Cell, Vol . 4, 59-67, January 1975, Copyright ©1975 by MIT
Further Evidence of Transcriptional and Translational Control of Histone Messenger RNA during the HeLa S3 Cycle Ted W . Borun*, Franco Gabriellit, Kozo Ajirom, Alfred'Zweidler#, and Corrado Baglionit *Fels Research Institute and Department of Biochemistry Temple University School of Medicine Philadelphia, Pennsylvania 19140 Department of Biology Massachusetts Institute of Technology Cambridge, Massachusetts 02139 *The Institute for Cancer Research Fox Chase Center for Cancer and Medical Sciences Philadelphia, Pennsylvania 19111
Summary Using a translational assay and new analytical procedures we have found that : -Histone mRNA can be detected both associated with polyribosomes and in the postribosomal supernatant of S phase HeLa S3 cells . -Inhibition of DNA replication by cytosine arabinoside treatment causes histone mRNA to completely disappear from polyribosomes, and little histone mRNA can be detected in the postribosomal supernatant of inhibited cells . These data indicate that histone mRNA does not accumulate in the cytoplasm after the inhibition of DNA replication . -Histone mRNA species cannot be detected in the postribosomal supernatant of G1 cells synchronized by selective detachment . This observation, together with the previous finding that histone mRNA is not present on G1 polyribosomes, is consistent with the idea of a transcriptional block in histone mRNA production and transport to the cytoplasm during G1 . Introduction Nuclear DNA replication and cytoplasmic histone synthesis occur at significant rates only during the S phase of the HeLa S3 cell cycle (Robbins and Borun, 1967) . Since histones are not synthesized in G1 cells prior to the initiation of DNA replication and histone synthesis in S phase cells stops within 30 min if DNA replication is inhibited, it is evident that these two macromolecular synthetic processes are tightly coupled (Robbins and Borun, 1967 ; Borun, Scharff, and Robbins, 1967 ; Gallwitz and Mueller, 1969) . The mechanisms which couple DNA and histone synthesis in the HeLa cell are unknown, but it seems probable that such mechanisms involve control of both the transcription and the translation of histone messenger RNA (mRNA) species (Borun et al ., 1967 ; Butler and Mueller, 1973) .
HeLa histone mRNA was discovered in labeling experiments that suggested that these RNA species sediment between 7S and 9S and are associated with small polyribosomes in cells actively synthesizing DNA (Robbins and Borun, 1967 ; Borun et al ., 1967 ; Gallwitz and Mueller, 1969) . A block in DNA replication not only inhibits histone polypeptide synthesis but also causes a parallel disappearance of labeled mRNA from polyribosomes . Other labeling experiments have indicated that histone mRNA species, unlike most other mRNA, do not contain poly A tracts at their 3' OH termini (Adesnik and Darnell, 1972 ; Greenberg and Perry, 1972) . Recent large scale experiments have made possible the physical isolation of quantities of polyribosomal histone mRNA species sufficient to permit their unequivocal identification by translation in heterologous cell-free protein synthesizing systems (Jacobs-Lorena, Baglioni, and Borun, 1972 ; Gallwitz and Briendl, 1972) . The in vitro translation assay provides an opportunity to investigate several additional aspects of the possible translational and transcriptional controls of HeLa histone mRNA and further characterize these RNA species . In this assay, various RNA preparations are added to an mRNA-dependent mouse-ascites cell-free protein synthesizing system . Measurement of the net stimulation of protein synthesis provides an index of the amount of mRNA in the RNA tested ; characterization of the cell-free synthetic products by various acrylamide gel electrophoresis procedures defines the number and kinds of translatable templates in the RNA preparation . Using this in vitro translation assay and a variety of procedures to fractionate RNA, we have investigated the distribution of histone mRNA in polyribosomes and postribosomal supernatants of G1 and S phase HeLa S3 cells . Parallel studies have been carried out on S phase cells treated with the inhibitor of DNA synthesis cytosine arabinoside . Results The Effect of a Block in DNA Replication on Translatable Polyribosomal Histone mRNA To better characterize histone mRNA species and the effect of a block in DNA replication on polyribosomal histone mRNA, polyribosomal RNA was extracted from HeLa S3 cells which were synchronized in S phase by double thymidine blockade (Stein and Borun, 1972) and from cells treated for 30-60 min with 40 µg/ml of cytosine arabinoside (C-ara) to block DNA replication . To facilitate subsequent analysis, this RNA was fractionated on sucrose gradients to obtain the material which sediments between the 4S and 18S peaks (4-1 8S RNA) .
Cell 60
In some experiments 4-18S RNA was also passed over oligo(dT)-cellulose to separate the RNA containing poly A tracts [poly(A)+] from the RNA which does not [poly(A)-], (Aviv and Leder, 1972) . Figure 1A shows the resolution of one A2 60 unit of control S phase polyribosomal 4-18S RNA which is poly(A)- resolved by electrophoresis on 20 cm analytical gels containing 6% acrylamide and run in 6 mm I .D . quartz tubes . The profile of one A260 of the poly(A) + fraction of this 4-18S RNA resolved under the same conditions is shown in Figure 1 B . Figure 1 C shows further resolution of the RNA in Figure 1A by electrophoresis for a total of 8 .5 hr at 250 volts . After this period of electrophoresis the 4S and 5S RNA have run off the gel and a 7S RNA peak has migrated to near the end of the gel . This 7S RNA is followed in turn by the peaks labeled a through d and by 18S RNA at the top of the gel . It can be seen in Figure 1 D that the peaks labeled a, b, c, and d disappear from the 4-18S polyribosomal RNA of cells treated with C-ara for 60 min to inhibit DNA replication . Subsequent experiments in this paper will show that these RNA species are histone mRNA or aggregates of such RNA . In contrast to the a-d peaks, the 7S and 18S RNA components of the 4-18S polyribosomal RNA of C-ara-treated cells are not affected by the inhibition of DNA replication . To demonstrate that the C-ara-sensitive polyribosomal RNA species are histone mRNA, large quantities (50-100 A260) of 4-18S RNA were isolated from the polyribosomes of control and C-ara-treated cells and fractionated on preparative 5 cm 8% acrylamide gels according to the procedure of Moriyama et al . (1969) as previously described (JacobsLorena et al ., 1972) . A 7-9S peak was obtained from both the control and C-ara-treated cells, but although both preparations came from the same number of cells, the 7-9S peak of C-ara cells contained 31 % as much RNA as the control . The stimulatory activity of RNA contained in these 7-9S peaks was assayed in the ascites cell-free sys-
tem at two different RNA concentrations (Table 1) . The 7-9S RNA isolated from S phase cells stimulated the cell-free system significantly, whereas the 7-9S RNA obtained from C-ara-treated cells did not . The small stimulation observed with this fraction is of the same order of magnitude as that obtained with noninformational RNA, like ribosomal RNA (Jacobs-Lorena and Baglioni, 1972a) . We have attributed this nonspecific stimulation to protection GELS RUN4 HOURS A.
GELS RUN8.5HWRS
Control 4 .185 PdyA(-) RNA
C. Control 4-18SPdyAF RNA
.8 7-9S
7-95
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7
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7S
/85
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18S
D.ToAa' C-ARA 4-PS RNA
Ccntrd4.18S PdyA(f)RNA
t
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4S
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- ~
/o cm
t F~
W
Figure 1 . Analytical Gel Profiles of 4-18S Polyribosomal RNA (A) Control poly(A)- RNA (Aviv and Leder, 1972) from S phase HeLa S3 cells run on 20 cm, 6% acrylamide gels containing 0 .2% bis acrylamide and Loening's Buffer E, containing 0 .2% SDS (Loening, 1969) . The gels were polymerized in 25 cm long 6 mm ID quartz gel tubes and run for 4 .5 hr at 250 volts . The gels were then scanned in an ISCO U .A.4 U .V . gel scanner at 260 nm . One A260 was run on the gel . (B) Control poly(A) + RNA from S cells run as in A, one A260 was run on the gel . (C) Control poly(A)- RNA from S cells run as in A but for a total of 8 .5 hr. (D) One A260 of total 4-18S polyribosomal RNA from S phase HeLa S3 cells pretreated for 60 min with 40 µg/ml cytosine arabinoside to inhibit DNA replication and run on a 6% analytical gel as in Figure 1C .
Table 1 . Protein Synthesis with 7-9S RNA of Polyribosomes of S Phase and Cytosine Arabinoside-Treated HeLa Cells RNA 7-9S of S Phase cells 7-9S of C-ara-treated cells Globin mRNA
Experiment 1 (10 pg/ml) net cpm (stimulation) 2,640 (1 .7) 995 (1 .2) 43,342 (6 .4)
Experiment 11 (70 µg/ml) net cpm (stimulation) 14,852 (3 .9) 4,132 (1 .8) 74,080 (15 .3)
The abbreviation C-ara is used to designate HeLa cells treated with cytosine arabinoside . The net cpm are calculated by subtracting the cpm incorporated in 1 hr at 30°C by the incubation without added RNA (3990 cpm in Experiment I and 5164 cpm in Experiment cpm + RNA II, respectively) from those incorporated in the same time by incubations with added RNA . The stimulation is calculated as cpm RNA' Each incubation contains 10 µl the indicated concentration of RNA, 1 µCi of 3H-amino acid labeling mixture (New England Nuclear) and the cold amino acids not present in this mixture . Duplicate aliquots have been sampled for counting, and the averages referred to 10 µl incubations are indicated .
