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In vitro translation of measles virus-induced RNA
VIROLOGY 96, 295-298 (1979)
In Vitro Translation
of Measles Virus-Induced
A. NIVELEAU’ Unite de Virologie
AND
Fondammtale et Appliquee, I, Place Pr Joseph Rena&,
RNA
T. F. WILD
INSERM-U.51,
Groupe de Recherches CNRS $$,
693’72Lyon Cedex 2, France
Accepted March 9, 1979
RNA was extracted from Vero cells infected with measles virus and tested for its ability to direct protein synthesis in the wheat germ cell-free translation system. When in vitro products were subjected to immunoprecipitation with immunoglobulins directed against measles virus four major bands were found to correspond to the viral proteins.
The possible involvement of measles virus in neurological diseases such as subacute sclerosing panencephalitis (SSPE) (1-3) and multiple sclerosis (4,5> provided a stimulus for research into the biology of this virus. All authors underline the major obstacles which are commonly met in the study of measles virus, such as the low yields of viral particles produced in cell culture and the inability to obtain virus preparations free from cellular contamination. Measles virus does not shut off host cell protein synthesis. Therefore, when the biosynthesis of viral proteins is investigated by polyacrylamide gel electrophoresis, the analysis of intracellular viral polypeptides is hampered by a prominent cellular background. However, the electrophoretic gel pattern of measles proteins has been investigated by several laboratories and similar results were obtained indicating that measles virus contains six or seven polypeptides (6-11). In order to gain further insight into the biology of measles virus, we examined the ability of RNA extracted from Vero cells infected with measles virus to direct the synthesis of viral proteins in the wheat germ cell-free system. We present here the results obtained in such experiments. RNA extracted from noninfected or measles (SSPE isolate Halle) virus-infected Vero cells either 24 or 36 hr after infection (m.o.i. = 1) was used to prime in vitro 1 To whom reprint requests should be addressed. 295
translation in the wheat germ system. Analysis on polyacrylamide slab gels showed that compared to the noninfected cells, RNA from cells 24 hr after infection gave several additional bands principally at 76K, 70K, 62K, 47K, 51K, and 3’7K (Fig. 1). In comparison to the noninfected cells certain bands were reduced in the infected cell RNA primed reactions. RNA from cells with an advanced viral cytopathic effect gave rise to only three major bands at 82K, 70K, and 60K. The overall efficiency of incorporation of [35S]methionine into proteins greater than 30K was much lower for the 36-hr RNA than the 24 hr, but greater amounts of low molecular weight proteins were found in the former. This may be a reflection of the extent of degradation of RNA in the infected cells. In order to determine the specificity of the proteins synthesized, the in vitro products primed by RNA extracted from cells 24 hr after infection were subjected to immunoprecipitation with immunoglobulins directed against measles virus. The immunoprecipitate was analyzed by electrophoresis on a 10% polyacrylamide slab gel (Fig. 2). Six major bands with molecular weights 76K, 70K, 63K, 47-51K, 42K, and 37K were detected. The results are summarized in Table 1. Although we did not detect the synthesis of an “LI’ polypeptide (200K) in the in vitro reaction it may be due to the degradation of RNA during extraction or the inability of the system to translate long mRNA molecules in spite of addition of polyamines (12, 13). 0042-6822/79/090295-04$02.00/0
Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
296
SHORT COMMUNICATIONS
76k 70k 62k 5lk 47k
37k
A BC
b
E
FIG. 1. SDS-polyacrylamide gel electrophoresis of [Wlmethionine-labeled products of cell-free protein synthesis in the presence of polyamines. Wheat germ extracts were prepared according to Marcu and Dudock (14). The only modification was the use of potassium acetate in place of potassium chloride. Standard reaction mixtures of 25 /Al contained 7.5 ~1 of wheat germ extract, 1 mM ATP, 0.02 mM GTP, 8.5 m&f ereatine phosphate, creatine kinase (final concentration 40 &ml), 23 m&f HEPES pH 7.6, 1 mM magnesium, 0.03 mM spermine, 0.3 n&f spermidine, 2 mM DTT, 25 fl of each amino acid (except methionine), 5 &i of r”S]methionine (specific activity > 400 Wmmol), and 5 pg of RNA. Mixtures were incubated at 23” for 90 min. To estimate incorporation, %l aliquots were precipitated with 5% cold TCA and boiled for 15 min. Samples were filtered on Millipore filters, washed with cold 5% TCA containing 1% unlabeled methionine, and counted in an Intertechnique liquid scintillation counter. In vitro synthesis products were analyzed by electrophoresis on SDS-containing slab gels according to Laemmli (15). Electrophoresis was carried
