Cell, Vol. 12,167-174,
September
1977, Copyright
0 1977 by MIT
Synthesis of Histone Messenger during the Cell Cycle Marialuisa Melli, Giovanni lnstitut fur Molekularbiologie der Universitat Zurich Winterthurerstrasse 266A 8057 Zurich, Switzerland
Spinelli II
and Eva Arnold
Summary Hybridization of HeLa cell RNA to the DNA of a recombinant phage containing sea urchin histone genes shows that histone mRNA of HeLa cells is synthesized throughout the entire cell cycle. Furthermore, histone messenger is synthesized in cells treated with cytosine arabinoside, an inhibitor of DNA duplication. Although histone RNA is found in the nucleus, the amount of cytoplasmic histone messenger is drastically reduced, in agreement with previous work. The absence of coupling between histone mRNA synthesis and DNA duplication shows that the regulatory process which determines the presence or absence of histone mRNA in the cytoplasm of HeLa cells is not at the transcriptional level. Introduction Several lines of evidence have suggested a coupling between histone mRNA synthesis and DNA duplication. The study of the synthesis of histone proteins in synchronized HeLa cells has shown that these proteins are synthesized exclusively during the S period of the cell cycle (Robbins and Borun, 1967; Gallwitz and Mueller, 1969; Butler and Mueller, 1973). An exception is found during the early developmental stages of amphibians and in sea urchin and amphibian oocytes (Cognetti, Spinelli and Vivoli, 1974; Adamson and Woodland, 1974). In oocytes, histone proteins are synthesized in the absence of DNAduplication. In fertilized amphibian eggs, histone proteins are synthesized in larger quantities than required by the newly synthesized DNA, and the excess histone proteins are stored. In agreement with the protein synthetic activity, histone mRNA is found in the cytoplasm of cells during the duplication of DNA. Inhibition of DNA duplication with cytosine arabinoside or hydroxyurea is accompanied by the decay of preexisting cytoplasmic histone mRNA (Robbins and Borun, 1967; Gallwitz and Mueller, 1969; Butler and Mueller, 1973). Under these conditions, no further accumulation of newly synthesized histone mRNA is observed. The cytoplasmic decay of histone mRNA of HeLa cells is rapid and it is associated with the end of histone protein synthesis (Gallwitz, 1975). Perry and Kelley (1973) have calculated a lifetime of ap-
RNA of HeLa Cells
proximately 11 hr for histone mRNA of mouse cells. This figure is in fairly good agreement with the length of the S period during the cell cycle. Although cytoplasmic histone mRNA and its products of translation have been thoroughly studied, little information is available on the synthesis of histone mRNA. In general, it has been assumed that the absence of histone mRNA in the cytoplasm of cells is due to a lack of synthesis of the RNA itself. This deduction seemed to be confirmed by experiments in which the availability of the histone genes for transcription during the cell cycle was studied in vitro. In these experiments, HeLa cell chromatin, native or reconstituted, was transcribed with E. coli RNA polymerase, and the RNA product was analyzed for the presence of histone mRNA (Stein et al., 1975). In this work, we have analyzed the synthesis of histone mRNA of HeLa cells in different periods of the cell cycle and after inhibition of DNA duplication with cytosine arabinoside. Histone mRNA sequences were detected by hybridization of histone gene DNA of sea urchin to poly(A)HeLa cell RNA (Melli et al., 1977). The results show that histone mRNA is synthesized throughout the cell cycle. RNA synthesis is not coupled to DNA duplication. In agreement with the published data, histone mRNA is found in the cytoplasm of HeLa cells only during the period of DNA synthesis. Results Hybridization of Ah22 DNA to RNA of HeLa Cells Fractionated on a Sucrose Density Gradient We have previously shown that histone mRNA of HeLa cells can be found as part of RNA of high molecular weight which probably is the first product of transcription of the histone genes (Melli et al., 1977). If the histone genes of HeLa cells are expressed only during the S period, high molecular weight histone RNA should be found only during the period of DNA duplication. To establish whether the synthesis of high molecular weight histone RNA is coupled to DNA duplication, we have analyzed HeLa cell RNA from synchronized cultures throughout the cell cycle. Histone mRNA sequences were detected by cross-hybridization of HeLa cell RNA to Ah22 DNA, a phage containing cloned Psammechinus miliaris sea urchin histone genes (Clarkson et al., 1976; Schaffner et al., 1976; Gross et al., 1976; Portmann, Schaffner and Birnstiel, 1976). HeLa cells synchronized by double thymidine block were labeled with 3H-uridine (Amersham; >20 Ci/mmole) during 60 min of incubation at 2, 5, 7, 9 and 12 hr after thymidine release. Cell synchrony was checked by incubating 0.5 ml of the cell culture for 10 min with 1 &i of
Cell 166
3H-thymidine. The incorporation of thymidine in TCA-precipitable counts indicated a peak of DNA synthesis 4-5 hr after thymidine release and an S period of approximately 9 hr. Tritiated thymidine incorporation reached a minimum between 10 and 13 hr after thymidine release. The residual incorporation was 57% of the maximum value observed at the peak of DNA duplication. Total RNA was extracted from the cells and centrifuged in 99% formamide, 2-10% sucrose density gradients. RNA fractions from the gradients were pooled as described in the legend to Figure 1 and passed through an oligo(dT)-cellulose column to separate poly(A)- RNA, which was then used for the hybridization reaction. An equal number of counts from each pool of RNA fractions was hybridized to 1 pg of Ah22 DNA (Melli et al., 1977). We have previously shown that under these conditions, hybridization is in excess of DNA, and that the
i
t
I
45s
285
18s
1 2
0
Figure 1. Hybridization Sucrose 99% Formamide
10 Fraction
20 no.
of Ah22 DNA to RNA Fractions Gradient
of a 2-10%
Total HeLa cell RNA was centrifuged in sucrose-formamide gradients for 22 hr at 39,000 rpm in the SW40 Spinco rotor at 23°C (Melli et al., 1977). The RNA fractions, pooled in seven samples as indicated in the figure, were hybridized to 1 pg of Ah22 DNA loaded on Millipore filters at the final concentration of 1.5 x lo6 cpm/ml, under the conditions described in Experimental Procedures. (- 0-) SH-RNA cpm/pI; the histograms indicate the hybrid radioactivity: the arrows indicate the position of the optical density peaks. The position of the 45s RNA precursor was obtained by cross-hybridizing Xenopus rDNA to HeLa RNA, as previously described (Melli et al., 1977). Total RNA of synchronized ceils labeled for 1 hr with 3H-uridine (a) 9 hr after release from double thymidine block and (b) 12 hr after release from double thymidine block.
