Messenger RNA Regulation in Human Diploid Fibroblast9 THOMAS H. MEEDEL AND ELLIOT M. LEVINE The Wistar lnstitute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, Pennsyluania 19104
ABSTRACT In resting, non-growing human diploid fibroblasts the amount of rRNA is reduced 1.8-fold, cytoplasmic polysoines are disaggregated, and the level of poly-A RNA (mRNA) is reduced 1.8-fold in relation to growing cells. The distribution of poly-A RNA is altered in resting, non-growing cells so that an average of 64% of the total cytoplasmic poly-A RNA sediments along with particles lighter than 80s (prepolysomal) in sucrose density gradients. By comparison, in growing cells only 30%of the cytoplasmic poly-A RNA sediments in the prepolysonial region. In SDS sucrose gradients, the sedimentation profile of the prepolysomal poly-A RNA from resting cells resembles that of polysonial poly-A RNA from those cells. In contrast, the average size of prepolysomal poly-A RNA from growing cells is much smaller than that of the polysomal poly-A RNA from those cells. These data are compatible with the possibility that resting cell prepolysomal poly-A is untranslated mRNA. Also consistent with this interpretation are experiments which demonstrate that one-quarter to one-third of the prepolysomal poly-A RNA of resting cells is recruited into polysomes in the presence of cycloheximide.
The ability of cultured fibroblasts to exist either as a logarithmically growing, or resting (essentially non-growing) population offers a model system for studying mechanisms of growth regulation, which may provide information relevent to processes that regulate growth in vitro. In fibroblasts and several other cell culture model systems, one difference between the two growth states is an alteration of protein synthesis activity which is reflected by changes in the macromolecular components of this system. In comparison to logarithmically growing cells, the protein synthetic activity of resting cells is reduced from three (Levine et al., '65; Stanners and Becker, '71; Rudland, '74) to six (Englehardt, '71) fold. Accompanying this decrease is a disaggregation of polyribosomes (Levine, '72, '74; Rudland, '74; Englehardt, '75), accelerated turnover of stable rRNA (Emerson, '71; Weber, '72; Abelson e t al., '74; Levine, '74) and a decrease in J. CELL. PHYSIOL., 90: 211-224.
DNA-dependent RNA polymerase activity (Ward and Plagemann, '69). Examination of messenger RNA as a component of the protein synthesis machinery has been facilitated by the discovery that most mRNAs contain a region of polyadenylic acid (Edmunds et al., '71; Lee e t al., '71; Darnell e t al., '71; Greenberg and Perry, '72). Comparisons of poly-A RNA levels (presumably mRNA) in growing and resting cells have yielded conflicting results. Johnson et al. ('74) determined that the amount of poly-A RNA was significantly (3-5-fold) reduced in resting (as compared to growing) cells. Rudland ('74), however, obReceived Feh. 27, '76. Accepted May 20, '76. 'This research was supported by US. Public Health Service Grants GM-20306, RR-05540, and CA-10915, and National Science Foundation Grant GB-35590. During the initial stages of this work T.H.M. was a predoctoral trainee supported by a U.S. Public Health Service Grant (2TIA1203) awarded to the Department of Microbiology of the University of Pennsyivania. The expert technical assistance of Mrs. Barbara Becker and Ms. Nancy Turck is gratefully acknowledged.
211
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served that the poly-A RNA content was similar in both growth states, but that a significant portion ofthe poly-A RNA was unassociated with polysomes in resting cells. These determinations of both poly-A RNA content and distribution were performed utilizing the permanent mouse cell lines 3T6 (Johnson et al., '74) or 3T3 (Johnson e t al., '74; Rudland, '74). By several criteria, 3T6 has been considered a transformed line (Todaro, '72), while t h e observation that 3T3 can cause tumors in nude (Boone, '75) and normal mice (Boone, personal communication) makes that cell line less than ideal as a model system for studying normal growth control. In view of this, we have chosen to study the metabolism of poly-A RNA in human diploid fibroblasts (HDF), derived from normal tissue. The observation that human diploid fibroblasts do not cause tumors in nude mice, when injected under conditions in which 3T3 cells do cause tumors, seems to support this choice (Boone, personal communication). The data presented below indicates there is a reduced amount of poly-A RNA in resting HDF (as compared to growing HDF), as well as an appreciable amount of non-polysome-associated poly-A RNA in the cytoplasm of resting cells. We interpret these results as compatible with processes that limit both the amount and the utilization of available mRNA in resting cells.
