Journal of Clinical Endocrinology and Metabolism Copyright © 1978 by The Endocrine Society
Vol. 46, No. 6 Printed in U.S.A.
Cell-Free Translation of Messenger RNA Extracted from a Human Insulinoma* M. ALAN PERMUTT,f ROSE CHYN, MARC GOLDFORD,* AND IRVING BOIME Departments of Medicine (M.A.P., R.C., M.G.), Obstetrics and Gynecology (I.B.), and Pharmacology (I.B.), Washington University School of Medicine, St. Louis, Missouri 63110 comigrated with bovine proinsulin and insulin. [ H]Leucine-labeled cell-free proteins were electrophoresed on NaDodSCvurea polyacrylamide slab gels. In the presence of insulinoma mRNA, discrete proteins of 25,000 and 11,500 mol wt were synthesized. The 11,500 mol wt protein was specifically immunoprecipitated with antiinsulin serum. Thus, cell-free translation of human insulinoma mRNA yields an immunoreactive insulin larger than proinsulin, which is the same size asfishand rat preproinsulins recently described. (J Clin Endocrinol Metab 46: 897, 1978)
ABSTRACT. Total nucleic acid has been extracted from a human pancreatic insulinoma. Purification of the messenger RNA (mRNA) fraction by oligo-dT cellulose chromatography yielded 200 jtig poly(A)-rich mRNA. This mRNA produced a 2-fold stimulation of protein synthesis in a wheat germ cell-free system. Analysis of the translation products by gel filtration chromatography (Biogel P-30) revealed nothing smaller than an acid-alcohol-soluble protein larger than bovine proinsulin. In contrast, insulinoma slices incubated with labeled amino acids synthesized smaller proteins which
T
HE TWO peptide chains of insulin, joined together by disulfide bridges, are derived from a single-chained polypeptide precursor, proinsulin (1-5). Steiner and associates were the first to demonstrate this by incubating radioactive amino acids with human insulinoma slices (1). Incorporation occurred first into proinsulin (9000 mol wt), which is converted within the pancreatic /?-cell to insulin and connecting peptide with a half-time of approximately 1 h. Insulin-related peptides larger than proinsulin have not been detected in biosynthetic experiments with insulinoma tissue or isolated islets. More recently, however, cell-free translation of messenger RNA (mRNA) derived from fish islets (6, 7), fetal bovine pancreas (8), and rat islets (9) resulted in the synthesis of proinsulin precursors approximately 11,000-12,000 mol wt.
Cell-free translation of RNA from human pancreatic islets has not been reported. Yip et al. translated RNA from human insulinomas in frog oocytes and obtained a heterogeneous mixture of immunoreactive insulin peptides (10). In this report we show that the poly (A) rich mRNA fraction derived from a human insulinoma directs the synthesis in a wheat germ cell-free system of a discrete protein having a molecular weight of 11,500, which is specifically bound by antiinsulin antibody. Materials and Methods 3
L-[ H]-Leucine (55 Ci/mmol) and L-[I4C]amino acid mixture (102-485 mCi/mmol) were obtained from New England Nuclear (Boston, MA); phenol was obtained from Fisher Scientific (St. Louis, MO) and redistilled before use; diethyl pyrocarbonate, NaDodSO4, and protein standards were obtained from Sigma Chemical (St. Louis, MO); oligo-dT cellulose was obtained from Collaborative Biochemical Corporation (Waltham, MA); sucrose (ribonuclease-free) and urea were obtained from Schwarz/Mann (Orangeburg, NY). Porcine proinsulin was supplied by Dr. Ronald Chance (Eli Lilly Co., Indianapolis, IN), and AIS (lot 526) was supplied by Dr. Peter Wright (Indiana University).
Received July 15,1977. Address requests for reprints to: Dr. M. Alan Permutt, Washington University School of Medicine, Metabolism Division, 660 South Euclid, St. Louis, Missouri 63110. * This work was supported by NIH Research Grants AM-16746 (M.A.P.) and AM-16865 (I.B.); Clinical Research Center Grant RR-00036, Division of Research Resources, NIH; and by a grant from the St. Louis Diabetic Children's Welfare Association. f Investigator for the Howard Hughes Medical Insti- Nucleic acid extraction tute, and current recipient of a Research Career Development Award (K04-AM00333). A t surgery, a t u m o r ( 2 x 2 c m ) w a s r e m o v e d 897
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 00:21 For personal use only. No other uses without permission. . All rights reserved.
898
PERMUTT ET AL.
