A2u-Globulin Mrna (Transcriptional Control/Steroid Hormone Action/Mediator Protein) CHING-LING C

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Proc. Nati. Acad. Sci. USA Vol. 76, No. 6, pp. 2669-2673, June 1979 Biochemistry Cycloheximide inhibition of hormonal induction of a2u-globulin mRNA (transcriptional control/steroid hormone action/mediator protein) CHING-LING C. CHEN AND PHILIP FEIGELSON Institute of Cancer Research and Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York, New York 10032 Communicated by Seymour Lieberman, March 19, 1979

ABSTRACT The induction of hepatic a2u-globulin synthesis methasone-induced hepatocytes comigrated in sodium dodecyl by glucocorticoids in isolated hepatocytes occurs via an increase sulfate (NaDodSO4)/polyacrylamide gels with authentic in the level of its mRNA as measured by cell-free translation and 20,000-dalton a2u-globulin. However, these dexamethasone- by hybridization to an a2u-globulin cDNA probe. To explore whether induction of this mRNA is a direct or an indirect con- induced hepatocytes, presumably through a hormonally evoked sequence of the interaction of the dexamethasone-receptor posttranslational modification, secrete into the suspension complex with the a2u-globulin genome, the requirement for medium two high molecular weight glycosylated forms of ongoing protein synthesis was examined. Concentrations of a2u-globulin (12). cycloheximide too low to prevent precursor incorporation into The induction of a2u-globulin mRNA by glucocorticoids total poly(A).containing RNA do prevent the hormonal induction manifests a lag of approximately 2 hr (12). This characteristic of a2u-globulin mRNA. Furthermore, incorporation of 3H- lag between glucocorticoid administration and a2u-globulin labeled amino acids into total protein was decreased by only mRNA induction seems longer than might be expected as a 40-50%, and the appearance of the dexamethasone-induced glycosylated forms of a2u-globulin was completely prevented consequence of a direct interaction between glucocorticoid- in these cycloheximide-treated hepatocytes. The results suggest receptor complex and chromatin. A question thus arises as to that the synthesis of a protein mediator(s) may be required for whether intervening events must precede the induction of the induction of a2u-globulin mRNA by glucocorticoids and that a2u-globulin mRNA in these cells. the steroid-receptor complex may not interact directly with the It has recently been found that glucocorticoid induction of a2u-globulin genome. rat liver tryptophan oxygenase mRNA in vivo is largely prevented if the rats are pretreated with protein synthesis inhibitors a2u-Globulin is present in the urine of male rats but not in the such as cycloheximide 30 min before hydrocortisone adminis- urine of female rats (1). This protein, which is synthesized in tration (13). These results suggest that the synthesis of inter- the parenchymal cells of male rat liver and represents ap- mediate protein(s) may be required for the induction of tryp- proximately 1% of hepatic protein synthesis, is secreted into the tophan oxygenase mRNA by glucocorticoids. A similar effect serum and excreted in the urine (2-4). In this laboratory, it has of protein synthesis inhibitors upon the hormonal induction of been shown that the in vivo regulation of the synthesis of this other specific mRNAs, such as ovalbumin and conalbumin, has protein by glucocorticoids (5), thyroid hormones (6), and an- been reported (14). To study whether the induction of a2u- drogens (7) occurs via modulation of the level of hepatic mRNA globulin mRNA by glucocorticoids requires a protein mediator, coding for ct2u-globulin, whereas pituitary growth hormone in vitro experiments were performed with hepatocyte sus- stimulates the synthesis of this protein translationally (8). pensions under carefully controlled experimental conditions. A hepatocyte suspension system that permits the study of hormonal control of (t2u-globulin synthesis under