amount of hybrid formed is proportionate to the percentage of complementary sequences present in the total population of RNA molecules. Figure 1 shows the results obtained after hybridizing RNA from HeLa cells harvested 9 and 12 hr after thymidine release to Ah22 DNA. The radioactive profile of the two RNA samples is similar to the profile of all other RNAs extracted from HeLa cells at different times of the S period (Melli et al., 1977). The distribution of the hybrid radioactivity is also very similar in all samples tested (data not shown). The same type of experiment was carried out with RNA of cells in which DNA synthesis was blocked with cytosine arabinoside (AraC) (data not shown). The distribution of the hybridization of the RNA fractions across a formamide gradient was similar to that shown in Figure 1, suggesting synthesis of histone RNA in the absence of DNA duplication (see following experiments). The persistence of histone RNA throughout the cell cycle and in the absence of DNA duplication could be explained in at least two ways. Incomplete synchrony of the cells would allow a continuous production of histone mRNA. Under these circumstances, histone mRNA should be found in the cytoplasm as well as in total RNA. Absence of coupling between DNA duplication and histone RNA synthesis would also produce the observed result. In this case, however, histone mRNA should not enter the cytoplasm of the cells: To distinguish between these possibilities, we compared the proportion of histone mRNA sequences present in total and cytoplasmic RNA of cells at different stages of the cell cycle. RNA synthesis in HeLa cells occurs throughout the entire interphase and halts at mitosis. The accumulation of RNA in the nucleus is constant during the entire synthetic period (Zetterberg, 1966), and there is no evidence of major qualitative or quantitative changes of the rate of RNA synthesis (Klevecz and Stubblefield, 1967; Pfeiffer and Tolmach, 1968; Pagoulatos and Darnell, 1970). We have measured the incorporation of 3H-uridine in total cellular RNA and nuclear RNA of cells pulse-labeled for 30 and 60 min (Table 1). The incorporation of radioactivity throughout the cell cycle and after inhibition of DNA synthesis with cytosine arabinoside is rather constant. A similar proportion of polyadenylated RNA is found in all samples tested, suggesting that the overall composition of the RNA population is not drastically changed, although qualitative variations cannot be excluded. In view of this result, we decided to compare the proportion of histone RNA sequences of cells pulse-labeled with 3H-uridine for 1 hr in the presence and in the absence of DNA duplication. The hybridization of a constant amount of hh22 DNA to increasing concentrations of poly(A)HeLa RNA gives a measure of the rela-
Synthesis 169
Table
of Histone
1. SH-Uridine
Hours after Thymidine Release
mRNA
Incorporation
of HeLa
Cells
during
the Cell Cycle
Total Cellular RNA (30 min Pulse) cpm x 1Om6
Poly(A)+
RNA
Nuclear RNA” (60 min Pulse) cpm x 1O-B
0.5
4.8
PoI~(A)~
16.0
4.2
18
4.15
16
4.0
23
0.5 + AraC
3.9
15.7
2
4.0
13.0
5
5.6
13.9
7
5.3
14.0
9
4.4
17.0
12
3.2
13.0
RNA
%
%
a Same RNA as that used in the experiment of Figure 2. HeLa cells synchronized by double thymidine block were washed with balanced salt solution and incubated in Eagle’s minimal essential medium with Eagle’s salts (Gibco, Grand Island, New York) supplemented with 5% each of calf and fetal calf serum. At the indicated times, the cells were concentrated to approximately 4 x lO”/ml and labeled for 30 or 60 min with 200 &i/ml of 3H-uridine (Amersham; 25 Ci/ mMole). When indicated, cytosine arabinoside was added to a final concentration of 40 *g/ml immediately after thymidine release. The nuclei were purified according to Penman (1966). Total RNAand nuclear RNA were extracted as described in Experimental Procedures. The numbers refer to 3H-uridine incorporation in IO6 cells.
tive proportion of histone RNA sequences present in the different cellular compartments during the cell cycle or after inhibition of DNA synthesis. In fact, the amount of hybrid obtained will depend upon the concentration of the histone RNA sequences in the total population of RNA under study (Bishop, 1969). Hybridization of Xh22 DNA with Nuclear RNA of HeLa Cells during and in the Absence of DNA Duplication We first compared the proportion of histone mRNA synthesized in synchronized cells during and in the absence of DNA duplication. It is known that cell synchrony obtained by double thymidine block usually involves approximately 80-85% of the cell population. Although more than 90% of the cells seem to be synchronous using the criterion of 3Hthymidine incorporation, the measure of the mitotic index might show a lower proportion of synchronized cells. To establish whether the same proportion of histone mRNA sequences are found in RNA of cells during DNA synthesis and in the absence of DNA duplication, we hybridized these RNAs to Ah22 DNA. Before hybridization, the RNA was degraded by alkaline treatment to a size of approximately 9s. The degradation of the RNA was carried out to avoid variations in the amount of the RNA-DNA hybrid due to the different size of the complementary RNA molecules rather than to the concentration of the RNA (Melli et al., 1977). Since breakage of the RNA in the cross-hybridization reaction results in a loss of hybrid radioactivity (Melli et al., 1977), the amount of DNA loaded on Millipore filters was increased to compensate for the loss of counts.
Increasing concentrations of nuclear RNA were incubated with duplicate Millipore filters loaded with 5 pg of Ah22 DNA each. The conditions of hybridization were as described in Experimental Procedures. Figure 2 shows the hybridization of Ah22 DNA with poly(A)- nuclear RNA of cells pulselabeled with 3H-uridine for 1 and 12 hr after thymidine release or treated with AraC to inhibit DNA duplication. At the concentrations of RNA used, the reaction is in the linear range. The control RNA seems to contain a slightly higher proportion of histone RNA sequences than the RNA from cells labeled in the absence of DNA duplication. No difference is observed between RNA from cells in G2 and RNA from cytosine arabinoside-treated cells. The proportion of histone mRNA sequences found in nuclear RNA of cells labeled in the absence of DNA duplication is only 19-20% lower than in control cells. Even assuming that 20% of the cells in both cultures were synthesizing DNA, it seems very improbable that they could synthesize 80% of the total mRNA produced by the same number of cells during the S period of the cell cycle. We therefore conclude that histone mRNA represents approximately the same proportion of the total labeled nuclear RNA of HeLa cells both during the period of DNA duplication and in the absence of DNA duplication. Hybridization of Xh22 DNA with Cytoplasmic and Total RNA from AraC-Treated Cells The absence of histone mRNA in the cytoplasm of HeLa cells treated with AraC or with hydroxyurea has been shown by different types of studies. The polysomal RNA of these cells does not contain an appreciable amount of sequences coding for his-
Cell 170
0
2 3H-RNA
J
4
2 3H-RNA Figure DNA
2. Hybridization
of Nuclear
cpm
xlO+ml
3H-RNA
of HeLa
Cells to Ah22
Approximately 9 x IO’ HeLa cells were synchronized by double thymidine block and divided into three equal samples. After thymidine release, one sample was incubated in fresh medium for approximately 30 min. The cells were then concentrated and incubated for 1 hr with 3H-uridine as described in the legend to Table 1, The second sample was incubated in fresh medium for 12 hr after thymidine release and labeled as described above. The third sample was incubated in fresh medium at the end of the double thymidine block with 40 pg/ml of cytosine arabinoside for 30 min. The cells were then labeled for 1 hr with 3H-uridine in a medium containing the same concentration of the drug. At the end of the incubation, the labeled cells were harvested and the nuclei were purified. Total nuclear RNA was extracted from the three different samples. The poly(A)RNA fraction obtained by affinity chromatography was alkali-degraded and hybridized to Ah22 DNA. The hybrid radioactivity refers to 5 pg DNA. RNA of cells labeled for 1 hr with 3H-uridine approximately 30 min after release from a double thymidine block (-•-), 12 hr after release (-0-), during AraC treatment (-W-).
