THE JOURNAI. OF BIOLOGICAL CHE~S~RY Vol. 246, No. 20, Issue of October 25, pp. 6159-6165, 1971

Printed in U.S.A.

Proteins Made in the Mammalian Cell Cycle*

(Received forrpublication, April 30, 1971)

THOUS 0. E'ox$ AND ARTHUR B. PMLDEE

From the Departments oj Biochemical Xciences and Biology, No$ett Laboratories, P&ceton University, Pri?zce- ton, New Jersey 08540

SUMMARY

DNA-binding proteins made by Chinese hamster ovary (CHO) cells at specific times in their cell cycle were chro- matographed on DNA-cellulose and analyzed with sodium dodecyl sulfate polyacrylamide electrophoresis. Some small proteins (less than 25,000 mol wt) were made in S (the period of DNA synthesis), but not during G1 (the period following mitosis and preceding DNA synthesis). These were relatively low in tryptophan and could in part be his- tones. Larger DNA-binding proteins were made equally in early and late interphase.

Proteins from cells labeled in the absence of calf serum during the S period had a different electrophoresis gel pattern compared to cells labeled in the presence of calf serum. Proteins from G1 cells labeled with or without serum had similar patterns. These results suggest that effects of serum are localized temporally in the cell cycle.

Comparing our results with those of Salas and Green (Nature (New Biol.), 229, 165 (1971)), we suggest that the Go state of nongrowing cells is distinct from the normal G1 phase of proliferating cells.

Mammaliall cells in culture proceed from one mitosis to the next by a poorly understood sequence of events. Relative to periods of mitosis (M phase) and chromosome replication (S phase) (l-3) the “gap ” int’ervals, G1 (postmitosis) and Gz (post- DXA synthesis), are less well defined (4-7). To investigate mo- lecular changes and functions during the mammalian cell cycle, we examined the synthesis of DNA-binding proteins in synchronous Chinese hamster ovary cells.

Proteins of two synchronous cultures were labeled with a [Y] and a [%I]amino acid, respectively, and were mised and chro- matographed on DNA-cellulose columns (8-10). Eluted pro- teins were separat.ed by sodium dodecyl sulfat’c polyacrylamide electrophoresis. Concurrent with our work (II), Salas and Green (la), using similar techniques, reported different DNA-

* This work was supported by United States Public Health Service Grant CA 11595, American Cancer Society Grant E-555, and United States Atomic Energy Commission Contract AT (30-l)-4108.

$ Predoctoral Fellow of the National Science Foundation. Present address; Department of Neuropathology, Harvard Medi- cal School, Boston, Massachusetts 02115.

binding proteins in growing and partially serum-deprived, non- growing mouse 31‘6 cells.

EXPERIMENT.4L I’ROCEDURE

Culture of Chinese Hamster Ovary Cell Lines-Chinese hamster ovary (CHO) cells (13) were provided by Drs. D. F. Petersen and R. A. Tobey of the Los Alamos Scientific Laboratory. The cells were maintained in Ham’s F-10 medium (14), which was modified to exclude calcium, heavy metal salts, and pyruvate, as required for harvesting mitotic cells by shaking (15). The bicarbonate- COZ buffer was also omitted, being replaced by 2 x lo- AI HEPESl buffer (Calbiochcm) with regular atmosphere. Cell growth for CHO’s was found to be the same with HEPES or bi- carbonate buffers. The medium was supplemented with calf se- rum (lo%), fetal calf serum (5$&) (both from Baltimore Biolog- ical Laboratory), and a penicillin-st.reptomycin solution (Grand Island Biological Company) at concentrations of 100 units and 100 pg per ml, respectively. Tests by two independent labora- tories of the frozen cell stocks and our own routine tests of growing stocks detected no contamination by l~leuropneumonia- like organisms (Pl’LO’s).