from the head of the pancreas of a patient with a previous history of fasting hypoglycemia and hyperinsulinemia. A small portion of the tumor was removed for pathological analysis and was subsequently reported to be an islet cell adenoma. Another piece was removed for incubation with L[14C]amino acids, and the remainder of the tumor was immediately dropped into liquid nitrogen, then stored at —70 C until extraction. Frozen tumor was crushed in a tissue pulverizer and the powder extracted in 0.1 M NaCl, 0.01 M Tris (pH 7.5), 0.01 M EDTA, 1% SDS, 0.05% heparin, and 0.1% diethylpyrocarbonate, and homogenized with an equal volume of buffer-saturated phenol-chloroform-isoamyl alcohol (1.0:1.0:0.1, vol/vol) with a motor-driven Potter-Elvejhem homogenizer. This mixture was shaken vigorously by hand for 15 min at room temperature, cooled to 4 C, and centrifuged at 10,000 X g for 10 min. The upper aqueous phase was removed from the phenol layer and reextracted three times until no visible interface remained. The aqueous phase was made 0.2 M with respect to NaCl, and one volume of 95% ethanol was added. DNA was removed by spooling with a glass rod, then another volume of 95% ethanol was added and the RNA was stored at —20 C overnight, collected at 30,000 X g for 10 min, and dissolved in sterile H2O. Sucrose gradient analysis of the RNA on 5-20% sucrose gradients was performed as previously described (11). Oligo-dT cellulose affinity chromatography Total nucleic acid, from which DNA had been partially removed, was chromatographed on oligodT cellulose by the method of Aviv and Leder (12). A total of 120 A26o U was applied to a 1-ml column on each of two occasions, yielding 2.7 and 1.7 A26o U adsorbed poly (A)-containing RNA (an A26o U is defined as that amount of RNA which has an absorbance of 1 when dissolved in 1 ml H2O and the light path is 1 cm). Cell-free protein synthesis Cell-free extracts from wheat germ were prepared as described by Roberts and Paterson (13), and protein synthesis was assayed according to the method of Aviv et al. (14). A typical reaction was performed in a final volume of 50 jul and contained 24 raM Hepes buffer (pH 7.0), 1 mM dithiothreitol, 1 mM ATP, 20 JUM GTP, 8 mM creatinine phosphate, 40 jug/ml creatinine phosphokinase, 64 mM KC1,1.7 mM MgCl2, 30-50 JUM 19 amino acids excluding leucine, 1-10 /xCi [3H]leucine, 10 /u.1 wheat germ S30 extract, 0.5-1.5 jug mRNA, and 400 JUM spermi-
JCE&M • 1978 Vol 46 • No 6
dine. Incubation was at 31 C for 90 min and was terminated by the addition of pancreatic ribonuclease. For NaDodSO4-urea-polyacrylamide gel electrophoresis, 10 \x\ reaction mixture was precipitated with an equal volume of cold 10% TCA and, after centrifugation, washed with ethanol and ether and air dried. To optimize detection of small peptides in the 2,000-20,000-mol wt range, NaDodSO4-polyacrylamide gel electrophoresis was performed by a modification of the method of Swank and Munkres (15). Slab gels were prepared of 17.5% acrylamide with 8 M urea and samples were electrophoresed with the discontinuous buffer system of Laemmli (16). Gels were sliced into 1.5-mm pieces, digested overnight in 1 ml 0.5 N NaOH at 37 C, neutralized with 1 ml 0.5 M HC1, added to 15 ml Instagel (Packard), and counted on a liquid scintillation counter. Protein standards of known molecular weight (17) were detected by staining with Coomassie blue [ovalbumin (43,000 mol wt), chymotrypsinogen (25,700 mol wt), myoglobin (17,200 mol wt), cytochrome c (12,300 mol wt), proinsulin (9,000 mol wt), and insulin /?-chain (3600 mol wt)]. Insulin when reduced with /?-mercaptoethanol separates into a- and /?-chains, and the a-chain is soluble in the stain so that only /J-chain remains (Permutt, M. A., unpublished observations). Radioactive proteins were also detected by impregnating the gels with PPO, drying (slab gel dryer; Hoefer Scientific Instruments, San Francisco), and exposing the gels to Kodak RP/R540/X-Omat film at -70 C in a cassette, according to the method of Bonner and Laskey (18). Exposure time was 9 days. Immunoprecipitation Reaction mixtures up to 50 ju.1 were diluted 10fold with a solution containing 0.15 M NaCl, 0.015 M NaH2PO4, 0.5% Triton X-100, and bovine serum albumin (0.1%, wt/vol). One hundred microliters of antiinsulin serum (diluted 1:100) was added, and after a 30-min incubation at 37 C, 100 jul goat antiguinea pig serum (1:10) was added. The reaction mixtures were incubated overnight at 4 C. The immunoprecipitates were washed twice with immunoprecipitation buffer and either analyzed by NaDodSO4-urea-polyacrylamide gel electrophoresis or dissolved in 1 ml NCS (Amersham/Searle, Clearbrook, IL) and counted in 10 ml Instagel. Labeling and extraction of insulinoma proteins Two slices of insulinoma (5-10 mg) were incubated in 0.5 ml Kreb's bicarbonate-buffered media with glucose (30 mg/dl) and L-[14C]amino acids (100 juCi/ml) 4 h at 37 C, with 95% O2-5% CO2, and
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 00:21 For personal use only. No other uses without permission. . All rights reserved.
899
CELL-FREE TRANSLATION OF INSULINOMA MRNA extracted in acid-alcohol, as previously described (4). Gel-filtration chromatography Acid-alcohol extracts (0.5 ml) of in vivo or cellfree synthesized labeled proteins were mixed with 1 mg carrier cytochrome c which served as an internal standard, and chromatographed on a BioGel P-30 (-400 mesh, Bio-Rad Laboratories, Richmond, CA) column (1.5 x 37.0 cm) in 2.5 M proprionic acid (9). The column was previously calibrated with bovine serum albumin (void volume), cytochrome c, and bovine proinsulin and insulin. One milliliter samples were collected and 50 /xl of each were counted in 5 ml Instagel.
Results Nucleic acid extraction
activity was retained. Immune precipitation of the cell-free proteins indicated that about 4% of the cell-free protein was specifically bound to antiinsulin serum (Table 1). Comparison of proteins synthesized by tumor slices and those in the cell-free system Insulinoma slices were incubated in L[14C]amino acids for 4 h, then homogenized in TCA, and the acid-alcohol soluble proteins were extracted. These were chromatographed on a Biogel P-30 column and major protein peaks were observed which coeluted with marker bovine proinsulin and insulin (Fig. IB). Cell-free translation of insulinoma mRNA with wheat germ extracts yielded labeled protein(s) which were similarly chromatographed on the Biogel P-30 column (Fig. 1A). Large proteins migrating in the void volume, plus a peak slightly smaller than cytochrome C were observed. In contrast to proteins synthesized by tumor slices, very little protein smaller than 12,000 mol wt was observed.
The presence of large quantities of pancreatic ribonuclease from contaminating acinar tissue made extraction of intact biologically active RNA difficult. The tumor used in these studies was obtained immediately after removal from the patient, frozen in liquid nitrogen, and later pulverized and immediately Analysis of products of cell-free translation extracted with phenol in the presence of riProteins synthesized in the presence of inbonuclease inhibitors. Approximately 10 mg total nucleic acid was extracted from the insulinoma. Sucrose gradient analysis of the 1 4 RNA indicated undegraded material, since the • \ 28S and 18S ribosomal RNA absorbance ratio 3 \ was approximately 2:1. After partial removal 2 of the viscous DNA, oligo-dT cellulose affinity chromatography yielded about 200 jug mRNA. 1 o This mRNA produced more than a 2-fold B S stimulation of incorporation by the cell-free v, 4 system as compared to that in the absence of 1 • mRNA (Table 1), indicating that biological 3 L
TABLE 1. Immunoprecipitation of cell-free translation products with antiinsulin serum
V
° Vcyto
2
1
Immunoprecipitate mRNA
TCA-insoluble
30
AIS
NS
(cpm/10nO 16,892 41,724 2747 1282 Aliquots of the [3H]leucine-labeled cell-free products were precipitated with cold TCA or immunoprecipitated with antiinsulin serum (AIS) or normal serum (NS) as described in Materials and Methods. The results are the average of duplicate samples which differed less than 10%.
