studies on the bacteriophage ms 2

Molec. gen. Genet. 117, 219--228 (1972) © by Springer-Verlag 1972

Studies on the Bacteriophage MS 2 X V I I . Suppressor-Sensitive Mutants of the A Protein Cistron

E. Vandamme, E. Remaut, M. van Montagu, and W. Fiers Laboratory of Molecular Biology, State University of Ghent, Belgium

Received April 5, 1972

Summary. The viral proteins synthesized in non-suppressor cells by amber mutants in the A protein eistron of the RNA bacteriophage MS2 were analyzed. Protein synthesis was studied in rifampicin-inhibited cultures and the labeled, viral proteins were separated on sodium dodecyl sulphate containing polyacrylamide gels. We found that 7 out of 19 mutants synthesized an A protein-fragment corresponding in length to 88 % of the wild-type A protein. This fragment was not incorporated into the defective particles formed by the mutants. 12 mutants synthesized no detectable amount of fragment. It was shown that the absence of fragment is not due to selective proteolytie breakdown.

Introduction

Nonsense codons (UAA, UAG and UGA) cause premature termination of the polypcptide chain specified by the cistron in which they occur. Working with amber mutants in the head protein of phage T4, Sarabhai et al. (1964) first demon- strated a colinearity between the length of the polypeptide-fragment produced and the site of the nonsense mutation. Similar results have been obtained with nonsense mutants in the alkaline phosphatase gene (Susuki and Garen, 1969) and in the fl-galactosidase gene of Escherichia coli (Morrison and Zipser, 1970).

In a system like RNA-bacteriophages, which does not permit analysis by recombination, characterisation of the nonsense-fragments provides in principle an alternative method of genetic mapping. With this aim, we have now analysed the polypeptide fragments synthesized by several amber mutants in the A protein of the RNA bacteriophage MS2. Somewhat unexpectedly we found no gradient in the size of the fragments produced, but rather two distinct classes of mutants, only one of which synthesized a detectable amount of A protein-fragment.

Materials and Methods

a) Chemicals and Media

L-[aH] leucine (1 C/mmole), L-[aH] histidine (0.5 C/mmole), L-14C] leucine (62 mC/mmole) ~nd L-[14C] histidine (57.8 mC/mmole), were all obtained from the Radioehemical Center, Amersham, Buckinghamshire, England. Rifampiein was a gift from Ciba, Basle, Switzerland and from Lepetit, Milan, Italy.

The composition of MS medium, Tris-yeast medium and Tris-amino acids medium has been described (Remaut and Fiefs, 1972). TPG was as described by Sinsheimer et al. (1962) with the addition of 20 [zg/ml of each of 20 L-amino acids.

b) Host Bacteria Strain Fll, an actinomycin-sensitive derivative of Q13 (Nozawa and Mizuno, 1968),

was obtained from Dr. Nozawa and was used as Su- host in the incorporation experiments with the mutants. CR63 and C3000 were used as Su + and Su- indicator strains for the amber mutants of the phage. All strains are derivatives of Esch~richia coli K12.

15 Molec. gen. Oenet. 117

220 E. Vandamme, E. Remaut, M. van Montagu, and W. Fiers:

c) Bacteriophage Mutants The MS2 mutants were isolated after mutagenesis of a purified phage suspension

(1014 p.f.u./ml) with nitrous acid or hydi'oxylamine. The nitrous acid mutagenesis was carried out at 37 ° C in a solution, containing 0.6 M-

sodium nitrite and 1 1V[-sodium acetate buffer, pH 4.6. The phage inactivation was approximately 3 log/hr. Samples were diluted in 1 M-glycine buffer, pH 8.5.

The hydroxylamine mutagenesis was carried out at 37 ° C in a solution, containing i M- NH~OH, 2 M-NaC1, 0.5 M-sodium phosphate and 0.05 M-~dgS04, adjusted to pH 7.5. The phage inactivation was approximately 6 log/hr. Samples were diluted in Tris-EDTA buffer, pH 7.5, containing 2% acetone. These samples were either used immediately for pouring masterplates or distributed in smaller portions and kept frozen.