The two polypeptides which after immunoprecipitation electrophoresed at 70K and 63K probably correspond to P and NP viral proteins, respectively. The highest molecular weight virus-induced protein (76K) may represent the nonglycosylated precursor of the hemagglutinin @OK) as no glycolysation takes place in the wheat germ system. A more affirmative designation awaits peptide mapping studies. The fourth polypeptide electrophoresed as a diffuse band with an apparent molecular weight of 47-51K. This protein was consistently detected in immunoprecipitates with immunoglobulins directed against measles virus in incubation mixtures programmed with RNA from infected cells but not in those directed by RNA from noninfected cells. This protein may correspond to the non glycosylated precursor F,, described by Graves et al. (IO), which would explain the absence of F, and Fz proteins. Peptide mapping will be necessary to confirm these possibilities. A fifth band with a molecular weight of 42K is most probably actin. Purified virus is usually associated with actin and gives rise to anti-a&in antibodies when inoculated into rabbits. Absorption studies with noninfected cells show it is difficult to completely deplete the serum of its antiactin activity., The last major band observed in the polyacrylamide gels migrated to the same position as the M protein (37K). Two major bands migrated ahead of the M protein and could be found in some preparations of purified viruses. They may represent degradation products. At present it is necessary to confirm the identity of the in vitro products by peptide on 10% acrylamide slab gels, at 4 mA/gel. After migration was completed, gels were treated for fluorography according to Bonner and Laskey (16). Samples are: (A) no exogenous RNA added; (B) RNA extracted from Vero cells infected 24 hr; (C) RNA extracted from noninfected Vero cells; (D) RNA extracted from Vero cells infected for 36 hr; (E), as in (D) but twice the amount of radioactivity. Virus-induced bands are indicated by arrows on the left. Molecular weights of standard proteins are indicated on the right (bovine serum albumin, 67K; aldolase, 40K; chymotrypsinogen A, 25 K).
SHORT COMMUNICATIONS
297 TABLE 1
67)
76k 70k 63k
COMPARISON OF IN VITRO PRODUCTS PRECIPITATED BY IMMUNOGLOBULINS DIRECTED AGAINST MEASLES VIRUS, WITH POLYPEPTIDES OBTAINED FROM VIRAL PARTICLES
H p
(67
Molecular weight
47/!?.1 k 41 4om
25b
Viral polypeptides
NP P4
k
37k
Fl M
if@ a 440
Old designation
*L
-
II
G (25
FIG. 2. (A) PAGE of [35S]methionine-labeled in vitro products immunoprecipitated with anti-measles immunoglobulins. Rabbits were immunized with purified measles virus given in two inoculations, with Freund’s complete adjuvant 3 weeks apart. The rabbits were bled 3 weeks after the second inoculation (HI titer: 16,000). Uninfected Vero cells were lysed by three successive cycles of freezing and thawing in PBS. Final concentration of protein was 0.9 mg/ml in a total volume of 10 ml as estimated by the technique of Kalb and Bernlohr (18). Enzacryl AH was activated following the manufacturer’s instructions. Lysed Vero cells were added to activated Enzacryl and gently rocked at 4” for 48 hr. The mixture was then filtered on a sintered-glass funnel and the resin washed with 0.1 M borate buffer, pH 8.8 until the effluent showed no absorption at 230 nm. Approximately ‘7 mg of proteins were fixed on 2.5 g of Enzacryl, here after referred to as EnzacrylVero. Total immunoglobulins were prepared from a rabbit immune serum according to Steinbuch and Audran (19). A solution of total immunoglobulins containing 20 mg/ml of proteins in a total volume of 2 ml of PBS was adsorbed on 2.5 g of Enzacryl-Vero resin equilibrated in the same buffer. Nonadsorbed material was recycled on a second identical column. The second effluent was concentrated to a volume of 2 ml by vacuum dialysis. After concentration, the solution was clarified by centrifugation at 10,OOOg for 15 min at 4”. For immunoprecipitation, the volume of the in vitro incubation mixtures was scaled up to 200 ~1. After incubation, samples were diluted to 1 ml with the immunoprecipitation buffer to give a final concentration of 20 mM phosphate, pH 7.6, 1% Nadeoxycholate, 1% Triton X-100. Samples were centrifuged
New designation
~ In vitro
Ref. (10) L P,
2
P2
NP
P,NP
4
P,O
Ref. (9)
Virus
L H P NP
200K 80K 70K 60-6213 55K
-
5
P5
F,”
M -
P,M -
M F.ZC
40-41K 37K 15K
products 76K 70K 63K 47-51K 42K 37K -
a Polypeptide (4) is usually found in small and varying amounts and thought not to be essential. * Polypeptide F, derived from F, is not glycosylated. c Polypeptide F2 derived from F,, is a carbohydraterich protein. Precursor F0 migrated in the region of NP when glycoproteins were labeled with glucosamine in pulse-chase experiments with infected cells protected from fusion.