tone proteins in an in vitro cell-free system. Pulselabeled polysomal or total cytoplasmic RNA from cells treated with AraC does not contain labeled 9s sequences which can be identified with histone mRNA (Robbins and Borun, 1967; Gallwitz and Mueller, 1969; Breindl and Gallwitz, 1973; Gallwitz, 1975). Using the technique of cross-hybridization to Xh22 DNA, we have compared the hybridization of histone mRNA obtained from the cytoplasm of AraC-treated cells with that of cytoplasmic histone mRNA from cells during the S period of the cell cycle. Figure 3 shows the results of such an experiment. The slope of the two curves is clearly differ-
4 cpmx10-6/m~
Figure 3. Hybridization of Cytoplasmic from AraC-Treated Cells to Ah22 DNA
and
The
of cytosine arabinoside [This treatment reincorporation into DNA,]
after sulted
cells
were
incubated
with
40 pg/ml
Total
Poly(A)-
RNA
release from double thymidine block. in 99% inhibition
of 3H-thymidine
After 30 min, ‘H-uridine was added and the cells were incubated further for 1 hr. The hybridization was carried out Millipore filters loaded with 2 pg of - Ah22 DNA with concentrations of RNA.
t-8-)
incubating increasing
total poly(A)- RNA + AraC; (-•-) cytoplasmic poly(A)-
RNA + AraC; (-0-) cytoplasmic labeled for 1 hr with 3H-uridine from double thymidine block.
poly(A)RNA from cells pulseapproximately 30 min after release
ent, suggesting that the proportion of cytoplasmic histone mRNA in cells where DNA duplication is inhibited is 20-30 times lower than in control cells. In accordance with the previous experiments, the hybridization of total poly(A)RNA from AraCtreated cells is approximately 5 times higher than the hybridization of cytoplasmic poly(A)- RNA from the same cells. These results show that in agreement with the published work, the presence of histone mRNA in the cytoplasm of HeLa cells is coupled with DNA duplication. Furthermore, they confirm that histone mRNA is synthesized in these cells, although it is not found in the cytoplasm. The absence of cytoplasmic histone messenger further confirms that the histone RNA found in the nuclei cannot be due to incomplete synchronization of the culture. Histone mRNA of Cytosine Arabinoside-Treated Cells after inhibition of Protein Synthesis with Cycloheximide Butler and Mueller (1973) have previously shown that the loss of cytoplasmic histone mRNA of cells
Synthesis 171
of Histone
mRNA
tions is shown in Figure 4. We have consistently observed that cycloheximide treatment inhibits the labeling of the RNA sedimenting in the 18s region of the gradient. This finding is in agreement with the inhibition of rRNA processing observed Pederson and Kumar (1971) in cells treated with cycloheximide. Aliquots of each RNA fraction from both gradients were used for the hybridization to 1 pg of Ah22 DNA loaded onto Millipore filters. The comparison of Figures 4a and 4b shows that de novo synthesized histone mRNA is found in the cytoplasm of cells in which protein synthesis is inhibited after block of DNA duplication. The result is in agreement with the work of Gallwitz (1975), who found protection of polysomal histone mRNA of HeLa cells if the two metabolic drugs were administered together to the cells. This observation suggests that nuclear histone RNA synthesized in the absence of DNA synthesis can enter the cytoplasm if protein synthesis is inhibited. The treatment with cycloheximide might block the synthe-
treated with hydroxyurea can be prevented by inhibition of protein synthesis with cycloheximide. HeLa cells treated with hydroxyurea continue to accumulate cytoplasmic histone mRNA if cycloheximide is added to the cell culture before the administration of hydroxyurea. These investigators have also suggested that inhibition of protein synthesis following the block of DNA synthesis does not restore the presence of histone mRNA in the cytoplasm of HeLa cells. Gallwitz (1975) found that protection of cytoplasmic histone mRNA occurs independent of the time of administration of cycloheximide relative to hydroxyurea. We have repeated this type of experiment using the technique of cross-hybridization as a method of detection of histone mRNA sequences in the cytoplasm of HeLa cells. AraC-treated cells 2 cycloheximide were labeled with 3H-uridine, and cytoplasmic RNA was purified and centrifuged in a 5-20% sucrose density gradient, as described in the legend to Figure 4. The radioactive profile of the two RNA prepara-
L”
Figure
4. Hybridization
of Cytoplasmic
RNA of AraC-Treated
10
”
Fraction Cells
20
no.
* Cycloheximide
to Ah22 DNA
Approximately 6 x IO’ HeLa cells from a synchronized culture were released from double thymidine block and incubated in fresh medium containing 40 pg/ml of cytosine arabinoside. After 20 min of incubation, the culture was divided into two equal aliquots, the ceils were concentrated to approximately 4 x lo8 cells per ml and cycloheximide at a concentration of IO rg/ml was added to one of the samples. After IO min of incubation, both cell cultures were labeled for 1 hr with 3H-uridine as described in Experimental Procedures. Total cytoplasmic RNA was centrifuged in a 5-20% sucrose density gradient containing 0.15 M NaCI, 0.01 M Tris (pH 7.5), 0.5% SDS and 0.005 M EDTAfor 12 hr at 27,000 rpm in an SW40 Spinco rotor at 23°C. The arrows indicate the position of the optical density markers. An aliquot of each RNA fraction from 8-24 (approximately 150 ~1 of 0.5 ml total volume) was hybridized to 1 pg of Ah22 DNA loaded on Millipore filters under the conditions described in Experimental Procedures. The RNA hybridized from each gradient corresponds to equal amounts of 16s optical density at 260 nm. (-0-) and (-•-) 3H-RNA cpm/5 ~1; (-• -) and (-A-) 3H-hybrid cpm. (a) + AraC; (b) + AraC + cycloheximide.