HeLa Histone mRNA 61
of residual endogenous mRNA (Jacobs-Lorena and Baglioni, 1972a) . These results are strengthened by the electrophoretic analysis of the product of translation of the 7-9S RNA (Figure 2) . The proteins synthesized with RNA isolated from control S phase cells co-migrate with marker histones(Figure2B) ; we have shown previously that this 7-9S RNA codes almost exclusively for histones by analyzing its translation product for tryptophan content (Jacobs-Lorena et al ., 1972) . The pattern of the product of 7-9S RNA isolated from C-ara-treated cells shows an increase over the
background observed in gels of control incubations without added RNA (Figure 2A) . However, while there are several poorly resolved peaks in the pattern, there is no indication that histone mRNA represents a significant component of this RNA . Also note the difference in the ordinates of Figure 2A
4
150
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.2
100 aU
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4
a
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600
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400
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200
20
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80
Figure 2 . Acrylamide Gel Electrophoresis of the Product of 7-9S Polyribosomal RNA of S Phase and Cytosine Arabinoside-Treated HeLa Cells The incubations are described in Table 1, Experiment II . The samples are treated as described in Experimental Procedures and analyzed on 15% gels (Panyim and Chalkley, 1969) . (A) Solid line, cell-free product of 7-9S RNA of C-ara-treated cells ; dotted line, no RNA added to the cell-free system . The two patterns have been superimposed by using marker 1 4C-labeled histones (arrows) to align them, The two arrows indicate the position of the marker histories which correspond to those shown in the bottom panel . (B) Solid line, cell-free product of 7-9S RNA from S phase cells ; dotted line, marker histones labeled in intact HeLa cells with 14C-lysine ; the peak migrating around Fraction 30 corresponds to F1 histones, whereas the other broad peak migrating at 50-60 corresponds to the other classes of histones, which are poorly resolved .
(-) top Figure 3 . Analytical Gel Scans of Postribosomal Supernatant RNA One A260 unit of RNA was analyzed as described in Figure 1 for 2 hr at 250 Volts . (Top panel) Control S phase postribosomal supernatant RNA . (Middle panel) RNA from the postribosomal supernatant of S phase cells treated for 60 min with 40 µg/ml of cytosine arabinoside . (Bottompanel) Poly(A)- (Aviv and Leder, 1972) 4-185 RNA from the postribosomal supernatant of G1 cells .
Cell 62
and 2B when comparing the amount of protein synthesized with equal quantities of control and C-ara 7-9S RNA in these experiments . These results confirm by a direct translation assay the conclusion reached previously by Borun et al . (1967) and Gallwitz and Mueller (1969) using 3 H-uridine-labeled material that histone mRNA disappears from S phase polyribosomes after a block in DNA replication . It is also apparent from the data in Figure 26 and previous data (Jacobs-Lorena et al ., 1972) that the 7-9S fraction of control S phase polyribosomal RNA prepared by the method of Moriyama et al . (1969) does contain the templates of all the principal classes of histone polypeptide but in an unresolved form . Subsequent work to be reported elsewhere has shown that the various histone mRNA species shown in Figure 1 C are eluted together in one peak which also contains a 7S RNA species . The 7-9S RNA fraction from the C-aratreated polyribosomes evidently consists almost entirely of this 7S RNA which has little template activity . Translation Assay of Histone mRNA in the Postribosomal Supernatant of Control, C-ara-Treated and G1 Cells In order to establish the fate of histone mRNA released from polyribosomes of C-ara-treated cells, we have isolated RNA from the postribosomal supernatant of these and control cells . In a parallel series of experiments, RNA has also been isolated from the postribosomal supernatant of cells synchronized in G1 . The RNA extracted from the postribosomal supernatant was fractionated by the same procedures
used for polyribosomal RNA . Figure 3 shows the resolution of one A260 unit of these supernatant RNA preparations resolved on analytical 20 cm 4% acrylamide gels for 2 hr at 250 volts . The gel pattern of RNA from S phase cells supernatant shows a double peak migrating at 7-9S similar to the double peak observed with polyribosomal RNA in Figure 1 . In the pattern of C-ara cells supernatant RNA only the 7S component is observed, whereas in the G1 cells supernatant RNA no distinct peak is observed migrating at 7S . In order to increase the sensitivity of this analysis we have used total supernatant RNA for control and C-ara cells, but have used for the G1 RNA analysis a 4S to 18S fraction obtained by preparative sucrose gradients . To establish whether the 7-9S RNA found in the supernatant is histone mRNA we have isolated large enough quantities of this RNA by preparative gel electrophoresis, as described above for polyribosomal RNA . The elution profile of S phase and C-ara-treated cells supernatant RNA shows a 7-9S peak, whereas no distinct 7-95 RNA peak is found in the G1 supernatant RNA . The RNA eluted in the region of 7-9S has been concentrated and used in translation experiments . The 7-9S RNA of the postribosomal supernatant of S phase cells causes a significant stimulation of the ascites cell-free system ; that obtained from the supernatant of C-ara treated cells gives a poor stimulation and that from G1 cells no significant stimulation (Table 2) . We have also tested these RNA preparations at different concentrations of RNA, ranging from 0 .05 to 0 .2 mg/mI of RNA .
Table 2 . Protein Synthesis with 7-9S RNA Isolated from Postribosomal Supernatant of HeLa Cells
Source of 7-9S RNA
Experiment 1 (0 .1 mg/ml) net cpm (stimulation)
S phase cells
3,500
(3 .1)
C-ara treated cells
1,255
(1 .9)
G1 phase cells
1,160
(1 .8)
13,990
(10 .1)
(Polyribosomes of S phase cells)
Experiment 11 (0 .2 mg/ml) net cpm (stimulation)
cpm in histones
12,460
(5 .3)
5960
4,864
(2 .1)
1210
In Experiment I each incubation mixture contains in a final vol of 10 µl between 0 .55 and 2 .04 µg of 7-9S RNA, 1 .3 µCi of 3 H-lysine (27 Ci/mmol) and all the components of the cell-free system described under Experimental Procedures . After 1 hr incubation at 30°C, 4 µl are sampled in duplicate . Each RNA fraction has been tested at two or three different concentrations within the linear range of response of the cell-free system shown in Figure 4 . The values reported here are the averages of different determinations normalized to 0 .1 mg/ml of added RNA and referred to a 10 µI incubation mixture . In Experiment II each incubation mixture contains in a final volume of 70 µl between 18 and 26 yg of 7-9S RNA, 10 µCi of 3H-lysine (29 Ci/mmol), and all the components of the cell-free system described under Experimental Procedures . The incubations have been carried out and analyzed as described above . The values reported have been normalized to a 0 .2 mg/mI of added RNA and referred to a 10 µl aliquot . Net cpm and stimulation have been defined in the legend of Table 1 . The cpm in histones have been determined by electrophoretic analysis of the translation product as described in the text . It should be pointed out that the incorporation of 3H-lysine and the corresponding stimulation of the cell-free system were better in Experiment 11 than in Experiment I, when referred to the concentration of RNA used . This is explained by the use of a different sample of the radioactive amino acid in these two experiments . Jacobs-Lorena and Baglioni (1972a) have reported previously that 3 H-lysine may inhibit the ascites cell-free system at high concentrations ; we have now found that different preparations of this amino acid obtained from New England Nuclear are incorporated by the cell-free system in a different way, though consistently within each sample .
HeLa Histone mRNA 63
In order to determine whether the cell-free system responds linearly to the input RNA in this concentration range, we have calibrated the system using polyribosomal 7-9S RNA obtained from S phase cells (Figure 4) . The response is linear up to a concentration of 0 .1 mg/ml and shows a plateau at higher concentrations . At 0 .2 mg/ml the polyribosomal 7-9S RNA is almost saturating the cell-free system . The cell-free product coded for by the supernatant 7-95 RNA preparations has been analyzed as described above for polyribosomal RNA (Figure 2) . Histone synthesis, as defined by co-electrophoresis of the cell-free product with marker histones, has been shown in this way for S phase and C-ara 7-9S RNA preparations . In order to analyze a sufficient amount of translation product of C-ara 7-9S supernatant RNA, which stimulates poorly the ascites cell-free system (Table 2), it has been necessary to apply to the gels a rather large aliquot of the incubation mixture . This overloading of the gels produces electropherograms with poor resolution, when compared to those obtained with S phase supernatant or polyribosomal 7-9S RNA . It has been possible, however, to estimate the relative amount of histone synthesized with the different 7-9S RNA preparations by measuring the radioactive protein present in the fractions corresponding to the major histone peaks (fractions 45-70 of Figure 2) and substracting the background observed in this region of the gels in the control incubations without added RNA . 15
15
I 0
to c a
These data (Table 2) suggest that translatable histone mRNA represents a larger proportion of the 7-9S RNA obtained from the supernatant of S phase cells than of that obtained from cells treated with C-ara . There is less 7-9S supernatant RNA in these cells, and it stimulates poorly protein synthesis and histone synthesis in particular . The 7-9S obtained from postribosomal supernatant of G1 cells does not significantly stimulate in vitro protein synthesis (Table 2) . In order to rule out the possibility that trace amounts of histone mRNA are present in G1 cells and that this mRNA is lost during preparative electrophoresis, we have also translated directly the RNA from postribosomal supernatant of G1 and S phase cells sedimenting between the 4S and the 18S peaks during sucrose gradient centrifugation (see Experimental Procedures) . Translation of the G1 cell RNA is rather poor, whereas that of S cell RNA is quite good (Table 3) . It is interesting to point out in this connection that the RNA isolated by sucrose gradient centrifugation only is found to stimulate protein synthesis more effectively than RNA isolated through several preparative steps . This agrees with similar observations made in isolating sea urchin histone mRNA (K .W . Gross, personal communication) . The products of the translation assays have been analyzed by electrophoresis on acrylamide gels using an improved procedure (Zweidler, 1973) that allows complete separation of the different histones (Figure 5) . The pattern obtained with S cell RNA corresponds very closely to all classes of marker histones, whereas no clear peak corresponding to any histone polypeptide is observed with the translation product of G1 postribosomal supernatant Table 3. Protein Synthesis with RNA Isolated from Gradient Fractions Source of 4-18S RNA
net cpm
(stimulation)
S cells postribosomal supernatant
8,966
(8 .0)
G1 cells postribosomal supernatant
2,411
(2 .1)
poly(A)-
33,541
(26 .7)
poly(A) +
1,750
(1 .9)
S cells polyribosomal :
Figure 4 . Response of the Ascites Cell-Free System to the Addition of Polyribosomal 7-9S RNA of S Phase Cells Each incubation contains in a final vol of 10 µl : 1 .3 µ Ci of 3H-lysine (27 Ci/mmole), the indicated amount of 7-9S RNA from the preparation shown in Figure 1 and all the other components of the cell-free system (see Experimental Procedures) . Duplicate samples are analyzed, and the average of the two determinations is reported ; duplicate determinations differ by less than 10% . The incorporation in the control without added RNA is 682 cpm ; these have been subtracted from the values plotted . The stimulation has been calculated by dividing the cpm obtained in an incubation with added RNA by the cpm in the incubation without added RNA .
