Printed in Sweden Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form resrrced
Experimental Cell Research 88 (1974) 319-326
UTILIZATION
OF PRE-EXISTING
CHROMOSOMAL
PROTEIN
MESSENGER SYNTHESIS
RNAs
FOR NONHISTONE
IN WI-38 HUMAN
DIPLOID
FIBROIiLASTS G. S. STEIN and D. E. BURTNER Vniuersity
of Florida,
Department of Biochemistry,
Gainesville,
Fla 32610, USA
SUMMARY Contact-inhibited monolayers of WI-38 human diploid fibroblasts show an increase in chromatin template activity within 1 h following stimulation to proliferate. Direct evidence that nonhistone chromosomal proteins are responsible for this increased transcriptional activity of the genome comes from previous studies which demonstrate that the template activity of chromatin reconstituted with nonhistone chromosomal proteins from WI-38 cells 1 h following stimulation is higher than the template activity of chromatin reconstituted with nonhistone chromosomal proteins from contact-inhibited cells. The present studies indicate that there is an increase in the amount of two specific newly synthesized molecular weight classes of nonhistone chromosomal proteins in chromatin 1 h following the stimulation of contact-inhibited WI-38 cells to proliferate and that the synthesis of these nonhistone chromosomal proteins occurs in the absence of new RNA synthesis. The possibility that such nonhistone chromosomal protein synthesis early during the prereplicative phase of the cell cycle may be regulated at the translational level is discussed.
When non-dividing cells are induced to proliferate by the application of specific stimuli, a complex and interdependent series of biochemical events are triggered which result in DNA replication and mitosis. Such modifications in the synthesis of macromolecules require alterations in the readout of information contained within the genome [ 1,2]. Evidence is rapidly accumulating which suggests that nonhistone chromosomal proteins may play a key role in the regulation of gene expression in eukaryotic cells [l-6] and specifically in the control of RNA synthesis during the cell cycle [I, 2, 7, 81. The stimulation of contact-inhibited monolayers of WI-38 human diploid fibroblasts to proliferate, by replacing exhausted growth medium with fresh medium containing 20 % fetal calf serum. leads to an increase in the
transcription of RNA within 1 h. This is reflected by a two-fold increase in uridine incorporation into whole cells as well as by a similar increase in the template activity of isolated chromatin [9]. Several lines of evidence suggest that the nonhistone chromosomal proteins associated with the genome of WI-38 fibroblasts 1 h following stimulation are responsible for the increased transcriptional activity observed at this time. An elevated rate of nonhistone chromosomal protein synthesis has been reported [lo]. More direct evidence that nonhistone chromosomal proteins function in such a regulatory capacity comes from the observation of Stein et al. [8] that the template activity of chromatin reconstituted with nonhistone chromosomal proteins from WI-38 cells 1 h following stimulation is higher than the Exptl Cell Res 88 (1974)
320 Stein and Burtner 1. DNA synthesis in WI-38 human diploid fibroblasts Table
Sample Contact-inhibited cells Cells 1 h following stimulation Cells 2 h following stimulation Cells 4 h following stimulation Cells 15 h following stimulation
cpm/lOO pg DNA
3H-Thymidinelabelled nuclei/ 1 000 cells
chromosomal proteins occurs in the absence of new RNA synthesis. The possibility that such nonhistone chromosomal protein synthesis early during the prereplicative phase of the cell cycle may be regulated at the translational level is discussed.