{
A
40 50 60 FRACTION NUMBER
FIG. 1. Comparison by gel filtration chromatography on a Biogel P-30 column of acid-alcohol-soluble labeled proteins synthesized with insulinoma mRNA in the cell-free system (A) and proteins synthesized by tumor slices (B). Protein synthesis, extraction, and chromatography are as described in Materials and Methods. Vo, VcYto, Vpi, and Vi, refer to the void volume, and the volume of distribution of cytochrome C, bovine proinsulin, and insulin, respectively.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 00:21 For personal use only. No other uses without permission. . All rights reserved.
sulinoma mRNA were electrophoresed on NaDodSO4-urea-polyacrylamide slab gels and detected by scintillation radioautography (Fig. 2). Major protein bands at approximately 25,000 and 11,500 were observed in the presence of mRNA (Fig. 2c), which were not observed when an equal amount of radioactive protein synthesized in the absence of mRNA was applied to the gel (Fig. 2d). Electrophoresis of labeled protein bound by antiinsulin serum showed enrichment of the 11,500-mol wt protein (Fig. 2e). Densitometric tracings of the total cell-free product and that bound by antiinsulin serum were compared. The 11,500mol wt protein comprised 17% of the total cellfree product, and 43% of the immune precipitate. The larger proteins precipitated by antiinsulin serum were found to be nonspecifically trapped in the immune precipitate, as treating cell-free products made in the absence of mRNA or presence of term placenta mRNA gave the same results (Fig. 3). Therefore, the only protein specifically bound by antiinsulin serum was the discrete band at 11,500 mol wt.
ova — chym —
myo — cytoc —
PI
-
B
-
Dye
a
b
o
d
e
FIG. 2. NaDodSCXt-urea-polyacrylamide slab gel electrophoresis of L-[3H]leucine-labeled products of cell-free synthesis detected by scintillation autoradiography as in Materials and Methods, a) Protein standards stained with Coomassie blue, ovalbumin (OVA, 43,000 mol wt); chymotrypsinogen (CHYMO, 25,700 mol wt); myoglobin (MYO, 17,200 mol wt); cytochrome C (CYTO, 12,300 mol wt); and /?-chain of insulin (3600 mol wt); b) porcine proinsulin (10 /xg) stained with Coomassie blue; c) products of cell-free translation plus insulinoma mRNA (18,800 cpm); d) minus mRNA (18,500 cpm); and e) an equal amount of 3H-protein as in (c) was immunoprecipitated with antiinsulin serum and electrophoresed.
0 Top
1978 No 6
JCE&M Vol46
PERMUTT ET AL.
900
10
20
30
40
t
Dye SLICE NUMBER
FIG. 3. NaDoSOt-urea polyacrylamide gel electrophoresis of antiinsulin immunoprecipitates of JH-protein synthesized in the cell-free system in the presence of insuli• ) , absence of mRNA (A A), and noma mRNA ( • presence of term human placenta mRNA (O O).
Discussion Biologically active mRNA has been extracted from a human insulinoma. Slices of the insulinoma synthesized both proinsulin, the 9000-mol wt single-chained precursor of insulin, and insulin (Fig. 1). In contrast, mRNA extracted from the tumor directed the synthesis of a protein immunologically related to proinsulin which was 20-30 amino acids larger than proinsulin, and no proteins smaller. Densitometric tracings of radioautographs of the cell-free products on NaDodSCXurea gels indicated that this protein comprised 17% of the total cell-free product. The observation that 4-5% of the total cell-free product is bound by antiinsulin serum (Table 1) is in agreement with that of Duguid et al. (19) who similarly noted that only about 20-30% of rat preproinsulin synthesized in a wheat germ system was precipitable by antiinsulin serum. Similarly, Sussman et al. (20) reported that pregrowth hormone synthesized in a cell-free system is only partially precipitated by antigrowth hormone antibody. A number of reports have suggested that large forms of immunoreactive insulin are obtained from tissue or serum extracts (21-25). The nature of these immunoreactive insulins has not been characterized beyond their ability to displace labeled insulin in an RIA. When tissue and serum extracts were examined under conditions which favor disaggregation (25), the large forms of immunoreactive insulin were converted to proinsulin and insulin. It is unlikely that the large forms of these
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 00:21 For personal use only. No other uses without permission. . All rights reserved.
CELL-FREE TRANSLATION OF INSULINOMA MRNA proteins are related to the product of cell-free translation of human islet RNA in this report, as the cell-free products were examined by NaDodSO4-urea gel electrophoresis in the presence of /?-mercaptoethanol. The existence of a proinsulin precursor has been unequivocably established by cell-free translation of rat mRNA (9,19). An additional 23 amino acid residues on the amino terminus of the /8-chain were observed. A partial sequence analysis of the preportion indicated a cluster of hydrophobic residues, similar to the sequence of the preforms of other secretory proteins (26-33). Cloning of the proinsulin gene in bacteria has confirmed an NH2-terminal extension on rat proinsulin (34). Acknowledgments The authors wish to thank Dr. Stephen Crespin for referral of the patient and Ms. Janie Pace for assistance in preparation of the manuscript.