Incubations were prepared as indicated in the legend of Table 2, in a final volume of 150 µl . The cpm were referred to 5 td aliquots . RNA concentrations were 0 .15 mg/ml in the experiments with polyribosomal RNA . The RNA has been prepared by centrifugation on sucrose gradients (see Experimental Procedures), by taking the fraction between the 4S and 18S peak but carefully excluding both these peaks. The polyribosomal RNA has been further passed through an oligo(dT)-cellulose column (Aviv and Leder, 1972) . The RNA not retained by the column is referred to as poly(A)- RNA and the RNA absorbed as poly(A) + RNA . The products of translation have been analyzed by gel electrophoresis as shown in Figure 5 . The results obtained with polyribosomal RNA are the average of two separate incubations using two independent preparations of RNA .
Cell 64
ACID-UREA-TRITON
ACID-UREA
OD
OD 2b
2b 2
Carrier Histories
Histones
2
1
2a2
I 1 2a1
3
I 1
i
3.2
U 20
40
8o
6o
cpm
b
mm ,-
4o
20
mm
cpm 600
300-
Supernatant 2a1
400
Supernatant
200 2b 3 2b 1 2a2 2a2 .1
200•
100
3.2
2a1 3 .1 2a2.2
20
40
S s 80
60
cpm 2b
Boo
40
20
0
60s
cpm
mRNA
mRNA
2b
400 PolyA polyA
polYA poIyA t
600
300
J
400-
200 3
2a1
2a2
1 2a1 200 i x
100
3.1
20
40
60
80
1
20
40
60S
HeLa Histone mRNA 65
RNA . We have included in Figure 5 the patterns obtained by the standard acid-urea electrophoresis . Outside of demonstrating the superiority of the acid-urea-triton method of analysis, these patterns are relevant for the identification of the different histone species by a standard procedure .
This identification is based only on the electrophoretic mobility of these proteins, and further studies are necessary to establish whether the mRNA for some of the minor histone species is relatively more abundant in the supernatant fraction . Discussion
Identification of Poly(A)- Histone mRNA Species RNA obtained from small polyribosomes of S phase cells by fractionation on sucrose gradients has been purified by chromatography on oligo(dT)cellulose by the method of Aviv and Leder (1972) . Only about 2% of the RNA is retained by oligo(dT)cellulose . Both the RNA not absorbed to the column and the RNA retained have been tested in the cellfree system . Excellent translation is obtained with the RNA which does not presumably contain poly(A), whereas the RNA retained by oligo(dT)cellulose is translated poorly (Table 3) . The translation products have been analyzed by gel electrophoresis (Figure 5, bottom panel) . The translation products of RNA not containing poly(A) migrate like all five major types of marker histones . A comparison between the products of translation of supernatant and polyribosomal 7-9S RNA can be made in Figure 5 . Relatively less histones F1, F3, and F2b are coded for by supernatant than by polyribosomal 7-9S RNA . These histones have a higher molecular weight than F2a1 and F2a2 . A possible explanation for this finding is that previously proposed for the presence of a-chain mRNA in the postribosomal supernatant of rabbit reticulocytes (Jacobs-Lorena and Baglioni, 1972b) . Smaller mRNAs, which are associated on the average with relatively fewer ribosomes, have a higher probability of being associated with no ribosomes at all by chance alone . This interpretation of the data is complicated by the difficulty of estimating with accuracy the amount of each histone synthesized, due to the presence of some minor peaks in the pattern of the product of supernatant 7-9S RNA . These peaks have tentatively been identified with minor histone species which can be observed in the pattern of carrier histones (top panel, Figure 5) .
We have established by a variety of procedures that practically no identifiable HeLa histone mRNA species remain associated with S phase polyribosomes after a block in DNA replication . In addition, we have observed that histone mRNA is present in the postribosomal supernatant of cells in S phase . This mRNA must represent a relatively sizable fraction of the histone mRNA present in these cells, though at present our methods of isolation and analysis do not allow us to estimate with accuracy the proportion of histone mRNA present in the supernatant relative to that associated with polyribosomes . The 7-9S RNA present in the supernatant may represent mRNA which is recirculating between polyribosomes and supernatant, as it has been suggested for the globin mRNA found in the postribosomal supernatant of rabbit reticulocytes (Jacobs-Lorena and Baglioni, 1972b) . Otherwise, it may represent mRNA of nuclear provenience, transiently present in the postribosomal supernatant before becoming associated with polyribosomes . Analyses of the RNA of C-ara treated cells indicate that, after a block in DNA synthesis, the histone mRNA released from polyribosomes does not accumulate in the postribosomal supernatant . These results are in agreement with previous suggestions that synthesis of new RNA is a prerequisite for histone synthesis after a reversible block in DNA synthesis (Gallwitz and Mueller, 1969) . The present results clearly indicate that RNA species which need to be synthesized under these conditions are histone mRNA . Analytical gels (Figure 3) indicate that there are definitely less histone mRNA-like species present in the postribosomal supernatant of C-aratreated cells . The small amount of histone mRNA found there by the translational assay probably ac-
Figure 5 . Acrylamide Gel Electrophoresis of the Translation Product of RNA Isolated by Sucrose Gradient The incubations are described in Table 3 . The samples are dialyzed against 0 .9 N acetic acid, centrifuged, and precipitated with 7 vol of acetone after addition of 0 .1 mg of carrier HeLa histones . The precipitate is dissolved in 8 M urea, 10% mercaptoethanol, 0 .001 Pyronin Y, and applied to 11 cm long gels containing 12% acrylamide, 0 .08% bis-acrylamide, 8 M urea, 6 mM Triton X-100, and 5% acetic acid . The gels are run for 4 hr at 200 Volts and then 0 .1 ml of 0 .5 M cysteamine is added for 30 min at 100 Volts . The samples are then electrophoresed for 12 .5 hr at 100 Volts. (Top panel) Scan of a gel of marker HeLa histones stained with 0 .4% amido black in 50% methanol, 10% acetic acid . (Middle panel) Translation products of the postribosomal supernatant of S phase cells (solid line) and G1 phase cells (dotted line) . Only the poly(A)- fraction of the G1 RNA was used for this assay . The gels are fractionated into 0 .5 mm slices with a Joyce-Label gel slicer and counted in Hydromix (Yorktown Research) . (Bottom panel) Translation products of S phase cells polyribosomal RNA not retained by oligo(dT)-cellulose (solid line) or absorbed to oligo(dT)-cellulose (dotted line) . The numbers above each peak refer to the different histone species (Zweidler, 1973) . The right hand panels are the same preparations resolved on acid-urea gels by the method of Panyim and Chalkley (1969) .