defined MATERIALS AND METHODS conditions in vitro has been developed (9, 10). Isolated hepa- Preparation and Incubation of Hepatocytes. Male tocytes synthesize and secrete (a2u-globulin and other hepatic Sprague-Dawley rats (280-320 g) were castrated and then, 14 proteins in vitro at an approximately linear rate throughout 30 days later, hepatocytes were prepared by a modified procedure hr of incubation. Hepatocytes isolated from animals in different of Seglen (9) involving the removal of Ca2+ from the liver fol- endocrine states synthesize a2u-globulin in vitro at rates con- lowed by recirculating perfusion with collagenase. The liver sistent with the hormonal effects upon its in vivo biosynthesis was then immediately flushed with calcium- and magne- (11). sium-free Hanks' balanced salt solution as described (11, 12). Increased synthesis of oau-globulin can be induced in vitro The liver cells were isolated, washed, and counted in a hemo- by addition of glucocorticoids to hepatocytes derived from male cytometer; viability was defined as the exclusion of trypan blue rats in which the rate of synthesis of this protein had been de- dye. The yield of cells was 7-9 X 108 cells per liver with viability pressed by adrenalectomy, castration, or estrogen pretreatment of greater than 90%. (11). The induction of (2,,-globulin synthesis by dexamethasone In a typical experiment for measuring the rate of total protein in such hepatocytes is highly specific and occurs by selective or a2u-globulin synthesis, 15 X 106 viable cells were suspended increase of aou-globulin mRNA in these cells, as measured by in 10 ml of Joklik-modified minimal essential medium (GIBCO) a wheat germ cell-free translational system and by hybridiza- containing 25 mM Hepes at pH 7.5 and 10% (vol. vol) fetal calf tion to a o2,1-globulin cDNA probe (12). The induced intra- serum (GIBCO). For mRNA studies, a large quantity of cells cellular (YoU-globulin or the translation product generated in was required to permit isolation of sufficient RNA. Cell sus- the wheat germ system using mRNA isolated from the dexa- pensions (50 ml containing 2.5-3.0 X 106 viable cells per ml) were incubated in 250-ml polypropylene flasks as described The publication costs of this article were defrayed in part by page above at 37°C under 95% 02/5% CO2 (11, 12). Hormones, in- charge payment. This article must therefore be hereby marked "ad- hibitors, or labeled precursors were added as indicated. vertisement' in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: NaDodSO4, sodium dodecdl sulfate. 2669 Downloaded by guest on October 1, 2021 2670 Biochemistry: Chen and Feigelson Proc. Natl. Acad. Sci. USA 76 (1979) Labeling of Hepatocytes. To determine the rate of synthesis and measurement of the amount of radioactivity in the aY2u- of total hepatic proteins or a2u-globulin, 15 X 106 hepatocytes globulin on the gel (6, 12). were incubated as described above. Dexamethasone or cycloheximide was added as indicated. After 8 hr of incubation, the RESULTS cells were washed and resuspended in leucine- and lysine-free To investigate the role of protein synthesis in a2u-globulin joklik-modified minimal essential medium containing 25 yCi mRNA induction by dexamethasone, experiments were per- (1 Ci = 3.7 X 1010becquerels) each of [3H]leucine and [3H]- formed with the protein synthesis inhibitor cycloheximide. A lysine per ml with dexamethasone and cycloheximide as indi- concentration of cycloheximide low enough to interfere only cated. The cells were chilled and harvested after 30 min of minimally with endogenous RNA synthesis was sought. precursor incorporation. Therefore, the effect of various levels of cycloheximide on total To evaluate the rate of synthesis of hepatic RNA, liver cells hepatocyte protein and RNA synthesis was studied. The degree (150 X 106) were incubated for 8 hr in the standard medium of inhibition of protein synthesis by cycloheximide increased supplemented with hormone or inhibitor as indicated. The cells