Harvesting Xitotic ClIO Cells by Shaking-Rounded metaphase cells were detached from monolayer cultures by shaking with a Precision equipoise reciprocating shaking machine, as described by Tobey, Anderson, and l’ctersen (15) after the method of Terasima and Tolmach (16). Suspensions of cells with mitotic indices of about 9O70 were routinely obtained from either Pyrex l-liter Blake bottles or polystyrene (Falcon) 250.ml T-75 flasks. The initial mitotic synchrony was usually determined by visual inspection of cells in the microscope. The large mitotic cells al- most all divided to G1 cells within 10 to 20 min. The degree of synchrony was determined in later parts of the cycle, during which S cells were labeled, in parallel with the protein-labeling experiments. Synchronous cells on glass cover slips were cul- tured continuously with [3H]tllymidinc (3 &i per ml) and were assayed for incorporation by autoradiography (17).

For culturing, harvested cells were left to settle and attach to 75.cm2 (T-75) polystyrene flasks. During the labeling interval, these cells were incubated in volumes of 2.5 t’o 4 ml of medium. This small volume was sufficient for culturing because the flasks were continuously rocked on a platform that allowed all cells to be bathed in a volume of liquid that could not cover a stationary flask.

1 The abbreviations used are: BEPES buffer, sodium 11’-2-hy- droxyethylpiperazine-iV’-2-ethanesulfonate; CHO, Chinese ham- ster ovary; SDS, sodium dodecyl sulfate.

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Proteins Made in Mammalian Cell Cycle Vol. 246, No. 20

Lubeling of Proteins with Radioactive Amino Acids--Labeling medium was prepared with dialyzed calf serum and dialyzed fetal calf serum. The following isotopes (New England Nuclear) and amounts were used: L-[4,5-3H]leucine (100 to 130 PC1 per ml, 6.0 to 7.0 X lO-6 M leucine), L-[U-14C]leucine (10 to 13 PCi per ml, 3.5 to 4.5 x lop5 M leucine), L-[G-3H]tryl~tophan (58 PC1 per ml, 8.0 x 10M6 M tryptophan), and L-[U-WIlysine (4.2 PCi per ml, 1.5 X 10e5 RI lysine).

Preparation of Protein Samples-To labeled cultures of CHO cells on polystyrene T-75 flasks were added 1 to 2 ml of a hypo- tonic buffer with 1 x 1OW M NaCl, 1 x 1O-2 M Tris-HCl (pH 8.0) at 4”, and 1.5 X 10m3 M MgClz (18). The cells were allowed to swell for several minutes, and were then placed at -20” to freeze. The samples were thawed, and enough solid NaCl was added to give a final salt concentration of 2.0 ~1. They were then frozen and thawed once more.

By the procedure of Yamamoto and Alberts (19), DNA was pelleted with a polyethylene glycol (PEG)-NaCl solution. One- half volume of a 42(r, (w/v) solution of PEG (Carbowax 6000, powdered, Union Carbide) in a buffer with 2 x 1OW M Tris-HCl, (pH 8.1) at 4”, 1 X 10W3 M EDTA, 1 X lo+ M 2-mercaptoethanol, and 2.0 M NaCl was added to the cell suspension. This gave a final concentration of 14% (w/v) polyethylene glycol and 2.0 M NaCl. After sitting at 4” for 2 hours the preparation was centri- fuged 15 min at 12,800 x g. The nearly DNA-free supernatant was dialyzed at 4” against 2 x 1OW M Tris-HCl (pH 8.7), 1 x 10e3 M EDTA, 1 X 1OW XI 2-mercaptoethanol, and 0.15 M NaCl to lower the NaCl concentration and remove magnesium ion. The dialyzed sample was centrifuged for 30 min at 39,000 rpm in a Spinco rotor 40 to obtain a particulate-free supernatant.

DNA-cellulose Chromatography-Munktell 410 cellulose, elec- trophoresis grade from Bio-Rad, was washed with ethanol (to remove pyridine), NaOH, and HCl, rinsed in distilled H20, and left with a gauze cover to dry. Approximately 30 g (about 75 ml) of the dried powder were sprinkled into a solution of 100 mg of calf thymus DNA (Worthington) in 50 ml of a buffer with 1 x IO+ M Tris (pH 7.4) and I X 1OW M EDTA. The resulting thick paste was allowed to dry under gauze. This partially dried DXA-cellulose was placed in a desiccator with vacuum and then left overnight with continuous vacuum pumping. The dried DT\‘X-cellulose was suspended in 100 ml of the Tris-EDTA buffer and placed in the cold room overnight. This cold slurry was washed three times by centrifugation to remove fines and un- bouud DNA.