Falcon 75 om2 flasks that contained 20 ml MEM supplemented with 10% fetal calf serum. Resting cultures were established by seeding 1 x lo4 cells/cm2 and refeeding cultures the second day after plating. Cells were considered to be in the resting state when no increase in population density was observed for three consecutive days. Growing cultures were obtained by seeding cells at 5 x 103/cm2, refeeding the second day after plating, and harvesting three days after seeding. Only logarithmically growing cultures were used for studies of growing cells. Cell numbers were measured with a Coulter Counter using trypsinized cell suspensions.
Cell fructionution, RNA isolation and purijication For preparation of total cytoplasmic RNA, a modification of the method of Johnson e t al. ('74) was used. Growth medium was removed from cultures and cell monolayers were washed with ice cold phosphate-buffered saline (PBS). The cells, still on the monolayer, were swelled in RSB (0.01 M NaC1,0.005 M Mg CI2,and 0.01 M Tris, p H 7.6; Warner e t al., '63). Triton X-100 was then added to 1% a n d t h e cells removed from the monolayer by pipetting buffer over them. At this point, most of the cells were lysed and the nuclei appeared free of adherant cytoplasmic "tags" by phase microscopy. The suspension was then stirred on a vortex mixer for 30 seconds and nuclei removed by sedimentation at 800 g for two minutes. The supernatant fluid from METHODS both resting and growing cells contained Cell culture greater than 85% of the total cellular Two HDF lines, WI-38 (Hayflick and RNA. The cytoplasmic supernatant was Moorehead, '61) and HEL (CCL 137, then adjusted to 0.1 M NaCI, 0.01 M American T y p e Culture Collection, EDTA, 0.01 M Tris, p H 7.4 and 0.5% SDS Rockville, Maryland) were employed in and the RNA was extracted by the these studies. Only cultures between phenol-chloroform method of Penman passages 16 and 26 judged mycoplasma ('69). free by agar plate cultivation (Hayflick, Cytoplasmic lysates to be analyzed by '65) and uridine phosphorylase assay sucrose gradient sedimentation were (Levine, '72, '74) were used. Cells were prepared as follows. Washed monolayers, grown at 37°C in a 5% CO, atmosphere in treated with crystalline trypsin (200 pgl
mRNA REGULATION IN HUMAN DIPLOID FIBROBLASTS
ml), were removed b y shaking the flask and immediately adding excess lima bean trypsin inhibitor. Cell clumps were dispersed by gentle pipetting and cells collected by centrifugation at 600 g for one minute. After washing with PBS, cell pellets were resuspended in polysome buffer (25 niM NaC1, 5 mM Mg Clz, 25 mM Tris, p H 7.4, plus 100 pg/ml heparin). Lysis of cells was accomplished by the addition of Triton X-100 to a concentration of 1% and intermittent stirring on the vortex mixer for ten minutes. Nuclei were removed from the lysed cell suspension by centrifugation at 800 g for two minutes. The cytoplasmic extract was layered directly on preformed gradients (11.0 ml; 15-45% sucrose in RSB plus 40 ,ug/ml heparin), and centrifuged in an SW-41 rotor at 38,000 rpm for two hours at 4°C. Gradients were collected through a Gilford continuous flow recording spectrophotometer and absorbance measured at 260 nm. Gradient fractions were precipitated overnight with 2 volumes of 95% ethanol at -2O”C, precipitates recovered by centrifugation, and RNA purified as detailed above.
213
formed between (3H)poly-U and poly-A: the RNAase resistant, TCA precipitable (3H) poly-U is a direct measure of the amount of poly-A present in the original samples (Bishop e t al., ’74).