901
13. ROBERTS, B. E., and B. M. PATERSON, Efficient translation of tobacco mosaic virus NA and rabbit globin 9S RNA in a cellfree system from commercial wheat germ, Proc Natl Acad Sci 70: 2303, 1973. 14. Aviv, H., I. BOIME, and P. LEDER, Protein synthesis directed by encephalomyocarditis virus RNA: properties of a transfer RNA-dependent system, Proc Natl Acad Sci 68: 2303, 1971. 15. SWANK, R. T., and K. D. MUNKRES, Molecular weight analysis of oligo-peptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate, Anal Biochem 39: 462, 1971. 16. LAEMMLI, U. K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature (Lond) 227: 680, 1970. 17. WEBER, K., and M. OSBORN, The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis, J Biol Chem 244: 4406, 1969. 18. BONNER, W. M., and R. A. LASKEY, A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels, Eur J Biochem 46: 83, 1974. 19. DUGUID, J. R., D. F. STEINER, and W. L. CHICK, Partial
purification and characterization of the mRNA for rat preproinsulin, Proc Natl Acad Sci 73: 3539, 1976. 20. SUSSMAN, P. M., R. J. TUSHIMSKI, and F. C. BANCROFT,
Pregrowth hormone: product of the translation in vitro of mRNA coding for growth hormone, Proc Natl Acad Sci 73: 29, 1976. 21. MELANI, F., W. G. RYAN, A. H. RUBENSTEIN, and D. F.
STEINER, Proinsulin secretion by a pancreatic beta-cell adenoma: proinsulin and C-peptide secretion, N Engl J Med 283: 713, 1970. 22. YALOW, R. S., and S. A. BERSON, Big big insulin, Metabolism 22: 703, 1973. 23. NUNES-CORREA, J., C. LOWRY, and P. H. SONKSEN, Presumed
References
insulinoma secreting a high-molecular-weight insulin analogue, Lancet 1: 837, 1974.
1. STEINER, D. F., and P. E. OYER, The biosynthesis of insulin and a probable precursor of insulin by a human islet cell adenoma, Proc Natl Acad Sci 57: 473, 1966.
24. BEISCHER, W., F. MELANI, L. KELLER, and E. F. PFEIFFER,
2. STEINER, D. F., D. D. CUNNINGHAM, L. SIGELMAN, and B.
25. PERMUTT, M., J. BIESBROECK, and R. CHYN, Characteristics
ATEN, Insulin biosynthesis: evidence for a precursor, Science 157: 697, 1967. 3. MORRIS, G. E., and A. KORNER, The effect of glucose on insulin biosynthesis by isolated islets of Langerhans of the rat, Biochim Biophys Ada 208: 404, 1970. 4. PERMUTT, M. A., and D. M. KIPNIS, Insulin biosynthesis. I. On the mechanism of glucose stimulation, J Biol Chem 247: 1194, 1972. 5. LIN, B. J., and R. E. HAIST, Insulin biosynthesis: effects of carbohydrates and related compounds, Can J Physiol Pharmacol AT: 791, 1969.
of high molecular weight insulins in insulinoma patients, J Clin Endocrinol Metab 44: 536, 1977.
Chromatographic heterogeneity of insulin extracted from insulinomas, J Clin Endocrinol Metab 40: 393, 1975.
26. KEMPER, B., J. F. HABENER, J. T. POTTS, J R . , and A. RICH,
Pre-proparathyroid hormone: fidelity of the translation of parathyroid messenger RNA by extracts of wheat germ, Biochemistry 15: 20, 1976. 27. SZCESNA, E., and I. BOIME, Messenger RNA dependent synthesis of authentic precursor to human placental lactogen: conversion to its mature hormonal form in ascites cell-free extracts, Proc Natl Acad Sci 73: 1179, 1976. 28. BOIME, I., D. MCWILLIAMS, E. SZCZESNA, and M. CAMEL,
Synthesis of human placental lactogen messenger RNA as a SZCZESNA, and D. MCWILLIAMS, Isolation of a biologically function of gestation, J Biol Chem 251: 820, 1975. active messenger RNA: preparation from fish pancreatic islets 29. MAURER, R. A., R. STONE, and J. GORSKI, Cell-free synthesis by oligo (2'-deoxythymidylic acid) affinity chromatography, of a large translation product of prolactin messenger RNA, J Ciba Found Symp 41: 97, 1976. Biol Chem 251: 2801, 1976. 7. SHIELDS, D., and G. BLOBEL, Cell-free synthesis offishpre- 30. EVANS, G. A., and M. G. ROSENFELD, Cell-free synthesis of a proinsulin, and processing by heterologous mammalian microprolactin precursor directed by mRNA from cultured rat somal membranes, Proc Natl Acad Sci 74: 2059, 1977. pituitary cells, J Biol Chem 251: 2842, 1976.
6. PERMUTT, M. A., J. BIESBROECK, R. CHYN, I. BOIME, E.
8. LOMEDICO, P. T., and G. F. SAUNDERS, Preparation of pan-
creatic mRNA: cell-free translation of an insulin-immunoreactive polypeptide, Nucleic Acids Res 3: 381, 1976. 9. CHAN, S. J., P. KEIM, and D. F. STEINER, Cell-free synthesis
of rat preproinsulins: characterization and partial amino acid sequence determination, Proc Natl Acad Sci 73: 1964, 1976. 10. YIP, C , C.-L. HEW, and H. Hsu, Translation of messenger ribonucleic acid from isolated pancreatic islets and human insulinomas, Proc Natl Acad Sci 72: 4777, 1975. 11. PERMUTT, M. A., and D. M. KIPNIS, Insulin biosynthesis. II. Effect of glucose on ribonucleic acid synthesis in isolated rat islets, J Biol Chem 247: 1200, 1972. 12. Aviv, H., and P. LEDER, Isolation of globin mRNA by affinity chromatography, Proc Natl Acad Sci 69: 1408, 1972.
31. SWAN, D., H. AVIV, and P. LEDER, Purification and properties
of biologically active messenger RNA for a myeloma light chain, Proc Natl Acad Sci #9: 1967, 1972. 32. MILSTEIN, C , G. G. BROWNLEE, T. M. HARRISON, and M. B.
MATHEWS, A possible precursor of immunoglobulin light chains, Nature (New Biol) 239: 117, 1972. 33. SCHECHTER, I., D. J. MCKEAN, R. GUYER, and W. TERRY,
Partial amino acid sequence of the precursor of immunoglobulin light chain programmed by messenger RNA in vitro, Science 187: 161, 1975. 34. ULLRICH, A., J. SHINE, J. CHIRGWIN, R. PICTET, E. TISCHER,
W. J. RUTTER, and H. M. GOODMAN, Rat insulin genes: construction of plasmids containing the coding sequences, Science 196: 1313, 1977.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 00:21 For personal use only. No other uses without permission. . All rights reserved.
