Volume 7 Number 1 1979
Nucleic Acids Research
Nonrepetitive DNA transcription in normal and regenerating rat liver
Leo J.Grady, Wayne P.Campbell and Arlene B.North Division of Laboratories and Research, New York State Department of Health, Empire State Plaza, Albany, NY 12201, USA
Received 12 April 1979 ABSTRACT
Initial RNA excess hybridization experiments employing total cell RNA and the complete complement of nonrepetitive DNA sequences showed no differences between normal and regenerating rat liver. However, when the DNA from the RNADNA hybrids was isolated and then reacted with homologous and heterologous RNAs the sensitivity of the assay was sufficiently improved to reveal that some of the nonrepetitive DNA transcripts present in normal liver are missing at 24 h and 48 h after a 70% partial hepatectomy. Additional experiments showed that while some of the missing sequences were common to both stages of regeneration, some were also different. The data thus suggest both quantitative and qualitative changes during liver regeneration in the population of RNA molecules transcribed from nonrepetitive DNA. INTRODUCTION
Nucleic acid hybridization techniques have been used by several laboratories to try to determine whether or not changes occur in the RNA species synthesized in liver following a partial hepatectomy. The original competition-hybridiza-
tion experiments, the results of which are now known to pertain only to repetitive DNA, produced conflicting results. Whereas Church and McCarthy obtained data from mice suggesting the synthesis of new classes of RNA molecules at various times during the regenerative process, Drews and Brawerman2 detected no differences in the RNA molecules present in normal and 12-h regenerating rat liver. The latter results have received recent support from an investigation by Greene and Fausto3 who employed saturation-hybridization experiments and found no evidence for large scale changes in repetitive DNA transcription at either 3 h or 6 h after partial hepatectomy. With regard to nonrepetitive DNA transcription, the saturation-hybridization data of Tedeschi et al. show that approximately 6% of these sequences are represented in the nuclear RNA of both normal and 12-h regenerating rat liver. Furthermore, these investigators were unable to detect any qualitative differences between the RNA species present in the two populations. In an earlier study employing cDNA, Colbert et al.5 observed no difference between normal and 0 Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
259
Nucleic Acids Research 12-h regenerating rat liver in the complexity of the polysomal poly(A+) RNA. However, in this case the actual sequences present appeared to differ by as much as 10 to 14%. One of the difficulties in studying nonrepetitive DNA transcription is that the proportion of these sequences transcribed is relatively small (usually less than 10%). Since the reproducibility of the experiments is seldom better than ± 0.5% and often closer to ± 1%, transcriptional changes equivalent to 1% or less of the nonrepetitive DNA are difficult to detect reliably. At the same time, 1% of rat nonrepetitive DNA contains enough information to code for approximately 20,000 average size proteins and thus a transcriptional change of this magnitude might reasonably be expected to have significant consequences in a cell. At present, the only approach to increasing the sensitivity of the hybridization assay that provides information on both the poly(A ) and poly(A ) RNA fractions involves isolating the DNA from total RNA-DNA hybrids and then using this DNA in further reactions with homologous and heterologous RNAs. When applied to liver, where about 6% of the nonrepetitive DNA is transcribed, the level of discrimination achieved can be increased better than tenfold. The present communication describes experiments in which this approach was successfully employed to reveal differences in nonrepetitive DNA transcription not only between sham-operated and regenerating liver, but also in regenerating liver at two different times following partial hepatectomy. MATERIALS AND METHODS Animals. Male, Fisher 344 rats were obtained from Charles River Laboratories. Prior to use they were held for at least ten days on a 12 h light-12 h dark schedule with food (Wayne Lab-Blox F6) and water always available. The animals were 120 days old when used. All surgical procedures were initiated 2 h before the end of a light cycle. RNA Purification. Rats were subjected either to a sham operation or to a 70% partial