To determine the amount of Dn’h bound to the cellulose, an aliquot of slurry was diluted and heated for 10 min at 98”. Ab- sorption at 260 nm of the supernatant indicated the amount of released DNA. A little over half of the original DNA stuck to the cellulose; there were 730 pg of DNA per ml of packed wet vol- ume. The DNA-cellulose was stored for more than 1 year as a frozen slurry at -20”. During this time the binding of DNA to cellulose apparently increased because less DNA4 was released by heating. iZssay of bound DSL1, using the diphenglamine color test for deoxyribose (20), indicated that the same amount of DNA was still present on the cellulose.

The DNAccllulose column had au inner diameter of 5 mm. A slurry iu dialysis buffer of 0.6 t,o 1.2 packed nrl of DNA-cellulose containing 440 t’o 880 pg of native calf thymus DKB was added. Most of the cellulose lvas alloned to settle; the fines were dis- carded. The ccllulosc was then washed with the buffer at 2 ml per hour with a Buchler Polystaltic pump, as were all further sol-

vent additions. Several hours before loading the sample, the dialysis buffer was removed and the column was equilibrated with 0.15 M NaCl elution buffer, consisting of dialysis buffer with 10% glycerol and 100 pg per ml of bovine albumin Fraction V (Sigma). Samples, usually 2 to 10 ml, were then added, followed by rinsing with at least 6 column volumes of the 0.15 M NaCl elution buffer. Proteins were eluted in two steps with 6 column volumes each of 0.60 M NaCl elution buffer and 2.0 M NaCl elution buffer.

Specificity of binding by proteins to the DNA portion, rather than the cellulose moiety, of DNA-cellulose was determined in several ways. In several experiments the protein extract was first run through a pure cellulose column before loading onto DNA-cellulose. In a modification of this procedure, a cellulose plug was placed directly over the DNA-cellulose plug in the same glass column and was then removed after loading but before elu- ting the proteins. In each case the cellulose was checked for ra- dioactivity and was found to have less than 0.2% of the total counts, Furthermore, experiments run without a prior cellulose column or plug did not yield different elution profiles or polyacryl- amide gel patterns. It is accordingly appropriate to refer to DNA-cellulose binding proteins as DNA-binding proteins.

Electrophoresis of Eluted Proteins on Polyacrylamide Gels- Eluted samples, usually 2 ml, were vacuum-dialyzed to remove salts and to concentrate the protein to about 0.1 ml. Two vol- umes of sample buffer containing 1 x 1OW M sodium phosphate buffer (pH 7.1), 0.5% SDS, and 19% glycerol were then added to the protein. Bromphenol blue as a migration marker was added, and the solution was heated at 98” for 2 min.

Electrophoresis gels, 0.6 x 8 cm, were made with 7.5y0 acryla- mide and 0.5y0 ethylene diacrylate, as cross-linking agent, by polymerizing with N , N , iv’, N-tetramethylethylenediamine and ammonium persulfate in 0.1 M sodium phosphate buffer, p1-I 7.1, containing 0.570 SDS (21). Samples were run at 3 ma per gel for 30 min to concentrate the protein into a narrow band and run it into the gel, and then electrophoresed at 5 to 8 ma per gel un- til the bromphenol blue (behaving as would a protein with mol wt 18,000 in these gels) had migrated three-quarters of the length of the gel toward the anode.

Gels were removed, frozen, and sliced (6 per cm) with a stack of razor blades, and each slice Jvas dissolved in 0.5 ml of 1.6 M

aqueous ammonia and was then counted in a l-dram vial (Mercer Glass Company) with 4 ml of Bray’s solution containing 4% Cab-0-Sil (21) with an Intertechnique liquid scintillation spec- trometer.

Known proteins were electrophoresed to obt’ain a standard linear relationship between the log of molecular weight and mi- gration distance (22-24). Bromphenol blue ran at nearly 18,000. Data were processed by computer, and all SDS gel patterns were normalized with bromphenol blue at a standard position and low molecular weight proteins to the right.