3 460
3
4237
1
3164
2
Cell culture
4592
5
Human diploid WI-38 fibroblasts [l l] were grown in monolayer culture in Eagle’s basal medium containing 10 % fetal calf serum (BME). The cells were incubated in a moist CO, incubator. Seven days after plating in 1 liter Blake culture flasks, the cells became confluent (in this paper such cells are referred to as ‘contact-inhibited’) and cellular proliferation was stimulated by discarding the exhausted growth medium and replacing it with fresh BME containing 20 % fetal calf serum (these cells will be referred to as serum-stimulated). The human dinloid WI-38 fibroblasts utilized in these studies ranged from passage 26 to 28. This is an important consideration since age-dependent modifications in the metabolism of chromosomal proteins have been observed in late passage human diploid fibroblasts [12]. The specific activity of DNA and the percentage of 3H-thymidine-labelled nuclei were determined at various times following serum stimulation. Table I indicates that at 1, 2 and 4 h the levels of DNA synthesis are not significantly different from those observed in contact-inhibited cells. In contrast, at 15 h, the cells are actively synthesizing DNA. This is in agreement with reports from several laboratories that DNA synthesis in serum-stimulated WI-38 human diploid fibroblasts occurs at this time [I 0. 151.
341 622
530
Cells were labelled with $H-thvmidine as described in Materials and Methods. To determine the rate of DNA synthesis (cpm/lOO ,ug DNA) cells were harvested and nuclei were isolated as described in Materials and Methods. Nuclei were washed twice with cold (4°C) 0.3 N perchloric acid and nucleic acids were extracted with hot (90°C) 1 N uerchloric acid. The amount of DNA present in the nucleic acid extracts was assayed by Burton’s modification of the diphenylamine reaction [13]. Each figure renresents an average of at least six determinations and the range of valies did not exceed 5 %. To determine the percentage of cells with 3H-thymidinelabelled nuclei, cells were harvested as described in Materials and Methods, smeared on acid washed microscope slides and prepared for autoradiography as described by Baserga [14]. Autoradiographs were exposed for 14 days and stained with hematoxylin following development. The values for 3H-thymidinelabelled nuclei/l 000 cells were obtained by counting 2 000 cells. Each value represents an average of 4 determinations and the range of values did not exceed 7 %.
MATERIALS
AND METHODS
Labelling cells with radio isotopes template activity of chromatin reconstituted with nonhistone chromosomal proteins from contact-inhibited cells. However, variations in the synthesis of defined classes of nonhistone chromosomal proteins in WI-38 fibroblasts I h following stimulation have not been demonstrated. The present studies indicate that there is an increase in the amount of two specific newly synthesized molecular weight classes of nonhistone chromosomal proteins in chromatin 1 h following the stimulation of contact-inhibited WI-38 cells to proliferate and that the synthesis of these nonhistone Exptl Cell Res 88 (1974)
The synthesis of nonhistone chromosomal proteins was assayed by labelling cells with SH-L-tryptophane. Medium was removed from the monolayer cultures and cells were incubated at 37°C for 3b min with L-ttyntophane-free BME containing 3H-L-tryptophane (IO -&iiml, 1.64 Ci/mM), and 2 % fetal calf serum. Isotope incorporation was terminated by pouring off the labelling medium and washing the monolayers 3 times with 50 ml of cold (4°C) Earle’s balanced salt solution. Labelling cells under these conditions does not stimulate cellular proliferation. When the growth medium which covers contact inhibited monolayers prior to labelling is replaced following incubation in tryptophane-free BME containing 3H-L-tryptophane and 2% fetal calf serum, neither DNA synthesis nor mitosis are induced. It should also be indicated that these labellina conditions do not interfere with nrocesses leading to DNA replication and mitosis since cells labelled 1 h following serum stimulation actively synthesize DNA at 15 h and divide mitotically at
Regulation of nonhistone chromosomal protein synthesis 24 h when stimulating medium is replaced at the termination of labelling. The svnthesis of DNA and RNA were assaved bv labelling cells for 30 min at 37°C with 3H-thymidine methvl (6 CiimM) and 3H-5-uridine (58 CiimM) respectively. 3H-Thymidine was added to the growth or stimulating medium to a final concentration of 1 ,&i/ml and 3H-uridine was added to a final concentration of 5 &i/ml. Labellina was terminated and the monolayers were washed as described above. 3H-L-Trvntoohane. 3H-thvmidine methvl and 3H-5uridine were purchased from New England Nuclear Corporation, Boston, Mass.