Cell 172
sis of a short-lived protein which specifically destroys or allows the decay of histone mRNA either in the nucleus or in the cytoplasm or in both cellular compartments. Hybridization of Cytoplasmic and Total RNA of Synchronized HeLa Cells 12 Hr after Thymidine Release Synchronized HeLa cells 12 hr after the release from double thymidine block were labeled for 1 hr with 3H-uridine. Total cellular RNA was extracted from half the culture, and cytoplasmic RNA was obtained from the remaining cells. The poly(A)fractions of the two RNA preparations were hybridized to Ah22 DNA as described in the legehd of Figure 5. By increasing the RNA concentration, the proportional hybridization of total cellular RNA is 5 times higher than that of cytoplasmic RNA (Figure 5). The comparison between the RNA-DNA hybridization curves of cytoplasmic RNA from cells in G2 and cells in S phase shows a drastic drop of the hybridization of RNA from G2 cells. The different slope of the two curves suggests that the relative proportion of histone RNA sequences is approximately 14 times higher in the cytoplasm of cells
during S phase as compared with 12 hr after thymidine release. The decrease of the concentration of histone messenger sequences in cells during G2 is less than that found in AraC-treated cells. This can be explained by the incomplete synchronization of the cells which is observed during the G2 period of the cell cycle. Probably cytosine arabinoside is comparatively more effective in producing inhibition of DNA duplication. These results confirm that HeLa cells 12 hr after thymidine release contain a very low concentration of histone mRNA in the cytoplasm and that histone RNA is present in the nucleus. Since cycloheximide was shown to increase the amount of cytopiasmic histone mRNA in AraC-treated cells, we investigated whether the same effect could be observed in cells from the G2 period of the cell cycle. A portion of the synchronized cell culture used for the experiments described above was incubated 12 hr after thymidine release, in a medium containing 10 pg/ml of cycloheximide. After 10 min of incubation at 37”C, 3H-uridine was added to the medium and the cells were labeled for 1 hr. The cytoplasmic poly(A)RNA of these cells was extracted and hybridized to Ah22 DNA. The results are shown in Figure 5. After cycloheximide treatment, the hybridization is approximately 4 times higher than that of RNA from untreated cells. The slope of this curve suggests, however, that the concentration of histone mRNA is approximately a quarter of that found in the cytoplasm of cells actively synthesizing DNA. We conclude that the inhibition of protein synthesis in cells during the G2 period of the cell cycle increases the amount of cytoplasmic histone mRNA. It does not, however, reestablish the same relative concentration of mRNA found in cells during the period of DNA duplication. Discussion
0
2
3H-RNA Figure 5. Hybridization Poly(A)RNA of Cells
4 cpmx10-6/
of Ah22 12 Hr after
ml
DNA to Cytoplasmic Thymidine Release
and
Total
The cells were pulsed for 1 hr with 3H-uridine, and the RNA was extracted and hybridized as described in Experimental Procedures. The hybrid radioactivity refers to 2 pg DNA. (-0-) total poly(A)RNA, 12 hr after thymidine release: (-A-) cytoplasmic poly(A)RNA, 12 hr after thymidine release: (-•-) cytoplasmic poly(A)RNA, 12. hr after thymidine release, labeled with 3H-uridine after inhibition of protein synthesis with cycloheximide; (-W) cytoplasmic poly(A)RNA from cells pulselabeled for 1 hr with 3H-uridine approximately 30 min after release from double thymidine block.
We have previously shown the specificity of the reaction between sea urchin histone genes and HeLa histone mRNA. The same degree of homology observed between HeLa histone mRNA and Echinus esculentus histone genes was found in studying the cross-hybridization of HeLa RNA with Psammechinus miliaris histone DNA sequences. The hybridization occurs only with the sea urchin gene sequences and in particular with the coding regions of the DNA (Melli et al., 1977). The validity of our approach is confirmed by the agreement between our results and the published data. The proportion of histone mRNA sequences found in the cytoplasm of cells labeled with 3H-uridine in the absence of DNA duplication is 5-7% of the control cells, labeled during the S period of the cell cycle. Furthermore, inhibition of protein syn-
Synthesis 173
of Histone
mRNA
thesis with cycloheximide increases the amount of histone mRNA in the cytoplasm of the same cells. Hodge et al. (1969) found that the treatment of HeLa cells with cycloheximide inhibits DNA duplication already 4 min after the addition of the drug to the culture. The coupling between DNA duplication and histone mRNA synthesis was in apparent contradiction with the claim that the inhibition of protein synthesis with cycloheximide restores the synthesis of histone mRNA. The continuous synthesis of histone RNA during the cell cycle offers an explanation for the effect of cycloheximide on HeLa cells. It has previously been shown that HeLa histone mRNA decays rapidly at the end of the S period and that cycloheximide prevents this breakdown. It is probable that the same catabolic mechanism is responsible for the decay of old and new histone mRNA. In other words, histone RNA is synthesized and processed during the entire cell cycle. During G2, the newly synthesized histone RNA molecules might enter the cytoplasm and be destroyed in the same way as the polysomal bound mRNA. The specific breakdown of this messenger might depend upon the synthesis of a short-lived protein and therefore be prevented by cycloheximide treatment. In this case, the regulatory step which quantitates the amount of histone mRNA in the cytoplasm of HeLa cells would be cytoplasmic. We cannot exclude a more complex process of regulation involving both cellular compartments, the nucleus and the cytoplasm. For example, a specific alteration of processing or transport of mRNA from the nucleus into the cytoplasm could stop the entrance of histone mRNA into the cytoplasm after the end of DNA duplication. The finding that histone mRNA synthesis and DNAduplication are not coupled is in disagreement with the conclusion of Stein et al. (1975). There is one major difference between the two types of experimental approaches. The data presented in this work concern the synthesis of histone mRNA in intact HeLa cells, whereas Stein et al. (1975) have been analyzing the synthesis of histone mRNA in vitro. The existence of a nuclease which specifically destroys histone mRNA in certain periods of the cell cycle could perhaps explain the divergent results. This nuclease could be compartmentalized in vivo, but could become free after lysis of the cells and catabolize histone mRNA as it is being synthesized. The problem of the purity of the hybridization probe used for the detection of the mRNA could also come into question. Transcriptional regulation is considered one of the means through which the cell adapts to different physiological conditions. The transcription of histone genes has been often quoted as an example of this type of control. A careful analysis of the
published work shows that there are in fact very few well documented cases of transcriptional control. Our work shows that the controlling step regulating the presence of histone mRNA in the cytoplasm of HeLa cells is not transcriptional and perhaps not even nuclear. Experimental
Procedures