For the isolation of amber mutants, the samples were plated on an Su-1 + strain. Individual plaques were transferred with a tooth-pick on Su-l+ and Su- plates. The amber mutants screened in that way were classified in three complementation groups (M. van Montagu, unpublished). All assembly (A protein) mutants have an e.o.p, of 1 on most of the weak amber and ochre suppressors relative to a strong Su-1 + strain. Coat protein or polymerase amber mutants on the other hand grow poorly if at all in weak suppressor strains.

The mutants used in this study are listed in Table 1.

d) Growth o/Mutant Phage Stocks An overnight culture of CR63 in MS medium was diluted 20-fold into 10 ml of fresh

MS medium and incubated at 37°C with vigorous a~ration. When the cells had reached a density of 1.10S/ml 2 m]VI-CaCle was added and the culture was infected with a single mutant plaque. 90 rain after infection 100 ~zg egg-white lysozyme/ml and 2.10 -3 M-EDTA were added. After standing at 4 ° C for at least 1 hr the cells were lysed by freezing and thawing twice in CO2-methanol. The mutant and the revertant titer were then determined on CR63 and C3000 respectively. All stocks used contained less than 1% revcrtants. The average mutant titer was 1.1011 p.f.u./ml.

e) Preparation o/Radioactive Proteins ]rom In/eeted Cells The conditions used for rifampiein-treatment and infection are described in detail else-

where (Remaut and Fiers, 1972). Schematically the procedure was the following: an expo- nentially growing F l l culture in Tris-yeast medium was infected with 10 mutant phage particles per bacterium and 4 min later 100 ~g rifampicin/ml was added. 5 rain later the cells were filtered off over Millipore and resuspended in Tris-amino acids medium containing 100 ~g rifampicin/ml. The cultures were then allowed to incorporate a radioactive amino acid from 20 to 60 min after infection. After lysis of the cells the labeled proteins were extracted with phenol and prepared for polyacrylamide gel electrophoresis as described pre- viously (Remaut and Fiers, 1972).

]) Polyacrylamide Gel Electrophoresis The labeled viral proteins were analysed on 10% polyacrylamide gels containing 0.1%

sodium dodecyl sulphate and 0.1 M-sodium phosphate buffer, pi t 7.2, according to Weber and Osborn (1969). The electrophoresis, the fractionation and the counting of the gels have been described (Remaut and Fiers, 1972).

Results

In order to de tec t v i rus- induced prote ins in the infected cells i t is necessary

to suppress host p ro te in synthesis e.g. wi th r ifampiein, an ant ib io t ic which

specifically inhibi ts the DNA-dependen t R N A polymerase ( t t a r t m a n n et al., 1967). Because of i ts increased r i fampic in-sens i t iv i ty s t ra in F l l was used as an Su- host in the exper iments wi th amber mutants . P re l iminary exper iments had shown

Nonsense Fragments of Amber Mutants of Bacteriophage MS2 221

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Fig. 1 a--e. Polyaerylamide gel eleetrophoresis of [14C] leueine-labeled proteins synthesized in a rifampiein-inhibited F l l culture infected with mutant am 906 (a), am 907 (b) or wild- type MS2 (e). The gels were run for 6 hr at 7 mA/tube. Eleegrophoresis was from the left (cathode) to the righlb (anode) in this and subsequent :figures. ° °, infected cells; o---% uninfected control. The radioaetivi V under the fastest moving peak is redrawn with a 4-fold scale reduction (°--°). This corresponds ~o the ordinal)e on the right hand side. The capital letters refer to the virus-induced proteins: repliease (R), A protein (A), A protein-fragment (A1)

and coat protein (C)

t h a t the infect ivi ty and drug-sensi t ivi ty of this s train were opt imal in a yeas t extract-enriched medium. Therefore the cells were infected and t rea ted with rifampicin in Tris-yeast med ium and subsequent ly t ransferred to a synthet ic medium for labeling of the virus-induced proteins. Each exper iment with a set of mu tan t s included an uninfected and a wild-type infected control. Under the conditions used the phage yield in the wild-type infected culture varied f rom 50 to 200 particles per bacterium.