Oligo dT cellulose chromatography Samples of RNA dissolved in 0.5 ml binding buffer (400 mM/NaCl, 10 mM Tris, p H 7.4, 0.5% SDS), were passed through a column containing 100 mg of oligo dT cellulose previously equilibrated with binding buffer. The eluate was recycled through the column an additional three times. The column was washed twice with 2 ml of binding buffer. The combined eluate constituted the “unbound fraction.” The column was repeatedly washed with 2 ml binding buffer until no significant radioactivity was eluted. The “bound fraction” was then eluted with three 1 ml washes of elution buffer (10 mM Tris, pH 7.4, 0.05% SDS). Carrier tRNA (50 pg/ml) and sodium chloride (to a concentration of 0.3 M) were added to the “bound fraction.” Both fractions were then precipitated by addition of 2 volumes of 95% ethanol. The precipitated RNA was allowed to ( 3 H ) poly-U hybridization analyses stand overnight at -2O”C, and collected (3H) poly-U hybridization analyses by centrifugation. were carried out by the method of Bishop et al. (’74). Samples of RNA in 2 x SSC Size estimation of steady state poly-A (0.4 M sodium chloride, 0.3 M sodium RNA citrate) were incubated with excess (3H) Size estimation was performed accordpoly-U (New England Nuclear, specific activity 4.5 Ci/mMole) at 45°C. After 120 ing to the method of McKnight and minutes, fractions were chilled at 4°C Schimke (’74). RNA samples were disand pancreatic RNAase was added to a solved in a buffer containing 0.5% SDS, 5 concentration of 50 pg/ml. Sixty minutes m M Tris HCl p H 7.5, 2.5 mM EDTA and later, 250 Fg of carrier bovine serum heated at 65°C for ten minutes. After albumin (BSA) was added and the frac- rapid chilling in an ice-salt bath, samples tion precipitated with trichloroacetic acid were layered directly on 11 ml, 15-3070 (TCA) at a final concentration of 5%. sucrose -SDS gradients with the above Samples were collected on millipore buffer composition. Gradients were cenfilters, washed with 5% TCA, dried, trifuged in an SW-41 rotor at 24,000 rpm dissolved in 5 ml scintillation fluid for 16 hours at 22°C. Gradient fractions (Liquifluor, New England Nuclear) and were adjusted to the ionic composition of assayed for radioactivity. Under these 2 x SSC and (3H) poly-U hybridization conditions double-stranded hybrids were studies performed as detailed previously.
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THOMAS H. MEEDEL AND ELLIOT M. LEVINE
Pol y-A sizing To 30 pg of RNA dissolved in DNAase buffer (100 mM NaCI, 15.0 mM MgC1, and 100 mM Tris, pII 7.4), 20 p g of DNAase were added and the sample incubated at 30°C for ten minutes. The buffer composition was then adjusted to 300 mM NaCl and 15 mM EDTA, and 5 units o f t , RNAase and 10 p g pancreatic RNAase were added. The sample was incubated at 37°C for 30 minutes, chilled and saturated with diethylpyrocarbonate. Carrier tRNA was then added and the sample precipitated by the addition of 2 volumes of ethanol. Precipitates were collected by centrifugation and resuspended in electrophoresis buffer (2 mM EDTA, 20 mM NaH,PO,, 0.2% SDS and 40 mM Tris pH 7.4). Samples were subjected to electrophoresis on 10% acrylamide gels as described by Hirsch and Penman ('73). Gels were sliced, and the RNA eluted from the slices in 2 x SSC at 37°C for 24 hours, and. aliquots of the
eluate removed for (3H) poly-U hybridization as described above. RESULTS
Cytoplasmic rRNA In earlier communications we demonstrated a disaggregation of free polysomes as growing cells entered quiescence (Levine et al., '65, '74). Figure 1 shows a similar study using a detergentcontaining buffer to extract free and membrane bound polysomes. As shown, there is a shift to smaller-sized polyribosomes (panel b) followed by a decrease in the amount of Azsoabsorbing polysomal material (panel c). In order to quantitate more precisely the amount of cellular ribosomal material, cytoplasmic extracts were prepared and adjusted to 2% SDS, and sedimented on SDS-sucrose gradients to display 18s and 28s RNA, which then were analyzed using a Gilford continuous flow recording spec-
E
g
I.0C
N
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0
z
a m a 0 rn
m a
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SEDIMENTATION
Fig. 1 SDG profiles of H D F at various growth stages. Cytoplasmic extracts were prepared from approximately 1.0 x lo' cells at various stages of growth as detailed in METHODS. The extracts were then layered on 11.0 ml, 15-45% sucrose gradients, and centrifuged for two hours at 4°C at 38,000 rpm in a SW-41 rotor. Gradients were analyzed at 260 nm using a Gilford continuous flow recording spectrophotometer. (a) Early log phase. (b) Late log phase. ( c ) Resting phase.