Cell 66
counts for a very small fraction of the histone mRNA present in the S phase cells at the time of the addition of C-ara to inhibit DNA replication . The absence of histone mRNA in the supernatant of G1 cells supports the previous suggestion that histone mRNA synthesis occurs only in S phase cells (Robbins and Borun, 1967 ; Spalding, Kaijwara, and Mueller, 1966) . These studies were based on labeling experiments and fractionation of newly synthesized polyribosomal RNA by polyacrylamide gel electrophoresis . In addition, polyribosomes of HeLa cells in G1 phase do not synthesize histones in a_ cell-free system and do not contain histone mRNA (Pederson and Robbins, 1970) . The present results allow us to confirm that histone mRNA is catabolyzed at the end of the S phase (Perry and Kelley, 1973) and is not preserved from one cell cycle to the next . One complication in the interpretation of the analysis by gel electrophoresis of histone mRNA is caused by the presence of the 7S RNA species . This RNA does not contain poly(A) since it is found in the poly(A)- fraction (Figure 1A and 1 D), is found in both polyribosomal and supernatant RNA, and is not translated by the ascites cellfree system . An untranslatable RNA of similar size has previously been found in the postribosomal supernatant of rabbit reticulocytes (Jacobs-Lorena and Baglioni, 1972b) . We believe that there is a 7S RNA which has no relationship to histone mRNA ; it seems possible that it is an RNA species of ribosomal origin . The presence of 7S RNA in the pattern of supernatant RNA of cells treated with C-ara (Figure 3, middle panel) is in agreement with this interpretation, though we cannot exclude that within this peak there is some histone mRNA which has been partially degraded or otherwise altered and hence is not translatable . This possibility is suggested by the curious observation of the disappearance of 7S RNA in the postribosomal supernatant of G1 cells (Figure 3, bottom panel) . A more trivial explanation, however, is that cells in G1 are in a different metabolic state and thus have no 7S of ribosomal origin in the postribosomal supernatant . The nature of the translational control of histone synthesis is still unclear . The normal lifetime of histone mRNA is of the order of hours (Liberti, Festucci, and Baglioni, 1973) and thus the rapid disappearance of histone mRNA after a block in DNA synthesis cannot be explained solely by a block in the transcription of histone mRNA followed by normal catabolism of the preexisting mRNA, although such a transcriptional block may occur under these conditions . Butler and Mueller (1973) have studied in some detail the translational control of HeLa histone synthesis . These authors have observed an increase in the cytoplasmic concentration of histones after a block in DNA synthesis ; this transient
increase and the loss of histone mRNA may be prevented by the addition of inhibitors of protein synthesis . The increase in the cytoplasmic concentration of histones may thus represent the first signal in a series of regulatory mechanisms that couple DNA replication and histone synthesis . Gabrielli and Baglioni (1974) have recently shown that addition of histones to cell-free systems does not inhibit specifically histone synthesis, but depresses protein synthesis in general . Histones thus do not act directly as translational repressors of their own synthesis . The nature of the translation control of histone synthesis remains unknown . Experimental Procedures Growth and Synchronization of Cells HeLa cells were grown in suspension cultures in modified Eagle's medium (MEM) as previously described (Eagle, 1959) . In different experiments from 14-28 I of cells were used to extract RNA from unsynchronized cells or from cells synchronized by a double thymidine block according to Stein and Borun (1972) . To block DNA synthesis, unsynchronized cells were incubated 30-60 min with 40 µg/ml of cytosine arabinoside . To obtain cells synchronized in G1, to 6 I of cells at a concentration of 5 x 10 5 ml were added 1 .2 I of warm Eagle's spinner medium (ESM) containing thymidine (Stein and Borun, 1972) to a final concentration of 2 mM . After 12 hr incubation the cells were collected by centrifugation, resuspended in 6 I of warm MEM medium and distributed in 60 Blake bottles. After 7-8 hr the cells which did not attach firmly were eliminated by four hard shakes and removal of the medium . Cell monolayers were washed with 40 ml of ESM . After 2 hr mitotic cells were detached by a gentle shake . These cells were incubated for 120 min in their medium and then collected by centrifugation . This procedure was repeated 6 times to obtain enough cells for RNA extraction from the postribosomal supernatant . Fractionation of Cells and Preparation of RNA The cells were collected by centrifugation and washed in cold Earle's saline . They were resuspended in approximately 1 :100 of the original volume of 10 mM NaCl, 10 mM Tris-HCI (pH 7 .4), and 1 .5 MM MgCl2 . The cells were homogenized in a Dounce homogenizer and the postmitochondrial supernatant was prepared by centrifugation for 10 min at 27,000 x g . Polyribosomes were prepared from postmitochondrial supernatant as previously described and extracted with phenol (JacobsLorena et al ., 1972) . The RNA was precipitated with ethanol ; it was then fractionated by zonal centrifugation and the RNA sedimenting between the 4S and the 18S peaks precipitated with 2 vol of ethanol . This RNA was then fractionated by preparative polyacrylamide gel electrophoresis in a Canalco apparatus following the procedure of Moriyama et al . (1969) and the conditions previously described by Jacobs-Lorena et al . (1972) . Postribosomal supernatant was prepared by 3 hr centrifugation at 15,000 x g of the postmitochondrial fraction . The postribosomal supernatant was made 2 .5% in sodium dodecyl sulphate, 1 mM in ZnCl 2 , 0 .1 mM in naphtalene disulphonic acid, and extracted with phenol (Jacobs-Lorena et al ., 1972) . The RNA was fractionated as described above. RNA was also fractionated by 18 hr centrifugation at 25,000 rpm on 15-30% sucrose gradients . Cell-Free Incubations The Krebs ascites cell-free system described by Mathews and Koerner (1970) has been used . The incubations contained 6/10 vol of ascites extract and 1/10 vol of the 10 fold concentrated
HeLa Histone mRNA 67
components of the cell-free system (ATP, GTP, creatine kinase, and creatine phosphate) plus unlabeled amino acids in the concentrations described by Jacobs-Lorena and Baglioni (1972a) when one labeled amino acid was used . The remaining 3/10 vol were used to add aqueous solutions of the RNA tested or water . One incubation without added RNA was used to determine the endogenous incorporation of the cell-free system . In general, ascites extracts were incubated for 1 hr at 30°C . Aliquots were sampled in duplicate and counted as described by Nishimura and Novelli (1964) . Radioactive amino acids were purchased from New England Nuclear . Analysis of the Products of Cell-Free Synthesis At the end of the incubation Mg-acetate was added to 8 mM and DNAase (Worthington) to 50 mg/ml . In later experiments this digestion was omitted . After 30 min incubation the samples were dialyzed against 0.9 N acetic acid and analyzed by polyacrylamide gel electrophoresis in acid-urea (Panyim and Chalkley, 1969) or acidurea-Triton (Zweidler, 1973) . The gels were fractionated in a Savant Autogeldivider or sliced and counted as previously described by Jacobs-Lorena et al . (1972) . Acknowledgments This work has been supported by research grants of the N .I .H . and N .S .F . to T . W . Borun, A . Zweidler, and C . Baglioni . An EMBO fellowship has provided partial support for F . Gabrielli . The authors would like to thank Dr . Robert Perry and Dawn Kelley for use of their oligo(dT)-cellulose column . Received June 27, 1974 ; revised August 23 and October 15, 1974 References Adesnik, M ., and Darnell, J . E ., Jr. (1972) . J . Mol . Biol . 67, 397-406 . Aviv, H ., and Leder, P . (1972) . Proc . Nat . Acad . Sci . USA 69, 14081412 . Borun, T. W ., Scharff, M . D ., and Robbins, E . (1967) . Proc . Nat . Acad . Sci . USA 58, 1977-1983 . Brendl, M ., and Gallwitz, D . (1973) . Eur . J . Biochem . 32, 381-391 . Butler, W . B ., and Mueller, G . C . (1973) . Biochim . Biophys . Acta 294, 481-496 . Eagle, H . (1959) Science 130, 432-434 . Gabrielli, F ., and Baglioni, C . (1974) . Eur . J . Biochem . 42, 121-128 . Gallwitz, D ., and Breindl, M . (1972) . Biochem . Biophys . Res . Commun . 47, 1106-1111 . Gallwitz, D ., and Mueller, G . C . (1969) . J . Biol . Chem . 244, 59475952 . Greenberg, J ., and Perry, R . P . (1972) . J . Mol . Biol . 72, 91-98 . Jacobs-Lorena, M ., and Baglioni, C . (1972a) . Biochemistry 11, 4970-4974 . Jacobs-Lorena, M ., and Baglioni, C . (1972b) . Proc . Nat . Acad . Sci . USA 69, 1425-1428 . Jacobs-Lorena, M ., Baglioni, C ., and Borun, T . W . (1972) . Proc . Nat . Acad . Sci . USA 69, 2095-2099 . Liberti, P ., Festucci, A., and Baglioni, C . (1973) . Mol . Biol . Rep . 1, 61-67 . Loening, U . F . (1969) . Biochem . J . 113, 131-138 . Mathews, M . B ., and Koerner, A . (1970) . Eur . J . Biochem . 17, 339343 . Moriyama, Y ., Hodnett, J . L ., Prestayko, A . W ., and Busch, H . (1969) . J . Mol . Biol . 39, 335-349 . Nishimura, S ., and Novelli, G . D . (1964) . Biochim . Biophys . Acta 80, 574-586 .
Panyim, S ., and Chalkley, R . (1969) . Arch . Biochem . Biophys . 130, 337-346 . Pederson, T ., and Robbins, E . (1970) . J . Cell . Biol . 45, 509-513 . Perry, R . P ., and Kelley, D . E . (1973) . J . Mol . Biol . 79, 681-696 . Robbins, E ., and Borun, T . W . (1967) . Proc . Nat . Acad . Sci . USA 57, 409-416 . Spalding, J ., Kaijwara, K ., and Mueller, G . C . (1966) . Proc. Nat . Acad . Sci . USA 56, 1535-1542 . Stein, G . S ., and Borun, T . W . (1972) . J . Cell . Biol . 52, 292-307 . Zweidler, A . (1973) . J . Cell . Biol . 59, 378a .