Rate of nonhistone chromosomal protein synthesis The rate of nonhistone chromosomal protein synthesis was determined by labelling monolayer cultures of cells at 37°C for 30 min with L-tryptophane-free BME containing 10 &i/ml 3H-L-tryptophane as described above. Incorporation of 3H-tryptophane into nonhistone chromosomal proteins under these conditions is linear for more than 60 min. Cells were then harvested. nuclei were isolated and chromatin was prepared. Chromatin was dissociated in 3 M NaCI, 5 M urea. 0.01 M Tris (nH 8.3) and DNA was nelleted by centrifugation at 150 000 g for 36 h. Since 96 % of the chromosomal proteins are found in the supernatant fraction [7], the amount of radioactivity in the supernatant was assayed by counting 0.1 ml aliquots in Triton-toluene liquid scintillation cocktail. The DNA in the pellet was hydrolysed for 2 h in 1 N perchloric acid at 90°C and the amount of DNA was determined by Burton’s modification of the diphenylamine reaction [13]. The data are expressed as cpm/mg DNA.
Rate of RNA synthesis Monolayer cultures of WI-38 human diploid fibroblasts were labelled with 3H-5-uridine as described above. RNA was extracted by the method of Scott, Fraccastoro & Taft [17] and the amount of RNA was determined by the two-wavelength method of Fleck & Munro [18]. Radioactivity incorporated into RNA was assayed by counting 0.1 ml aliquots of extracted RNA in 10 ml of Triton-toluene liauid scintillation cocktail [19]. The data is expressed as cpm//cg RNA.
Isolation of nuclei and chromatin Nuclei and chromatin were isolated at 4°C as described previously [8]. Cells were harvested by scraping the monolayers with a rubber policeman, washed 3 times with 80 vol of Earle balanced salt solution and lysed with 80 vol of 80 mM NaCI, 20 mM EDTA, 1 % Triton X-100 (pH 7.2). Nuclei were pelleted by centrifugation at 1 000 g for 4 min and washed 3 times with the lysing medium in a similar manner. This was followed by 2 washes with 0.15 M NaCI, 0.01 M Tris (pH 8.0). The nuclei were pelleted by centrifugation at 1 500 g for 3 min following each
321
wash. Nuclei isolated in this manner are free of cytoplasmic contamination when examined by phase contrast microscopy. Nuclei were lysed in 60 vol of distilled water by gentle homogenization. The chromatin was allowed to swell in an ice bath for 30 min and was then pelleted by centrifugation at 20 000 g for 20 min. The protein/DNA ratios of the chromatin preparations from contact-inhibited and stimulated WI-38 cells were 1.8 and the nonhistone chromosomal proteimhistone ratios were 1.4. Quantitation of the nucleic acid and nrotein components of the chromatin preparations was carried out as described nreviously bv Stein et al. 1161. Although components of the nuclear membrane may be associated with the chromatin nrenaration, this would be restricted to the inner portion of the nuclear envelone since nuclei were isolated with Triton X-100 which removes the outer layer of the nuclear envelope. If segments of the inner nuclear membrane do, in fact, -remain associated with the isolated genome, they may have a functional significance rather than being artifactual.
SDS polyacrylamide gel electrophoresis of chromosomal proteins Chromatin was dissociated in 1 % SDS, 1 % /3mercaptoethanol, 0.01 M sodium phosphate (pH 7.0), heated at 60°C for 60 min and dialysed against 0.1 % SDS, 0.1 % p-mercaptoethanol, 0.01 M sodium phosphate (pH 7.0) for 12 h at 22°C. Sucrose was added to a final concentration of 15 % and 0.1 ml aliquots containing 75 /Ag of chromosomal proteins were electrophoresed on 0.6 by 15 cm, 7.5 % polyacrylamide gels containing 0.1 % SDS. Electrophoresis was carried out for 11 h at 4 mA/gel in a running buffer of 0.1 % SDS, 0.1 M sodium phosphate (pH 7.0) at 22°C. Details of the procedure have been reported [20]. Gels were fractionated mechanically and collected in liquid scintillation counting vials containing 10 ml of Triton X-100 toluene cocktail [19].