Vol. 46, No. 6 Printed in U.S.A.
Cell-Free Translation of Messenger RNA Extracted from a Human Insulinoma* M. ALAN PERMUTT,f ROSE CHYN, MARC GOLDFORD,* AND IRVING BOIME Departments of Medicine (M.A.P., R.C., M.G.), Obstetrics and Gynecology (I.B.), and Pharmacology (I.B.), Washington University School of Medicine, St. Louis, Missouri 63110 comigrated with bovine proinsulin and insulin. [ H]Leucine-labeled cell-free proteins were electrophoresed on NaDodSCvurea polyacrylamide slab gels. In the presence of insulinoma mRNA, discrete proteins of 25,000 and 11,500 mol wt were synthesized. The 11,500 mol wt protein was specifically immunoprecipitated with antiinsulin serum. Thus, cell-free translation of human insulinoma mRNA yields an immunoreactive insulin larger than proinsulin, which is the same size asfishand rat preproinsulins recently described. (J Clin Endocrinol Metab 46: 897, 1978)
ABSTRACT. Total nucleic acid has been extracted from a human pancreatic insulinoma. Purification of the messenger RNA (mRNA) fraction by oligo-dT cellulose chromatography yielded 200 jtig poly(A)-rich mRNA. This mRNA produced a 2-fold stimulation of protein synthesis in a wheat germ cell-free system. Analysis of the translation products by gel filtration chromatography (Biogel P-30) revealed nothing smaller than an acid-alcohol-soluble protein larger than bovine proinsulin. In contrast, insulinoma slices incubated with labeled amino acids synthesized smaller proteins which
T
HE TWO peptide chains of insulin, joined together by disulfide bridges, are derived from a single-chained polypeptide precursor, proinsulin (1-5). Steiner and associates were the first to demonstrate this by incubating radioactive amino acids with human insulinoma slices (1). Incorporation occurred first into proinsulin (9000 mol wt), which is converted within the pancreatic /?-cell to insulin and connecting peptide with a half-time of approximately 1 h. Insulin-related peptides larger than proinsulin have not been detected in biosynthetic experiments with insulinoma tissue or isolated islets. More recently, however, cell-free translation of messenger RNA (mRNA) derived from fish islets (6, 7), fetal bovine pancreas (8), and rat islets (9) resulted in the synthesis of proinsulin precursors approximately 11,000-12,000 mol wt.
Cell-free translation of RNA from human pancreatic islets has not been reported. Yip et al. translated RNA from human insulinomas in frog oocytes and obtained a heterogeneous mixture of immunoreactive insulin peptides (10). In this report we show that the poly (A) rich mRNA fraction derived from a human insulinoma directs the synthesis in a wheat germ cell-free system of a discrete protein having a molecular weight of 11,500, which is specifically bound by antiinsulin antibody. Materials and Methods 3
L-[ H]-Leucine (55 Ci/mmol) and L-[I4C]amino acid mixture (102-485 mCi/mmol) were obtained from New England Nuclear (Boston, MA); phenol was obtained from Fisher Scientific (St. Louis, MO) and redistilled before use; diethyl pyrocarbonate, NaDodSO4, and protein standards were obtained from Sigma Chemical (St. Louis, MO); oligo-dT cellulose was obtained from Collaborative Biochemical Corporation (Waltham, MA); sucrose (ribonuclease-free) and urea were obtained from Schwarz/Mann (Orangeburg, NY). Porcine proinsulin was supplied by Dr. Ronald Chance (Eli Lilly Co., Indianapolis, IN), and AIS (lot 526) was supplied by Dr. Peter Wright (Indiana University).
Received July 15,1977. Address requests for reprints to: Dr. M. Alan Permutt, Washington University School of Medicine, Metabolism Division, 660 South Euclid, St. Louis, Missouri 63110. * This work was supported by NIH Research Grants AM-16746 (M.A.P.) and AM-16865 (I.B.); Clinical Research Center Grant RR-00036, Division of Research Resources, NIH; and by a grant from the St. Louis Diabetic Children's Welfare Association. f Investigator for the Howard Hughes Medical Insti- Nucleic acid extraction tute, and current recipient of a Research Career Development Award (K04-AM00333). A t surgery, a t u m o r ( 2 x 2 c m ) w a s r e m o v e d 897
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 00:21 For personal use only. No other uses without permission. . All rights reserved.
898
PERMUTT ET AL.