Further Evidence of Transcriptional and Translational Control of Histone Messenger RNA during the HeLa S3 Cycle Ted W . Borun*, Franco Gabriellit, Kozo Ajirom, Alfred'Zweidler#, and Corrado Baglionit *Fels Research Institute and Department of Biochemistry Temple University School of Medicine Philadelphia, Pennsylvania 19140 Department of Biology Massachusetts Institute of Technology Cambridge, Massachusetts 02139 *The Institute for Cancer Research Fox Chase Center for Cancer and Medical Sciences Philadelphia, Pennsylvania 19111
Summary Using a translational assay and new analytical procedures we have found that : -Histone mRNA can be detected both associated with polyribosomes and in the postribosomal supernatant of S phase HeLa S3 cells . -Inhibition of DNA replication by cytosine arabinoside treatment causes histone mRNA to completely disappear from polyribosomes, and little histone mRNA can be detected in the postribosomal supernatant of inhibited cells . These data indicate that histone mRNA does not accumulate in the cytoplasm after the inhibition of DNA replication . -Histone mRNA species cannot be detected in the postribosomal supernatant of G1 cells synchronized by selective detachment . This observation, together with the previous finding that histone mRNA is not present on G1 polyribosomes, is consistent with the idea of a transcriptional block in histone mRNA production and transport to the cytoplasm during G1 . Introduction Nuclear DNA replication and cytoplasmic histone synthesis occur at significant rates only during the S phase of the HeLa S3 cell cycle (Robbins and Borun, 1967) . Since histones are not synthesized in G1 cells prior to the initiation of DNA replication and histone synthesis in S phase cells stops within 30 min if DNA replication is inhibited, it is evident that these two macromolecular synthetic processes are tightly coupled (Robbins and Borun, 1967 ; Borun, Scharff, and Robbins, 1967 ; Gallwitz and Mueller, 1969) . The mechanisms which couple DNA and histone synthesis in the HeLa cell are unknown, but it seems probable that such mechanisms involve control of both the transcription and the translation of histone messenger RNA (mRNA) species (Borun et al ., 1967 ; Butler and Mueller, 1973) .
HeLa histone mRNA was discovered in labeling experiments that suggested that these RNA species sediment between 7S and 9S and are associated with small polyribosomes in cells actively synthesizing DNA (Robbins and Borun, 1967 ; Borun et al ., 1967 ; Gallwitz and Mueller, 1969) . A block in DNA replication not only inhibits histone polypeptide synthesis but also causes a parallel disappearance of labeled mRNA from polyribosomes . Other labeling experiments have indicated that histone mRNA species, unlike most other mRNA, do not contain poly A tracts at their 3' OH termini (Adesnik and Darnell, 1972 ; Greenberg and Perry, 1972) . Recent large scale experiments have made possible the physical isolation of quantities of polyribosomal histone mRNA species sufficient to permit their unequivocal identification by translation in heterologous cell-free protein synthesizing systems (Jacobs-Lorena, Baglioni, and Borun, 1972 ; Gallwitz and Briendl, 1972) . The in vitro translation assay provides an opportunity to investigate several additional aspects of the possible translational and transcriptional controls of HeLa histone mRNA and further characterize these RNA species . In this assay, various RNA preparations are added to an mRNA-dependent mouse-ascites cell-free protein synthesizing system . Measurement of the net stimulation of protein synthesis provides an index of the amount of mRNA in the RNA tested ; characterization of the cell-free synthetic products by various acrylamide gel electrophoresis procedures defines the number and kinds of translatable templates in the RNA preparation . Using this in vitro translation assay and a variety of procedures to fractionate RNA, we have investigated the distribution of histone mRNA in polyribosomes and postribosomal supernatants of G1 and S phase HeLa S3 cells . Parallel studies have been carried out on S phase cells treated with the inhibitor of DNA synthesis cytosine arabinoside . Results The Effect of a Block in DNA Replication on Translatable Polyribosomal Histone mRNA To better characterize histone mRNA species and the effect of a block in DNA replication on polyribosomal histone mRNA, polyribosomal RNA was extracted from HeLa S3 cells which were synchronized in S phase by double thymidine blockade (Stein and Borun, 1972) and from cells treated for 30-60 min with 40 µg/ml of cytosine arabinoside (C-ara) to block DNA replication . To facilitate subsequent analysis, this RNA was fractionated on sucrose gradients to obtain the material which sediments between the 4S and 18S peaks (4-1 8S RNA) .
Cell 60
In some experiments 4-18S RNA was also passed over oligo(dT)-cellulose to separate the RNA containing poly A tracts [poly(A)+] from the RNA which does not [poly(A)-], (Aviv and Leder, 1972) . Figure 1A shows the resolution of one A2 60 unit of control S phase polyribosomal 4-18S RNA which is poly(A)- resolved by electrophoresis on 20 cm analytical gels containing 6% acrylamide and run in 6 mm I .D . quartz tubes . The profile of one A260 of the poly(A) + fraction of this 4-18S RNA resolved under the same conditions is shown in Figure 1 B . Figure 1 C shows further resolution of the RNA in Figure 1A by electrophoresis for a total of 8 .5 hr at 250 volts . After this period of electrophoresis the 4S and 5S RNA have run off the gel and a 7S RNA peak has migrated to near the end of the gel . This 7S RNA is followed in turn by the peaks labeled a through d and by 18S RNA at the top of the gel . It can be seen in Figure 1 D that the peaks labeled a, b, c, and d disappear from the 4-18S polyribosomal RNA of cells treated with C-ara for 60 min to inhibit DNA replication . Subsequent experiments in this paper will show that these RNA species are histone mRNA or aggregates of such RNA . In contrast to the a-d peaks, the 7S and 18S RNA components of the 4-18S polyribosomal RNA of C-ara-treated cells are not affected by the inhibition of DNA replication . To demonstrate that the C-ara-sensitive polyribosomal RNA species are histone mRNA, large quantities (50-100 A260) of 4-18S RNA were isolated from the polyribosomes of control and C-ara-treated cells and fractionated on preparative 5 cm 8% acrylamide gels according to the procedure of Moriyama et al . (1969) as previously described (JacobsLorena et al ., 1972) . A 7-9S peak was obtained from both the control and C-ara-treated cells, but although both preparations came from the same number of cells, the 7-9S peak of C-ara cells contained 31 % as much RNA as the control . The stimulatory activity of RNA contained in these 7-9S peaks was assayed in the ascites cell-free sys-
tem at two different RNA concentrations (Table 1) . The 7-9S RNA isolated from S phase cells stimulated the cell-free system significantly, whereas the 7-9S RNA obtained from C-ara-treated cells did not . The small stimulation observed with this fraction is of the same order of magnitude as that obtained with noninformational RNA, like ribosomal RNA (Jacobs-Lorena and Baglioni, 1972a) . We have attributed this nonspecific stimulation to protection GELS RUN4 HOURS A.
GELS RUN8.5HWRS
Control 4 .185 PdyA(-) RNA
C. Control 4-18SPdyAF RNA
.8 7-9S
7-95
d .4
a b. 45'
7
Q
5S
7S
/85
c
7S
18S
D.ToAa' C-ARA 4-PS RNA
Ccntrd4.18S PdyA(f)RNA
t
.8
7S
4S
(+l
- ~
/o cm
t F~
W
Figure 1 . Analytical Gel Profiles of 4-18S Polyribosomal RNA (A) Control poly(A)- RNA (Aviv and Leder, 1972) from S phase HeLa S3 cells run on 20 cm, 6% acrylamide gels containing 0 .2% bis acrylamide and Loening's Buffer E, containing 0 .2% SDS (Loening, 1969) . The gels were polymerized in 25 cm long 6 mm ID quartz gel tubes and run for 4 .5 hr at 250 volts . The gels were then scanned in an ISCO U .A.4 U .V . gel scanner at 260 nm . One A260 was run on the gel . (B) Control poly(A) + RNA from S cells run as in A, one A260 was run on the gel . (C) Control poly(A)- RNA from S cells run as in A but for a total of 8 .5 hr. (D) One A260 of total 4-18S polyribosomal RNA from S phase HeLa S3 cells pretreated for 60 min with 40 µg/ml cytosine arabinoside to inhibit DNA replication and run on a 6% analytical gel as in Figure 1C .
Table 1 . Protein Synthesis with 7-9S RNA of Polyribosomes of S Phase and Cytosine Arabinoside-Treated HeLa Cells RNA 7-9S of S Phase cells 7-9S of C-ara-treated cells Globin mRNA
Experiment 1 (10 pg/ml) net cpm (stimulation) 2,640 (1 .7) 995 (1 .2) 43,342 (6 .4)
Experiment 11 (70 µg/ml) net cpm (stimulation) 14,852 (3 .9) 4,132 (1 .8) 74,080 (15 .3)
The abbreviation C-ara is used to designate HeLa cells treated with cytosine arabinoside . The net cpm are calculated by subtracting the cpm incorporated in 1 hr at 30°C by the incubation without added RNA (3990 cpm in Experiment I and 5164 cpm in Experiment cpm + RNA II, respectively) from those incorporated in the same time by incubations with added RNA . The stimulation is calculated as cpm RNA' Each incubation contains 10 µl the indicated concentration of RNA, 1 µCi of 3H-amino acid labeling mixture (New England Nuclear) and the cold amino acids not present in this mixture . Duplicate aliquots have been sampled for counting, and the averages referred to 10 µl incubations are indicated .