Fig. 1 shows the results of a typical exper iment with mutan t s am 906 and am 907. I n the wild-type infected culture (Fig. 1 e) three viral proteins were synthesized which have been identified as: the viral replicase (R), the A protein (A) and the coat protein (C) (Nathans et al., 1966; Vifiuela et al., 1967; and our unpubl ished results). Mutants am 906 and am 907 (Fig. l a and b) both failed to synthesize in tac t A protein. Following hlfection with m u t a n t am 907, however, a new protein was formed (A 1 in Fig. 1 b) which had a slightly higher eleetrophoretic mobil i ty t han nat ive A protein. I n all probabi l i ty this peak represents the nonsense polypept ide- f ragment of the A protein synthesized by m u t a n t am 907. This polypept ide contained histidine and therefore was no t an

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Fraction number Fig. 2a- -c . Coelectrophoresis of [aH] leucine-labeled proteins of wild-type MS2 with proteins of mutant am 302. The gels were run for 20 hr at 5 mA/tube. The coat protein has run off the gels. • ., infected cells; o - -% uninfected controls, a, a m 302-induced proteins; b, coelectrophoresis of a m 302- and wild-type-induced proteins; c, wild-type-induced proteins

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ReLative mobi[ity Fig. 3. Molecular weight estimate for the A protein-fragment by electrophoresis in polyaerylamide gels in the presence of sodium dodecyl sulphate. The protein bands were stained with Coomassie brilliant blue as described by Chrambach et a l . (1967). The distances migrated by the known marker proteins relative to that of eytochrome e are plotted against thelogarithm of their molecular weights and a straight line is fit ted to the points. The marker proteins are: bovine serum albumin (1), ovalbumin (2), alcoholdehydrogenase from liver, monomer (3), aleoholdehydrogenase from yeast, monomer (d), pepsin (5), ehymotrypsinogen (6) and eytochrome e (7). The molecular weight of the marker proteins are taken from Weber and Osborn (1969) and Dunker and Rueekert (1969). The molecular weights of the A protein (A) and the coat protein (G) were determined by coelectrophoresis with these marker proteins, that of the A protein-fragment (At) was inferred from its position relative to the A protein and

the coat protein


Table 1. Summary of the properties of A protein mutants

Mutant Mutagen Duration of Synthesis- number mutagenesis level

(rain) of fragment

100 302 303 304A 306 307 309 604 606 615 616 620 900 902 906 907 911 913 920

nitrous acid hydroxylamine hydroxylamine hydroxylamine hydroxylamine hydroxylamine hydroxylamine hydroxylamine hydroxylamine hydroxylamine hydroxylamine hydroxylalnine nitrous acid nitrous acid nitrous acid nitrous acid nitrous acid nitrous acid nitrous acid

60 1.6 a 30 0.9 30 n.s.b 30 n.s. 30 0.7 30 n.s. 30 n.s. 60 n.s. 60 0.6 60 n.s. 60 n.s. 60 n.s. 180 n.s. 180 1.0 1 8 0 n . s .

180 1.0 180 n.s. 180 n.s. 180 1.3

a The level of synthesis of the A protein-fragment is expressed as the ratio of the molar amount of fragment synthesized in mutant-infected cells to the molar amount of wild-type A protein synthesized in MS2-infected cells. The radioactivity incorporated was normalized with respect to the synthesis of coat protein. I t is assumed that the level of coat protein synthesis is not affected by the mutation and can therefore be taken as a measure for the overall protein synthesis-capacity of the infected cells. b n.s. means: no fragment found.

oligomer of the coat protein, which lacks this amino acid (L ine t al. , 1967). B y coelectrophoresis, during a longer time, with wild-type A protein (Fig. 2) it was shown to be clearly dist inct f rom the latter. Comparing its electrophoretic mobil i ty on sodium dodecyl sulphate containing gels with the mobil i ty of known marker proteins (Fig. 3), we calculated a molecular weight of about 36000 daltons. Using the same method we found for the complete A protein a value of 42000 daltons.

