Proc. Natl. Acad. Sci. USA Vol. 74, No. 6, pp. 2475-2479, June 1977
Cell Biology
Globin RNA synthesis in vitro by isolated erythroleukemic cell nuclei: Direct evidence for increased transcription during erythroid differentiation (mercurinucleotides/mercaptans/affinity chromatography/gene regulation)
STUART H. ORKIN* AND PAUL S. SWERDLOW The Division of Hematology-Oncology of the Department of Medicine, Children's Hospital Medical Center, and the Sidney Farber Cancer Institute, and the Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115
Communicated by Arthur B. Pardee, March 31, 1977
Murine erythroleukemic cells accumulate ABSTRACT cytoplasmic globin mRNA during differentiation induced in tissue culture by dimethyl sulfoxide. Cellular accumulation of globin RNA may reflect transcriptional activation of the globin genes and/or posttranscriptional stabilization of globin RNA during differentiation. To evaluate possible transcriptional controls directly, globin RNA synthesis by isolated erythroleukemic cell nuclei was studied. Conditions were established for optimal nuclear RNA synthesis in vitro in the presence of a mercurinucleotide (Hg-CTP). Mercurated RNA synthesized in vitro was purified free of endogenous RNA by affinity chromatography on sulfhydrylSepharose, and analyzed for the presence of newly synthesized globin RNA sequences by molecular hybridization to globin complementary [32PJDNA. The results demonstrate markedly increased synthesis of globin RNA by nuclei isolated from dimethyl sulfoxide-treated cells, even within 5 min of nuclear transcription in vitro. These findings are most consistent with transcriptional activation of the globin genes upon induction of differentiation.
Murine erythroleukemic cells capable of differentiation in vitro response to various inducers (1) constitute an important, well-characterized system for the study of the cellular and in
molecular events associated with erythrocyte development. When induced with dimethyl sulfoxide, these cells accumulate substantial quantities of cytoplasmic globin messenger RNA (2-4). Whether this reflects transcriptional activation of the globin genes upon induction, posttranscriptional stabilization of globin RNA sequences, or a combination of these mechanisms, is unresolved. In this report we provide direct evidence for transcriptional activation of the globin genes in induced cells by study of globin RNA synthesis by isolated nuclei. Newly synthesized RNA made by isolated nuclei in vitro in the presence of a mercurinucleotide (5, 6) and an appropriate mercaptan was purified free of endogenous RNA by affinity chromatography (7) and analyzed for the presence of globin RNA sequences by hybridization to globin complementary DNA (cDNA). The results demonstrate increased synthesis of globin RNA in nuclei isolated from induced erythroleukemic cells. MATERIALS AND METHODS
Cell Culture. Murine erythroleukemic cells of a long-term stock of T3C12 (2) capable of extensive differentiation in response to dimethyl sulfoxide (3, 4) were maintained in Dulbecco's modified Eagle's medium containing 10% heatinactivated fetal calf serum. Erythroid maturation was induced passage
Abbreviations: cDNA, complementary DNA; Hg-RNA, mercurated RNA. * To whom all correspondence should be addressed. 2475
with 1.5% (vol/vol) dimethyl sulfoxide as previously described
(4).