RESULTS Nonhistone chromosomal protein in WI-38 cells 1 h following serum stimulation
synthesis
Initially, the rates of nonhistone chromosomal protein synthesis in contact-inhibited WI-38 human diploid fibroblasts and in cells 1 h following serum stimulation were compared. Cells were labelled for 30 min with 3H-L-tryptophane. Nuclei were isolated and chromatin was prepared as described in Materials and Methods. The data in table 2 indicate that a three-fold increase in the rate of 3H-tryptophane incorporation into Exptl Cell Res 8% (1974)
322
Stein and Burtner
Table 2. Nonhistone chromosomal protein synthesis in WI-38 human diploid fibroblasts Sample
cpm/mg DNA
(a) Contact-inhibited cells (b) 1 h following stimulation (c) Actinomycin-treated and stimulated 1 h
17 800 51 310 49 220
The rate of nonhistone chromosomal protein synthesis was determined: (a) in contact-inhibited cells, (6) 1 h following serum stimulation of contactinhibited cells; (c) 1 h following stimulation of contact-inhibited cells which were treated with actinomycin D (1 ,ug/ml) for 30 min and then serum stimulated in the presence of the antimetabolite. Cells were labelled for 30 min with 3H-L-tryptophane (I 0 &i/ml). All procedures are described in Materials and Methods. Each value represents an average of at least five determinations and the range of values did not exceed 5 Y,.
chromosomal proteins occurs I h following stimulation. Since histones do not contain tryptophane residues the 3H-tryptophane incorporation solely reflects nonhistone chromosomal protein synthesis. It should be indicated that fluctuations in the size and specific activity of the amino acid precursor pools do not exceed 15 % 1 h after contactinhibited WI-38 cells are stimulated to proliferate, further supporting an increased rate of nonhistone chromosomal protein synthesis at this time. Table 2 also shows that the increased rate of nonhistone chromosomal protein synthesis at 1 h is insensitive to the inhibition of RNA synthesis. Contact-inhibited WI-38 cells treated with actinomycin D (1 fig/ml) for 30 min and then stimulated in the presence of actinomycin D exhibit a rate of nonhistone chromosomal protein synthesis 1 h following stimulation which is identical to that observed in cells stimulated in the absence of the antimetabolite. Table 3 indicates that the concentration of actinomycin D used in these experiments is effective in suppressing RNA synthesis in contact-inhibited cells as Exptl Cell Res 88 (1974)
well as in cells 1 h following stimulation by 98.1 and 98.2 % respectively. Cells pretreated with actinomycin D for 30 min and then stimulated in the presence of the drug show a similar inhibition of RNA synthesis (98.7) 1 h following stimulation (table 3). While Rovera & Baserga [lo] also observe an increased rate of nonhistone chromosomal protein synthesis in WI-38 human diploid fibroblasts 1 h following stimulation, they do not report insensitivity of such synthesis to inhibition of transcription. Synthesis of specific molecular weight classes of nonhistone chromosomal proteins in WI-38 cells 1 h following serum stimulation To determine whether variations in the synthesis of specific classes of nonhistone chromosomal proteins occur 1 h following serum stimulation of contact-inhibited WI-38 human diploid fibroblasts to proliferate, cells were labelled with 3H-tryptophane for Table 3. Effect of actinomycin D (A MD) on RNA synthesis in WI-38 human diploid fibroblasts Sample
cpm//dg RNA
(a) Contact-inhibited cells Contact-inhibited plus AMD (b) 1 h following stimulation 1 h following stimulation plus AMD (c) AMD-treated and stimulated Ih
3 771 73 8 605 108 115
The effect of actinomycin D (I pg/ml) on the rate of RNA synthesis was determined: (a) in contactinhibited cells; (b) 1 h following serum stimulation of contact-inhibited cells. The rate of RNA synthesis 1 h following serum stimulation of contact-inhibited cells which were treated with actinomycin D (1 pg/ml) for 30 min and then stimulated in the presence of the antimetabolite was also determined (c). Cells were labelled for 30 min with 3H-5-uridine (5 &i/ml). All procedures are described in Materials and Methods. Each value represents an average of at least five determinations and the range of values did not exceed 5 %.