Cell Growth, Cell Synchronization, Labeling of the RNA and RNA Extraction HeLa cells were synchronized, grown and labeled as previously described (Wilson and Melli, 1977). Cell synchrony was checked in each experiment by pulsing with 3H-thymidine aliquots of the cell cultures throughout the cell cycle. After labeling, the cells were washed twice in basic salt solution, and total RNA (Melli et al., 1977) or cytoplasmic RNA was extracted. To purify cytoplasmic RNA, the cells were suspended in RSB [lo mM NaCl, 1.5 mM MgCI, and 10 mM Tris (pH 7.5)] and lysed in Nonidet Sp-40. Nuclei and mitochondriae were removed, and proteins were digested with proteinase K in 2% SDS. The procedure was the same as that described for the extraction of total RNA (Curtis and Weissmann, 1976). Poly(A)RNA was obtained by affinity chromatography through oligo(dT)-cellulose (Collaborative Research). Centrifugation of RNA in 99% Formamide Sucrose Density Gradients The centrifugation was carried out according to Melli et al. (1977). RNA-DNA Hybridization Ah22 DNA was purified according to R. Portmann, G. Sogo. T. Koller and W. Zillig (manuscript in preparation). RNA-DNA hybridization was carried out as described (M. Melli et al., 1977). Millipore filters loaded with 1-5 pg Ah22 DNA were incubated with HeLa cells RNA in 0.2 ml 2 x SSC at 65°C overnight. Duplicate filters carrying approximately 5 pg of bacterial DNA were used to control the background radioactivity which was subtracted from each sample. Acknowledgments We are grateful to Prof. M. Birnstiel for reading the manuscript and providing us with the Ah22 phages (Clarkson et al., 1976). We thank Miss A. Binkert for growing Ah22 phages. G. Spinelli was a recipient of a long-term EMBO fellowship. This work was supported by a grant from the Swiss National Science Foundation. Received
February
25, 1977;
revised
April
19, 1977
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September
1977, Copyright
0 1977 by MIT
Synthesis of Histone Messenger during the Cell Cycle Marialuisa Melli, Giovanni lnstitut fur Molekularbiologie der Universitat Zurich Winterthurerstrasse 266A 8057 Zurich, Switzerland
Spinelli II
and Eva Arnold
Summary Hybridization of HeLa cell RNA to the DNA of a recombinant phage containing sea urchin histone genes shows that histone mRNA of HeLa cells is synthesized throughout the entire cell cycle. Furthermore, histone messenger is synthesized in cells treated with cytosine arabinoside, an inhibitor of DNA duplication. Although histone RNA is found in the nucleus, the amount of cytoplasmic histone messenger is drastically reduced, in agreement with previous work. The absence of coupling between histone mRNA synthesis and DNA duplication shows that the regulatory process which determines the presence or absence of histone mRNA in the cytoplasm of HeLa cells is not at the transcriptional level. Introduction Several lines of evidence have suggested a coupling between histone mRNA synthesis and DNA duplication. The study of the synthesis of histone proteins in synchronized HeLa cells has shown that these proteins are synthesized exclusively during the S period of the cell cycle (Robbins and Borun, 1967; Gallwitz and Mueller, 1969; Butler and Mueller, 1973). An exception is found during the early developmental stages of amphibians and in sea urchin and amphibian oocytes (Cognetti, Spinelli and Vivoli, 1974; Adamson and Woodland, 1974). In oocytes, histone proteins are synthesized in the absence of DNAduplication. In fertilized amphibian eggs, histone proteins are synthesized in larger quantities than required by the newly synthesized DNA, and the excess histone proteins are stored. In agreement with the protein synthetic activity, histone mRNA is found in the cytoplasm of cells during the duplication of DNA. Inhibition of DNA duplication with cytosine arabinoside or hydroxyurea is accompanied by the decay of preexisting cytoplasmic histone mRNA (Robbins and Borun, 1967; Gallwitz and Mueller, 1969; Butler and Mueller, 1973). Under these conditions, no further accumulation of newly synthesized histone mRNA is observed. The cytoplasmic decay of histone mRNA of HeLa cells is rapid and it is associated with the end of histone protein synthesis (Gallwitz, 1975). Perry and Kelley (1973) have calculated a lifetime of ap-
RNA of HeLa Cells
proximately 11 hr for histone mRNA of mouse cells. This figure is in fairly good agreement with the length of the S period during the cell cycle. Although cytoplasmic histone mRNA and its products of translation have been thoroughly studied, little information is available on the synthesis of histone mRNA. In general, it has been assumed that the absence of histone mRNA in the cytoplasm of cells is due to a lack of synthesis of the RNA itself. This deduction seemed to be confirmed by experiments in which the availability of the histone genes for transcription during the cell cycle was studied in vitro. In these experiments, HeLa cell chromatin, native or reconstituted, was transcribed with E. coli RNA polymerase, and the RNA product was analyzed for the presence of histone mRNA (Stein et al., 1975). In this work, we have analyzed the synthesis of histone mRNA of HeLa cells in different periods of the cell cycle and after inhibition of DNA duplication with cytosine arabinoside. Histone mRNA sequences were detected by hybridization of histone gene DNA of sea urchin to poly(A)HeLa cell RNA (Melli et al., 1977). The results show that histone mRNA is synthesized throughout the cell cycle. RNA synthesis is not coupled to DNA duplication. In agreement with the published data, histone mRNA is found in the cytoplasm of HeLa cells only during the period of DNA synthesis. Results Hybridization of Ah22 DNA to RNA of HeLa Cells Fractionated on a Sucrose Density Gradient We have previously shown that histone mRNA of HeLa cells can be found as part of RNA of high molecular weight which probably is the first product of transcription of the histone genes (Melli et al., 1977). If the histone genes of HeLa cells are expressed only during the S period, high molecular weight histone RNA should be found only during the period of DNA duplication. To establish whether the synthesis of high molecular weight histone RNA is coupled to DNA duplication, we have analyzed HeLa cell RNA from synchronized cultures throughout the cell cycle. Histone mRNA sequences were detected by cross-hybridization of HeLa cell RNA to Ah22 DNA, a phage containing cloned Psammechinus miliaris sea urchin histone genes (Clarkson et al., 1976; Schaffner et al., 1976; Gross et al., 1976; Portmann, Schaffner and Birnstiel, 1976). HeLa cells synchronized by double thymidine block were labeled with 3H-uridine (Amersham; >20 Ci/mmole) during 60 min of incubation at 2, 5, 7, 9 and 12 hr after thymidine release. Cell synchrony was checked by incubating 0.5 ml of the cell culture for 10 min with 1 &i of
Cell 166
3H-thymidine. The incorporation of thymidine in TCA-precipitable counts indicated a peak of DNA synthesis 4-5 hr after thymidine release and an S period of approximately 9 hr. Tritiated thymidine incorporation reached a minimum between 10 and 13 hr after thymidine release. The residual incorporation was 57% of the maximum value observed at the peak of DNA duplication. Total RNA was extracted from the cells and centrifuged in 99% formamide, 2-10% sucrose density gradients. RNA fractions from the gradients were pooled as described in the legend to Figure 1 and passed through an oligo(dT)-cellulose column to separate poly(A)- RNA, which was then used for the hybridization reaction. An equal number of counts from each pool of RNA fractions was hybridized to 1 pg of Ah22 DNA (Melli et al., 1977). We have previously shown that under these conditions, hybridization is in excess of DNA, and that the
i
t
I
45s
285
18s
1 2
0
Figure 1. Hybridization Sucrose 99% Formamide
10 Fraction
20 no.
of Ah22 DNA to RNA Fractions Gradient
of a 2-10%
Total HeLa cell RNA was centrifuged in sucrose-formamide gradients for 22 hr at 39,000 rpm in the SW40 Spinco rotor at 23°C (Melli et al., 1977). The RNA fractions, pooled in seven samples as indicated in the figure, were hybridized to 1 pg of Ah22 DNA loaded on Millipore filters at the final concentration of 1.5 x lo6 cpm/ml, under the conditions described in Experimental Procedures. (- 0-) SH-RNA cpm/pI; the histograms indicate the hybrid radioactivity: the arrows indicate the position of the optical density peaks. The position of the 45s RNA precursor was obtained by cross-hybridizing Xenopus rDNA to HeLa RNA, as previously described (Melli et al., 1977). Total RNA of synchronized ceils labeled for 1 hr with 3H-uridine (a) 9 hr after release from double thymidine block and (b) 12 hr after release from double thymidine block.