Isolation of Nuclei. Washed cell pellets were suspended in 0.3 M sucrose (Schwarz/Mann ultrapure) containing 2 mM Mg(OAc)2, 3 mM CaCI2, 10 mM Tris-HCl at pH 8.0, and 2 mM 2-mercaptoethanol. After addition of Triton X-100 to 0.25%, the cell suspension was stirred on a vortex mixer for 30 sec. The crude nuclei were gently sedimented at 1000 X g for 5 min at 40, and then resuspended in the same buffer minus Triton X100. An equal volume of 2 M sucrose containing 5 mM Mg(OAc)2, 10 mM Tris at pH 8.0, and 2 mM mercaptoethanol was immediately added, and the mixture was homogenized with five strokes of a tight-fitting Teflon-glass homogenizer. Purified nuclei were sedimented through a cushion of 2 M sucrose containing 2 mM Mg(OAc)2, 3 mM CaCl2, and 10 mM Tris at pH 8.0 by centrifugation at 25,000 rpm in a Beckman SW-40 rotor for 45 min at 40. The nuclear pellet was resuspended in 25% (vol/vol) glycerol/5 mM Mg(OAc)2/50 mM Tris at pH 8.0/2 mM mercaptoethanol at 1 to 2 X 108 nuclei per ml, and frozen in liquid nitrogen. RNA Synthesis by Isolated Nuclei. Conditions for RNA synthesis were modified slightly from those described for other systems (8, 9). Each reaction mixture contained 50 mM N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) at pH 8.0, 5 mM Mg(OAc)2, 1 mM MnCl2, 150 mM KCI, 12.5% glycerol, 4 mM phosphoenolpyruvate, pyruvate kinase (3 units/ml), 0.185 mM of three ribonucleoside triphosphates, 0.037 mM of the fourth ribonucleoside triphosphate labeled with 3H, and approximately 5 X 107 rapidly thawed nuclei per ml. Mercurinucleotide, either Hg-CTP or Hg-UTP (P.L. Biochemicals), replaced the corresponding unmodified nucleotide at the same concentration in the reaction mixture when RNA containing mercury (Hg-RNA) was prepared. Mercaptans, as noted below, were present at 10 mM final concentration in the reaction mixture. The reaction mixture was incubated at 230 with intermittent, gentle shaking and aliquots were withdrawn at appropriate times, spotted on Whatman 3MM paper, and processed as described by Ernest et al. (9) to monitor RNA synthesis. No adjustment was made for background cpm at time zero. Preparative Isolation of RNA after Nuclear Synthesis. After incubation, nuclei were diluted 10-fold with 20 mM Tris at pH 7.5/50 mM NaCl/1 mM EDTA/1% sodium dodecyl sulfate and extracted vigorously in a vortex mixer with an equal volume of 600 phenol previously equilibrated with Tris buffer. The aqueous phase was reextracted three times with chloroform/isoamyl alcohol (24:1 vol/vol), and precipitated with ethanol. In early experiments samples were treated with RNase-free DNase and reprecipitated prior to affinity chromatography. In more recent experiments DNase treatment was
2476
Cell Biology: Orkin and Swerdlow
Proc. Nati. Acad. Sci. USA 74 (1977)
16
101 0
14-
L
0.
cm8
0
12
x
E
0.
10
0. Q)
~~~0~
0
0
E.1
0
0~
a.
Q-
HS
2
0
c.1020
3
0
4
0 2 0
10 30 20 Time, min
FIG. 1. Time course of nuclear RNA synthesis in vitro. Control erythroleukemic cell nuclei were incubated in the complete reaction mixture with [3H]UTP as the labeled ribonucleoside triphosphate. Incorporation at 30 min was 30.6 pmol of UTP per 106 nuclei.
omitted without any consequence with respect to purification of the mercurated RNA. Affinity Purification of Hg-RNA. Sulfhydryl-Sepharose-6B was prepared precisely as described by Dale and Ward (7), who used the method of Cuatrecasas (10). Each batch of sulfhydryl-Sepharose was tested for its ability to bind samples of Hg-RNA and for nonspecific adsorption of nucleic acid by passage of radioactive, nonmercurated RNA. The sulfhydryl supports used in this work had immeasurably low nonspecific binding, less than one part in 105. Prior to each use the Sepharose was briefly washed with 20 mM Tris at pH 7.5/0.1 M NaCl containing 0.1 M mercaptoethanol, and then repeatedly washed with buffer lacking the mercaptan. A 5-ml packed column was equilibrated with buffer containing 0.25% sodium dodecyl sulfate, and the RNA was applied after brief heating at 65° in this buffer. The column was washed with approximately 5 column volumes of this buffer, and then 50 column volumes of buffer lacking sodium dodecyl sulfate. Hg-RNA bound to the column was eluted with addition of buffer containing mercaptan. Fractions containing eluted Hg-RNA were pooled, carrier Escherichta coli tRNA (100 ,g) was added, and the RNA was precipitated with ethanol. Precipitated RNA was resuspended in water and frozen in liquid nitrogen prior to molecular hybridization. Preparation of Globin mRNA and cDNA. Pure mouse globin mRNA was prepared from ex-breeder CD-1 mice (Charles River Breeding Laboratories) by oligo(dT)-cellulose chromatography and sucrose gradient centrifugation as previously described (4). Globin cDNA was synthesized in 25 Al using 50 mM KCI; 50 mM Tris at pH 8.3; 8 mM MgCl2; 10 mM dithiothreitol; 1 mM each dATP, dCTP, and dTTP; 40 jig of oligo(dT) per ml; 100 jig of actinomycin D per ml; 150 MCi of [32P]dGTP (115-135 Ci/mmol, New England Nuclear); 1 mg of mRNA; and approximately 10 units of RNA-dependent DNA polymerase (reverse transcriptase) from avian myeloblastosis virus at 370 for 30 min. cDNA was purified as before (4). The specific activity of cDNA was approximately 1.5 X 108
cpm/Ag.