HeLa Histone mRNA 61
of residual endogenous mRNA (Jacobs-Lorena and Baglioni, 1972a) . These results are strengthened by the electrophoretic analysis of the product of translation of the 7-9S RNA (Figure 2) . The proteins synthesized with RNA isolated from control S phase cells co-migrate with marker histones(Figure2B) ; we have shown previously that this 7-9S RNA codes almost exclusively for histones by analyzing its translation product for tryptophan content (Jacobs-Lorena et al ., 1972) . The pattern of the product of 7-9S RNA isolated from C-ara-treated cells shows an increase over the
background observed in gels of control incubations without added RNA (Figure 2A) . However, while there are several poorly resolved peaks in the pattern, there is no indication that histone mRNA represents a significant component of this RNA . Also note the difference in the ordinates of Figure 2A
4
150
A
.2
100 aU
M
14 50 -
A dOA
4
a
ti4,~'
~ ~A4Q b°
t•
~dy p A
a
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O
N
7S
Q
U
600
60
400
40
I 4-18S FRKT
M
200
20
20
40 60 Fractions
80
Figure 2 . Acrylamide Gel Electrophoresis of the Product of 7-9S Polyribosomal RNA of S Phase and Cytosine Arabinoside-Treated HeLa Cells The incubations are described in Table 1, Experiment II . The samples are treated as described in Experimental Procedures and analyzed on 15% gels (Panyim and Chalkley, 1969) . (A) Solid line, cell-free product of 7-9S RNA of C-ara-treated cells ; dotted line, no RNA added to the cell-free system . The two patterns have been superimposed by using marker 1 4C-labeled histones (arrows) to align them, The two arrows indicate the position of the marker histories which correspond to those shown in the bottom panel . (B) Solid line, cell-free product of 7-9S RNA from S phase cells ; dotted line, marker histones labeled in intact HeLa cells with 14C-lysine ; the peak migrating around Fraction 30 corresponds to F1 histones, whereas the other broad peak migrating at 50-60 corresponds to the other classes of histones, which are poorly resolved .
(-) top Figure 3 . Analytical Gel Scans of Postribosomal Supernatant RNA One A260 unit of RNA was analyzed as described in Figure 1 for 2 hr at 250 Volts . (Top panel) Control S phase postribosomal supernatant RNA . (Middle panel) RNA from the postribosomal supernatant of S phase cells treated for 60 min with 40 µg/ml of cytosine arabinoside . (Bottompanel) Poly(A)- (Aviv and Leder, 1972) 4-185 RNA from the postribosomal supernatant of G1 cells .
Cell 62
and 2B when comparing the amount of protein synthesized with equal quantities of control and C-ara 7-9S RNA in these experiments . These results confirm by a direct translation assay the conclusion reached previously by Borun et al . (1967) and Gallwitz and Mueller (1969) using 3 H-uridine-labeled material that histone mRNA disappears from S phase polyribosomes after a block in DNA replication . It is also apparent from the data in Figure 26 and previous data (Jacobs-Lorena et al ., 1972) that the 7-9S fraction of control S phase polyribosomal RNA prepared by the method of Moriyama et al . (1969) does contain the templates of all the principal classes of histone polypeptide but in an unresolved form . Subsequent work to be reported elsewhere has shown that the various histone mRNA species shown in Figure 1 C are eluted together in one peak which also contains a 7S RNA species . The 7-9S RNA fraction from the C-aratreated polyribosomes evidently consists almost entirely of this 7S RNA which has little template activity . Translation Assay of Histone mRNA in the Postribosomal Supernatant of Control, C-ara-Treated and G1 Cells In order to establish the fate of histone mRNA released from polyribosomes of C-ara-treated cells, we have isolated RNA from the postribosomal supernatant of these and control cells . In a parallel series of experiments, RNA has also been isolated from the postribosomal supernatant of cells synchronized in G1 . The RNA extracted from the postribosomal supernatant was fractionated by the same procedures
used for polyribosomal RNA . Figure 3 shows the resolution of one A260 unit of these supernatant RNA preparations resolved on analytical 20 cm 4% acrylamide gels for 2 hr at 250 volts . The gel pattern of RNA from S phase cells supernatant shows a double peak migrating at 7-9S similar to the double peak observed with polyribosomal RNA in Figure 1 . In the pattern of C-ara cells supernatant RNA only the 7S component is observed, whereas in the G1 cells supernatant RNA no distinct peak is observed migrating at 7S . In order to increase the sensitivity of this analysis we have used total supernatant RNA for control and C-ara cells, but have used for the G1 RNA analysis a 4S to 18S fraction obtained by preparative sucrose gradients . To establish whether the 7-9S RNA found in the supernatant is histone mRNA we have isolated large enough quantities of this RNA by preparative gel electrophoresis, as described above for polyribosomal RNA . The elution profile of S phase and C-ara-treated cells supernatant RNA shows a 7-9S peak, whereas no distinct 7-95 RNA peak is found in the G1 supernatant RNA . The RNA eluted in the region of 7-9S has been concentrated and used in translation experiments . The 7-9S RNA of the postribosomal supernatant of S phase cells causes a significant stimulation of the ascites cell-free system ; that obtained from the supernatant of C-ara treated cells gives a poor stimulation and that from G1 cells no significant stimulation (Table 2) . We have also tested these RNA preparations at different concentrations of RNA, ranging from 0 .05 to 0 .2 mg/mI of RNA .
Table 2 . Protein Synthesis with 7-9S RNA Isolated from Postribosomal Supernatant of HeLa Cells
Source of 7-9S RNA
Experiment 1 (0 .1 mg/ml) net cpm (stimulation)
S phase cells
3,500
(3 .1)
C-ara treated cells
1,255
(1 .9)
G1 phase cells
1,160
(1 .8)
13,990
(10 .1)
(Polyribosomes of S phase cells)
Experiment 11 (0 .2 mg/ml) net cpm (stimulation)
cpm in histones
12,460
(5 .3)
5960
4,864
(2 .1)
1210
In Experiment I each incubation mixture contains in a final vol of 10 µl between 0 .55 and 2 .04 µg of 7-9S RNA, 1 .3 µCi of 3 H-lysine (27 Ci/mmol) and all the components of the cell-free system described under Experimental Procedures . After 1 hr incubation at 30°C, 4 µl are sampled in duplicate . Each RNA fraction has been tested at two or three different concentrations within the linear range of response of the cell-free system shown in Figure 4 . The values reported here are the averages of different determinations normalized to 0 .1 mg/ml of added RNA and referred to a 10 µI incubation mixture . In Experiment II each incubation mixture contains in a final volume of 70 µl between 18 and 26 yg of 7-9S RNA, 10 µCi of 3H-lysine (29 Ci/mmol), and all the components of the cell-free system described under Experimental Procedures . The incubations have been carried out and analyzed as described above . The values reported have been normalized to a 0 .2 mg/mI of added RNA and referred to a 10 µl aliquot . Net cpm and stimulation have been defined in the legend of Table 1 . The cpm in histones have been determined by electrophoretic analysis of the translation product as described in the text . It should be pointed out that the incorporation of 3H-lysine and the corresponding stimulation of the cell-free system were better in Experiment 11 than in Experiment I, when referred to the concentration of RNA used . This is explained by the use of a different sample of the radioactive amino acid in these two experiments . Jacobs-Lorena and Baglioni (1972a) have reported previously that 3 H-lysine may inhibit the ascites cell-free system at high concentrations ; we have now found that different preparations of this amino acid obtained from New England Nuclear are incorporated by the cell-free system in a different way, though consistently within each sample .