as the concentration of this inhibitor was increased. Hepatocyte were collected, resuspended in fresh medium containing hor- RNA synthesis was affected to a lesser degree at each level of mone or inhibitor, and pulse-labeled with ['4C]orotic acid (2.5 cycloheximide (Fig. 1). Some decrease in total RNA synthesis ttCi/ml) for 60 min. was expected because this inhibitor is known to cause a decrease Determination of Total Hepatic Protein and a2u-Globulin in RNA polymerase I transcription of nucleolar ribosomal DNA Synthesis. Liver cells labeled with [3H]leucine and [3H]lysine (18, 19). were harvested and lysed by sonication and detergent treatment The effect of cycloheximide on intracellular ca2u-globulin (1% Triton X-100/1% sodium deoxycholate) (11, 12). The synthesis and the endogenous level of functional a2u-globulin 3H-labeled cell cytosols were prepared by centrifugation at mRNA in these cells was also studied. Increasing concentrations 105,000 X g for 60 min. Incorporation into total protein was of cycloheximide resulted in progressively decreased a2u-glo- measured by applying 1O0Ul of 3H-labeled cytosol onto What- bulin synthesis in hepatocytes. Cycloheximide caused negligible man 3MM filter paper discs that were immersed in 10% tri- loss of translatable a2u-globulin mRNA. Thus, although the chloroacetic acid containing 10 mM unlabeled amino acids and intracellular synthesis of ao2u-globulin and total protein were processed as described (11, 12). greatly inhibited as the concentration of cycloheximide in- The newly synthesized a2u-globulin was isolated by immu- creased, the translational levels of endogenous a2u-globulin noprecipitation of 3H-labeled cytosols or medium with anti- mRNA and total mRNA essentially were unaffected (Fig. 1). a2u-globulin (11, 12). The dissolved immunoprecipitates were This system permits exploration of the role of protein synthesis subjected to NaDodSO4/polyacrylamide gel electrophoresis. during the induction of a2u-globulin mRNA by glucocorti- Radioactivity in the a2u-globulin peaks on the gel was quanti- coids. tated as described (11, 12). The effect of various concentrations of cycloheximide on the Isolation of Total Hepatic RNA and mRNA from Cells. dexamethasone induction of synthesis of a2u-globulin mRNA Total hepatic RNA was extracted as described (12). Cells and a2u-globulin protein was studied. As previously shown (12), (150-200 X 106) were homogenized in sodium acetate/Na- in the absence of cycloheximide, dexamethasone induced the DodSO4/EDTA buffer and extracted with phenol and chlo- synthesis of a2u-globulin with a concomitant increase of a2u- roform as described (12, 15, 16). The RNA in the aqueous phase globulin mRNA (Table 1; Fig. 2 A and B). Cycloheximide at was precipitated at -20°C with 2 vol of ethanol containing 0.05 ,ug/ml inhibited protein synthesis by only 40% but com- LiCl. The yield of RNA by this procedure was 2-2.5 mg/108 cells. Contaminating DNA was removed by digestion with DNase (12). The poly(A)-containing RNA was isolated by chromatog- 120 raphy of isolated total RNA on oligo(dT)-cellulose columns as described by Aviv and Leder (17). The isolated RNA was dis- 100 solved in 1 mM EDTA/10 mM Tris, pH 7.2/0.25% NaDodSO4. After being heated at 85°C for 5 min, the RNA solution was brought to 0.5 M NaCl and slowly passed twice through an ol- 80 igo(dT)-cellulose column. After the oligo(dT)-cellulose column C was washed with application buffer, poly(A)-containing RNA COI60 was eluted with 1mM EDTA/10 mM Tris, pH 7.2/0.05% Na- 0 DodSO4. The poly(A)-containing RNA was precipitated with ethanol/LiCl as described above. 