amount of hybrid formed is proportionate to the percentage of complementary sequences present in the total population of RNA molecules. Figure 1 shows the results obtained after hybridizing RNA from HeLa cells harvested 9 and 12 hr after thymidine release to Ah22 DNA. The radioactive profile of the two RNA samples is similar to the profile of all other RNAs extracted from HeLa cells at different times of the S period (Melli et al., 1977). The distribution of the hybrid radioactivity is also very similar in all samples tested (data not shown). The same type of experiment was carried out with RNA of cells in which DNA synthesis was blocked with cytosine arabinoside (AraC) (data not shown). The distribution of the hybridization of the RNA fractions across a formamide gradient was similar to that shown in Figure 1, suggesting synthesis of histone RNA in the absence of DNA duplication (see following experiments). The persistence of histone RNA throughout the cell cycle and in the absence of DNA duplication could be explained in at least two ways. Incomplete synchrony of the cells would allow a continuous production of histone mRNA. Under these circumstances, histone mRNA should be found in the cytoplasm as well as in total RNA. Absence of coupling between DNA duplication and histone RNA synthesis would also produce the observed result. In this case, however, histone mRNA should not enter the cytoplasm of the cells: To distinguish between these possibilities, we compared the proportion of histone mRNA sequences present in total and cytoplasmic RNA of cells at different stages of the cell cycle. RNA synthesis in HeLa cells occurs throughout the entire interphase and halts at mitosis. The accumulation of RNA in the nucleus is constant during the entire synthetic period (Zetterberg, 1966), and there is no evidence of major qualitative or quantitative changes of the rate of RNA synthesis (Klevecz and Stubblefield, 1967; Pfeiffer and Tolmach, 1968; Pagoulatos and Darnell, 1970). We have measured the incorporation of 3H-uridine in total cellular RNA and nuclear RNA of cells pulse-labeled for 30 and 60 min (Table 1). The incorporation of radioactivity throughout the cell cycle and after inhibition of DNA synthesis with cytosine arabinoside is rather constant. A similar proportion of polyadenylated RNA is found in all samples tested, suggesting that the overall composition of the RNA population is not drastically changed, although qualitative variations cannot be excluded. In view of this result, we decided to compare the proportion of histone RNA sequences of cells pulse-labeled with 3H-uridine for 1 hr in the presence and in the absence of DNA duplication. The hybridization of a constant amount of hh22 DNA to increasing concentrations of poly(A)HeLa RNA gives a measure of the rela-
Synthesis 169
Table
of Histone
1. SH-Uridine
Hours after Thymidine Release
mRNA
Incorporation
of HeLa
Cells
during
the Cell Cycle
Total Cellular RNA (30 min Pulse) cpm x 1Om6
Poly(A)+
RNA
Nuclear RNA” (60 min Pulse) cpm x 1O-B
0.5
4.8
PoI~(A)~
16.0
4.2
18
4.15
16
4.0
23
0.5 + AraC
3.9
15.7
2
4.0
13.0
5
5.6
13.9
7
5.3
14.0
9
4.4
17.0
12
3.2
13.0
RNA
%
%
a Same RNA as that used in the experiment of Figure 2. HeLa cells synchronized by double thymidine block were washed with balanced salt solution and incubated in Eagle’s minimal essential medium with Eagle’s salts (Gibco, Grand Island, New York) supplemented with 5% each of calf and fetal calf serum. At the indicated times, the cells were concentrated to approximately 4 x lO”/ml and labeled for 30 or 60 min with 200 &i/ml of 3H-uridine (Amersham; 25 Ci/ mMole). When indicated, cytosine arabinoside was added to a final concentration of 40 *g/ml immediately after thymidine release. The nuclei were purified according to Penman (1966). Total RNAand nuclear RNA were extracted as described in Experimental Procedures. The numbers refer to 3H-uridine incorporation in IO6 cells.
tive proportion of histone RNA sequences present in the different cellular compartments during the cell cycle or after inhibition of DNA synthesis. In fact, the amount of hybrid obtained will depend upon the concentration of the histone RNA sequences in the total population of RNA under study (Bishop, 1969). Hybridization of Xh22 DNA with Nuclear RNA of HeLa Cells during and in the Absence of DNA Duplication We first compared the proportion of histone mRNA synthesized in synchronized cells during and in the absence of DNA duplication. It is known that cell synchrony obtained by double thymidine block usually involves approximately 80-85% of the cell population. Although more than 90% of the cells seem to be synchronous using the criterion of 3Hthymidine incorporation, the measure of the mitotic index might show a lower proportion of synchronized cells. To establish whether the same proportion of histone mRNA sequences are found in RNA of cells during DNA synthesis and in the absence of DNA duplication, we hybridized these RNAs to Ah22 DNA. Before hybridization, the RNA was degraded by alkaline treatment to a size of approximately 9s. The degradation of the RNA was carried out to avoid variations in the amount of the RNA-DNA hybrid due to the different size of the complementary RNA molecules rather than to the concentration of the RNA (Melli et al., 1977). Since breakage of the RNA in the cross-hybridization reaction results in a loss of hybrid radioactivity (Melli et al., 1977), the amount of DNA loaded on Millipore filters was increased to compensate for the loss of counts.
Increasing concentrations of nuclear RNA were incubated with duplicate Millipore filters loaded with 5 pg of Ah22 DNA each. The conditions of hybridization were as described in Experimental Procedures. Figure 2 shows the hybridization of Ah22 DNA with poly(A)- nuclear RNA of cells pulselabeled with 3H-uridine for 1 and 12 hr after thymidine release or treated with AraC to inhibit DNA duplication. At the concentrations of RNA used, the reaction is in the linear range. The control RNA seems to contain a slightly higher proportion of histone RNA sequences than the RNA from cells labeled in the absence of DNA duplication. No difference is observed between RNA from cells in G2 and RNA from cytosine arabinoside-treated cells. The proportion of histone mRNA sequences found in nuclear RNA of cells labeled in the absence of DNA duplication is only 19-20% lower than in control cells. Even assuming that 20% of the cells in both cultures were synthesizing DNA, it seems very improbable that they could synthesize 80% of the total mRNA produced by the same number of cells during the S period of the cell cycle. We therefore conclude that histone mRNA represents approximately the same proportion of the total labeled nuclear RNA of HeLa cells both during the period of DNA duplication and in the absence of DNA duplication. Hybridization of Xh22 DNA with Cytoplasmic and Total RNA from AraC-Treated Cells The absence of histone mRNA in the cytoplasm of HeLa cells treated with AraC or with hydroxyurea has been shown by different types of studies. The polysomal RNA of these cells does not contain an appreciable amount of sequences coding for his-
Cell 170
0
2 3H-RNA
J
4
2 3H-RNA Figure DNA
2. Hybridization
of Nuclear
cpm
xlO+ml
3H-RNA
of HeLa
Cells to Ah22
Approximately 9 x IO’ HeLa cells were synchronized by double thymidine block and divided into three equal samples. After thymidine release, one sample was incubated in fresh medium for approximately 30 min. The cells were then concentrated and incubated for 1 hr with 3H-uridine as described in the legend to Table 1, The second sample was incubated in fresh medium for 12 hr after thymidine release and labeled as described above. The third sample was incubated in fresh medium at the end of the double thymidine block with 40 pg/ml of cytosine arabinoside for 30 min. The cells were then labeled for 1 hr with 3H-uridine in a medium containing the same concentration of the drug. At the end of the incubation, the labeled cells were harvested and the nuclei were purified. Total nuclear RNA was extracted from the three different samples. The poly(A)RNA fraction obtained by affinity chromatography was alkali-degraded and hybridized to Ah22 DNA. The hybrid radioactivity refers to 5 pg DNA. RNA of cells labeled for 1 hr with 3H-uridine approximately 30 min after release from a double thymidine block (-•-), 12 hr after release (-0-), during AraC treatment (-W-).
