10 Ml) contained 0.5 M NaCl, 25 mM N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid (Hepes), pH 6.8, 0.5 mM EDTA, 50% formamide (Eastman), 5 jug of E. coli 5S RNA (Miles), 0.7 Mug of heat-denatured E. coli DNA, and either 0.5-1 ng of cRNA, 1 ng of cDNA, or 0.5 ng of cRNAr. After preincubation for 10 min at 900, the tubes were incubated at 430 for 48 hr, which was sufficient to allow completion of all hybridization reactions (26). Reactions containing cDNA were analyzed with micrococcal nuclease as described previously (16, 27) except that each tube was emptied into 1.05 ml of 50 mM Tris-HCl, pH 8.3, 0.4 M NaCl, 10 mM MgCl2, 0.1 mM CaC12. Two aliquots of 500 Ml were taken for assay and to one was added 40 Mg of micrococcal nuclease. After incubation for 60 min at 370, the samples were precipitated with 2 ml of cold 60% trichloroacetic acid for 20 min at 40 and collected on nitrocellulose filters. After washing with 10 ml of cold 10% trichloroacetic acid, radioactivity was measured by liquid scintillation spectrometry. Reactions containing cRNA or cRNAr were analyzed with RNase A (Worthing-
-~~~~~~~~
60 \. tO 40 0
20 -
KIs1
0
100.4j\
B
cRNAr
80\% _j60 z
Z40
N
JR-0~~~~~~~~~ 20
10
20 30 40 50 RIBONUCLEASE A (Lg/ml)
60
FIG. 1. Specificity of RNase A for single-stranded cRNA and
cRNAr. (A) Hybridization reactions containing 1 ng of rabbit [:H]cRNA (0), or cRNA and 2 ng of [32P]cDNA (-) were incubated 48 hr at 430 and analyzed with the specified concentratiintif RNase A. (B) Hybridization reactions containing 2000 cpm of [HIcRNAr were frozen immediately after heating 10 min at 900 (0) or frozen after incubation for 48 hr at 430 (O). The reactions were analyzed with the specified concentrations of RNase A.
ton) in a similar fashion, except that the hydrolysis mixtur contained 10 mM Tris-HCI, pH 7.4, 0.5 M NaCl, and 5 m EDTA, incubation was for 30 min at 370, and precipitatioi was with 2 ml of cold 15% trichloroacetic acid for 20 min a 4°. Results were either plotted directly or corrected for self, annealing of the cDNA or cRNA and normalized to 100% as described by Young et al. (28). RESULTS
Synthesis of cRNA and analysis of cRNA*
Biochemistry
Strand-selective transcription of globin genes in rabbit erythroid cells and chromatin (cDNA/RNA nucleotidyltransferase/DNA-RNA hybridization/mRNA/nuclear RNA)
G. N. WILSON, A. W. STEGGLES, AND A. W. NIENHUIS Molecular Hematology Branch, National Heart and Lung Institute, National Institutes of Health, Bethesda, Maryland 20014
Communicated by Donald S. Fredrickson, September 19,1975 In order to investigate the symmetry of gloABSTRACT bin gene transcription, complementary RNA (cRNA) was synthesized using rabbit globin complementary DNA (cDNA) as a template for Escherichia coli DNA-dependent RNA polymerase (RNA nucleotidyltransferase). The cRNA hybridized specifically to its own cDNA template but not to sheep cDNA, rabbit globin mRNA, or poly(dT). Hybridization studies with cRNA demonstrated that RNA sequences transcribed from the DNA strand complementary to the globin gene region (anti-strand) were not present in cellular, total nuclear, or fractionated nuclear RNA from rabbit marrow. Such sequences were detected in RNA transcribed from rabbit marrow chromatin by E. coli or sheep liver RNA polymerases, but amounted to less than 50% of the globin mRNA sequences present in the same transcript. The evidence indicates that globin mRNA transcription is predominantly DNA strand specific.