2 15
mRNA REGULATION IN HUMAN D I PLO I D FIBROBLASTS
represented in figure 1. The polysome associated (>80s) and prepolysome asLevels of rRNA in growing and resting cells sociated regions (
ABSTRACT In resting, non-growing human diploid fibroblasts the amount of rRNA is reduced 1.8-fold, cytoplasmic polysoines are disaggregated, and the level of poly-A RNA (mRNA) is reduced 1.8-fold in relation to growing cells. The distribution of poly-A RNA is altered in resting, non-growing cells so that an average of 64% of the total cytoplasmic poly-A RNA sediments along with particles lighter than 80s (prepolysomal) in sucrose density gradients. By comparison, in growing cells only 30%of the cytoplasmic poly-A RNA sediments in the prepolysonial region. In SDS sucrose gradients, the sedimentation profile of the prepolysomal poly-A RNA from resting cells resembles that of polysonial poly-A RNA from those cells. In contrast, the average size of prepolysomal poly-A RNA from growing cells is much smaller than that of the polysomal poly-A RNA from those cells. These data are compatible with the possibility that resting cell prepolysomal poly-A is untranslated mRNA. Also consistent with this interpretation are experiments which demonstrate that one-quarter to one-third of the prepolysomal poly-A RNA of resting cells is recruited into polysomes in the presence of cycloheximide.
The ability of cultured fibroblasts to exist either as a logarithmically growing, or resting (essentially non-growing) population offers a model system for studying mechanisms of growth regulation, which may provide information relevent to processes that regulate growth in vitro. In fibroblasts and several other cell culture model systems, one difference between the two growth states is an alteration of protein synthesis activity which is reflected by changes in the macromolecular components of this system. In comparison to logarithmically growing cells, the protein synthetic activity of resting cells is reduced from three (Levine et al., '65; Stanners and Becker, '71; Rudland, '74) to six (Englehardt, '71) fold. Accompanying this decrease is a disaggregation of polyribosomes (Levine, '72, '74; Rudland, '74; Englehardt, '75), accelerated turnover of stable rRNA (Emerson, '71; Weber, '72; Abelson e t al., '74; Levine, '74) and a decrease in J. CELL. PHYSIOL., 90: 211-224.
DNA-dependent RNA polymerase activity (Ward and Plagemann, '69). Examination of messenger RNA as a component of the protein synthesis machinery has been facilitated by the discovery that most mRNAs contain a region of polyadenylic acid (Edmunds et al., '71; Lee e t al., '71; Darnell e t al., '71; Greenberg and Perry, '72). Comparisons of poly-A RNA levels (presumably mRNA) in growing and resting cells have yielded conflicting results. Johnson et al. ('74) determined that the amount of poly-A RNA was significantly (3-5-fold) reduced in resting (as compared to growing) cells. Rudland ('74), however, obReceived Feh. 27, '76. Accepted May 20, '76. 'This research was supported by US. Public Health Service Grants GM-20306, RR-05540, and CA-10915, and National Science Foundation Grant GB-35590. During the initial stages of this work T.H.M. was a predoctoral trainee supported by a U.S. Public Health Service Grant (2TIA1203) awarded to the Department of Microbiology of the University of Pennsyivania. The expert technical assistance of Mrs. Barbara Becker and Ms. Nancy Turck is gratefully acknowledged.
211
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THOMAS H. MEEDEL AND ELLIOT M. LEVINE
served that the poly-A RNA content was similar in both growth states, but that a significant portion ofthe poly-A RNA was unassociated with polysomes in resting cells. These determinations of both poly-A RNA content and distribution were performed utilizing the permanent mouse cell lines 3T6 (Johnson et al., '74) or 3T3 (Johnson e t al., '74; Rudland, '74). By several criteria, 3T6 has been considered a transformed line (Todaro, '72), while t h e observation that 3T3 can cause tumors in nude (Boone, '75) and normal mice (Boone, personal communication) makes that cell line less than ideal as a model system for studying normal growth control. In view of this, we have chosen to study the metabolism of poly-A RNA in human diploid fibroblasts (HDF), derived from normal tissue. The observation that human diploid fibroblasts do not cause tumors in nude mice, when injected under conditions in which 3T3 cells do cause tumors, seems to support this choice (Boone, personal communication). The data presented below indicates there is a reduced amount of poly-A RNA in resting HDF (as compared to growing HDF), as well as an appreciable amount of non-polysome-associated poly-A RNA in the cytoplasm of resting cells. We interpret these results as compatible with processes that limit both the amount and the utilization of available mRNA in resting cells.