from the head of the pancreas of a patient with a previous history of fasting hypoglycemia and hyperinsulinemia. A small portion of the tumor was removed for pathological analysis and was subsequently reported to be an islet cell adenoma. Another piece was removed for incubation with L[14C]amino acids, and the remainder of the tumor was immediately dropped into liquid nitrogen, then stored at —70 C until extraction. Frozen tumor was crushed in a tissue pulverizer and the powder extracted in 0.1 M NaCl, 0.01 M Tris (pH 7.5), 0.01 M EDTA, 1% SDS, 0.05% heparin, and 0.1% diethylpyrocarbonate, and homogenized with an equal volume of buffer-saturated phenol-chloroform-isoamyl alcohol (1.0:1.0:0.1, vol/vol) with a motor-driven Potter-Elvejhem homogenizer. This mixture was shaken vigorously by hand for 15 min at room temperature, cooled to 4 C, and centrifuged at 10,000 X g for 10 min. The upper aqueous phase was removed from the phenol layer and reextracted three times until no visible interface remained. The aqueous phase was made 0.2 M with respect to NaCl, and one volume of 95% ethanol was added. DNA was removed by spooling with a glass rod, then another volume of 95% ethanol was added and the RNA was stored at —20 C overnight, collected at 30,000 X g for 10 min, and dissolved in sterile H2O. Sucrose gradient analysis of the RNA on 5-20% sucrose gradients was performed as previously described (11). Oligo-dT cellulose affinity chromatography Total nucleic acid, from which DNA had been partially removed, was chromatographed on oligodT cellulose by the method of Aviv and Leder (12). A total of 120 A26o U was applied to a 1-ml column on each of two occasions, yielding 2.7 and 1.7 A26o U adsorbed poly (A)-containing RNA (an A26o U is defined as that amount of RNA which has an absorbance of 1 when dissolved in 1 ml H2O and the light path is 1 cm). Cell-free protein synthesis Cell-free extracts from wheat germ were prepared as described by Roberts and Paterson (13), and protein synthesis was assayed according to the method of Aviv et al. (14). A typical reaction was performed in a final volume of 50 jul and contained 24 raM Hepes buffer (pH 7.0), 1 mM dithiothreitol, 1 mM ATP, 20 JUM GTP, 8 mM creatinine phosphate, 40 jug/ml creatinine phosphokinase, 64 mM KC1,1.7 mM MgCl2, 30-50 JUM 19 amino acids excluding leucine, 1-10 /xCi [3H]leucine, 10 /u.1 wheat germ S30 extract, 0.5-1.5 jug mRNA, and 400 JUM spermi-
JCE&M • 1978 Vol 46 • No 6
dine. Incubation was at 31 C for 90 min and was terminated by the addition of pancreatic ribonuclease. For NaDodSO4-urea-polyacrylamide gel electrophoresis, 10 \x\ reaction mixture was precipitated with an equal volume of cold 10% TCA and, after centrifugation, washed with ethanol and ether and air dried. To optimize detection of small peptides in the 2,000-20,000-mol wt range, NaDodSO4-polyacrylamide gel electrophoresis was performed by a modification of the method of Swank and Munkres (15). Slab gels were prepared of 17.5% acrylamide with 8 M urea and samples were electrophoresed with the discontinuous buffer system of Laemmli (16). Gels were sliced into 1.5-mm pieces, digested overnight in 1 ml 0.5 N NaOH at 37 C, neutralized with 1 ml 0.5 M HC1, added to 15 ml Instagel (Packard), and counted on a liquid scintillation counter. Protein standards of known molecular weight (17) were detected by staining with Coomassie blue [ovalbumin (43,000 mol wt), chymotrypsinogen (25,700 mol wt), myoglobin (17,200 mol wt), cytochrome c (12,300 mol wt), proinsulin (9,000 mol wt), and insulin /?-chain (3600 mol wt)]. Insulin when reduced with /?-mercaptoethanol separates into a- and /?-chains, and the a-chain is soluble in the stain so that only /J-chain remains (Permutt, M. A., unpublished observations). Radioactive proteins were also detected by impregnating the gels with PPO, drying (slab gel dryer; Hoefer Scientific Instruments, San Francisco), and exposing the gels to Kodak RP/R540/X-Omat film at -70 C in a cassette, according to the method of Bonner and Laskey (18). Exposure time was 9 days. Immunoprecipitation Reaction mixtures up to 50 ju.1 were diluted 10fold with a solution containing 0.15 M NaCl, 0.015 M NaH2PO4, 0.5% Triton X-100, and bovine serum albumin (0.1%, wt/vol). One hundred microliters of antiinsulin serum (diluted 1:100) was added, and after a 30-min incubation at 37 C, 100 jul goat antiguinea pig serum (1:10) was added. The reaction mixtures were incubated overnight at 4 C. The immunoprecipitates were washed twice with immunoprecipitation buffer and either analyzed by NaDodSO4-urea-polyacrylamide gel electrophoresis or dissolved in 1 ml NCS (Amersham/Searle, Clearbrook, IL) and counted in 10 ml Instagel. Labeling and extraction of insulinoma proteins Two slices of insulinoma (5-10 mg) were incubated in 0.5 ml Kreb's bicarbonate-buffered media with glucose (30 mg/dl) and L-[14C]amino acids (100 juCi/ml) 4 h at 37 C, with 95% O2-5% CO2, and
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 00:21 For personal use only. No other uses without permission. . All rights reserved.
899
CELL-FREE TRANSLATION OF INSULINOMA MRNA extracted in acid-alcohol, as previously described (4). Gel-filtration chromatography Acid-alcohol extracts (0.5 ml) of in vivo or cellfree synthesized labeled proteins were mixed with 1 mg carrier cytochrome c which served as an internal standard, and chromatographed on a BioGel P-30 (-400 mesh, Bio-Rad Laboratories, Richmond, CA) column (1.5 x 37.0 cm) in 2.5 M proprionic acid (9). The column was previously calibrated with bovine serum albumin (void volume), cytochrome c, and bovine proinsulin and insulin. One milliliter samples were collected and 50 /xl of each were counted in 5 ml Instagel.
Results Nucleic acid extraction
activity was retained. Immune precipitation of the cell-free proteins indicated that about 4% of the cell-free protein was specifically bound to antiinsulin serum (Table 1). Comparison of proteins synthesized by tumor slices and those in the cell-free system Insulinoma slices were incubated in L[14C]amino acids for 4 h, then homogenized in TCA, and the acid-alcohol soluble proteins were extracted. These were chromatographed on a Biogel P-30 column and major protein peaks were observed which coeluted with marker bovine proinsulin and insulin (Fig. IB). Cell-free translation of insulinoma mRNA with wheat germ extracts yielded labeled protein(s) which were similarly chromatographed on the Biogel P-30 column (Fig. 1A). Large proteins migrating in the void volume, plus a peak slightly smaller than cytochrome C were observed. In contrast to proteins synthesized by tumor slices, very little protein smaller than 12,000 mol wt was observed.
The presence of large quantities of pancreatic ribonuclease from contaminating acinar tissue made extraction of intact biologically active RNA difficult. The tumor used in these studies was obtained immediately after removal from the patient, frozen in liquid nitrogen, and later pulverized and immediately Analysis of products of cell-free translation extracted with phenol in the presence of riProteins synthesized in the presence of inbonuclease inhibitors. Approximately 10 mg total nucleic acid was extracted from the insulinoma. Sucrose gradient analysis of the 1 4 RNA indicated undegraded material, since the • \ 28S and 18S ribosomal RNA absorbance ratio 3 \ was approximately 2:1. After partial removal 2 of the viscous DNA, oligo-dT cellulose affinity chromatography yielded about 200 jug mRNA. 1 o This mRNA produced more than a 2-fold B S stimulation of incorporation by the cell-free v, 4 system as compared to that in the absence of 1 • mRNA (Table 1), indicating that biological 3 L
TABLE 1. Immunoprecipitation of cell-free translation products with antiinsulin serum
V
° Vcyto
2
1
Immunoprecipitate mRNA
TCA-insoluble
30
AIS
NS
(cpm/10nO 16,892 41,724 2747 1282 Aliquots of the [3H]leucine-labeled cell-free products were precipitated with cold TCA or immunoprecipitated with antiinsulin serum (AIS) or normal serum (NS) as described in Materials and Methods. The results are the average of duplicate samples which differed less than 10%.