RESULTS

Attainment of Xynchrony-Harvested cells had an initial rnitot’ic index of 80 to 95%. Kcarly all of these became G1 cells within 30 min after settling on the surface of the culture flask. Syn- chrony of CHO cells quickly diminished, however, because the G1 lengths of cells in a population varied over a 4-fold range (17). Fig. 1 shows the progression of cells through the cycle.

Knowing t’he average lengths of G1, S, and GZ in CHO cells (4.8, 4.1, and 2.7 hours, respectively (25)), Fig. 1 allowed deter- mination of how synchronous the cells were in different parts of

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80- ‘ii 2 Y 60-

i 40-

20

! 2 4 6 8 10 12 14 16 18 20 TIME IN CULTURE (HOURS)

FIG. 1. Synchrony of Gr and S cells during labeling experiments. The bars are the labeling intervals for Gr and S. Cells that have entered S (circles) were assayed as those that contained nuclear grains in autoradiographs after they were grown continuously with tritiated thymidine. The dashed line represents cells that had reached the next mitosis (metaphase). The onset of Gz (squares) has been estimated by assuming that it is parallel to metaphase and precedes it by about 3 hours (25).

the cycle. (It must be noted that the times quoted above were calculated with nonsynchronous growing cells. Published data (25) and our data (Fig. 1) with synchronous cells, however, indi- cate lengthened average phase intervals: Gr, 7 to 8 hours, and S plus Gz, 7 to 9 hours. For estimation of asynchrony, we have assumed that the relative phase lengths have remained nearly the same.) Cells were grown with radioactive amino acids during the intervals, Gr and S, as indicated by bars in Fig. 1. Greater than 90% Gr cells were labeled with amino acids in the first in- terval, but approximately 40% Gr, Gz, and M cells were present when cells were labeled in the second interval. Fig. 1 also indi- cates that labeling done in this manner could not give proteins labeled mostly during Gz since synchrony was poor during Gz (at about 9 hours), and because Gz is relatively short compared to Gr and S.

Synchronous cells labeled in the above manner yield quite pure patterns for proteins synthesized during the Gr interval. Pro- teins made uniquely in S and Gz can then be seen as qualitatively new species. But proteins made uniquely in Gr would be less easily identified since some G1 cells were always present in the labeling intervals for S and Gz. Consequently, they should be identified by quantitative differences rather than qualitative ones.

Labeling Proteins in Synchronous G1 and S Cells-Proteins labeled in Gr cells (1 to 3+ hours postmitosis) with rH]leucine and in S cells (8 to 103 hours postmitosis) with [14C]leucine were co- chromatographed on DNA-cellulose columns (Fig. 2). Proteins which did not bind, and ran through the native DNA-cellulose column (Fig. 2a) at 0.15 M NaCl, were then chromatographed on a denatured calf thymus DNA-cellulose column (Fig. 2b). The fractions of the total extract which did bind to native and de- natured DNA are summarized in Table I.

Qualitative differences among the proteins from the elutions shown in Fig. 2 (and Table I) were determined on 7.5% poly- acrylamide gels containing 0.5% SDS. These patterns (Fig. 3) show 10 to 20 peaks of proteins binding to native DNA in each of the two salt eluates. During S one or more classes of proteins with molecular weights below 25,000 were uniquely synthesized.

NAA”E DNA COLUMNi 0151 06 120

NC CM)

/ b / I

DENATURED DNA COLUMN

0 10 20 0 10 20 ELUATE VOLUME, ml ELUATE VOLUME, ml

FIG. 2. Elution profiles from native and denatured DNA-cellu- lose of labeled G1 and S proteins. Proteins were loaded onto the of columns in 0.15 M NaCl buffer and were eluted with 0.6 M and 2.0 M NaCl buffers. Of each fraction, 20~1 were counted in 3 ml Bray’s scintillation fluid. Cells were labeled during G1 (1 to 3; hours postmitosis) with [aH]leucine (0) and during S (8 to 103 hours postmitosis) with [l%]leucine (A). These are total counts and have not been normalized as in Table I. a, elution of proteins from native DNA-cellulose. b, elution from denatured DNA- cellulose of proteins that did not bind to native DNA-cellulose.