hepatectomy under light ether anesthesia. At 2.5 h and 48 h posthepatectomy they were again anesthetized with ether and the liver removed into ice-cold buffer (0.01 M sodium acetate, pH 5.0, containing 0.1 M NaCl and 125 pg/ml heparin). The livers from 3 to 6 rats were pooled for each RNA extraction. After two washeswith cold buffer, the livers were disrupted with a Virtis S-23 homogenizer for 1 min at a setting of 60. The homogenate was made 0.5% with sodium lauryl sulfate (SLS) and the RNA purified by hot phenol extraction as previously described.6 In this instance, the DNase used in RNA purification was Sigma DN Cl and was first passed over agarose-UMP and then treated
260
Nucleic Acids Research with bentonite to remove residual RNase.7 Glycogen was eliminated using a-amylase which had been treated with bentonite to remove contaminating RNase.8 At least one sample of RNA was also prepared for each condition using the CsCl technique of Chikaraishi et al.9 In Vitro Labeling of DNA. Unlabeled rat liver DNA was isolated from A Cot 20,000 nondisrupted tissue by the procedure of McCarthy and Hoyer. The in repetitive DNA fraction was prepared as in an earlier publication. vitro labeling reaction contained, in 0.5 ml of phosphate buffer, pH 7.6:
7.5 pg of Cot 20,000 DNA, 2 x 10 M each of dATP, dGTP, dCTP (P-L Biochemicals), 0.01 M MgCl2, 0.5 mCi 3H-TTP (New England Nuclear, sp. act. = 90 to 100 Ci/ mmole), 40 units of E. coli DNA polymerase I (Worthington Biochemical, Lot No. 07N88 or No. 08F31). The reaction was assembled at 00C, then incubated at 12 0C. The extent of label incorporation was followed by diluting one microliter aliquot into 0.20 ml 0.25 M EDTA containing 50 pg of unlabeled salmon DNA and then passing it over a column of Sephadex G-50 coarse equilibrated with 0.03 M PB (PB = phosphate buffer containing equimolar amounts of Na2HP04 and NaH2P04 The counts eluting in the void volume gave an indication of the amount of labeling which had taken place. This procedure is quite rapid and can be completed within 30 min. When the amount of label incorporated was equivalent to 50% substitution (the actual time varied from 6 to 24 h depending on the lot of enzyme used), the reaction was terminated by adding 0.24 ml, 0.25 M EDTA and heating at 680C for 10 min. After adding 50 pg of sheared, unlabeled E. coli DNA the reaction was passed over a column of Sephadex G-50 fine equilibrated with 0.03 M PB and the material eluting in the void volume was collected. This material was heated in a boiling water bath for 5 min, quickly cooled in an ice bath, and passed at room temperature over a 1 cc column of hydroxylapatite (Bio-Rad, DNA grade) equilibrated with 0.03 M PB. The column was washed three times with 1 ml of 0.12 M PB at room temperature. The temperature of the column was then raised to 600C and single-strand DNA eluted by washing once with 1.0 ml 0.12 M PB + 0.1% SLS and nine times with 1.0 ml of 0.12 M PB without SLS. The specific activity of the final DNA preparations was 1.5 to 2.0 x 107 cpm/pug. Isolation of DNA Transcribed in Rat Liver. The general approach to isolating the nonrepetitive DNA sequences transcribed in rat liver was similar
employed by Galau et al. with the sea urchin. Total liver RNA (1.2 mg) was mixed with in vitro labeled nonrepetitive DNA (1.2 pg) in 0.1 ml of 0.4 M PB containing 0.01 M EDTA and 0.1% SLS. The mixture was layered with paraffin oil, heated in a boiling water bath for 2 min and then incubated at 600C for 120 h. At this time, the sample was diluted to 0.12 M PB at 600C and singleto that
261
Nucleic Acids Research strand DNA separated from RNA-DNA and DNA-DNA hybrids on hydroxylapatite as described elsewhere.6 To remove SLS, the fraction containing the nucleic acid hybrids was passed over Sephadex G-25 equilibrated with 0.12 M PB, then RNA was eliminated by treating with 50 pg/ml of RNase (heat-treated before use to inactivate DNase) for 10 h at 550C. Next, SLS was added to 0.05% and the solution extracted with an equal volume of chloroform-isoamyl alcohol (24:1, v/v). The aqueous layer was passed over hydroxylapatite at 600C to separate the DNA rendered single-strandby the RNase treatment from the DNA-DNA hybrids. The single-strand DNA was concentrated, treated briefly with-proteinase K (Beckman Instrument Company) to remove any trace of RNase, and incubated a second time with RNA exactly as described above. The RNA-DNA hybrids were again separated from single-strand DNA on hydroxylapatite at 600C and the RNA eliminated by bringing the solution