{
A
40 50 60 FRACTION NUMBER
FIG. 1. Comparison by gel filtration chromatography on a Biogel P-30 column of acid-alcohol-soluble labeled proteins synthesized with insulinoma mRNA in the cell-free system (A) and proteins synthesized by tumor slices (B). Protein synthesis, extraction, and chromatography are as described in Materials and Methods. Vo, VcYto, Vpi, and Vi, refer to the void volume, and the volume of distribution of cytochrome C, bovine proinsulin, and insulin, respectively.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 00:21 For personal use only. No other uses without permission. . All rights reserved.
sulinoma mRNA were electrophoresed on NaDodSO4-urea-polyacrylamide slab gels and detected by scintillation radioautography (Fig. 2). Major protein bands at approximately 25,000 and 11,500 were observed in the presence of mRNA (Fig. 2c), which were not observed when an equal amount of radioactive protein synthesized in the absence of mRNA was applied to the gel (Fig. 2d). Electrophoresis of labeled protein bound by antiinsulin serum showed enrichment of the 11,500-mol wt protein (Fig. 2e). Densitometric tracings of the total cell-free product and that bound by antiinsulin serum were compared. The 11,500mol wt protein comprised 17% of the total cellfree product, and 43% of the immune precipitate. The larger proteins precipitated by antiinsulin serum were found to be nonspecifically trapped in the immune precipitate, as treating cell-free products made in the absence of mRNA or presence of term placenta mRNA gave the same results (Fig. 3). Therefore, the only protein specifically bound by antiinsulin serum was the discrete band at 11,500 mol wt.
ova — chym —
myo — cytoc —
PI
-
B
-
Dye
a
b
o
d
e
FIG. 2. NaDodSCXt-urea-polyacrylamide slab gel electrophoresis of L-[3H]leucine-labeled products of cell-free synthesis detected by scintillation autoradiography as in Materials and Methods, a) Protein standards stained with Coomassie blue, ovalbumin (OVA, 43,000 mol wt); chymotrypsinogen (CHYMO, 25,700 mol wt); myoglobin (MYO, 17,200 mol wt); cytochrome C (CYTO, 12,300 mol wt); and /?-chain of insulin (3600 mol wt); b) porcine proinsulin (10 /xg) stained with Coomassie blue; c) products of cell-free translation plus insulinoma mRNA (18,800 cpm); d) minus mRNA (18,500 cpm); and e) an equal amount of 3H-protein as in (c) was immunoprecipitated with antiinsulin serum and electrophoresed.
0 Top
1978 No 6
JCE&M Vol46
PERMUTT ET AL.
900
10
20
30
40
t
Dye SLICE NUMBER
FIG. 3. NaDoSOt-urea polyacrylamide gel electrophoresis of antiinsulin immunoprecipitates of JH-protein synthesized in the cell-free system in the presence of insuli• ) , absence of mRNA (A A), and noma mRNA ( • presence of term human placenta mRNA (O O).
Discussion Biologically active mRNA has been extracted from a human insulinoma. Slices of the insulinoma synthesized both proinsulin, the 9000-mol wt single-chained precursor of insulin, and insulin (Fig. 1). In contrast, mRNA extracted from the tumor directed the synthesis of a protein immunologically related to proinsulin which was 20-30 amino acids larger than proinsulin, and no proteins smaller. Densitometric tracings of radioautographs of the cell-free products on NaDodSCXurea gels indicated that this protein comprised 17% of the total cell-free product. The observation that 4-5% of the total cell-free product is bound by antiinsulin serum (Table 1) is in agreement with that of Duguid et al. (19) who similarly noted that only about 20-30% of rat preproinsulin synthesized in a wheat germ system was precipitable by antiinsulin serum. Similarly, Sussman et al. (20) reported that pregrowth hormone synthesized in a cell-free system is only partially precipitated by antigrowth hormone antibody. A number of reports have suggested that large forms of immunoreactive insulin are obtained from tissue or serum extracts (21-25). The nature of these immunoreactive insulins has not been characterized beyond their ability to displace labeled insulin in an RIA. When tissue and serum extracts were examined under conditions which favor disaggregation (25), the large forms of immunoreactive insulin were converted to proinsulin and insulin. It is unlikely that the large forms of these
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 00:21 For personal use only. No other uses without permission. . All rights reserved.
CELL-FREE TRANSLATION OF INSULINOMA MRNA proteins are related to the product of cell-free translation of human islet RNA in this report, as the cell-free products were examined by NaDodSO4-urea gel electrophoresis in the presence of /?-mercaptoethanol. The existence of a proinsulin precursor has been unequivocably established by cell-free translation of rat mRNA (9,19). An additional 23 amino acid residues on the amino terminus of the /8-chain were observed. A partial sequence analysis of the preportion indicated a cluster of hydrophobic residues, similar to the sequence of the preforms of other secretory proteins (26-33). Cloning of the proinsulin gene in bacteria has confirmed an NH2-terminal extension on rat proinsulin (34). Acknowledgments The authors wish to thank Dr. Stephen Crespin for referral of the patient and Ms. Janie Pace for assistance in preparation of the manuscript.