40 The synthesis of total hepatic RNA or poly(A)-containing RNA was determined by isolation of '4C-labeled total RNA or 20 poly(A)-containing RNA from hepatocytes that had been labeled with [14C]orotic acid for 60 min. Aliquots of labeled RNA were assayed for radioactivity by scintillation techniques. -2 -1 0 1 2 Measurement of a2u-Globulin mRNA. The level of func- log cycloheximide (jig/ml) tional a2u-globulin mRNA was quantiated by translation of FIG. 1. Effect of varying concentrations of cycloheximide on the poly(A)-containing RNA or total RNA in a wheat germ cell-free synthesis of total protein (0), a2u-globulin (0), and total RNA (A) protein-synthesizing system containing [3H]leucine and [3H]- in hepatocytes and on their mRNA translational activities [02u- lysine. The synthesis of total released chains was measured by globulin mRNA (o), and total released chains (m)J. The control for total protein synthesis was 2.7 X 106 cpm/15 X 106 viable cells; for trichloroacetic acid precipitation on filter paper discs (12). The a2u-globulin synthesis it was 24 X 103 cpm/15 X 106 viable cells. The level of a20-globulin mRNA was quantitated by immunopre- control for synthesis of total RNA was 6.3 X 105 cpm/mg of RNA. The cipitation of 3H-labeled released chains with anti-a20-globulin control for a2u-globulin mRNA was 2790 cpm/106 cpm of total re- followed by NaDodSO4/polyacrylamide gel electrophoresis leased chain. Downloaded by guest on October 1, 2021 Biochemistry: Chen and Feigelson Proc. Natl. Acad. Sci. USA 76 (1979) 2671

Table 1. Effect of cycloheximide on dexamethone inductiQn<;f a2u-glgbulin synthesis and its mRNA activity % of control Treatment* Intracellular synthesist mRNA translationt

CHX, Total (t2u- % a2u- Total released (ru- Dex Mg/ml protein Globulin globulin chains Globulin - 0 100 100 100 100 100 + 0 93 176 189 92 190 - 0.05 61 63 104 102 103 + 0.05 57 63 110 95 113 - 0.5 20 21 102 105 101 + 0.5 17 18 106 101 98 - 5.0 6 7 102 90 97 + 5.0 7 8 103 97 95 * Dex, dexamethasone at 0.1 MM; CHX, cycloheximide. t Hepatocytes (15 X 106) were incubated with 0.1OAM dexamethasone or cycloheximide or both for 8 hr. The cells were then pulse-labeled with [3H]leucine and [3H]lysine for 30 min. Total protein and a2u-globulin were isolated and quantitated as described in Materials and Methods. Control values: for total protein synthesis, 3.68 X 106 cpm/15 X 106 viable cells; for a2u-globulin synthesis, 25,800 cpm/15 X 106 viable cells; for a2u-globulin synthesis, 0.7% of total protein synthesis. RNA was isolated from incubated hepatocytes (150 X 106) and its translational activity was assayed in the wheat germ cell-free system. The mRNA translational activities for total protein and a2u-globulin were quantitated. Control values: for total released chains, 1.9 X 106 cpm/220 Mg of total RNA; for ac2u-globulin mRNA, 2580 cpm in a2u-globulin per 1 X 106 cpm of total released chains. pletely prevented the hormonal induction of intracellular To ascertain that the inhibitory effect of cycloheximide on a2u-globulin synthesis. Furthermore, when the mRNA isolated the hormonal induction of a2u-globulin mRNA was not due to from these cells was translated in the wheat germ cell-free a generalized impairment of mRNA synthesis, the synthesis of system, it was found that this low level of cycloheximide es- total RNA and total poly(A)-containing RNA was examined. sentially completely prevented the induction of a2u-globulin Cycloheximide moderately affected ['4C]orotic acid incorpo- mRNA by dexamethasone (Table 1; Fig. 2 C and D). This de- ration into total RNA and poly(A)-containing RNA; at 0.05 pendence upon unimpaired protein synthesis suggests that the ,ug/ml, it caused only a 10% diminution in the synthesis of total synthesis of one or more intermediate proteins is required to RNA and poly(A)-containing RNA (Table 2). In coitrast, the mediate steroidal induction of a2u-globulin mRNA. induction of a2u-globulin mRNA by dexamethasone was essentially completely inhibited (Table 1; Fig. 2). Dexamethasone