24
48
72
Time in Me2SO, hr
Molecular Hybridization. Cytoplasmic RNA-cDNA hybridization for quantitation of accumulated globin RNA was
FIG. 2. Nuclear RNA synthesis by nuclei isolated from dimethyl sulfoxide-treated cells. Nuclei were prepared from cells treated 49 hr and 72 hr with dimethyl sulfoxide (Me2SO) and compared with control nuclei with respect to in vitro RNA synthesis. Values are expressed as pmol of UTP incorporated per 106 nuclei over 5-min incubation.
performed as before (4). Pure globin RNA hybridized with [32P]cDNA with RNA concentration X time for 50% hybridization, Crtl/2 = 5 X 10-4 sec X moles of nucleotide per liter. 3H-Labeled Hg-RNA samples were incubated at 650 for 24 hr in a 10-Ml total volume containing 0.6 M NaCl, 20 mM Tris at pH 7.5, an additional 5 Mg of E. coli tRNA, and 3-5 pg of [32P]cDNA. A standard reference curve of the hybridization of pure globin RNA with cDNA was determined in parallel with each assay. After incubation for 24 hr, the extent of hybridization of the [32P]cDNA was assayed by digestion with Si nuclease (4). RESULTS RNA Synthesis by Erythroleukemic Cell Nuclei. Isolated nuclei from control erythroleukemic cells were active in RNA synthesis in vntro (Fig. 1). Incorporation of [3HIUTP continued for 60 min and averaged 30 pmol per 106 nuclei over the initial 30-min period, similar to that observed in other efficient nuclear systems (8, 9, 11, 12). Incorporation was sensitive to inhibition by pancreatic RNase or actinomycin D (not shown) and dependent on the presence of all four nucleoside triphosphates (see below). Nuclei isolated from dimethyl sulfoxide-treated erythroleukemic cells were less active than those from uninduced cells (Fig. 2), consistent with the progressive reduction in total RNA synthesis observed in induced cell cultures (13). Synthesis of Hg-RNA. Incorporation of mercurinucleotides into RNA or DNA by E. coli polymerases is totally dependent on the presence of a mercaptan to act as a mercury ligand (5). There is considerable specificity with respect to the appropriate mercaptan for a given polymerase (5). Analysis of the mercaptan requirements of eukaryotic polymerases has not been reported in detail. Smith and Huang (11) observed that RNA synthesis by myeloma nuclei was approximately 60% of control rates when Hg-UTP was used in place of UTP in the presence of mercaptoethanol, but less than 10% in the presence of dithiothreitol or in the absence of any mercaptan. Our initial experiments with erythroleukemic cell nuclei, Hg-UTP, and mercaptoethanol demonstrated more limited RNA synthesis relative to controls and prompted closer examination of the mercaptan requirements in this eukaryotic system. Representative results for RNA synthesis in the presence
Cell Biology: Orkin and Swerdlow .