Falcon 75 om2 flasks that contained 20 ml MEM supplemented with 10% fetal calf serum. Resting cultures were established by seeding 1 x lo4 cells/cm2 and refeeding cultures the second day after plating. Cells were considered to be in the resting state when no increase in population density was observed for three consecutive days. Growing cultures were obtained by seeding cells at 5 x 103/cm2, refeeding the second day after plating, and harvesting three days after seeding. Only logarithmically growing cultures were used for studies of growing cells. Cell numbers were measured with a Coulter Counter using trypsinized cell suspensions.
Cell fructionution, RNA isolation and purijication For preparation of total cytoplasmic RNA, a modification of the method of Johnson e t al. ('74) was used. Growth medium was removed from cultures and cell monolayers were washed with ice cold phosphate-buffered saline (PBS). The cells, still on the monolayer, were swelled in RSB (0.01 M NaC1,0.005 M Mg CI2,and 0.01 M Tris, p H 7.6; Warner e t al., '63). Triton X-100 was then added to 1% a n d t h e cells removed from the monolayer by pipetting buffer over them. At this point, most of the cells were lysed and the nuclei appeared free of adherant cytoplasmic "tags" by phase microscopy. The suspension was then stirred on a vortex mixer for 30 seconds and nuclei removed by sedimentation at 800 g for two minutes. The supernatant fluid from METHODS both resting and growing cells contained Cell culture greater than 85% of the total cellular Two HDF lines, WI-38 (Hayflick and RNA. The cytoplasmic supernatant was Moorehead, '61) and HEL (CCL 137, then adjusted to 0.1 M NaCI, 0.01 M American T y p e Culture Collection, EDTA, 0.01 M Tris, p H 7.4 and 0.5% SDS Rockville, Maryland) were employed in and the RNA was extracted by the these studies. Only cultures between phenol-chloroform method of Penman passages 16 and 26 judged mycoplasma ('69). free by agar plate cultivation (Hayflick, Cytoplasmic lysates to be analyzed by '65) and uridine phosphorylase assay sucrose gradient sedimentation were (Levine, '72, '74) were used. Cells were prepared as follows. Washed monolayers, grown at 37°C in a 5% CO, atmosphere in treated with crystalline trypsin (200 pgl
mRNA REGULATION IN HUMAN DIPLOID FIBROBLASTS
ml), were removed b y shaking the flask and immediately adding excess lima bean trypsin inhibitor. Cell clumps were dispersed by gentle pipetting and cells collected by centrifugation at 600 g for one minute. After washing with PBS, cell pellets were resuspended in polysome buffer (25 niM NaC1, 5 mM Mg Clz, 25 mM Tris, p H 7.4, plus 100 pg/ml heparin). Lysis of cells was accomplished by the addition of Triton X-100 to a concentration of 1% and intermittent stirring on the vortex mixer for ten minutes. Nuclei were removed from the lysed cell suspension by centrifugation at 800 g for two minutes. The cytoplasmic extract was layered directly on preformed gradients (11.0 ml; 15-45% sucrose in RSB plus 40 ,ug/ml heparin), and centrifuged in an SW-41 rotor at 38,000 rpm for two hours at 4°C. Gradients were collected through a Gilford continuous flow recording spectrophotometer and absorbance measured at 260 nm. Gradient fractions were precipitated overnight with 2 volumes of 95% ethanol at -2O”C, precipitates recovered by centrifugation, and RNA purified as detailed above.
213
formed between (3H)poly-U and poly-A: the RNAase resistant, TCA precipitable (3H) poly-U is a direct measure of the amount of poly-A present in the original samples (Bishop e t al., ’74).