TABLE I Per cent of total protein eluted

Soluble, and nearly DNA-free, protein in buffer containing 0.15 M NaCl was applied to a native calf thymus DNA-cellulose column. Most of that extract did not bind and was then applied to a denatured calf thymus DNA-cellulose column. The respec- tive fractions (of proteins labeled in G1 and S cells) that did bind and that were then eluted with buffers containing 0.6 M NaCl or 2.0 M NaCl are indicated in the table. The total percentages account for the fractions of the extract that bound to either native or denatured DNA; very little residual radioactivity was detected on DNA-cellulose after it had been washed with 2.0 M NaCI.

Native DNA Denatured DNA Phase Total

0.6 M NaCl 2.0 xi NaCl 0.6 x NaCl 2.0 x NaCl -~____~

Gl 1.8 1.0 1.2 0.4 4.4 S 2.1 1.6 1.4 0.5 5.6

The large and intermediate proteins were made in nearly equal amounts and ratios during G1 and S. The over-all profiles of radioactivity and the large differences seen among small proteins of G1 and S cells were seen in all experiments. Some variations, however, in relative heights of major peaks were seen among ex- periments. Some differences in single gel slices, as Fraction 7 in Fig. 3b, were not seen, repeatedly.

Three to five major peaks of proteins were eluted from de- natured DNA (Fig. 3, c and d). Little variation in synthesis between G1 and S was detected. The protein peak eluted at 0.6 M NaCl is similar to a protein fraction from 3T3 mouse cells, which was isolated by similar methods and was under investiga- tion prior to our study.2

Proteins, from Gr and S cells, that bind to DNA between 0.05 M NaCl and 0.15 M NaCl were also studied. Proteins in 0.15 M

NaCl were prepared and chromatographed on native DNA-cellu- lose, as above. Proteins that did not bind to the column in 0.15 M NaCl were then dialyzed against buffer containing 0.05 M

NaCl, were centrifuged to obtain a particulate-free supernatant,

2 G. Herrick and B. Alberts, unpublished results.

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6162 Proteins Made in Mammalian Cell Cycle Vol. 246, No. 20

and were then chromatographed on native DNA-cellulose. A protein fraction was eluted with 0.15 M NaCl buffer. The pro- teins that did not bind to this column were rechromatographed on denatured DNA-cellulose. The SDS polyacrylamide profiles of these protein fractions are in Fig. 4.

PROTEINS ELUTED FROM NATl”E DNA WITH 0 5 M NaCl

90,000 49,000 27,000 ~10,000 MOLECULAR WEIGHT (APPROXIMATE)

1 b

These proteins (Fig. 4) that bind between 0.05 M NaCl and 0.15 M NaCl appear to be very similar for G1 and S. Only a small difference was observed for two peaks of large proteins that bind to native DNA. A similar protein peak as observed be- tween 0.15 M NaCl and 0.6 M NaCl (Fig. 3~) was eluted specifi- cally from denatured DNA.

Labeling of S Proteins with Lysine and Tryptophan-Differen- tial amino acid labeling of S cells was done to characterize the S-enriched proteins eluted between 0.6 M NaCl and 2.0 M NaCl. Because histones contain lysine but contain no more than trace amounts of tryptophan (26), cells in S (4 to 12 hours after mitosis) were simultaneously labeled with [3H]tryptophan and [14C]lysine. Proteins from these cells were prepared as above in 0.15 M NaCl and were chromatographed on native DNA-cellulose. The frac- tion removed between 0.6 M NaCl and 2.0 M NaCl was then analyzed by SDS polyacrylamide electrophoresis (Fig. 5).