tone proteins in an in vitro cell-free system. Pulselabeled polysomal or total cytoplasmic RNA from cells treated with AraC does not contain labeled 9s sequences which can be identified with histone mRNA (Robbins and Borun, 1967; Gallwitz and Mueller, 1969; Breindl and Gallwitz, 1973; Gallwitz, 1975). Using the technique of cross-hybridization to Xh22 DNA, we have compared the hybridization of histone mRNA obtained from the cytoplasm of AraC-treated cells with that of cytoplasmic histone mRNA from cells during the S period of the cell cycle. Figure 3 shows the results of such an experiment. The slope of the two curves is clearly differ-
4 cpmx10-6/m~
Figure 3. Hybridization of Cytoplasmic from AraC-Treated Cells to Ah22 DNA
and
The
of cytosine arabinoside [This treatment reincorporation into DNA,]
after sulted
cells
were
incubated
with
40 pg/ml
Total
Poly(A)-
RNA
release from double thymidine block. in 99% inhibition
of 3H-thymidine
After 30 min, ‘H-uridine was added and the cells were incubated further for 1 hr. The hybridization was carried out Millipore filters loaded with 2 pg of - Ah22 DNA with concentrations of RNA.
t-8-)
incubating increasing
total poly(A)- RNA + AraC; (-•-) cytoplasmic poly(A)-
RNA + AraC; (-0-) cytoplasmic labeled for 1 hr with 3H-uridine from double thymidine block.
poly(A)RNA from cells pulseapproximately 30 min after release
ent, suggesting that the proportion of cytoplasmic histone mRNA in cells where DNA duplication is inhibited is 20-30 times lower than in control cells. In accordance with the previous experiments, the hybridization of total poly(A)RNA from AraCtreated cells is approximately 5 times higher than the hybridization of cytoplasmic poly(A)- RNA from the same cells. These results show that in agreement with the published work, the presence of histone mRNA in the cytoplasm of HeLa cells is coupled with DNA duplication. Furthermore, they confirm that histone mRNA is synthesized in these cells, although it is not found in the cytoplasm. The absence of cytoplasmic histone messenger further confirms that the histone RNA found in the nuclei cannot be due to incomplete synchronization of the culture. Histone mRNA of Cytosine Arabinoside-Treated Cells after inhibition of Protein Synthesis with Cycloheximide Butler and Mueller (1973) have previously shown that the loss of cytoplasmic histone mRNA of cells
Synthesis 171
of Histone
mRNA
tions is shown in Figure 4. We have consistently observed that cycloheximide treatment inhibits the labeling of the RNA sedimenting in the 18s region of the gradient. This finding is in agreement with the inhibition of rRNA processing observed Pederson and Kumar (1971) in cells treated with cycloheximide. Aliquots of each RNA fraction from both gradients were used for the hybridization to 1 pg of Ah22 DNA loaded onto Millipore filters. The comparison of Figures 4a and 4b shows that de novo synthesized histone mRNA is found in the cytoplasm of cells in which protein synthesis is inhibited after block of DNA duplication. The result is in agreement with the work of Gallwitz (1975), who found protection of polysomal histone mRNA of HeLa cells if the two metabolic drugs were administered together to the cells. This observation suggests that nuclear histone RNA synthesized in the absence of DNA synthesis can enter the cytoplasm if protein synthesis is inhibited. The treatment with cycloheximide might block the synthe-
treated with hydroxyurea can be prevented by inhibition of protein synthesis with cycloheximide. HeLa cells treated with hydroxyurea continue to accumulate cytoplasmic histone mRNA if cycloheximide is added to the cell culture before the administration of hydroxyurea. These investigators have also suggested that inhibition of protein synthesis following the block of DNA synthesis does not restore the presence of histone mRNA in the cytoplasm of HeLa cells. Gallwitz (1975) found that protection of cytoplasmic histone mRNA occurs independent of the time of administration of cycloheximide relative to hydroxyurea. We have repeated this type of experiment using the technique of cross-hybridization as a method of detection of histone mRNA sequences in the cytoplasm of HeLa cells. AraC-treated cells 2 cycloheximide were labeled with 3H-uridine, and cytoplasmic RNA was purified and centrifuged in a 5-20% sucrose density gradient, as described in the legend to Figure 4. The radioactive profile of the two RNA prepara-
L”
Figure
4. Hybridization
of Cytoplasmic
RNA of AraC-Treated
10
”
Fraction Cells
20
no.
* Cycloheximide
to Ah22 DNA
Approximately 6 x IO’ HeLa cells from a synchronized culture were released from double thymidine block and incubated in fresh medium containing 40 pg/ml of cytosine arabinoside. After 20 min of incubation, the culture was divided into two equal aliquots, the ceils were concentrated to approximately 4 x lo8 cells per ml and cycloheximide at a concentration of IO rg/ml was added to one of the samples. After IO min of incubation, both cell cultures were labeled for 1 hr with 3H-uridine as described in Experimental Procedures. Total cytoplasmic RNA was centrifuged in a 5-20% sucrose density gradient containing 0.15 M NaCI, 0.01 M Tris (pH 7.5), 0.5% SDS and 0.005 M EDTAfor 12 hr at 27,000 rpm in an SW40 Spinco rotor at 23°C. The arrows indicate the position of the optical density markers. An aliquot of each RNA fraction from 8-24 (approximately 150 ~1 of 0.5 ml total volume) was hybridized to 1 pg of Ah22 DNA loaded on Millipore filters under the conditions described in Experimental Procedures. The RNA hybridized from each gradient corresponds to equal amounts of 16s optical density at 260 nm. (-0-) and (-•-) 3H-RNA cpm/5 ~1; (-• -) and (-A-) 3H-hybrid cpm. (a) + AraC; (b) + AraC + cycloheximide.