Table 1 summarizes the results obtained in analysing the viral proteins synthesized in rifampicin-inhibited F l l cultures infected with 19 independent ly isolated A protein mutants . All of the mu tan t s fell in either of two classes:

1. 12 mu tan t s showed a pa t t e rn of protein synthesis similar to t ha t observed with m u t a n t a m 906. There was no detectable synthesis of an A protein-fragment. A polypept ide-f ragment with a molecular weight of about 14000 would coelectro- phorese with the coat protein and thus escape detection. However , when the m u t a n t proteins were labeled with histidine (absent f rom the coat protein), we still found no radioact iv i ty in this region of the gels. Pre l iminary experiments, in which the infected cultures were pulse-labeled for 4 min and the viral proteins immedia te ly ex t rac ted with phenol, showed essentially the same pa t t e rn of protein synthesis as presented in Fig. l a . This result indicates t ha t the absence of flag-

224 E. Vandamme, E. Remaut, M. van ~¢Iontagu, and W. Fiers:

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Fig. 4a--d. Coelectrophoresis of [3H] leucine-labeled proteins of mutant a m 302 with [14C] leueine-labeled proteins of mutants a m 306 (a), a m 100 (b), a m 907 (e) or a m 606 (d). The gels were run for 22 hr at 5 mA/tube. Fractions 1 to 17 are not shown in the graphs. There was no radioactivity above the background level in this region of the gel. The coat protein has run off. o--o, [3H] radioactivity, • °, [14C] radioactivity. The arrows point to the position of wild-type A protein. (The peak seen around fraction 35 is due to residual bacterial

protein synthesis. I t was also present in uninfected controls, not shown on the graphs)

merit is p robably no t due to proteoly~ic cleavage of the nonsense polypept ide and subsequent loss during the isolation procedure.

2. The 7 remaining mutan t s synthesized an A prote in-f ragment with a mobil i ty equal to t ha t of the f ragment formed by m u t a n t am 907. [SH] leucine-labeled f ragment f rom m u t a n t a m 302 coeleetrophoresed in long runs with [14C] leucine- labeled f ragments f rom 4 other mu tan t s (Fig. 4). We have fur ther compared the molar amoun t of f ragment synthesized by the various mu tan t s (see Table 1). Relat ive to the amoun t of coat protein formed the synthesis of the A protein- f ragment was within the limits of experimental error equal for all mu tan t s and was the same as the molar synthesis of wild-type A protein.

Finally, we have determined whether the A prote in-f ragment is incorpora ted into the defective particles which are formed when a m b e r mutan t s in the A pro-


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Fig. 5a--c. Polyacrylamide gel electrophoresis of [all] leucinc-labeled proteins present in purified defective particles. _An F11 culture in TPG +amino acids was infected with mutants am 302 or am 606 and incubated in the presence of 20 ~C [~H] leucine/ml. The cells were allowed to lyse and the defective particles were purified essentially according to Fiers et al. (1965). The proteins present in the particles were extracted with phenol and electrophoresed on polyacrylamide gels for 6 hr at 7 mA/tube, a, defective particles of am 302, b, defective particles of am 606, e, wild-type virus particles. The radioactivity under the coat protein-

peak was redrawn with a 10-fold scale reduction ( . . . . )

rein infect an Su- strain (Lodish et al., 1965; geisenberg , 1966; Argetsinger and Gussin, i966). We have analysed the proteins present in purified defective par- t ides formed after infection with m u t a n t a m 302 or a m 606. F r o m the results shown in Fig. 5 it is clear t h a t the A protein-fragment synthesized i n v ivo by these mutan t s is no t incorporated to a significant extent into defective particles.