Proc. Natl. Acad. SCt. USA 74 (1977)
2477
A
8
oa0
7
I
2
D-a1) 4-
T
CA
o
o4 x
,-
c x'
CL-
g' 5
E
0'- E j a.
a, u
mE
'aI
I
co20 10
20
10
20
Time, mmn
FIG. 3. Mercaptan requirements for nuclear RNA synthesis in the presence of mercurinucleotide. (A) RNA synthesis in the presence of: CTP plus 10 mM mercaptoethanol (0); Hg-CTP plus 10 mM thioglycerol (0); Hg-CTP plus 10 mM mercaptoethanol (A); Hg-CTP without added mercaptan (1); neither CTP nor Hg-CTP (X). Synthesis was monitored by incorporation of [3HJUTP. No difference in control RNA synthesis with CTP was noted with 10 mM thioglycerol replacing 10 mM mercaptoethanol. (B) RNA synthesis in the presence of: UTP plus 10 mM mercaptoethanol (0); Hg-UTP plus 10 mM mercaptoethanol (A); Hg-UTP plus 10 mM thioglycerol (0); Hg-UTP without added mercaptan (3). Incorporation of [3H]GTP was used to monitor RNA synthesis. No difference in control RNA synthesis with UTP was noted when thioglycerol replaced mercaptoethanol.
of Hg-UTP or Hg-CTP with mercaptoethanol or thioglycerol as the mercaptan are shown in Fig. 3. With either mercaptan, RNA synthesis was less reduced with substitution of Hg-CTP for CTP than it was with Hg-UTP for UTP. Furthermore, thioglycerol permitted considerably greater RNA synthesis than mercaptoethanol in the presence of Hg-CTP. Incorporation was totally dependent on the presence of an active mercaptan and on the presence of all four nucleoside triphosphates. The relative rates of RNA synthesis in the presence of these and other mercaptans with Hg-CTP and Hg-UTP are summarized in Table 1. In general, RNA synthesis was greater with Hg-CTP as the mercurinucleotide. With either mercurinucleotide, however, Table 1. Relative nuclear RNA synthesis in the presence of a mercurinucleotide and various mercaptans
Mercurinucleotide Mercaptan
Hg-UTP
Hg-CTP
Dithiothreitol 2-Mercaptoethanol Thioglycerol Thioglucose Mercaptosuccinic acid 2-Mercaptoethylamine None added
100 33 30 80 10 32
Cell Biology
Globin RNA synthesis in vitro by isolated erythroleukemic cell nuclei: Direct evidence for increased transcription during erythroid differentiation (mercurinucleotides/mercaptans/affinity chromatography/gene regulation)
STUART H. ORKIN* AND PAUL S. SWERDLOW The Division of Hematology-Oncology of the Department of Medicine, Children's Hospital Medical Center, and the Sidney Farber Cancer Institute, and the Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115
Communicated by Arthur B. Pardee, March 31, 1977
Murine erythroleukemic cells accumulate ABSTRACT cytoplasmic globin mRNA during differentiation induced in tissue culture by dimethyl sulfoxide. Cellular accumulation of globin RNA may reflect transcriptional activation of the globin genes and/or posttranscriptional stabilization of globin RNA during differentiation. To evaluate possible transcriptional controls directly, globin RNA synthesis by isolated erythroleukemic cell nuclei was studied. Conditions were established for optimal nuclear RNA synthesis in vitro in the presence of a mercurinucleotide (Hg-CTP). Mercurated RNA synthesized in vitro was purified free of endogenous RNA by affinity chromatography on sulfhydrylSepharose, and analyzed for the presence of newly synthesized globin RNA sequences by molecular hybridization to globin complementary [32PJDNA. The results demonstrate markedly increased synthesis of globin RNA by nuclei isolated from dimethyl sulfoxide-treated cells, even within 5 min of nuclear transcription in vitro. These findings are most consistent with transcriptional activation of the globin genes upon induction of differentiation.