3.C caused a slight increase in the incorporation of [14CJorotic acid A B into poly(A)- RNA which is compatible with the known aug- mentation of ribosomal RNA synthesis by this steroid (20, 21). The translational activity of total poly(A)-containing RNA 2.0 obtained from hepatocytes treated with cycloheximide was identical to that of poly(A)-containing RNA isolated from control or from hormone-treated cells (Table 1). We have reported earlier that dexamethasone induces the 010 appearance of two high molecular weight glycosylated forms of a2u-globulin in hepatocyte media (12). At 0.05 ,ug/ml cy- E Table 2. Effect of cycloheximide on synthesis of RNA in hepatocytes C D Treatment* Intracellular synthesis, % of controlt CHX, Total Poly(A)+ Poly(A)- E 2.0 11 Dex Mg/rml RNA RNA RNA - .0O 100 100 100

1.0 + 0 120 108 125 - 0.05 89 92 89 + 0.05 93 9:3 96 - 0.5 6.3 70 63 + 0.5 68 71 67 0 10 20 30 40 0 10 20 30 40 - 5.0 5:3 59 52 Slice + 5.0 55 62 55 F I. 2. Electrophoresis of a2u-globulin synthesized in hepatocytes * Dex, dexamethasone at 0.1 MM; CHX, cycloheximide. by pulse-labeling and synthesized by mRNA in the wheat germ cell- Hepatocytes (150 X 106) were incubated as described in Table 1 for free translational system. Hepatocytes (15 X 10fi) were incubated with 8 hr. The cells were resuspended in fresh media again containing no addition (A), with 0.1 MM dexamethasone (B), with cycloheximide the indicated levels of dexamethasone or cycloheximide and incu- at 0.05 Mg/ml (C), or with cycloheximide (0.05 Mg/ml) and 0.1 MM bated with [14C]orotic acid (2.5,uCi/ml) for 60 min. The RNA was dexamethasone for 8 hr (D). The cells were then pulse-labeled with isolated by phenol/chloroform extraction. The RNAs were separated P:3HIleucine and [:3H]lysine for 30 min as described in Fig. 1. The in- on oligo(dT)-cellulose columns. Control values: for total RNA intracellular a2u-globulin (0) was isolated and the ca2u-globulin mRNA corporation, 6.63 x 105 cpm; for poly(A)+ RNA incorporation, 0.323 (0) was translated as described in Fig. 1. X 10- cpm; for poly(A)- RNA incorporation, 5.93 X 10; cpm. Downloaded by guest on October 1, 2021 2672 Biochemistry: Chen and Feigelson Proc. Natl. Acad. Sci. USA 76 (1979) cDNA hybridization probes creates the opportunity to explore whether similar complexities exist during hormonal induction of specific mRNA species in mammals. It recently has been shown that pretreatment of animals with cycloheximide largely prevents the hydrocortisone induction of hepatic tryptophan oxygenase mRNA (13). Similarly, the induction of both ovalbumin and conalbumin mRNAs by either estrogen or proges- terone in the chicken oviduct explant culture system is prevented by translational inhibitors (14). In contrast, the induction of mouse mammary tumor viral RNA by glucocorticoids is not inhibited by either puromycin or cycloheximide (26). In the present studies, the in vitro induction of a2u-globulin E 0 ,_ t l mRNA by dexamethasone was completely prevented by cy- C D cloheximide. To minimize possible nonspecific toxic effects of 2.0 this translational inhibitor on RNA synthesis and other cell functions, a low concentration of cycloheximide, 0.05 mg/ml, was chosen. At this concentration, cycloheximide completely prevented the induction of synthesis of a2u-globulin and its 1.0 mRNA by glucocorticoids. This level of inhibitor does not affect the hepatocyte synthesis of total RNA or poly(A)-containing RNA. Likewise, the translational activity of endogenous total hepatic mRNA or that of the uninduced a2u-globulin mRNA 0 1 0 20 30 40 0 1 0 20 30 40 is unaffected by cycloheximide. These findings suggest that Slice cycloheximide blocks the de novo synthesis of some protein(s) FIG. 3. Electrophoresis of dexamethasone-induced glycosylated that participates in the dexamethasone induction of a2u-glo- forms of a2u-globulin from media of hepatocytes incubated with or bulin mRNA. The appearance of the inducible high molecular without cycloheximide. Hepatocytes (15 X 106) were incubated for weight glycosylated forms of a2u-globulin in the medium of 12 hr with [3H]leucine (12.5 piCi/ml) and no addition (A), 0.1 ,gM dexamethasone-treated hepatocytes is also inhibited by this low dexamethasone (B), cycloheximide at. 0.05 ,g/ml (C), or cyclohexi- concentration of cycloheximide. mide at 0.05 jg/ml plus 0.1 tiM dexamethasone (D). Labeled a2u- The phenomenon described above is reminiscent of the globulin was isolated for assay. Arrows indicate position of authentic earlier demonstration (21, 27) that a-amanitin or cycloheximide 20,000-dalton a2u-globulin. prevents the RNA polymerase I-catalyzed increase of ribosomal RNA synthesis induced by glucocorticoids (21, 27). It was suggested that the glucocorticoid-receptor complex interacts cloheximide decreased the synthesis of ca2u-globulin only by with the hepatic genome to augment the a-amanitin-sensitive, 50% (Fig. 2; Table 1) but it completely prevents the appearance RNA polymerase II-catalyzed transcriptional synthesis of of the two dexamethasone-inducible high molecular weight mRNA(s) which were translated, in a process sensitive to cy- forms of a2u-globulin (Fig. 3). Thus, this aspect of dexameth- cloheximide, to one or more polypeptides necessary for the asone induction is also obviated by low levels of cycloheximide, binding of RNA polymerase I to the nucleolar ribosomal DNA presumably as a consequence of the translational inhibitor template (21, 27). The hormonally induced polypeptide(s) were preventing the normal hormonal increase in a2u-globulin therefore proposed as mediators responsible for the increased mRNA activity. RNA polymerase I activity and ribosomal RNA synthesis. A similar process is indicated by the present studies on the glu- DISCUSSION cocorticoid induction of a2u-globulin mRNA. In both instances Our previous studies indicated that glucocorticoids induce an the hormone acts at one part of the genome to initiate events increase in the synthesis of intracellular a2u1-globulin in hepa- that result in transcription of other genes. Thus, the steroid- tocytes as a consequence of the hormone-induced increase in receptor complex may interact at a specific gene locus to induce a2u-globulin mRNA as measured both by a cell-free transla- transcriptional synthesis of mediator protein mRNA whose tional assay and by a specific (12u-globulin cDNA probe (12). translation to "mediator protein" is cycloheximide sensitive. This in vitro hormonal induction of synthesis of a2u-globulin The mediator protein may in turn accelerate (y2t,-globulin gene and its mRNA manifests a lag of approximately 2 hr (11, 12). expression. Therefore, the effect of a steroid hormone need not Earlier studies showed that saturation of glucocorticoid re- be directly upon the structural gene coding for hormonally ceptors by hydrocortisone and their translocation to the nucleus inducible mRNA species. The hormone may induce the syn- is complete within 30 min (22). Therefore, the existence of a thesis of species of mRNA that in turn controls transcription of 2-hr delay suggests that there may be intermediate processes other genes. It is of interest to note that hormonal control of a between the binding of steroid receptors to chromatin and the mediator protein that regulates the transcription of more than induction of this specific mRNA. Evidence for such indirect one structural gene would serve to coordinate the derepression phenomena is also provided by studies showing that the insect of clusters of genes during endocrine regulation. hormone ecdysone causes puffing on the polytenic salivary gland chromosomes of Chironomus (23, 24) and Drosophila The skillful technical assistance of Ms. Julie Kwang is gratefully (25). This hormone stimulated the incorporation of radioactive acknowledged. This work was supported in part by Grant CA 22376 Institutes of uridine into RNA at specific sites on these chromosomes, causing from the National Health. at these loci in a puffing temporarily defined sequence. Fur- 1. Roy, A. K. & Neuhaus, 0. W. (1966) Proc. Soc. Exp. Biol. Aled. thermore, puromycin was shown to affect "late" puffing but 121, 894-899. not "early" puffing (23-25). 2. Roy, A. K. & Neuhaus. 0. W. (1966) Biochim. Biophys. Acta 127, The development of heterogeneous translational systems and 82-87. 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