mapping. We are also using the system of microinjection into Xenopus oocytes as a system which permits glycosylation and cleavage. at 120,OOOgfor 60 min. Supernatants were reacted with rabbit anti-measles immunoglobulins (prepared as described above) for 1 hr at 37” and overnight at 4”. Samples received an appropriate amount of anti-rabbit IgG serum and the mixture was incubated for a further 2 hr at 37” and 4 h at 4”. The immunoprecipitates were collected by centrifugation at 12,000g for 5 min at 4”. Pellets were resuspended in the same buffer and washed by three successive cycles of centrifugation. Finally, pellets were resuspended in the dissociating sample buffer and boiled for 3 min before analyzing on slab gels. The designated molecular weights of the immunoprecipitated products were determined from the marker protein as in Fig. 1. (B) PAGE [35S]methionine-labeled purified virus. Measles virus was purified ae already described (17). The polypeptides are identified according to the designation of Graves et al. (10).
298
SHORT
COMMUNICATIONS
REFERENCES 1. CONNOLLY, J. H., ALLEN, I. V., HURWITZ, L. J., and MILUR, I. H. D.,Lancet 1,542-544 (1967). 2. HORTA-BARBOSA, L., FUCCILLO, D. A., ZEMAN, W., and SEVER, J. L., Nature (London) 221, 974 (1969). 3. PAYNE, F. E., BAUBLOS, V. V., and ITASHI, H. H., N. Engl. J. Med. 281, 585-589 (1969). 4. SEVER, J. L., and ZEMAN, W., Neurology 18, (Pt. 2), 1-192 (1967). 5. MARTIN, S. J., and FRASER, K. B. (eds.), Med. Microbial. Immunol. 160.73-250 (1974). 6. HALL, W. W., and MARTIN, S. J., J. Gen. Virol. 22, 363-374 (1974). 7. MOUNTCASTLE, W. E., and CHOPPIN, P. W., Virology 78, 463-474 (1978). 8. TYRRELL, D. L. J., and NORRBY, E., J. Gen. Viral. 39, 219-229 (1978). 9. WESCHLER, S. L., and FIELDS, B. N., J. Viral. 25, 285-297 (1978). 10. GRAVES, M. C., SILVER, S. M., and CHOPPIN, P. W., Virology 86, 254-263 (1978).
11. HARDWICK, J. M., and BUSSELL, R. H. J. Viral. 25, 687-692 (1978). 12. ATKINS, J. F., LEWIS, J. B., ANDERSON, C. W., and GESTELAND, R. F., J. Biol. Chem. 250, 5688-5695 (1975). IS. HUNTER, A. R., FARRELL, P. J., JACKSON, R. J., and HUNT, T., Eur. J. Biochem. 75, 149- 157 (1977). 14. MARCU, K., and DUDOCK, B., Nucleic Acids Res. 1, 1385-1397 (1974). 15. LAEMMLI, U. K., Nature (1970).
(London)
227, 680-685
16. BONNER, W. M., and LASKEY, R. A., Eur. Biochem. 46, 83-88 (1974).
J.
17. WILD, T. F., and GREENLAND, T., lntwuirology 11, 275-281 (1979). 18. KALB JR., V. F. K., and BERNLOHR, R. W., Anal. Biochem. 82.362-371 (1977). 19. STEINBUCH, M., and AUDRAN, R., Arch. them. Biophys. 134, 279-284 (1969).