Cell 172
sis of a short-lived protein which specifically destroys or allows the decay of histone mRNA either in the nucleus or in the cytoplasm or in both cellular compartments. Hybridization of Cytoplasmic and Total RNA of Synchronized HeLa Cells 12 Hr after Thymidine Release Synchronized HeLa cells 12 hr after the release from double thymidine block were labeled for 1 hr with 3H-uridine. Total cellular RNA was extracted from half the culture, and cytoplasmic RNA was obtained from the remaining cells. The poly(A)fractions of the two RNA preparations were hybridized to Ah22 DNA as described in the legehd of Figure 5. By increasing the RNA concentration, the proportional hybridization of total cellular RNA is 5 times higher than that of cytoplasmic RNA (Figure 5). The comparison between the RNA-DNA hybridization curves of cytoplasmic RNA from cells in G2 and cells in S phase shows a drastic drop of the hybridization of RNA from G2 cells. The different slope of the two curves suggests that the relative proportion of histone RNA sequences is approximately 14 times higher in the cytoplasm of cells
during S phase as compared with 12 hr after thymidine release. The decrease of the concentration of histone messenger sequences in cells during G2 is less than that found in AraC-treated cells. This can be explained by the incomplete synchronization of the cells which is observed during the G2 period of the cell cycle. Probably cytosine arabinoside is comparatively more effective in producing inhibition of DNA duplication. These results confirm that HeLa cells 12 hr after thymidine release contain a very low concentration of histone mRNA in the cytoplasm and that histone RNA is present in the nucleus. Since cycloheximide was shown to increase the amount of cytopiasmic histone mRNA in AraC-treated cells, we investigated whether the same effect could be observed in cells from the G2 period of the cell cycle. A portion of the synchronized cell culture used for the experiments described above was incubated 12 hr after thymidine release, in a medium containing 10 pg/ml of cycloheximide. After 10 min of incubation at 37”C, 3H-uridine was added to the medium and the cells were labeled for 1 hr. The cytoplasmic poly(A)RNA of these cells was extracted and hybridized to Ah22 DNA. The results are shown in Figure 5. After cycloheximide treatment, the hybridization is approximately 4 times higher than that of RNA from untreated cells. The slope of this curve suggests, however, that the concentration of histone mRNA is approximately a quarter of that found in the cytoplasm of cells actively synthesizing DNA. We conclude that the inhibition of protein synthesis in cells during the G2 period of the cell cycle increases the amount of cytoplasmic histone mRNA. It does not, however, reestablish the same relative concentration of mRNA found in cells during the period of DNA duplication. Discussion
0
2
3H-RNA Figure 5. Hybridization Poly(A)RNA of Cells
4 cpmx10-6/
of Ah22 12 Hr after
ml
DNA to Cytoplasmic Thymidine Release
and
Total
The cells were pulsed for 1 hr with 3H-uridine, and the RNA was extracted and hybridized as described in Experimental Procedures. The hybrid radioactivity refers to 2 pg DNA. (-0-) total poly(A)RNA, 12 hr after thymidine release: (-A-) cytoplasmic poly(A)RNA, 12 hr after thymidine release: (-•-) cytoplasmic poly(A)RNA, 12. hr after thymidine release, labeled with 3H-uridine after inhibition of protein synthesis with cycloheximide; (-W) cytoplasmic poly(A)RNA from cells pulselabeled for 1 hr with 3H-uridine approximately 30 min after release from double thymidine block.
We have previously shown the specificity of the reaction between sea urchin histone genes and HeLa histone mRNA. The same degree of homology observed between HeLa histone mRNA and Echinus esculentus histone genes was found in studying the cross-hybridization of HeLa RNA with Psammechinus miliaris histone DNA sequences. The hybridization occurs only with the sea urchin gene sequences and in particular with the coding regions of the DNA (Melli et al., 1977). The validity of our approach is confirmed by the agreement between our results and the published data. The proportion of histone mRNA sequences found in the cytoplasm of cells labeled with 3H-uridine in the absence of DNA duplication is 5-7% of the control cells, labeled during the S period of the cell cycle. Furthermore, inhibition of protein syn-
Synthesis 173
of Histone
mRNA
thesis with cycloheximide increases the amount of histone mRNA in the cytoplasm of the same cells. Hodge et al. (1969) found that the treatment of HeLa cells with cycloheximide inhibits DNA duplication already 4 min after the addition of the drug to the culture. The coupling between DNA duplication and histone mRNA synthesis was in apparent contradiction with the claim that the inhibition of protein synthesis with cycloheximide restores the synthesis of histone mRNA. The continuous synthesis of histone RNA during the cell cycle offers an explanation for the effect of cycloheximide on HeLa cells. It has previously been shown that HeLa histone mRNA decays rapidly at the end of the S period and that cycloheximide prevents this breakdown. It is probable that the same catabolic mechanism is responsible for the decay of old and new histone mRNA. In other words, histone RNA is synthesized and processed during the entire cell cycle. During G2, the newly synthesized histone RNA molecules might enter the cytoplasm and be destroyed in the same way as the polysomal bound mRNA. The specific breakdown of this messenger might depend upon the synthesis of a short-lived protein and therefore be prevented by cycloheximide treatment. In this case, the regulatory step which quantitates the amount of histone mRNA in the cytoplasm of HeLa cells would be cytoplasmic. We cannot exclude a more complex process of regulation involving both cellular compartments, the nucleus and the cytoplasm. For example, a specific alteration of processing or transport of mRNA from the nucleus into the cytoplasm could stop the entrance of histone mRNA into the cytoplasm after the end of DNA duplication. The finding that histone mRNA synthesis and DNAduplication are not coupled is in disagreement with the conclusion of Stein et al. (1975). There is one major difference between the two types of experimental approaches. The data presented in this work concern the synthesis of histone mRNA in intact HeLa cells, whereas Stein et al. (1975) have been analyzing the synthesis of histone mRNA in vitro. The existence of a nuclease which specifically destroys histone mRNA in certain periods of the cell cycle could perhaps explain the divergent results. This nuclease could be compartmentalized in vivo, but could become free after lysis of the cells and catabolize histone mRNA as it is being synthesized. The problem of the purity of the hybridization probe used for the detection of the mRNA could also come into question. Transcriptional regulation is considered one of the means through which the cell adapts to different physiological conditions. The transcription of histone genes has been often quoted as an example of this type of control. A careful analysis of the
published work shows that there are in fact very few well documented cases of transcriptional control. Our work shows that the controlling step regulating the presence of histone mRNA in the cytoplasm of HeLa cells is not transcriptional and perhaps not even nuclear. Experimental
Procedures
Cell Growth, Cell Synchronization, Labeling of the RNA and RNA Extraction HeLa cells were synchronized, grown and labeled as previously described (Wilson and Melli, 1977). Cell synchrony was checked in each experiment by pulsing with 3H-thymidine aliquots of the cell cultures throughout the cell cycle. After labeling, the cells were washed twice in basic salt solution, and total RNA (Melli et al., 1977) or cytoplasmic RNA was extracted. To purify cytoplasmic RNA, the cells were suspended in RSB [lo mM NaCl, 1.5 mM MgCI, and 10 mM Tris (pH 7.5)] and lysed in Nonidet Sp-40. Nuclei and mitochondriae were removed, and proteins were digested with proteinase K in 2% SDS. The procedure was the same as that described for the extraction of total RNA (Curtis and Weissmann, 1976). Poly(A)RNA was obtained by affinity chromatography through oligo(dT)-cellulose (Collaborative Research). Centrifugation of RNA in 99% Formamide Sucrose Density Gradients The centrifugation was carried out according to Melli et al. (1977). RNA-DNA Hybridization Ah22 DNA was purified according to R. Portmann, G. Sogo. T. Koller and W. Zillig (manuscript in preparation). RNA-DNA hybridization was carried out as described (M. Melli et al., 1977). Millipore filters loaded with 1-5 pg Ah22 DNA were incubated with HeLa cells RNA in 0.2 ml 2 x SSC at 65°C overnight. Duplicate filters carrying approximately 5 pg of bacterial DNA were used to control the background radioactivity which was subtracted from each sample. Acknowledgments We are grateful to Prof. M. Birnstiel for reading the manuscript and providing us with the Ah22 phages (Clarkson et al., 1976). We thank Miss A. Binkert for growing Ah22 phages. G. Spinelli was a recipient of a long-term EMBO fellowship. This work was supported by a grant from the Swiss National Science Foundation. Received
February
25, 1977;
revised
April
19, 1977
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