Cl t

i

-I MOLECULAR WEIGHT (APPROXIMATE)

042

PROTEINS ELUTED I FROM DENATURED DNA % WITH 0 15 M NaCl

I 3 088 8

2

i

90.000 49.000 *7.000 ~10,000 MOLECULAR WEIGHT (APPROXIMATE)

FIG. 4. SDS polyacrylamide gel profiles of proteins bound to DNA-cellulose between 0.05 M NaCl and 0.15 M NaCl. a, proteins eluted from native DNA. b, proteins eluted from denatured DNA. Circles represent [zH]leucine (Gt) and triangles represent [*%]leucine (S). Approximate molecular weights are plotted on the abscissa. Counts are expressed as per cent of total in the soluble extract. Total single isotope counts per min were between 2,000 and 10,000 for these gels.

FIQ. 3. SDS polyacrylamide gel profiles of proteins eluted in Fig. 2. a, proteins eluted from native DNA-cellulose with 0.6 M NaCl. b, proteins eluted from native DNA-cellulose with 2.0 M NaCl. c, proteins eluted from denatured DNA-cellulose with 0.6 M NaCl. d, proteins eluted from denatured DNA-cellulose with 2.0 M NaCI. Circles represent [3H]leucine (GI) and triangles represent [Wlleucine (S). Approximate molecular weights are plotted on the abscissa. Counts are expressed as per cent of total in the soluble extract. Total single isotope counts per min were between 1,700 and 12,000 for these gels.

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S-CELL PROTEINS ELUTED FROM NATIVE DNA WITH 2.0 M NoCl

90,000 49,000 27,000 ~10,000 MOLECULAR WEIGHT (APPROXIMATE)

FIG. 5. SDS polyacrylamide gel profile of proteins from S cells labeled with [l%]lysine (A) and PH]tryptophan (0). Counts are expressed as per cent of total in the soluble extract. Total iso- tope counts per min were 9000 for [l%]lysine and 3000 for f3H]tryp- tophan.

Proteins with large and intermediate sizes incorporated trypto- phan in similar proportion to lysine and leucine (Fig. 5 and com- pare Fig. 3b). The small proteins (less than about 25,000 mol wt) contained relatively little tryptophan, although some pro- teins in that region did incorporate tryptophan. This result is consistent with the suggestion that some of these proteins are histones. The small peak of tryptophan in this region may cor- respond either to other proteins containing tryptophan that are made during S or to proteins made during both G1 and S. We were not able to get these cultures of synchronous cells to in- corporate enough [l%]tryptophan to check the latter possibility directly in a double label experiment,

The profile of incorporated lysine in proteins made in S cells is nearly the same as that for leucine (compare Figs. 36 and 5). The protein peak corresponding to about 35,000 molecular weight, however, was significantly higher when cells were labeled with lysine. The same result was obtained for S cells labeled simultaneously with [l*C]lysine and t3H]leucine. This peak may contain some lysine-rich histones. Elgin and Bonner (27) found that lysine-rich rat liver histones migrated to this position in SDS polyacrylamide gels, whereas the other histones migrated to positions that indicate molecular weights less than 25,000. Fur- thermore, Gurley and Hardin (28) reported for CHO cells that the lysine to leucine ratio was 6.7 for the lysine-rich histone frac- tion and only 0.8 to 1.7 for the other histones.

Labeling Proteins in Synchronous Cells in Absence of Calf Serum-Because serum is required for growth of CHO cells, we examined the effects of serum deprivation during the labeling periods in G1 and S. Cells in four intervals of the cycle were ex- amined. Cells in early and late G1 (1 to 33 hours and 4 to 6+ hours postmitosis) were labeled in the absence of serum with [3H]leucine and [YJleucine, respectively. In parallel, other cells in early and late “S” (7 to 9s hours and 103 to 13 hours postmito- sis) were labeled with the two isotopes. Two separate soluble protein samples were prepared from the mixture of early and late G1 cells and the mixture of early and late “S” cells. After bind- ing to native calf thymus DNA-cellulose columns in 0.08 M NaCl, the proteins were eluted stepwise with 0.15 M NaCl, 0.6 M NaCl, and 2.0 M NaCl buffers. The elution profiles for rH]leucine and [l*C]leucine were the same for the two G1 intervals, and likewise for the two S intervals. Therefore, to allow comparison of G1 with S, only the profiles of [3H]leucine have been plotted (Fig. 6).