While the selection of one strand of DNA as a template for RNA synthesis is a hallmark of transcription in prokaryotes, the rule of asymmetric transcription in higher organisms has not been established. The most definitive evidence concerns the transcription of the clustered and moderately reiterated ribosomal genes of Xenopus laevis, where hybridization to separate strands of ribosomal DNA (1) and electron microscopic evidence (2) conclusively demonstrate asymmetric transcription of ribosomal RNA precursor. Symmetrical transcription has been documented, however, in HeLa cell mitochondria (3) and during the maturation of animal viruses (4, 5). Of great interest is the symmetry of transcription of single-copy eukaryotic DNA. The fact that eukaryotic mRNA is predominantly single-stranded (6) does not rule out the transient existence of RNA transcribed from the DNA strand complementary to unique gene regions (anti-strand transcript) as an intermediate in processing or as a template for putative reverse transcriptases or RNA replicases (7). With regard to the former possibility, sequences complementary to polysomal RNA have been identified in HeLa cell nuclear RNA (8). Double-stranded RNA has also been detected in nuclear RNA (9, 10) but its renaturation properties favor hairpin configurations of single RNA molecules rather than true symmetrical intermediates (11). Globin genes are present in one or only a few copies in several species (12, 13) and thus are suitable for investigating the symmetry of transcription of single copy genes. In this report, we describe the synthesis of a specific hybridization probe for detection of RNA sequences transcribed from the DNA strand complementary to the globin genes, document the absence of such sequences in rabbit marrow cells, and Abbreviations: cDNA, DNA complementary to globin mRNA; cRNA, RNA complementary to cDNA; cRNAr, RNA complementary to rabbit globin mRNA; Hepes, N-hydroxyethylpiperazineN'-2-ethanesulfonic acid.
4835
examine the symmetry of globin gene transcription in a chromatin-primed cell-free system.
METHODS
Preparation of cDNA. Rabbit and sheep globin mRNA were prepared as described previously (14, 15). Rabbit and sheep cDNAs were synthesized as before (16) but purified by a modification of the method of Harrison et al. (17). The reaction mixtures (1 ml) were centrifuged through Sephadex G-25 in a 10 ml syringe, adjusted to 0.1 M NaOH, 0.9 M NaCI, 0.01 M EDTA, and applied to a 12 ml 5-30% sucrose gradient containing the same buffer. After centrifugation at 40,000 rpm for 18 hr at 20° in the Spinco SW A1 rotor, fractions were collected and monitored for their content of [3H]or [32P]cDNA. Those fractions containing cDNA of greater size than 300 nucleotides by comparison to an Escherichia coli DNA standard were pooled, neutralized with HCI, mixed with S A260 units of yeast tRNA (Miles), and precipitated with two volumes of ethanol at -20°. The precipitate was collected by centrifugation, dissolved in distilled water, refiltered on Sephadex G-25, and lyophilized before use. Preparation of cRNA. Ribonucleic acid complementary to sheep or rabbit cDNA (cRNA) was synthesized essentially as described (18, 19) in a reaction mixture (1 ml) containing 50 4mol of Tris-HCI, pH 7.9, 3 ,umol of MgCI2, 8 pgmol of KCI, 1.2 ,mol of 2-mercaptoethanol, 5% (vol/vol) glycerol, 0.01 ,umol of [3H]UTP (30 Ci/mmol; Schwarz/Mann), 0.2 ,umol of ATP, GTP, and CTP (Schwarz/Mann), 50 ng of [32PlcDNA (1000 cpm/ng) and 100 units of E. coli RNA polymerase (RNA nucleotidyltransferase; Grand Island Biologicals). After incubation for 30 min at 370, 2 ,umol of CaCl2 and 20 ,ug of DNase I (Worthington) were added and the mixture incubated for 10 min at 370. Sodium dodecyl sulfate was added to 0.25% and the mixture was extracted at room temperature with an equal volume of phenol saturated with 0.5 M KC1. The aqueous phase was purified by Sephadex G-50 column chromatography in distilled water and lyophilized. Approximately 250 ng of [3H]cRNA (2000 cpm/ ng) were recovered, containing less than 0.1 ng (100 cpm) of the cDNA template. Sizing of the cRNA's was accomplished by sodium dodecyl sulfate-polyacrylamide electrophoresis (14) or by sucrose gradient centrifugation in 70% formamide
(29).
Ribonucleic acid complementary to rabbit globin mRNA (cRNAr) was synthesized as previously described (20, 21) in a 1 ml reaction mixture containing 100 ,umol of Tris-HCI, pH 7.9, 3 ,umol of MnCl2, 10 ,mol of KC1, 1.2 ,mol of 2mercaptoethanol, 5% (vol/vol) glycerol, 0.01 ,lmol of [3H]UTP (30 Ci/mmol), 0.2 ,gmol of ATP, GTP, and CTP, 1 ,ug of globin mRNA, and 20 units of Micrococcus lysodeikticus RNA polymerase (Miles). After incubation and extrac-
4836
Proc. Nat. Acad. Sci. USA 72 (1975)
Biochemistry: Wilson et al.