to 0.3 N NaOH for 90 min. After neutralization, the DNA recovered was used as the DNA expressed in liver (E-DNA) under the conditions represented by the RNA employed in the hybridization reactions. Hybridization Conditions. RNA-DNA reactions used 1.2 mg of RNA and 0.001 lig of in vitro labeled DNA (either E-DNA or total nonrepetitive DNA, depending on the experiment) in 0.1 ml of 0.4 M PB containing 0.01 M EDTA and 0.1% SLS. The mixtures were incubated at 600C in polypropylene micro test tubes (Bio-Rad) and were layered with paraffin oil to prevent evaporation. The tubes were rinsed with sterile distilled water containing 0.1% diethylpyrocarbonate before use. At various times, 15 pl samples were removed, diluted to 0.12 M PB + 0.1% SLS at 600C and passed over hydroxylapatite at 600C. The procedure for determining the distribution of radioactivity between single-strand and hybridized DNA has been published.13 Control reactions containing DNA alone never showed significant reaction (Cot = 210) of unlabeled DNA in mixtures with 0.5 pg/ml of sheared, unfractionated DNA labeled in vivo with The first two reactions contained 0.12 M PB and 0.01 M EDTA and the latter 0.9 M PB and 0.01 M EDTA. At selected times, samples were diluted to 0.12 M PB + 0.1% SLS and the degree of reassociation assayed on hydroxylapatite at 60 0C. The nature of the in vitro labeled expressed DNA isolated after two incubations with RNA was ascertained in a similar manner, but with 0.01 lig/ml of E-DNA as the labeled component.
3H-thymidine.
RESULTS
Rat Liver DNA. The reassociation of total rat DNA is illustrated in Figure 1. About 70% of the DNA is comprised of nonrepetitive sequences which = 2,600. These results agree very well with those of Colbert et have a Cot
al.5 Proportion of Nonrepetitive DNA Transcribed. The extent to which nonrepetitive DNA is transcribed in the liver of sham-operated rats and in regenerating liver at 2.5 and 48 h after a 70% partial hepatectomy is shown in Figure 2. Although the data presented were all obtained using DNA labeled in vitro, no difference was observed in similar experiments using nonrepetitive That in vitro labeled DNA is sequences isolated from in vivo labeled DNA.
In,
100
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80-
2
60
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40 Lu
Nucleic Acids Research
Nonrepetitive DNA transcription in normal and regenerating rat liver
Leo J.Grady, Wayne P.Campbell and Arlene B.North Division of Laboratories and Research, New York State Department of Health, Empire State Plaza, Albany, NY 12201, USA
Received 12 April 1979 ABSTRACT
Initial RNA excess hybridization experiments employing total cell RNA and the complete complement of nonrepetitive DNA sequences showed no differences between normal and regenerating rat liver. However, when the DNA from the RNADNA hybrids was isolated and then reacted with homologous and heterologous RNAs the sensitivity of the assay was sufficiently improved to reveal that some of the nonrepetitive DNA transcripts present in normal liver are missing at 24 h and 48 h after a 70% partial hepatectomy. Additional experiments showed that while some of the missing sequences were common to both stages of regeneration, some were also different. The data thus suggest both quantitative and qualitative changes during liver regeneration in the population of RNA molecules transcribed from nonrepetitive DNA. INTRODUCTION
Nucleic acid hybridization techniques have been used by several laboratories to try to determine whether or not changes occur in the RNA species synthesized in liver following a partial hepatectomy. The original competition-hybridiza-
tion experiments, the results of which are now known to pertain only to repetitive DNA, produced conflicting results. Whereas Church and McCarthy obtained data from mice suggesting the synthesis of new classes of RNA molecules at various times during the regenerative process, Drews and Brawerman2 detected no differences in the RNA molecules present in normal and 12-h regenerating rat liver. The latter results have received recent support from an investigation by Greene and Fausto3 who employed saturation-hybridization experiments and found no evidence for large scale changes in repetitive DNA transcription at either 3 h or 6 h after partial hepatectomy. With regard to nonrepetitive DNA transcription, the saturation-hybridization data of Tedeschi et al. show that approximately 6% of these sequences are represented in the nuclear RNA of both normal and 12-h regenerating rat liver. Furthermore, these investigators were unable to detect any qualitative differences between the RNA species present in the two populations. In an earlier study employing cDNA, Colbert et al.5 observed no difference between normal and 0 Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