m 5,000 2,500 tg;% 1 .YT 1 ~42 1 ,.,o ! Per Cenf of

0.08 1 0.15 ( 0.6 12.0 NoCl IM)

ELUATE VOLUME. ml

1 1.16 1 2.13 1 821 PerCentofExtrocl

0081 015 1 06 120 NaCl(M)

i ELUATE VOLUME, ml

FIG. 6. Elution profiles from DNA-cellulose of proteins from cells labeled in the absence of serum during GI and S. Proteins were bound to columns in 0.08 M NaCl and were eluted with 0.15 M NaCl, 0.6 M NaCI, and 2.0 M NaCl buffers. Of each fraction, 20 ~1 were counted in 3 ml of Bray’s scintillation fluid. Because the elution patterns of [WI?]leucine are similar, only the patterns for [3H]leucine are presented to allow comparison of G1 and S. a, cells labeled without serum during GI (1 to 33 hours postmitosis). b, cells labeled without serum during S (104 to 13 hours postmito- sis).

7.49 ‘, iI 2.0 5.95 li 2.0 :lj&-- ::;w

84.000 43.000 20,000 84,000 43,000 20,000

MOLECULAR WEIGHT (APPROXIMATE) MOLECULAR WEIGHT (APPROXIMATE)

FIG. 7. SDS polyacrylamide gel profiles of proteins eluted in Fig. 6. a, G1 proteins eluted with each of three NaCl concentra- tions were electrophoresed. b, S proteins eluted with each of three NaCl concentrations were electrophoresed. Solid lines represent [3H]leucine and broken lines represent [l%]leucine. Ap- proximate molecular weights are on the abscissa. Counts, ex- pressed as per cent of total gel counts, are on the ordinate. Total single isotope countsper min were between 600 and 15,000 for these gels.

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The eluted peaks were then analyzed by electrophoresis in poly- acrylamide gels containing 0.5% SDS (Fig. 7).

In the absence of calf serum numerous proteins were different’ly synthesized during G1 and S. Differences were not, however, observed between early and late G, intervals, or between early and late S intervals. Between G1 and S, the 0.6 RI XaCl and 2.0 JI KaCl SDS polyacrylamide elcctrophoresis profiles appeared markedly different; the 0.15 M iSaC eluates were more similar. These differences between G1 and S were also seen in the column elution patt,crns (Fig. 6). The 0.6 M fraction, which qualitatively exhibited the most differences between CL and S (Fig. 7), was considerably increased in relative magnitude during S (Fig. 6). These qualitative and quantitative differences were in contrast to proteins labeled in cells provided &h the normal amount of serum, for which only the small S proteins were cyclically pro- duced.

The protein differences observed between labeling cells with or without, serum were mostly seen in S (compare Figs. 3, 4, and 7). Relatively few changes occurred in G1, except for quantita- tive change5 in middle-sized proteins in the 2.0 RI KaCl eluate. The differences when S cells were lab&d without serum consisted primarily of the addition of large and intermediat,e sized proteins in the 0.6 JI XaCl eluate. Proteins in the 2.0 RZ KaCl eluates, made during S, were less different with and without serum; the differences between G1 and S in this fraction were similar t’o those seen in the presence of serum.

DISCUSSION

The larger DNAbinding proteins synthesized by G1 nnd S cells were similar, but one or more classes of small prot’eins were labeled primarily during S. These S-enriched proteins had molecular weights around 15,000, ranging from about 10,000 to 25,000 as determiued by SDS polyacrylamide clcctrophoresis.

Histone synthesis and DNA4 synthesis have been shown in previous investigat.ions (7, 28-30) to be largely coincident. This relationship and the small size of most histones (27) suggest that the S-enriched DNA-binding proteins may be histories. How- ever, Elgin and Banner (27) have also found that some nonhis- tone chromosomal prot,eins migrate in SDS polyacrylamide gels to the same region (less than 25,000 mol wt) as do most, histones and as do the S-enriched proteins that we see. Since the small nonhistoric and historic chromoaomal proteins may serve com- plemelltary functions in t.he cell nucleus, they may both be syn- thesized during S. We therefore attempted to learn whether any of the S-enriched proteins were histones.