Regulation of north&one chromosomalprotein synthesis 323 b
1
I 5
5
10
10
15
15
M
2C
25
25
30
30
35
35
40
4
45
~5
50
ZC
55
55
60
60
65
65
30 min. Chromatin was then isolated, dissociated, and 3H-tryptophane-labelled chromosomal proteins were fractionated electrophoretically according to molecular weight on SDS polyacrylamide gels. Since histones do not contain 3H-tryptophane residues, the distribution of radioactivity into the various molecular weight classes of chromosomal proteins (fig. 1) solely reflects
5
10
15
20
2s
3c
35
40
4.5
50
55
60
65
Fig. 1. Abscissa: fraction; ordinate: % cpm. (a) SDS-polyacrylamide gel electrophoretic profiles of 3H-L-tryptophane-1abelled chromosomal proteins from contact-inhibited monolayers of WI-38 human diploid fibroblasts. Cells were labelled for 30 min with 3H-L-tryptophane (I .64 ,&i/ml). Nuclei were isolated, chromatin was prepared and dissociated and chromosomal proteins were fractionated electrophoretically on SDS-polyacrylamide gels as described in Materials and Methods; (b) SDSpolyacrylamide gel electrophoretic profile of 3H-~tryptophane-labelled chromosomal proteins from WI-38 human diploid fibroblasts I h following stimulation. The same procedures described in (a) were used; (c) SDS-polyacrylamide gel electrophoretic profile of 3H-rAryptophane-1abelled chromosomal proteins from WI-38 human diploid fibroblasts I h following stimulation. The same procedures described in (a) were used except the contact-inhibited cells were treated with actinomycin D (1 pg/ml) for 30 min and then stimulated for 1 h in the presence of the antimetabolite.
nonhistone chromosomal protein synthesis. Polypeptides with a molecular weight of 23 000 migrate between fractions 60 to 65 in the gels illustrated in fig. 1. A comparison of fig. 1 a and b indicate that there are pronounced modifications in the nonhistone chromosomal proteins synthesized and associated with the genome of contact-inhibited WI-38 cells (fig. la) Exptl Cell Res 88 (1974)
324 Stein and But-her and WI-38 cells 1 h following stimulation (fig. lb). An increased synthesis of nonhistone chromosomal polypeptides which migrate between fractions l-5 (molecular weight greater than 120 000) and fractions 45-55 (35 000 molecular weight) are evident in the stimulated cells. The polypeptides which migrate between fractions l-5 and 45-55 represent 25 and 14% respectively of the nonhistone chromosomal protein synthesis in WI-38 human diploid fibroblasts 1 h following serum stimulation. The present data do not exclude the possibility that these 2 molecular weight classes of nonhistone chromosomal proteins which are synthesized and associated with the genome of WI-38 cells 1 h following serum stimulation may also be synthesized in unstimulated WI-38 cells. Perhaps following serum stimulation there is an increased recruitment of these nonhistone chromosomal proteins from a newly synthesized pool contained within the cytoplasm or nucleoplasm, the size of which may not be greater in stimulated than in unstimulated cells. Fig. lc shows the distribution of 3Htryptophane into the various molecular weight classes of nonhistone chromosomal polypeptides synthesized and associated with the genome in WI-38 cells pretreated with actinomycin D and stimulated for 1 h in the presence of the drug. Consistent with insensitivity of the increased nonhistone chromosomal protein synthesis which occurs at 1 h in serum stimulated WI-38 cells to inhibition of transcription, actinomycin D was ineffective in suppressing the synthesis of nonhistone chromosomal proteins which migrate between fractions l-5 and 45-55. These findings suggest that two molecular weight classes of nonhistone chromosomal proteins whose synthesis is induced 1 h following stimulation of contact-inhibited Exptl Cell Res 88 (1974)
cells to proliferate RNA templates.