Murine erythroleukemic cells capable of differentiation in vitro response to various inducers (1) constitute an important, well-characterized system for the study of the cellular and in
molecular events associated with erythrocyte development. When induced with dimethyl sulfoxide, these cells accumulate substantial quantities of cytoplasmic globin messenger RNA (2-4). Whether this reflects transcriptional activation of the globin genes upon induction, posttranscriptional stabilization of globin RNA sequences, or a combination of these mechanisms, is unresolved. In this report we provide direct evidence for transcriptional activation of the globin genes in induced cells by study of globin RNA synthesis by isolated nuclei. Newly synthesized RNA made by isolated nuclei in vitro in the presence of a mercurinucleotide (5, 6) and an appropriate mercaptan was purified free of endogenous RNA by affinity chromatography (7) and analyzed for the presence of globin RNA sequences by hybridization to globin complementary DNA (cDNA). The results demonstrate increased synthesis of globin RNA in nuclei isolated from induced erythroleukemic cells. MATERIALS AND METHODS
Cell Culture. Murine erythroleukemic cells of a long-term stock of T3C12 (2) capable of extensive differentiation in response to dimethyl sulfoxide (3, 4) were maintained in Dulbecco's modified Eagle's medium containing 10% heatinactivated fetal calf serum. Erythroid maturation was induced passage
Abbreviations: cDNA, complementary DNA; Hg-RNA, mercurated RNA. * To whom all correspondence should be addressed. 2475
with 1.5% (vol/vol) dimethyl sulfoxide as previously described
(4).
Isolation of Nuclei. Washed cell pellets were suspended in 0.3 M sucrose (Schwarz/Mann ultrapure) containing 2 mM Mg(OAc)2, 3 mM CaCI2, 10 mM Tris-HCl at pH 8.0, and 2 mM 2-mercaptoethanol. After addition of Triton X-100 to 0.25%, the cell suspension was stirred on a vortex mixer for 30 sec. The crude nuclei were gently sedimented at 1000 X g for 5 min at 40, and then resuspended in the same buffer minus Triton X100. An equal volume of 2 M sucrose containing 5 mM Mg(OAc)2, 10 mM Tris at pH 8.0, and 2 mM mercaptoethanol was immediately added, and the mixture was homogenized with five strokes of a tight-fitting Teflon-glass homogenizer. Purified nuclei were sedimented through a cushion of 2 M sucrose containing 2 mM Mg(OAc)2, 3 mM CaCl2, and 10 mM Tris at pH 8.0 by centrifugation at 25,000 rpm in a Beckman SW-40 rotor for 45 min at 40. The nuclear pellet was resuspended in 25% (vol/vol) glycerol/5 mM Mg(OAc)2/50 mM Tris at pH 8.0/2 mM mercaptoethanol at 1 to 2 X 108 nuclei per ml, and frozen in liquid nitrogen. RNA Synthesis by Isolated Nuclei. Conditions for RNA synthesis were modified slightly from those described for other systems (8, 9). Each reaction mixture contained 50 mM N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) at pH 8.0, 5 mM Mg(OAc)2, 1 mM MnCl2, 150 mM KCI, 12.5% glycerol, 4 mM phosphoenolpyruvate, pyruvate kinase (3 units/ml), 0.185 mM of three ribonucleoside triphosphates, 0.037 mM of the fourth ribonucleoside triphosphate labeled with 3H, and approximately 5 X 107 rapidly thawed nuclei per ml. Mercurinucleotide, either Hg-CTP or Hg-UTP (P.L. Biochemicals), replaced the corresponding unmodified nucleotide at the same concentration in the reaction mixture when RNA containing mercury (Hg-RNA) was prepared. Mercaptans, as noted below, were present at 10 mM final concentration in the reaction mixture. The reaction mixture was incubated at 230 with intermittent, gentle shaking and aliquots were withdrawn at appropriate times, spotted on Whatman 3MM paper, and processed as described by Ernest et al. (9) to monitor RNA synthesis. No adjustment was made for background cpm at time zero. Preparative Isolation of RNA after Nuclear Synthesis. After incubation, nuclei were diluted 10-fold with 20 mM Tris at pH 7.5/50 mM NaCl/1 mM EDTA/1% sodium dodecyl sulfate and extracted vigorously in a vortex mixer with an equal volume of 600 phenol previously equilibrated with Tris buffer. The aqueous phase was reextracted three times with chloroform/isoamyl alcohol (24:1 vol/vol), and precipitated with ethanol. In early experiments samples were treated with RNase-free DNase and reprecipitated prior to affinity chromatography. In more recent experiments DNase treatment was
2476
Cell Biology: Orkin and Swerdlow
Proc. Nati. Acad. Sci. USA 74 (1977)
16
101 0
14-
L
0.
cm8
0
12
x
E
0.