Because histones do not contain tryptophan, the incorporation of [31-I]trylptophan into the S-enriched proteins was determined (Fig. 5). Hi&ones should not contain incorporated tryptophan. The low amount of relative tryptophan incorporation suggests that these proteins are partially histones. Evidence that these proteins are basic would also be necessary to show that they are histoues, since some nonhistone DSAbinding proteins with small sizes could also lack tryptophan. Our data do not permit us to determine what proportions of the S proteins are, respectively, histone and nonhistone.

When SDS polyacrylamide patterns of proteins from cells la- beled with and mit.lzout serum were compared (Figs. 3, 4, and 7), several differences were observed. Additional int.ermediate and large sized DXA-binding proteins were observed for S cells labeled without serum. This apparent increaycd product,ion may represent the accumulation of precursor molecules Klien serum

is present, such proteins might either become degraded and lose affinity for DNA or become parts of larger st.ructures, such as nuclear membrane. Nuclear growth has been shown to occur

almost exclusively during S (31) and may include DNA-binding components. Furt,hermorc, Amos, Hoyt, and Horisberger (32) have shown that the movement of lysine-labeled proteins from the nuclei of chick embryo fibroblasts is serumdependent. These two observations suggest that general nuclear growth is restricted to the 8 period and requires serum in the growth me- dium.

Although the syntheses of proteins in cells grown mith and without serum were not assayed simultaneously in double label expcrimcnta, an indirect comparison of the two types of results appears valid for several reasons. First, the column elution pat- terns for proteins of G1 and S cells grown with label iu the absence of serum were strikingly different, and reproducibly so. In toll- trast, the column elution pat,terns were qualitatively the same for proteins of G1 and S cells grown with label in the presence of serum; t,his was seen reproducibly in many single and double label experiments. Furthermore, two respective sets of clectro- phoretic patterns were seen in all experiments conducted either with or without serum present.

Several further qualifications are necessary n-hen evaluating these protein synthesis experiments based on the incorporation of labeled amino acids. To allow absolute comparison of syn- thetic patterns, t’ransport rates and pool sizes of precursors must be the same for the tTy\-o cell populations. Significant changes during the cell cycle iu t’ransport rates and pool sizes have not% beeu reported. In any event, an over-all change should not alter relative peak heights in cell fractions.

Different rates of specific protein turnover between G1 and S would alter the relative incorporation of amino acids by proteins made throughout the cell cycle. Thus the observed differences in labeling of a particular protein could represent either cyclic synthesis with a constant rat’e of turnover or continuous synthesis with relatively higher turnover during the remainder of the cell cycle (or both). Uoth mechanisms could provide the same rcgu- lation of function; therefore, both could be physiologically sig- nificant.

Snlas and Green (12) have reported differences for DSAbind- ing proteins synthesized in growing versus serurn-dcpriced, rcst- ing 3T6 mouse cells. L1.e did not find differences between Gl and S for proteins bindin,, v at the same NaCl concent,rntions, to both native and denatured calf thymus DNA. 11-c interpret the resting state obtained by Salas and Green as representing Go, a state of i~oul)i’oliferatioii (33). Therefore, those two types of experiments suggest that the synthesis of DN,Lbinding proteins differs for Go and G1. This conclusion indicates that Go and G1 differ qualitatively in their metabolic patterns. Go does not simply represent a prolonged G1 period with reduced metabolic activities; it is qualitatively, as well as quantitatively (34), distinct from G1.

Serum effects on the synthesis of DNAbinding proteins first exhibit themselves in S. We have cxarnilled the effects of serum deprivation for periods of a few hours during G1 and S. By a different approach, Salas and Green (12) examined the effects of different serum concentrations on nongrowing cells. Both results suggest that when the serum supplied to cells is reduced protein production is altered during S. These alterations may then lead to the resting state that follows. Go could result from cells en- tering an S state with altered protein production.

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Issue of October 25, 1971 T. 0. Fox ntrcl A. B. I-'artlee 61%

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Thomas O. Fox and Arthur B. PardeeProteins Made in the Mammalian Cell Cycle

1971, 246:6159-6165.J. Biol. Chem. 

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