Bio-
In Vitro Translation
of Measles Virus-Induced
A. NIVELEAU’ Unite de Virologie
AND
Fondammtale et Appliquee, I, Place Pr Joseph Rena&,
RNA
T. F. WILD
INSERM-U.51,
Groupe de Recherches CNRS $$,
693’72Lyon Cedex 2, France
Accepted March 9, 1979
RNA was extracted from Vero cells infected with measles virus and tested for its ability to direct protein synthesis in the wheat germ cell-free translation system. When in vitro products were subjected to immunoprecipitation with immunoglobulins directed against measles virus four major bands were found to correspond to the viral proteins.
The possible involvement of measles virus in neurological diseases such as subacute sclerosing panencephalitis (SSPE) (1-3) and multiple sclerosis (4,5> provided a stimulus for research into the biology of this virus. All authors underline the major obstacles which are commonly met in the study of measles virus, such as the low yields of viral particles produced in cell culture and the inability to obtain virus preparations free from cellular contamination. Measles virus does not shut off host cell protein synthesis. Therefore, when the biosynthesis of viral proteins is investigated by polyacrylamide gel electrophoresis, the analysis of intracellular viral polypeptides is hampered by a prominent cellular background. However, the electrophoretic gel pattern of measles proteins has been investigated by several laboratories and similar results were obtained indicating that measles virus contains six or seven polypeptides (6-11). In order to gain further insight into the biology of measles virus, we examined the ability of RNA extracted from Vero cells infected with measles virus to direct the synthesis of viral proteins in the wheat germ cell-free system. We present here the results obtained in such experiments. RNA extracted from noninfected or measles (SSPE isolate Halle) virus-infected Vero cells either 24 or 36 hr after infection (m.o.i. = 1) was used to prime in vitro 1 To whom reprint requests should be addressed. 295
translation in the wheat germ system. Analysis on polyacrylamide slab gels showed that compared to the noninfected cells, RNA from cells 24 hr after infection gave several additional bands principally at 76K, 70K, 62K, 47K, 51K, and 3’7K (Fig. 1). In comparison to the noninfected cells certain bands were reduced in the infected cell RNA primed reactions. RNA from cells with an advanced viral cytopathic effect gave rise to only three major bands at 82K, 70K, and 60K. The overall efficiency of incorporation of [35S]methionine into proteins greater than 30K was much lower for the 36-hr RNA than the 24 hr, but greater amounts of low molecular weight proteins were found in the former. This may be a reflection of the extent of degradation of RNA in the infected cells. In order to determine the specificity of the proteins synthesized, the in vitro products primed by RNA extracted from cells 24 hr after infection were subjected to immunoprecipitation with immunoglobulins directed against measles virus. The immunoprecipitate was analyzed by electrophoresis on a 10% polyacrylamide slab gel (Fig. 2). Six major bands with molecular weights 76K, 70K, 63K, 47-51K, 42K, and 37K were detected. The results are summarized in Table 1. Although we did not detect the synthesis of an “LI’ polypeptide (200K) in the in vitro reaction it may be due to the degradation of RNA during extraction or the inability of the system to translate long mRNA molecules in spite of addition of polyamines (12, 13). 0042-6822/79/090295-04$02.00/0
Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
296
SHORT COMMUNICATIONS
76k 70k 62k 5lk 47k
37k
A BC
b
E
FIG. 1. SDS-polyacrylamide gel electrophoresis of [Wlmethionine-labeled products of cell-free protein synthesis in the presence of polyamines. Wheat germ extracts were prepared according to Marcu and Dudock (14). The only modification was the use of potassium acetate in place of potassium chloride. Standard reaction mixtures of 25 /Al contained 7.5 ~1 of wheat germ extract, 1 mM ATP, 0.02 mM GTP, 8.5 m&f ereatine phosphate, creatine kinase (final concentration 40 &ml), 23 m&f HEPES pH 7.6, 1 mM magnesium, 0.03 mM spermine, 0.3 n&f spermidine, 2 mM DTT, 25 fl of each amino acid (except methionine), 5 &i of r”S]methionine (specific activity > 400 Wmmol), and 5 pg of RNA. Mixtures were incubated at 23” for 90 min. To estimate incorporation, %l aliquots were precipitated with 5% cold TCA and boiled for 15 min. Samples were filtered on Millipore filters, washed with cold 5% TCA containing 1% unlabeled methionine, and counted in an Intertechnique liquid scintillation counter. In vitro synthesis products were analyzed by electrophoresis on SDS-containing slab gels according to Laemmli (15). Electrophoresis was carried