259
Nucleic Acids Research 12-h regenerating rat liver in the complexity of the polysomal poly(A+) RNA. However, in this case the actual sequences present appeared to differ by as much as 10 to 14%. One of the difficulties in studying nonrepetitive DNA transcription is that the proportion of these sequences transcribed is relatively small (usually less than 10%). Since the reproducibility of the experiments is seldom better than ± 0.5% and often closer to ± 1%, transcriptional changes equivalent to 1% or less of the nonrepetitive DNA are difficult to detect reliably. At the same time, 1% of rat nonrepetitive DNA contains enough information to code for approximately 20,000 average size proteins and thus a transcriptional change of this magnitude might reasonably be expected to have significant consequences in a cell. At present, the only approach to increasing the sensitivity of the hybridization assay that provides information on both the poly(A ) and poly(A ) RNA fractions involves isolating the DNA from total RNA-DNA hybrids and then using this DNA in further reactions with homologous and heterologous RNAs. When applied to liver, where about 6% of the nonrepetitive DNA is transcribed, the level of discrimination achieved can be increased better than tenfold. The present communication describes experiments in which this approach was successfully employed to reveal differences in nonrepetitive DNA transcription not only between sham-operated and regenerating liver, but also in regenerating liver at two different times following partial hepatectomy. MATERIALS AND METHODS Animals. Male, Fisher 344 rats were obtained from Charles River Laboratories. Prior to use they were held for at least ten days on a 12 h light-12 h dark schedule with food (Wayne Lab-Blox F6) and water always available. The animals were 120 days old when used. All surgical procedures were initiated 2 h before the end of a light cycle. RNA Purification. Rats were subjected either to a sham operation or to a 70% partial hepatectomy under light ether anesthesia. At 2.5 h and 48 h posthepatectomy they were again anesthetized with ether and the liver removed into ice-cold buffer (0.01 M sodium acetate, pH 5.0, containing 0.1 M NaCl and 125 pg/ml heparin). The livers from 3 to 6 rats were pooled for each RNA extraction. After two washeswith cold buffer, the livers were disrupted with a Virtis S-23 homogenizer for 1 min at a setting of 60. The homogenate was made 0.5% with sodium lauryl sulfate (SLS) and the RNA purified by hot phenol extraction as previously described.6 In this instance, the DNase used in RNA purification was Sigma DN Cl and was first passed over agarose-UMP and then treated
260
Nucleic Acids Research with bentonite to remove residual RNase.7 Glycogen was eliminated using a-amylase which had been treated with bentonite to remove contaminating RNase.8 At least one sample of RNA was also prepared for each condition using the CsCl technique of Chikaraishi et al.9 In Vitro Labeling of DNA. Unlabeled rat liver DNA was isolated from A Cot 20,000 nondisrupted tissue by the procedure of McCarthy and Hoyer. The in repetitive DNA fraction was prepared as in an earlier publication. vitro labeling reaction contained, in 0.5 ml of phosphate buffer, pH 7.6:
7.5 pg of Cot 20,000 DNA, 2 x 10 M each of dATP, dGTP, dCTP (P-L Biochemicals), 0.01 M MgCl2, 0.5 mCi 3H-TTP (New England Nuclear, sp. act. = 90 to 100 Ci/ mmole), 40 units of E. coli DNA polymerase I (Worthington Biochemical, Lot No. 07N88 or No. 08F31). The reaction was assembled at 00C, then incubated at 12 0C. The extent of label incorporation was followed by diluting one microliter aliquot into 0.20 ml 0.25 M EDTA containing 50 pg of unlabeled salmon DNA and then passing it over a column of Sephadex G-50 coarse equilibrated with 0.03 M PB (PB = phosphate buffer containing equimolar amounts of Na2HP04 and NaH2P04 The counts eluting in the void volume gave an indication of the amount of labeling which had taken place. This procedure is quite rapid and can be completed within 30 min. When the amount of label incorporated was equivalent to 50% substitution (the actual time varied from 6 to 24 h depending on the lot of enzyme used), the reaction was terminated by