10
0. Q)
~~~0~
0
0
E.1
0
0~
a.
Q-
HS
2
0
c.1020
3
0
4
0 2 0
10 30 20 Time, min
FIG. 1. Time course of nuclear RNA synthesis in vitro. Control erythroleukemic cell nuclei were incubated in the complete reaction mixture with [3H]UTP as the labeled ribonucleoside triphosphate. Incorporation at 30 min was 30.6 pmol of UTP per 106 nuclei.
omitted without any consequence with respect to purification of the mercurated RNA. Affinity Purification of Hg-RNA. Sulfhydryl-Sepharose-6B was prepared precisely as described by Dale and Ward (7), who used the method of Cuatrecasas (10). Each batch of sulfhydryl-Sepharose was tested for its ability to bind samples of Hg-RNA and for nonspecific adsorption of nucleic acid by passage of radioactive, nonmercurated RNA. The sulfhydryl supports used in this work had immeasurably low nonspecific binding, less than one part in 105. Prior to each use the Sepharose was briefly washed with 20 mM Tris at pH 7.5/0.1 M NaCl containing 0.1 M mercaptoethanol, and then repeatedly washed with buffer lacking the mercaptan. A 5-ml packed column was equilibrated with buffer containing 0.25% sodium dodecyl sulfate, and the RNA was applied after brief heating at 65° in this buffer. The column was washed with approximately 5 column volumes of this buffer, and then 50 column volumes of buffer lacking sodium dodecyl sulfate. Hg-RNA bound to the column was eluted with addition of buffer containing mercaptan. Fractions containing eluted Hg-RNA were pooled, carrier Escherichta coli tRNA (100 ,g) was added, and the RNA was precipitated with ethanol. Precipitated RNA was resuspended in water and frozen in liquid nitrogen prior to molecular hybridization. Preparation of Globin mRNA and cDNA. Pure mouse globin mRNA was prepared from ex-breeder CD-1 mice (Charles River Breeding Laboratories) by oligo(dT)-cellulose chromatography and sucrose gradient centrifugation as previously described (4). Globin cDNA was synthesized in 25 Al using 50 mM KCI; 50 mM Tris at pH 8.3; 8 mM MgCl2; 10 mM dithiothreitol; 1 mM each dATP, dCTP, and dTTP; 40 jig of oligo(dT) per ml; 100 jig of actinomycin D per ml; 150 MCi of [32P]dGTP (115-135 Ci/mmol, New England Nuclear); 1 mg of mRNA; and approximately 10 units of RNA-dependent DNA polymerase (reverse transcriptase) from avian myeloblastosis virus at 370 for 30 min. cDNA was purified as before (4). The specific activity of cDNA was approximately 1.5 X 108
cpm/Ag.
24
48
72
Time in Me2SO, hr
Molecular Hybridization. Cytoplasmic RNA-cDNA hybridization for quantitation of accumulated globin RNA was
FIG. 2. Nuclear RNA synthesis by nuclei isolated from dimethyl sulfoxide-treated cells. Nuclei were prepared from cells treated 49 hr and 72 hr with dimethyl sulfoxide (Me2SO) and compared with control nuclei with respect to in vitro RNA synthesis. Values are expressed as pmol of UTP incorporated per 106 nuclei over 5-min incubation.