901
13. ROBERTS, B. E., and B. M. PATERSON, Efficient translation of tobacco mosaic virus NA and rabbit globin 9S RNA in a cellfree system from commercial wheat germ, Proc Natl Acad Sci 70: 2303, 1973. 14. Aviv, H., I. BOIME, and P. LEDER, Protein synthesis directed by encephalomyocarditis virus RNA: properties of a transfer RNA-dependent system, Proc Natl Acad Sci 68: 2303, 1971. 15. SWANK, R. T., and K. D. MUNKRES, Molecular weight analysis of oligo-peptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate, Anal Biochem 39: 462, 1971. 16. LAEMMLI, U. K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature (Lond) 227: 680, 1970. 17. WEBER, K., and M. OSBORN, The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis, J Biol Chem 244: 4406, 1969. 18. BONNER, W. M., and R. A. LASKEY, A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels, Eur J Biochem 46: 83, 1974. 19. DUGUID, J. R., D. F. STEINER, and W. L. CHICK, Partial
purification and characterization of the mRNA for rat preproinsulin, Proc Natl Acad Sci 73: 3539, 1976. 20. SUSSMAN, P. M., R. J. TUSHIMSKI, and F. C. BANCROFT,
Pregrowth hormone: product of the translation in vitro of mRNA coding for growth hormone, Proc Natl Acad Sci 73: 29, 1976. 21. MELANI, F., W. G. RYAN, A. H. RUBENSTEIN, and D. F.
STEINER, Proinsulin secretion by a pancreatic beta-cell adenoma: proinsulin and C-peptide secretion, N Engl J Med 283: 713, 1970. 22. YALOW, R. S., and S. A. BERSON, Big big insulin, Metabolism 22: 703, 1973. 23. NUNES-CORREA, J., C. LOWRY, and P. H. SONKSEN, Presumed
References
insulinoma secreting a high-molecular-weight insulin analogue, Lancet 1: 837, 1974.
1. STEINER, D. F., and P. E. OYER, The biosynthesis of insulin and a probable precursor of insulin by a human islet cell adenoma, Proc Natl Acad Sci 57: 473, 1966.
24. BEISCHER, W., F. MELANI, L. KELLER, and E. F. PFEIFFER,
2. STEINER, D. F., D. D. CUNNINGHAM, L. SIGELMAN, and B.
25. PERMUTT, M., J. BIESBROECK, and R. CHYN, Characteristics
ATEN, Insulin biosynthesis: evidence for a precursor, Science 157: 697, 1967. 3. MORRIS, G. E., and A. KORNER, The effect of glucose on insulin biosynthesis by isolated islets of Langerhans of the rat, Biochim Biophys Ada 208: 404, 1970. 4. PERMUTT, M. A., and D. M. KIPNIS, Insulin biosynthesis. I. On the mechanism of glucose stimulation, J Biol Chem 247: 1194, 1972. 5. LIN, B. J., and R. E. HAIST, Insulin biosynthesis: effects of carbohydrates and related compounds, Can J Physiol Pharmacol AT: 791, 1969.
of high molecular weight insulins in insulinoma patients, J Clin Endocrinol Metab 44: 536, 1977.
Chromatographic heterogeneity of insulin extracted from insulinomas, J Clin Endocrinol Metab 40: 393, 1975.
26. KEMPER, B., J. F. HABENER, J. T. POTTS, J R . , and A. RICH,
Pre-proparathyroid hormone: fidelity of the translation of parathyroid messenger RNA by extracts of wheat germ, Biochemistry 15: 20, 1976. 27. SZCESNA, E., and I. BOIME, Messenger RNA dependent synthesis of authentic precursor to human placental lactogen: conversion to its mature hormonal form in ascites cell-free extracts, Proc Natl Acad Sci 73: 1179, 1976. 28. BOIME, I., D. MCWILLIAMS, E. SZCZESNA, and M. CAMEL,
Synthesis of human placental lactogen messenger RNA as a SZCZESNA, and D. MCWILLIAMS, Isolation of a biologically function of gestation, J Biol Chem 251: 820, 1975. active messenger RNA: preparation from fish pancreatic islets 29. MAURER, R. A., R. STONE, and J. GORSKI, Cell-free synthesis by oligo (2'-deoxythymidylic acid) affinity chromatography, of a large translation product of prolactin messenger RNA, J Ciba Found Symp 41: 97, 1976. Biol Chem 251: 2801, 1976. 7. SHIELDS, D., and G. BLOBEL, Cell-free synthesis offishpre- 30. EVANS, G. A., and M. G. ROSENFELD, Cell-free synthesis of a proinsulin, and processing by heterologous mammalian microprolactin precursor directed by mRNA from cultured rat somal membranes, Proc Natl Acad Sci 74: 2059, 1977. pituitary cells, J Biol Chem 251: 2842, 1976.
6. PERMUTT, M. A., J. BIESBROECK, R. CHYN, I. BOIME, E.
8. LOMEDICO, P. T., and G. F. SAUNDERS, Preparation of pan-
creatic mRNA: cell-free translation of an insulin-immunoreactive polypeptide, Nucleic Acids Res 3: 381, 1976. 9. CHAN, S. J., P. KEIM, and D. F. STEINER, Cell-free synthesis
of rat preproinsulins: characterization and partial amino acid sequence determination, Proc Natl Acad Sci 73: 1964, 1976. 10. YIP, C , C.-L. HEW, and H. Hsu, Translation of messenger ribonucleic acid from isolated pancreatic islets and human insulinomas, Proc Natl Acad Sci 72: 4777, 1975. 11. PERMUTT, M. A., and D. M. KIPNIS, Insulin biosynthesis. II. Effect of glucose on ribonucleic acid synthesis in isolated rat islets, J Biol Chem 247: 1200, 1972. 12. Aviv, H., and P. LEDER, Isolation of globin mRNA by affinity chromatography, Proc Natl Acad Sci 69: 1408, 1972.
31. SWAN, D., H. AVIV, and P. LEDER, Purification and properties
of biologically active messenger RNA for a myeloma light chain, Proc Natl Acad Sci #9: 1967, 1972. 32. MILSTEIN, C , G. G. BROWNLEE, T. M. HARRISON, and M. B.
MATHEWS, A possible precursor of immunoglobulin light chains, Nature (New Biol) 239: 117, 1972. 33. SCHECHTER, I., D. J. MCKEAN, R. GUYER, and W. TERRY,
Partial amino acid sequence of the precursor of immunoglobulin light chain programmed by messenger RNA in vitro, Science 187: 161, 1975. 34. ULLRICH, A., J. SHINE, J. CHIRGWIN, R. PICTET, E. TISCHER,
W. J. RUTTER, and H. M. GOODMAN, Rat insulin genes: construction of plasmids containing the coding sequences, Science 196: 1313, 1977.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 20 November 2015. at 00:21 For personal use only. No other uses without permission. . All rights reserved.