The two polypeptides which after immunoprecipitation electrophoresed at 70K and 63K probably correspond to P and NP viral proteins, respectively. The highest molecular weight virus-induced protein (76K) may represent the nonglycosylated precursor of the hemagglutinin @OK) as no glycolysation takes place in the wheat germ system. A more affirmative designation awaits peptide mapping studies. The fourth polypeptide electrophoresed as a diffuse band with an apparent molecular weight of 47-51K. This protein was consistently detected in immunoprecipitates with immunoglobulins directed against measles virus in incubation mixtures programmed with RNA from infected cells but not in those directed by RNA from noninfected cells. This protein may correspond to the non glycosylated precursor F,, described by Graves et al. (IO), which would explain the absence of F, and Fz proteins. Peptide mapping will be necessary to confirm these possibilities. A fifth band with a molecular weight of 42K is most probably actin. Purified virus is usually associated with actin and gives rise to anti-a&in antibodies when inoculated into rabbits. Absorption studies with noninfected cells show it is difficult to completely deplete the serum of its antiactin activity., The last major band observed in the polyacrylamide gels migrated to the same position as the M protein (37K). Two major bands migrated ahead of the M protein and could be found in some preparations of purified viruses. They may represent degradation products. At present it is necessary to confirm the identity of the in vitro products by peptide on 10% acrylamide slab gels, at 4 mA/gel. After migration was completed, gels were treated for fluorography according to Bonner and Laskey (16). Samples are: (A) no exogenous RNA added; (B) RNA extracted from Vero cells infected 24 hr; (C) RNA extracted from noninfected Vero cells; (D) RNA extracted from Vero cells infected for 36 hr; (E), as in (D) but twice the amount of radioactivity. Virus-induced bands are indicated by arrows on the left. Molecular weights of standard proteins are indicated on the right (bovine serum albumin, 67K; aldolase, 40K; chymotrypsinogen A, 25 K).
SHORT COMMUNICATIONS
297 TABLE 1
67)
76k 70k 63k
COMPARISON OF IN VITRO PRODUCTS PRECIPITATED BY IMMUNOGLOBULINS DIRECTED AGAINST MEASLES VIRUS, WITH POLYPEPTIDES OBTAINED FROM VIRAL PARTICLES
H p
(67
Molecular weight
47/!?.1 k 41 4om
25b
Viral polypeptides
NP P4
k
37k
Fl M
if@ a 440
Old designation
*L
-
II
G (25
FIG. 2. (A) PAGE of [35S]methionine-labeled in vitro products immunoprecipitated with anti-measles immunoglobulins. Rabbits were immunized with purified measles virus given in two inoculations, with Freund’s complete adjuvant 3 weeks apart. The rabbits were bled 3 weeks after the second inoculation (HI titer: 16,000). Uninfected Vero cells were lysed by three successive cycles of freezing and thawing in PBS. Final concentration of protein was 0.9 mg/ml in a total volume of 10 ml as estimated by the technique of Kalb and Bernlohr (18). Enzacryl AH was activated following the manufacturer’s instructions. Lysed Vero cells were added to activated Enzacryl and gently rocked at 4” for 48 hr. The mixture was then filtered on a sintered-glass funnel and the resin washed with 0.1 M borate buffer, pH 8.8 until the effluent showed no absorption at 230 nm. Approximately ‘7 mg of proteins were fixed on 2.5 g of Enzacryl, here after referred to as EnzacrylVero. Total immunoglobulins were prepared from a rabbit immune serum according to Steinbuch and Audran (19). A solution of total immunoglobulins containing 20 mg/ml of proteins in a total volume of 2 ml of PBS was adsorbed on 2.5 g of Enzacryl-Vero resin equilibrated in the same buffer. Nonadsorbed material was recycled on a second identical column. The second effluent was concentrated to a volume of 2 ml by vacuum dialysis. After concentration, the solution was clarified by centrifugation at 10,OOOg for 15 min at 4”. For immunoprecipitation, the volume of the in vitro incubation mixtures was scaled up to 200 ~1. After incubation, samples were diluted to 1 ml with the immunoprecipitation buffer to give a final concentration of 20 mM phosphate, pH 7.6, 1% Nadeoxycholate, 1% Triton X-100. Samples were centrifuged
New designation
~ In vitro
Ref. (10) L P,
2
P2
NP
P,NP
4
P,O
Ref. (9)
Virus
L H P NP
200K 80K 70K 60-6213 55K
-
5
P5
F,”
M -
P,M -
M F.ZC
40-41K 37K 15K
products 76K 70K 63K 47-51K 42K 37K -
a Polypeptide (4) is usually found in small and varying amounts and thought not to be essential. * Polypeptide F, derived from F, is not glycosylated. c Polypeptide F2 derived from F,, is a carbohydraterich protein. Precursor F0 migrated in the region of NP when glycoproteins were labeled with glucosamine in pulse-chase experiments with infected cells protected from fusion.