HeLa Histone mRNA 63
In order to determine whether the cell-free system responds linearly to the input RNA in this concentration range, we have calibrated the system using polyribosomal 7-9S RNA obtained from S phase cells (Figure 4) . The response is linear up to a concentration of 0 .1 mg/ml and shows a plateau at higher concentrations . At 0 .2 mg/ml the polyribosomal 7-9S RNA is almost saturating the cell-free system . The cell-free product coded for by the supernatant 7-95 RNA preparations has been analyzed as described above for polyribosomal RNA (Figure 2) . Histone synthesis, as defined by co-electrophoresis of the cell-free product with marker histones, has been shown in this way for S phase and C-ara 7-9S RNA preparations . In order to analyze a sufficient amount of translation product of C-ara 7-9S supernatant RNA, which stimulates poorly the ascites cell-free system (Table 2), it has been necessary to apply to the gels a rather large aliquot of the incubation mixture . This overloading of the gels produces electropherograms with poor resolution, when compared to those obtained with S phase supernatant or polyribosomal 7-9S RNA . It has been possible, however, to estimate the relative amount of histone synthesized with the different 7-9S RNA preparations by measuring the radioactive protein present in the fractions corresponding to the major histone peaks (fractions 45-70 of Figure 2) and substracting the background observed in this region of the gels in the control incubations without added RNA . 15
15
I 0
to c a
These data (Table 2) suggest that translatable histone mRNA represents a larger proportion of the 7-9S RNA obtained from the supernatant of S phase cells than of that obtained from cells treated with C-ara . There is less 7-9S supernatant RNA in these cells, and it stimulates poorly protein synthesis and histone synthesis in particular . The 7-9S obtained from postribosomal supernatant of G1 cells does not significantly stimulate in vitro protein synthesis (Table 2) . In order to rule out the possibility that trace amounts of histone mRNA are present in G1 cells and that this mRNA is lost during preparative electrophoresis, we have also translated directly the RNA from postribosomal supernatant of G1 and S phase cells sedimenting between the 4S and the 18S peaks during sucrose gradient centrifugation (see Experimental Procedures) . Translation of the G1 cell RNA is rather poor, whereas that of S cell RNA is quite good (Table 3) . It is interesting to point out in this connection that the RNA isolated by sucrose gradient centrifugation only is found to stimulate protein synthesis more effectively than RNA isolated through several preparative steps . This agrees with similar observations made in isolating sea urchin histone mRNA (K .W . Gross, personal communication) . The products of the translation assays have been analyzed by electrophoresis on acrylamide gels using an improved procedure (Zweidler, 1973) that allows complete separation of the different histones (Figure 5) . The pattern obtained with S cell RNA corresponds very closely to all classes of marker histones, whereas no clear peak corresponding to any histone polypeptide is observed with the translation product of G1 postribosomal supernatant Table 3. Protein Synthesis with RNA Isolated from Gradient Fractions Source of 4-18S RNA
net cpm
(stimulation)
S cells postribosomal supernatant
8,966
(8 .0)
G1 cells postribosomal supernatant
2,411
(2 .1)
poly(A)-
33,541
(26 .7)
poly(A) +
1,750
(1 .9)
S cells polyribosomal :
Figure 4 . Response of the Ascites Cell-Free System to the Addition of Polyribosomal 7-9S RNA of S Phase Cells Each incubation contains in a final vol of 10 µl : 1 .3 µ Ci of 3H-lysine (27 Ci/mmole), the indicated amount of 7-9S RNA from the preparation shown in Figure 1 and all the other components of the cell-free system (see Experimental Procedures) . Duplicate samples are analyzed, and the average of the two determinations is reported ; duplicate determinations differ by less than 10% . The incorporation in the control without added RNA is 682 cpm ; these have been subtracted from the values plotted . The stimulation has been calculated by dividing the cpm obtained in an incubation with added RNA by the cpm in the incubation without added RNA .
Incubations were prepared as indicated in the legend of Table 2, in a final volume of 150 µl . The cpm were referred to 5 td aliquots . RNA concentrations were 0 .15 mg/ml in the experiments with polyribosomal RNA . The RNA has been prepared by centrifugation on sucrose gradients (see Experimental Procedures), by taking the fraction between the 4S and 18S peak but carefully excluding both these peaks. The polyribosomal RNA has been further passed through an oligo(dT)-cellulose column (Aviv and Leder, 1972) . The RNA not retained by the column is referred to as poly(A)- RNA and the RNA absorbed as poly(A) + RNA . The products of translation have been analyzed by gel electrophoresis as shown in Figure 5 . The results obtained with polyribosomal RNA are the average of two separate incubations using two independent preparations of RNA .
Cell 64
ACID-UREA-TRITON
ACID-UREA
OD
OD 2b
2b 2
Carrier Histories
Histones
2
1
2a2
I 1 2a1
3
I 1
i
3.2
U 20
40
8o
6o
cpm
b
mm ,-
4o
20
mm
cpm 600
300-
Supernatant 2a1
400
Supernatant
200 2b 3 2b 1 2a2 2a2 .1
200•
100
3.2
2a1 3 .1 2a2.2
20
40
S s 80
60
cpm 2b
Boo
40
20
0
60s
cpm
mRNA
mRNA
2b
400 PolyA polyA
polYA poIyA t
600
300
J
400-
200 3
2a1
2a2
1 2a1 200 i x
100
3.1
20
40
60
80
1
20
40
60S
HeLa Histone mRNA 65
RNA . We have included in Figure 5 the patterns obtained by the standard acid-urea electrophoresis . Outside of demonstrating the superiority of the acid-urea-triton method of analysis, these patterns are relevant for the identification of the different histone species by a standard procedure .
This identification is based only on the electrophoretic mobility of these proteins, and further studies are necessary to establish whether the mRNA for some of the minor histone species is relatively more abundant in the supernatant fraction . Discussion
Identification of Poly(A)- Histone mRNA Species RNA obtained from small polyribosomes of S phase cells by fractionation on sucrose gradients has been purified by chromatography on oligo(dT)cellulose by the method of Aviv and Leder (1972) . Only about 2% of the RNA is retained by oligo(dT)cellulose . Both the RNA not absorbed to the column and the RNA retained have been tested in the cellfree system . Excellent translation is obtained with the RNA which does not presumably contain poly(A), whereas the RNA retained by oligo(dT)cellulose is translated poorly (Table 3) . The translation products have been analyzed by gel electrophoresis (Figure 5, bottom panel) . The translation products of RNA not containing poly(A) migrate like all five major types of marker histones . A comparison between the products of translation of supernatant and polyribosomal 7-9S RNA can be made in Figure 5 . Relatively less histones F1, F3, and F2b are coded for by supernatant than by polyribosomal 7-9S RNA . These histones have a higher molecular weight than F2a1 and F2a2 . A possible explanation for this finding is that previously proposed for the presence of a-chain mRNA in the postribosomal supernatant of rabbit reticulocytes (Jacobs-Lorena and Baglioni, 1972b) . Smaller mRNAs, which are associated on the average with relatively fewer ribosomes, have a higher probability of being associated with no ribosomes at all by chance alone . This interpretation of the data is complicated by the difficulty of estimating with accuracy the amount of each histone synthesized, due to the presence of some minor peaks in the pattern of the product of supernatant 7-9S RNA . These peaks have tentatively been identified with minor histone species which can be observed in the pattern of carrier histones (top panel, Figure 5) .
We have established by a variety of procedures that practically no identifiable HeLa histone mRNA species remain associated with S phase polyribosomes after a block in DNA replication . In addition, we have observed that histone mRNA is present in the postribosomal supernatant of cells in S phase . This mRNA must represent a relatively sizable fraction of the histone mRNA present in these cells, though at present our methods of isolation and analysis do not allow us to estimate with accuracy the proportion of histone mRNA present in the supernatant relative to that associated with polyribosomes . The 7-9S RNA present in the supernatant may represent mRNA which is recirculating between polyribosomes and supernatant, as it has been suggested for the globin mRNA found in the postribosomal supernatant of rabbit reticulocytes (Jacobs-Lorena and Baglioni, 1972b) . Otherwise, it may represent mRNA of nuclear provenience, transiently present in the postribosomal supernatant before becoming associated with polyribosomes . Analyses of the RNA of C-ara treated cells indicate that, after a block in DNA synthesis, the histone mRNA released from polyribosomes does not accumulate in the postribosomal supernatant . These results are in agreement with previous suggestions that synthesis of new RNA is a prerequisite for histone synthesis after a reversible block in DNA synthesis (Gallwitz and Mueller, 1969) . The present results clearly indicate that RNA species which need to be synthesized under these conditions are histone mRNA . Analytical gels (Figure 3) indicate that there are definitely less histone mRNA-like species present in the postribosomal supernatant of C-aratreated cells . The small amount of histone mRNA found there by the translational assay probably ac-
Figure 5 . Acrylamide Gel Electrophoresis of the Translation Product of RNA Isolated by Sucrose Gradient The incubations are described in Table 3 . The samples are dialyzed against 0 .9 N acetic acid, centrifuged, and precipitated with 7 vol of acetone after addition of 0 .1 mg of carrier HeLa histones . The precipitate is dissolved in 8 M urea, 10% mercaptoethanol, 0 .001 Pyronin Y, and applied to 11 cm long gels containing 12% acrylamide, 0 .08% bis-acrylamide, 8 M urea, 6 mM Triton X-100, and 5% acetic acid . The gels are run for 4 hr at 200 Volts and then 0 .1 ml of 0 .5 M cysteamine is added for 30 min at 100 Volts . The samples are then electrophoresed for 12 .5 hr at 100 Volts. (Top panel) Scan of a gel of marker HeLa histones stained with 0 .4% amido black in 50% methanol, 10% acetic acid . (Middle panel) Translation products of the postribosomal supernatant of S phase cells (solid line) and G1 phase cells (dotted line) . Only the poly(A)- fraction of the G1 RNA was used for this assay . The gels are fractionated into 0 .5 mm slices with a Joyce-Label gel slicer and counted in Hydromix (Yorktown Research) . (Bottom panel) Translation products of S phase cells polyribosomal RNA not retained by oligo(dT)-cellulose (solid line) or absorbed to oligo(dT)-cellulose (dotted line) . The numbers above each peak refer to the different histone species (Zweidler, 1973) . The right hand panels are the same preparations resolved on acid-urea gels by the method of Panyim and Chalkley (1969) .