adding 0.24 ml, 0.25 M EDTA and heating at 680C for 10 min. After adding 50 pg of sheared, unlabeled E. coli DNA the reaction was passed over a column of Sephadex G-50 fine equilibrated with 0.03 M PB and the material eluting in the void volume was collected. This material was heated in a boiling water bath for 5 min, quickly cooled in an ice bath, and passed at room temperature over a 1 cc column of hydroxylapatite (Bio-Rad, DNA grade) equilibrated with 0.03 M PB. The column was washed three times with 1 ml of 0.12 M PB at room temperature. The temperature of the column was then raised to 600C and single-strand DNA eluted by washing once with 1.0 ml 0.12 M PB + 0.1% SLS and nine times with 1.0 ml of 0.12 M PB without SLS. The specific activity of the final DNA preparations was 1.5 to 2.0 x 107 cpm/pug. Isolation of DNA Transcribed in Rat Liver. The general approach to isolating the nonrepetitive DNA sequences transcribed in rat liver was similar
employed by Galau et al. with the sea urchin. Total liver RNA (1.2 mg) was mixed with in vitro labeled nonrepetitive DNA (1.2 pg) in 0.1 ml of 0.4 M PB containing 0.01 M EDTA and 0.1% SLS. The mixture was layered with paraffin oil, heated in a boiling water bath for 2 min and then incubated at 600C for 120 h. At this time, the sample was diluted to 0.12 M PB at 600C and singleto that
261
Nucleic Acids Research strand DNA separated from RNA-DNA and DNA-DNA hybrids on hydroxylapatite as described elsewhere.6 To remove SLS, the fraction containing the nucleic acid hybrids was passed over Sephadex G-25 equilibrated with 0.12 M PB, then RNA was eliminated by treating with 50 pg/ml of RNase (heat-treated before use to inactivate DNase) for 10 h at 550C. Next, SLS was added to 0.05% and the solution extracted with an equal volume of chloroform-isoamyl alcohol (24:1, v/v). The aqueous layer was passed over hydroxylapatite at 600C to separate the DNA rendered single-strandby the RNase treatment from the DNA-DNA hybrids. The single-strand DNA was concentrated, treated briefly with-proteinase K (Beckman Instrument Company) to remove any trace of RNase, and incubated a second time with RNA exactly as described above. The RNA-DNA hybrids were again separated from single-strand DNA on hydroxylapatite at 600C and the RNA eliminated by bringing the solution to 0.3 N NaOH for 90 min. After neutralization, the DNA recovered was used as the DNA expressed in liver (E-DNA) under the conditions represented by the RNA employed in the hybridization reactions. Hybridization Conditions. RNA-DNA reactions used 1.2 mg of RNA and 0.001 lig of in vitro labeled DNA (either E-DNA or total nonrepetitive DNA, depending on the experiment) in 0.1 ml of 0.4 M PB containing 0.01 M EDTA and 0.1% SLS. The mixtures were incubated at 600C in polypropylene micro test tubes (Bio-Rad) and were layered with paraffin oil to prevent evaporation. The tubes were rinsed with sterile distilled water containing 0.1% diethylpyrocarbonate before use. At various times, 15 pl samples were removed, diluted to 0.12 M PB + 0.1% SLS at 600C and passed over hydroxylapatite at 600C. The procedure for determining the distribution of radioactivity between single-strand and hybridized DNA has been published.13 Control reactions containing DNA alone never showed significant reaction (Cot = 210) of unlabeled DNA in mixtures with 0.5 pg/ml of sheared, unfractionated DNA labeled in vivo with The first two reactions contained 0.12 M PB and 0.01 M EDTA and the latter 0.9 M PB and 0.01 M EDTA. At selected times, samples were diluted to 0.12 M PB + 0.1% SLS and the degree of reassociation assayed on hydroxylapatite at 60 0C. The nature of the in vitro labeled expressed DNA isolated after two incubations with RNA was ascertained in a similar manner, but with 0.01 lig/ml of E-DNA as the labeled component.
3H-thymidine.
RESULTS
Rat Liver DNA. The reassociation of total rat DNA is illustrated in Figure 1. About 70% of the DNA is comprised of nonrepetitive sequences which = 2,600. These results agree very well with those of Colbert et have a Cot
al.5 Proportion of Nonrepetitive DNA Transcribed. The extent to which nonrepetitive DNA is transcribed in the liver of sham-operated rats and in regenerating liver at 2.5 and 48 h after a 70% partial hepatectomy is shown in Figure 2. Although the data presented were all obtained using DNA labeled in vitro, no difference was observed in similar experiments using nonrepetitive That in vitro labeled DNA is sequences isolated from in vivo labeled DNA.
In,
100
z
tpA
80-
2
60
z
40 Lu