Oligo dT cellulose chromatography Samples of RNA dissolved in 0.5 ml binding buffer (400 mM/NaCl, 10 mM Tris, p H 7.4, 0.5% SDS), were passed through a column containing 100 mg of oligo dT cellulose previously equilibrated with binding buffer. The eluate was recycled through the column an additional three times. The column was washed twice with 2 ml of binding buffer. The combined eluate constituted the “unbound fraction.” The column was repeatedly washed with 2 ml binding buffer until no significant radioactivity was eluted. The “bound fraction” was then eluted with three 1 ml washes of elution buffer (10 mM Tris, pH 7.4, 0.05% SDS). Carrier tRNA (50 pg/ml) and sodium chloride (to a concentration of 0.3 M) were added to the “bound fraction.” Both fractions were then precipitated by addition of 2 volumes of 95% ethanol. The precipitated RNA was allowed to ( 3 H ) poly-U hybridization analyses stand overnight at -2O”C, and collected (3H) poly-U hybridization analyses by centrifugation. were carried out by the method of Bishop et al. (’74). Samples of RNA in 2 x SSC Size estimation of steady state poly-A (0.4 M sodium chloride, 0.3 M sodium RNA citrate) were incubated with excess (3H) Size estimation was performed accordpoly-U (New England Nuclear, specific activity 4.5 Ci/mMole) at 45°C. After 120 ing to the method of McKnight and minutes, fractions were chilled at 4°C Schimke (’74). RNA samples were disand pancreatic RNAase was added to a solved in a buffer containing 0.5% SDS, 5 concentration of 50 pg/ml. Sixty minutes m M Tris HCl p H 7.5, 2.5 mM EDTA and later, 250 Fg of carrier bovine serum heated at 65°C for ten minutes. After albumin (BSA) was added and the frac- rapid chilling in an ice-salt bath, samples tion precipitated with trichloroacetic acid were layered directly on 11 ml, 15-3070 (TCA) at a final concentration of 5%. sucrose -SDS gradients with the above Samples were collected on millipore buffer composition. Gradients were cenfilters, washed with 5% TCA, dried, trifuged in an SW-41 rotor at 24,000 rpm dissolved in 5 ml scintillation fluid for 16 hours at 22°C. Gradient fractions (Liquifluor, New England Nuclear) and were adjusted to the ionic composition of assayed for radioactivity. Under these 2 x SSC and (3H) poly-U hybridization conditions double-stranded hybrids were studies performed as detailed previously.
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THOMAS H. MEEDEL AND ELLIOT M. LEVINE
Pol y-A sizing To 30 pg of RNA dissolved in DNAase buffer (100 mM NaCI, 15.0 mM MgC1, and 100 mM Tris, pII 7.4), 20 p g of DNAase were added and the sample incubated at 30°C for ten minutes. The buffer composition was then adjusted to 300 mM NaCl and 15 mM EDTA, and 5 units o f t , RNAase and 10 p g pancreatic RNAase were added. The sample was incubated at 37°C for 30 minutes, chilled and saturated with diethylpyrocarbonate. Carrier tRNA was then added and the sample precipitated by the addition of 2 volumes of ethanol. Precipitates were collected by centrifugation and resuspended in electrophoresis buffer (2 mM EDTA, 20 mM NaH,PO,, 0.2% SDS and 40 mM Tris pH 7.4). Samples were subjected to electrophoresis on 10% acrylamide gels as described by Hirsch and Penman ('73). Gels were sliced, and the RNA eluted from the slices in 2 x SSC at 37°C for 24 hours, and. aliquots of the
eluate removed for (3H) poly-U hybridization as described above. RESULTS
Cytoplasmic rRNA In earlier communications we demonstrated a disaggregation of free polysomes as growing cells entered quiescence (Levine et al., '65, '74). Figure 1 shows a similar study using a detergentcontaining buffer to extract free and membrane bound polysomes. As shown, there is a shift to smaller-sized polyribosomes (panel b) followed by a decrease in the amount of Azsoabsorbing polysomal material (panel c). In order to quantitate more precisely the amount of cellular ribosomal material, cytoplasmic extracts were prepared and adjusted to 2% SDS, and sedimented on SDS-sucrose gradients to display 18s and 28s RNA, which then were analyzed using a Gilford continuous flow recording spec-
E
g
I.0C
N
w
0
z
a m a 0 rn
m a
.7:
.5c
.2!
SEDIMENTATION
Fig. 1 SDG profiles of H D F at various growth stages. Cytoplasmic extracts were prepared from approximately 1.0 x lo' cells at various stages of growth as detailed in METHODS. The extracts were then layered on 11.0 ml, 15-45% sucrose gradients, and centrifuged for two hours at 4°C at 38,000 rpm in a SW-41 rotor. Gradients were analyzed at 260 nm using a Gilford continuous flow recording spectrophotometer. (a) Early log phase. (b) Late log phase. ( c ) Resting phase.
2 15
mRNA REGULATION IN HUMAN D I PLO I D FIBROBLASTS
represented in figure 1. The polysome associated (>80s) and prepolysome asLevels of rRNA in growing and resting cells sociated regions (