Cell 66
counts for a very small fraction of the histone mRNA present in the S phase cells at the time of the addition of C-ara to inhibit DNA replication . The absence of histone mRNA in the supernatant of G1 cells supports the previous suggestion that histone mRNA synthesis occurs only in S phase cells (Robbins and Borun, 1967 ; Spalding, Kaijwara, and Mueller, 1966) . These studies were based on labeling experiments and fractionation of newly synthesized polyribosomal RNA by polyacrylamide gel electrophoresis . In addition, polyribosomes of HeLa cells in G1 phase do not synthesize histones in a_ cell-free system and do not contain histone mRNA (Pederson and Robbins, 1970) . The present results allow us to confirm that histone mRNA is catabolyzed at the end of the S phase (Perry and Kelley, 1973) and is not preserved from one cell cycle to the next . One complication in the interpretation of the analysis by gel electrophoresis of histone mRNA is caused by the presence of the 7S RNA species . This RNA does not contain poly(A) since it is found in the poly(A)- fraction (Figure 1A and 1 D), is found in both polyribosomal and supernatant RNA, and is not translated by the ascites cellfree system . An untranslatable RNA of similar size has previously been found in the postribosomal supernatant of rabbit reticulocytes (Jacobs-Lorena and Baglioni, 1972b) . We believe that there is a 7S RNA which has no relationship to histone mRNA ; it seems possible that it is an RNA species of ribosomal origin . The presence of 7S RNA in the pattern of supernatant RNA of cells treated with C-ara (Figure 3, middle panel) is in agreement with this interpretation, though we cannot exclude that within this peak there is some histone mRNA which has been partially degraded or otherwise altered and hence is not translatable . This possibility is suggested by the curious observation of the disappearance of 7S RNA in the postribosomal supernatant of G1 cells (Figure 3, bottom panel) . A more trivial explanation, however, is that cells in G1 are in a different metabolic state and thus have no 7S of ribosomal origin in the postribosomal supernatant . The nature of the translational control of histone synthesis is still unclear . The normal lifetime of histone mRNA is of the order of hours (Liberti, Festucci, and Baglioni, 1973) and thus the rapid disappearance of histone mRNA after a block in DNA synthesis cannot be explained solely by a block in the transcription of histone mRNA followed by normal catabolism of the preexisting mRNA, although such a transcriptional block may occur under these conditions . Butler and Mueller (1973) have studied in some detail the translational control of HeLa histone synthesis . These authors have observed an increase in the cytoplasmic concentration of histones after a block in DNA synthesis ; this transient
increase and the loss of histone mRNA may be prevented by the addition of inhibitors of protein synthesis . The increase in the cytoplasmic concentration of histones may thus represent the first signal in a series of regulatory mechanisms that couple DNA replication and histone synthesis . Gabrielli and Baglioni (1974) have recently shown that addition of histones to cell-free systems does not inhibit specifically histone synthesis, but depresses protein synthesis in general . Histones thus do not act directly as translational repressors of their own synthesis . The nature of the translation control of histone synthesis remains unknown . Experimental Procedures Growth and Synchronization of Cells HeLa cells were grown in suspension cultures in modified Eagle's medium (MEM) as previously described (Eagle, 1959) . In different experiments from 14-28 I of cells were used to extract RNA from unsynchronized cells or from cells synchronized by a double thymidine block according to Stein and Borun (1972) . To block DNA synthesis, unsynchronized cells were incubated 30-60 min with 40 µg/ml of cytosine arabinoside . To obtain cells synchronized in G1, to 6 I of cells at a concentration of 5 x 10 5 ml were added 1 .2 I of warm Eagle's spinner medium (ESM) containing thymidine (Stein and Borun, 1972) to a final concentration of 2 mM . After 12 hr incubation the cells were collected by centrifugation, resuspended in 6 I of warm MEM medium and distributed in 60 Blake bottles. After 7-8 hr the cells which did not attach firmly were eliminated by four hard shakes and removal of the medium . Cell monolayers were washed with 40 ml of ESM . After 2 hr mitotic cells were detached by a gentle shake . These cells were incubated for 120 min in their medium and then collected by centrifugation . This procedure was repeated 6 times to obtain enough cells for RNA extraction from the postribosomal supernatant . Fractionation of Cells and Preparation of RNA The cells were collected by centrifugation and washed in cold Earle's saline . They were resuspended in approximately 1 :100 of the original volume of 10 mM NaCl, 10 mM Tris-HCI (pH 7 .4), and 1 .5 MM MgCl2 . The cells were homogenized in a Dounce homogenizer and the postmitochondrial supernatant was prepared by centrifugation for 10 min at 27,000 x g . Polyribosomes were prepared from postmitochondrial supernatant as previously described and extracted with phenol (JacobsLorena et al ., 1972) . The RNA was precipitated with ethanol ; it was then fractionated by zonal centrifugation and the RNA sedimenting between the 4S and the 18S peaks precipitated with 2 vol of ethanol . This RNA was then fractionated by preparative polyacrylamide gel electrophoresis in a Canalco apparatus following the procedure of Moriyama et al . (1969) and the conditions previously described by Jacobs-Lorena et al . (1972) . Postribosomal supernatant was prepared by 3 hr centrifugation at 15,000 x g of the postmitochondrial fraction . The postribosomal supernatant was made 2 .5% in sodium dodecyl sulphate, 1 mM in ZnCl 2 , 0 .1 mM in naphtalene disulphonic acid, and extracted with phenol (Jacobs-Lorena et al ., 1972) . The RNA was fractionated as described above. RNA was also fractionated by 18 hr centrifugation at 25,000 rpm on 15-30% sucrose gradients . Cell-Free Incubations The Krebs ascites cell-free system described by Mathews and Koerner (1970) has been used . The incubations contained 6/10 vol of ascites extract and 1/10 vol of the 10 fold concentrated
HeLa Histone mRNA 67
components of the cell-free system (ATP, GTP, creatine kinase, and creatine phosphate) plus unlabeled amino acids in the concentrations described by Jacobs-Lorena and Baglioni (1972a) when one labeled amino acid was used . The remaining 3/10 vol were used to add aqueous solutions of the RNA tested or water . One incubation without added RNA was used to determine the endogenous incorporation of the cell-free system . In general, ascites extracts were incubated for 1 hr at 30°C . Aliquots were sampled in duplicate and counted as described by Nishimura and Novelli (1964) . Radioactive amino acids were purchased from New England Nuclear . Analysis of the Products of Cell-Free Synthesis At the end of the incubation Mg-acetate was added to 8 mM and DNAase (Worthington) to 50 mg/ml . In later experiments this digestion was omitted . After 30 min incubation the samples were dialyzed against 0.9 N acetic acid and analyzed by polyacrylamide gel electrophoresis in acid-urea (Panyim and Chalkley, 1969) or acidurea-Triton (Zweidler, 1973) . The gels were fractionated in a Savant Autogeldivider or sliced and counted as previously described by Jacobs-Lorena et al . (1972) . Acknowledgments This work has been supported by research grants of the N .I .H . and N .S .F . to T . W . Borun, A . Zweidler, and C . Baglioni . An EMBO fellowship has provided partial support for F . Gabrielli . The authors would like to thank Dr . Robert Perry and Dawn Kelley for use of their oligo(dT)-cellulose column . Received June 27, 1974 ; revised August 23 and October 15, 1974 References Adesnik, M ., and Darnell, J . E ., Jr. (1972) . J . Mol . Biol . 67, 397-406 . Aviv, H ., and Leder, P . (1972) . Proc . Nat . Acad . Sci . USA 69, 14081412 . Borun, T. W ., Scharff, M . D ., and Robbins, E . (1967) . Proc . Nat . Acad . Sci . USA 58, 1977-1983 . Brendl, M ., and Gallwitz, D . (1973) . Eur . J . Biochem . 32, 381-391 . Butler, W . B ., and Mueller, G . C . (1973) . Biochim . Biophys . Acta 294, 481-496 . Eagle, H . (1959) Science 130, 432-434 . Gabrielli, F ., and Baglioni, C . (1974) . Eur . J . Biochem . 42, 121-128 . Gallwitz, D ., and Breindl, M . (1972) . Biochem . Biophys . Res . Commun . 47, 1106-1111 . Gallwitz, D ., and Mueller, G . C . (1969) . J . Biol . Chem . 244, 59475952 . Greenberg, J ., and Perry, R . P . (1972) . J . Mol . Biol . 72, 91-98 . Jacobs-Lorena, M ., and Baglioni, C . (1972a) . Biochemistry 11, 4970-4974 . Jacobs-Lorena, M ., and Baglioni, C . (1972b) . Proc . Nat . Acad . Sci . USA 69, 1425-1428 . Jacobs-Lorena, M ., Baglioni, C ., and Borun, T . W . (1972) . Proc . Nat . Acad . Sci . USA 69, 2095-2099 . Liberti, P ., Festucci, A., and Baglioni, C . (1973) . Mol . Biol . Rep . 1, 61-67 . Loening, U . F . (1969) . Biochem . J . 113, 131-138 . Mathews, M . B ., and Koerner, A . (1970) . Eur . J . Biochem . 17, 339343 . Moriyama, Y ., Hodnett, J . L ., Prestayko, A . W ., and Busch, H . (1969) . J . Mol . Biol . 39, 335-349 . Nishimura, S ., and Novelli, G . D . (1964) . Biochim . Biophys . Acta 80, 574-586 .
Panyim, S ., and Chalkley, R . (1969) . Arch . Biochem . Biophys . 130, 337-346 . Pederson, T ., and Robbins, E . (1970) . J . Cell . Biol . 45, 509-513 . Perry, R . P ., and Kelley, D . E . (1973) . J . Mol . Biol . 79, 681-696 . Robbins, E ., and Borun, T . W . (1967) . Proc . Nat . Acad . Sci . USA 57, 409-416 . Spalding, J ., Kaijwara, K ., and Mueller, G . C . (1966) . Proc. Nat . Acad . Sci . USA 56, 1535-1542 . Stein, G . S ., and Borun, T . W . (1972) . J . Cell . Biol . 52, 292-307 . Zweidler, A . (1973) . J . Cell . Biol . 59, 378a .