mapping. We are also using the system of microinjection into Xenopus oocytes as a system which permits glycosylation and cleavage. at 120,OOOgfor 60 min. Supernatants were reacted with rabbit anti-measles immunoglobulins (prepared as described above) for 1 hr at 37” and overnight at 4”. Samples received an appropriate amount of anti-rabbit IgG serum and the mixture was incubated for a further 2 hr at 37” and 4 h at 4”. The immunoprecipitates were collected by centrifugation at 12,000g for 5 min at 4”. Pellets were resuspended in the same buffer and washed by three successive cycles of centrifugation. Finally, pellets were resuspended in the dissociating sample buffer and boiled for 3 min before analyzing on slab gels. The designated molecular weights of the immunoprecipitated products were determined from the marker protein as in Fig. 1. (B) PAGE [35S]methionine-labeled purified virus. Measles virus was purified ae already described (17). The polypeptides are identified according to the designation of Graves et al. (10).
298
SHORT
COMMUNICATIONS
REFERENCES 1. CONNOLLY, J. H., ALLEN, I. V., HURWITZ, L. J., and MILUR, I. H. D.,Lancet 1,542-544 (1967). 2. HORTA-BARBOSA, L., FUCCILLO, D. A., ZEMAN, W., and SEVER, J. L., Nature (London) 221, 974 (1969). 3. PAYNE, F. E., BAUBLOS, V. V., and ITASHI, H. H., N. Engl. J. Med. 281, 585-589 (1969). 4. SEVER, J. L., and ZEMAN, W., Neurology 18, (Pt. 2), 1-192 (1967). 5. MARTIN, S. J., and FRASER, K. B. (eds.), Med. Microbial. Immunol. 160.73-250 (1974). 6. HALL, W. W., and MARTIN, S. J., J. Gen. Virol. 22, 363-374 (1974). 7. MOUNTCASTLE, W. E., and CHOPPIN, P. W., Virology 78, 463-474 (1978). 8. TYRRELL, D. L. J., and NORRBY, E., J. Gen. Viral. 39, 219-229 (1978). 9. WESCHLER, S. L., and FIELDS, B. N., J. Viral. 25, 285-297 (1978). 10. GRAVES, M. C., SILVER, S. M., and CHOPPIN, P. W., Virology 86, 254-263 (1978).
11. HARDWICK, J. M., and BUSSELL, R. H. J. Viral. 25, 687-692 (1978). 12. ATKINS, J. F., LEWIS, J. B., ANDERSON, C. W., and GESTELAND, R. F., J. Biol. Chem. 250, 5688-5695 (1975). IS. HUNTER, A. R., FARRELL, P. J., JACKSON, R. J., and HUNT, T., Eur. J. Biochem. 75, 149- 157 (1977). 14. MARCU, K., and DUDOCK, B., Nucleic Acids Res. 1, 1385-1397 (1974). 15. LAEMMLI, U. K., Nature (1970).
(London)
227, 680-685
16. BONNER, W. M., and LASKEY, R. A., Eur. Biochem. 46, 83-88 (1974).
J.
17. WILD, T. F., and GREENLAND, T., lntwuirology 11, 275-281 (1979). 18. KALB JR., V. F. K., and BERNLOHR, R. W., Anal. Biochem. 82.362-371 (1977). 19. STEINBUCH, M., and AUDRAN, R., Arch. them. Biophys. 134, 279-284 (1969).
Bio-