tion of the RNA product as described for cRNA, approximately 20 ng of [3H]cRNAr was recovered as an unresolved mixture with its globin mRNA template. The cRNAr was prepared to provide a test of resistance of RNA duplexes to RNase. Preparation of Rabbit Marrow Cellular and Nuclear RNA. Marrow cells from 1 to 2 kg phenylhydrazine-stimulated rabbits (15) were suspended in NCTC-109 (Grand Island Biologicals), filtered through four layers of cheesecloth (Chickopee Mills), and centrifuged at 1000 X g for 10 min. Ninety-five percent of the cells were erythroid precursors as determined by cytological study. Cellular RNA was extracted from 1 g of washed rabbit marrow cells by the method of Penman (22). Nuclei were prepared from 5 g of cells using the citric acid method described by Knowler et al. (23). Nuclear RNA was extracted and fractionated on sodium dodecyl sulfate for formamide sucrose gradients by the method of Lanyon et al. (24). Preparation and Transcription of Rabbit Marrow Chromatin. Rabbit marrow chromatin was prepared as described previously (16) and transcribed within 2 hr of isolation. The standard transcription reaction (2 ml) contained 30 mM Tris-HCl, pH 7.9, 100 mM KC1, 3 mM MgCl2, 3 mM MnCl2, 1.2 mM 2-mercaptoethanol, 5-15% glycerol, 2 mM ATP, GTP, and CTP, 0.4 mM [32P]UTP (0.1 mCi/mmol), rabbit marrow chromatin containing 500 ,ug of DNA, and either 200 units of E. coli RNA polymerase or 50 units of sheep liver RNA polymerase II prepared as described previously (16). After incubation for 1 hr at 370, the amount of RNA synthesized was estimated by removing aliquots for precipitation with 10% trichloroacetic acid. For control reactions without polymerase, 5-50 Ag of [32P]RNA carrier purified from a standard transcription reaction containing 50 jg of sonicated E. coli DNA (Worthington) and 200 units of E. coli RNA polymerase was added to ensure accurate comparison with the reaction containing enzyme. After centrifugation at 4° for 10 min at 12,000 X g, the procedure of Penman (22) was used to extract RNA from the supernatant with 70-80% yield of acid-insoluble radioactivity. The extracted RNA was purified by Sephadex G-50 chromatography and lyophilized. The purified fraction contained 5075% of the initial acid-insoluble radioactivity (60-100 Mg by 32p cpm, 80-120 Ag by orcinol assay) and less than 0.2 Mg of DNA. Hybridization Assays. The procedure was similar to that described by Harrison et al. (25). Reactions (10 Ml) contained 0.5 M NaCl, 25 mM N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid (Hepes), pH 6.8, 0.5 mM EDTA, 50% formamide (Eastman), 5 jug of E. coli 5S RNA (Miles), 0.7 Mug of heat-denatured E. coli DNA, and either 0.5-1 ng of cRNA, 1 ng of cDNA, or 0.5 ng of cRNAr. After preincubation for 10 min at 900, the tubes were incubated at 430 for 48 hr, which was sufficient to allow completion of all hybridization reactions (26). Reactions containing cDNA were analyzed with micrococcal nuclease as described previously (16, 27) except that each tube was emptied into 1.05 ml of 50 mM Tris-HCl, pH 8.3, 0.4 M NaCl, 10 mM MgCl2, 0.1 mM CaC12. Two aliquots of 500 Ml were taken for assay and to one was added 40 Mg of micrococcal nuclease. After incubation for 60 min at 370, the samples were precipitated with 2 ml of cold 60% trichloroacetic acid for 20 min at 40 and collected on nitrocellulose filters. After washing with 10 ml of cold 10% trichloroacetic acid, radioactivity was measured by liquid scintillation spectrometry. Reactions containing cRNA or cRNAr were analyzed with RNase A (Worthing-
-~~~~~~~~
60 \. tO 40 0
20 -
KIs1
0
100.4j\
B
cRNAr
80\% _j60 z
Z40
N
JR-0~~~~~~~~~ 20
10
20 30 40 50 RIBONUCLEASE A (Lg/ml)
60
FIG. 1. Specificity of RNase A for single-stranded cRNA and
cRNAr. (A) Hybridization reactions containing 1 ng of rabbit [:H]cRNA (0), or cRNA and 2 ng of [32P]cDNA (-) were incubated 48 hr at 430 and analyzed with the specified concentratiintif RNase A. (B) Hybridization reactions containing 2000 cpm of [HIcRNAr were frozen immediately after heating 10 min at 900 (0) or frozen after incubation for 48 hr at 430 (O). The reactions were analyzed with the specified concentrations of RNase A.
ton) in a similar fashion, except that the hydrolysis mixtur contained 10 mM Tris-HCI, pH 7.4, 0.5 M NaCl, and 5 m EDTA, incubation was for 30 min at 370, and precipitatioi was with 2 ml of cold 15% trichloroacetic acid for 20 min a 4°. Results were either plotted directly or corrected for self, annealing of the cDNA or cRNA and normalized to 100% as described by Young et al. (28). RESULTS
Synthesis of cRNA and analysis of cRNA*