may utilize
pre-existing
DISCUSSION Results presented indicate that two defined molecular weight classes of nonhistone chromosomal proteins which are synthesized and associated with the genome of WI-38 cells 1 h following serum stimulation are not actively synthesized components of the genome in contact-inhibited cells. Previous studies have directly shown that the nonhistone chromosomal proteins which comprise the genome of WI-38 cells 1 h following serum stimulation are responsible for the increased template activity of chromatin which is observed at this time [8]. One can therefore speculate that the two molecular weight classes of nonhistone chromosomal proteins which are synthesized and associated with the genome of WI-38 cells 1 h following serum stimulation may, at least in part, be responsible for the activation of gene readout which is apparent early during the prereplicative phase of the cell cycle. Perhaps this modification in the elaboration of information by the genome at 1 h triggers the complex and interdependent series of biochemical events which lead to initiation of DNA replication at 12 h and mitosis at 24 h. Evidence is also presented which suggests that the two classes of nonhistone chromosomal proteins whose synthesis is apparent 1 h following the stimulation of contactinhibited WI-38 human diploid fibroblasts to proliferate occurs in the absence of DNA dependent RNA synthesis. This may be interpreted to indicate that these actinomycin D insensitive nonhistone chromosomal proteins are synthesized on pre-existing mRNA templates transcribed and processed when the cells are contact-inhibited and then
Regulation
of nonhistone chromosomal protein synthesis
activated following serum stimulation. An alternative explanation would be that processing, either in the nucleus or in the cytoplasm, of the precursors of the messenger RNAs for these nonhistone chromosomal proteins is not completed until the cells are stimulated to proliferate. Although one can consider the possibility that regions of the genome containing the nucleotide sequences which code for these nonhistone chromosomal proteins are arranged in such a manner that they are inaccessible to actinomycin D, this explanation does not seem very viable. Our experiments do not directly indicate the half-lives of mRNAs which code for nonhistone chromosomal proteins synthesized during the prereplicative phase of the cell cycle in WI-38 cells. Since relatively longlived mRNAs are the rule rather than the exception in eukaryotic cells, the postulation of post-transcriptional regulation of nonhistone chromosomal protein synthesis does not require pre-existing mRNAs of exceptional stability. Such RNA, even if ‘unstable’ in the conventional sense (half-lives of 2-3 h) could still support the increased rate of protein synthesis 1-2 h later via a translational control mechanism. The activation and utilization of preexisting mRNAs in eukaryotic cells is not restricted to the mRNA templates for nonhistone chromosomal proteins which are synthesized early during the prereplicative phase of the cell cycle in WI-38 cells. Rather, the initial increased rate of nonhistone chromosomal protein synthesis during the first segment of G 1 in mouse salivary gland cells stimulated to proliferate by isoproterenol [21], as well as the synthesis of several major classes of nonhistone chromosomal proteins during the prereplicative phase of the cell cycle in continuously dividing HeLa S, cells [22], have been shown to exhibit insensitivity to actinomycin D. An-
325