performed as before (4). Pure globin RNA hybridized with [32P]cDNA with RNA concentration X time for 50% hybridization, Crtl/2 = 5 X 10-4 sec X moles of nucleotide per liter. 3H-Labeled Hg-RNA samples were incubated at 650 for 24 hr in a 10-Ml total volume containing 0.6 M NaCl, 20 mM Tris at pH 7.5, an additional 5 Mg of E. coli tRNA, and 3-5 pg of [32P]cDNA. A standard reference curve of the hybridization of pure globin RNA with cDNA was determined in parallel with each assay. After incubation for 24 hr, the extent of hybridization of the [32P]cDNA was assayed by digestion with Si nuclease (4). RESULTS RNA Synthesis by Erythroleukemic Cell Nuclei. Isolated nuclei from control erythroleukemic cells were active in RNA synthesis in vntro (Fig. 1). Incorporation of [3HIUTP continued for 60 min and averaged 30 pmol per 106 nuclei over the initial 30-min period, similar to that observed in other efficient nuclear systems (8, 9, 11, 12). Incorporation was sensitive to inhibition by pancreatic RNase or actinomycin D (not shown) and dependent on the presence of all four nucleoside triphosphates (see below). Nuclei isolated from dimethyl sulfoxide-treated erythroleukemic cells were less active than those from uninduced cells (Fig. 2), consistent with the progressive reduction in total RNA synthesis observed in induced cell cultures (13). Synthesis of Hg-RNA. Incorporation of mercurinucleotides into RNA or DNA by E. coli polymerases is totally dependent on the presence of a mercaptan to act as a mercury ligand (5). There is considerable specificity with respect to the appropriate mercaptan for a given polymerase (5). Analysis of the mercaptan requirements of eukaryotic polymerases has not been reported in detail. Smith and Huang (11) observed that RNA synthesis by myeloma nuclei was approximately 60% of control rates when Hg-UTP was used in place of UTP in the presence of mercaptoethanol, but less than 10% in the presence of dithiothreitol or in the absence of any mercaptan. Our initial experiments with erythroleukemic cell nuclei, Hg-UTP, and mercaptoethanol demonstrated more limited RNA synthesis relative to controls and prompted closer examination of the mercaptan requirements in this eukaryotic system. Representative results for RNA synthesis in the presence
Cell Biology: Orkin and Swerdlow .
Proc. Natl. Acad. SCt. USA 74 (1977)
2477
A
8
oa0
7
I
2
D-a1) 4-
T
CA
o
o4 x
,-
c x'
CL-
g' 5
E
0'- E j a.
a, u
mE
'aI
I
co20 10
20
10
20
Time, mmn
FIG. 3. Mercaptan requirements for nuclear RNA synthesis in the presence of mercurinucleotide. (A) RNA synthesis in the presence of: CTP plus 10 mM mercaptoethanol (0); Hg-CTP plus 10 mM thioglycerol (0); Hg-CTP plus 10 mM mercaptoethanol (A); Hg-CTP without added mercaptan (1); neither CTP nor Hg-CTP (X). Synthesis was monitored by incorporation of [3HJUTP. No difference in control RNA synthesis with CTP was noted with 10 mM thioglycerol replacing 10 mM mercaptoethanol. (B) RNA synthesis in the presence of: UTP plus 10 mM mercaptoethanol (0); Hg-UTP plus 10 mM mercaptoethanol (A); Hg-UTP plus 10 mM thioglycerol (0); Hg-UTP without added mercaptan (3). Incorporation of [3H]GTP was used to monitor RNA synthesis. No difference in control RNA synthesis with UTP was noted when thioglycerol replaced mercaptoethanol.
of Hg-UTP or Hg-CTP with mercaptoethanol or thioglycerol as the mercaptan are shown in Fig. 3. With either mercaptan, RNA synthesis was less reduced with substitution of Hg-CTP for CTP than it was with Hg-UTP for UTP. Furthermore, thioglycerol permitted considerably greater RNA synthesis than mercaptoethanol in the presence of Hg-CTP. Incorporation was totally dependent on the presence of an active mercaptan and on the presence of all four nucleoside triphosphates. The relative rates of RNA synthesis in the presence of these and other mercaptans with Hg-CTP and Hg-UTP are summarized in Table 1. In general, RNA synthesis was greater with Hg-CTP as the mercurinucleotide. With either mercurinucleotide, however, Table 1. Relative nuclear RNA synthesis in the presence of a mercurinucleotide and various mercaptans
Mercurinucleotide Mercaptan
Hg-UTP
Hg-CTP
Dithiothreitol 2-Mercaptoethanol Thioglycerol Thioglucose Mercaptosuccinic acid 2-Mercaptoethylamine None added
100 33 30 80 10 32