other striking, although unrelated, example of the activation and utilization of preexisting mRNAs in eukaryotic cells is that of the mRNAs utilized for protein synthesis during the initial stages of embryonic development [23]. The concept of nonhistone chromosomal proteins as activators of gene expression early during the prereplicative phase of the cell cycle in WI-38 human diploid fibroblasts is by no means unique. Such reasoning is consistant with several lines of evidence which suggest that nonhistone chromosomal proteins play a key role in the regulation of gene expression during the cell cycle of continuously dividing cells and a variety of quiescent cell populations which are stimulated to proliferate. Significant variations in the rates of synthesis [lo, 21, 24-261 and turnover [27] of these proteins during defined periods of the cell cycle have been reported. Differences in the phosphorylation and dephosphorylation of nonhistone chromosomal proteins throughout the cell cycle have been observed [28, 291. Furthermore, unlike the histones whose synthesis is restricted to S phase and tightly coupled with DNA replication [30], the synthesis of nonhistone chromosomal proteins occurs independent of concomitant DNA synthesis [24, 261. Modifications in the metabolism of nonhistone chromosomal proteins during the cell cycle of cells infected and transformed by RNA [31] as well as DNA [32-351 oncogenic viruses have also been described. More direct evidence that nonhistone chromosomal proteins are involved in the regulation of DNA dependent RNA synthesis during the cell cycle comes from studies which show that (a) chromatin reconstituted with cell cycle stage-specific nonhistone chromosomal proteins exhibits a template activity characteristic of the native chromatin from which these proteins are isolated [7, 81 and Exptl Cell Res 88 (1974
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(b) nonhistone chromosomal proteins may regulate transcription during the cell cycle by mediating the binding of histones to DNA [36]. However, the precise manner in which nonhistone chromosomal proteins interact with the nucleic acid and protein components of the genome to direct the transcription of defined sequences remains to be elucidated. These studies were supported by the following research grants: GB 38349 from the NSF, F73UF from the ACS and GM 20535 from the NIH.
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14. Baserga, R, Methods in cancer research (ed H
Busch) vol. 1, p. 45. Academic Press, New York (1967). 15. Rhode, S L & Ellem, K A 0, Exptl cell res 53 (1968) 184. t6. Stein, G S, Wang, P L & Adelman, R C, Exptl geront 8 (1973) 123. 17. Scott, J P, Fraccastoro, A P & Taft, E B, J histochem cytochem 4 (1956) 1. 1s. Fleck, A & Munro, H N, Biochim biophys acta 55 (1962) 571. t9, Patterson, M S & Green, R C, Anal them 37 (1965) 854. 20. Bhorjee, J & Pederson, T, Biochemistry 12 (1973) 2766. 21. Stein, G S & Baserga, R, J biol them 245 (I 970) 6097. 22. Stein, G S & Matthews, D, Science 181 (1973) 71. 23. Gross, P R & Cousineau, G H, J cell biol 19 (1963) 260. 24. Stein, G S & Borun, T W, J cell biol 52 (1972) 292. 25. Levy, R, Levy, S, Rosenberg, S & Simpson, R, Biochemistry 12 (1973) 224. 26. Stein, G S & Thrall, C L, FEBS letters 34 (1973) 35. 27. Borun, T W & Stein, G S, J cell biol 52 (1972) 308. 28. Platz, R, Stein, G S & Kleinsmith, L J, Biochem biophys res comm 51 (1973) 735. 29. Karn, J, Johnson, E M, Vidai, G & Allfrey, V G, J biol them 249 (1974) 667. 30. Robbins, E & Borun, T W, Proc natl acad sci US 57 (1967) 409. 31. Stein, G S, Moscovici, G, Moscovici, C & Mon, M, FEBS letters 38 (1974) 295. 32. Krause, M 0 & Stein, G S, Biochem biophys comm 59 (1954) 796. 33. Rovera, G, Baserga, R & Defendi, V, Nature new biol 237 (1972) 240. 34. Ledinko, L, Virology 54 (1973) 294. 35. Zardi, L, Lin, J & Baserga, R, Nature new biol 245 (1973) 211. 36. Stein, G S, Hunter, G & Lavie, L, Biochem j 139 (1974) 71.
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Keceived March 27, 1974 Revised version received May 13, 1974