Discussion

We have analysed the virus-induced proteins synthesized in rifampiein- t rea ted Su- cultures following infection with MS2 a m b e r mutan t s in the A protein cistron. Wi th respect to the synthesis of an A protein fragment , 19 independent ly isolated mu tan t s could be classified in two groups: 1. 7 out of 19 synthesized a polypept ide-f ragment with a molecular weight of about 36000 daltons, corresponding in chain-length to 88% of the wild-type A protein. The fragments produced by several of these mutan t s coelectrophoresed on polyacrylamidc gels, indicating tha t the muta t ion is either at the same or at very closely linked sites


in the cistron. The molar amount of fragment formed in mutant-infected cells was roughly equal to the molar amount of native A protein in wild-type-infected cells. 2. The remaining 12 mutants showed no detectable synthesis of a nonsense- fragment of the A protein. I t has been shown that nonsense-fragments of the fl-galactosidase (Goldsehmidt, 1970) and of the lac repressor (P la t t e t al., 1970) may be subject to proteolytie breakdown in the cell. Such a mechanism could evidently cause the loss of unstable A protein-fragments. Experiments in which the viral proteins were pulse-labeled for 4 rain and immediately extracted with phenol have indicated, however, tha t proteolytic cleavage of the type mentioned above is not likely to be responsible for the absence of fragment in these mutants. Indeed, the extraction procedure used here is known to recover extremely unstable tRNA precursor molecules with a half life of less than 3 rain (Altman, 1971). The half lives reported for the fl-galactosidase-fragmcnt and the lay repressor- fragment are 7.4 and 20 mln respectively. In order to explain the absence of fragment in our mutants on the basis of proteolytic degradation one would have to invoke a type of proteolysis exceedingly faster than the one responsible for the breakdown of the bacterial (mutant) proteins.

As may be seen from Table 1 the occurrence of mutants in the two groups was not biased neither by the choice of the mutagen (hydroxylamine or nitrous acid) nor by the time of mutagenesis. A protein mutants which produce a polypeptide-fragment with properties similar to tha t found in mutant am 907 have been reported for MS2 (Vifiuela et al., 1968; Nathans et al., 1969) and for the related phage f2 (Lodish and Robertson, 1969).

Two hypotheses can be considered to explain our results. I t may be that only a few sites in the A protein cistron can effectively be mutagenized to an amber eodon. One of these would then be rather distal in the cistron (leading to the production of the 36000 daltons fragment) whereas the other(s) would be so proximal to the initiation site that the resulting polypeptide-fragment is too small to be detected in the gels. There are several albeit weak arguments against the occurrence of only two putative "ho t spots". Considering the mutagenie action of the mutagens used, the amber codon is most probably derived from the triplet CAG (coding for glutamine) by a C to U transition. Analysis of the amino acid composition of the A protein of phage MS2 (van de Kerckhove and van Montagu, personal communication) and of the related phage R17 (Steitz, 1968) reveals approximately 33 glutamic acid residues. I t seems reasonable to assume that at least half of these are present in the polypeptide chain as glutamine, so that many potentially amber-generating sites are expected. I t may be recalled tha t in the coat protein 6 glutamines are present and that 4 of the corresponding eodons can mutate to UAG. Moreover, the high frequency with which amber mutants in the A protein were found (Fiers et al., 1969) would favor the existence of many mutation sites rather than a few "ho t spots".

In an alternative model to explain the absence of fragment we would postulate tha t the level of synthesis of most fragments is too low to be detectable on the gels. This situation would then be analogous to tha t observed with amber mutants in the coat protein cistron, where no coat protein-fragment was found in mutant- infected Su- cells (Sugiyama et al., 1969). Similarly, with some mutants in the fi-galaetosidase gene the amount of fragment formed was as low as 1% of the


wi ld - type level (Morrison and Zipser, 1970). I n f avor of th is hypo thes i s is the f inding t h a t a U G A - m u t a n t , which m u s t have ar i sen b y ano the r mutagen ic even t t h a n the amber mutan t s , equa l ly fai ls to synthes ize an A p ro t e in - f r agmen t ( Remau t a n d Fiers , 1972). The n a t u r e of such a hypo the t i c a l mechan i sm responsible for avo id ing the synthes is of useless nonsense- f ragments , is a t p resen t no t clear.

F u r t h e r expe r imen t s will be needed to decide be tween these possibil i t ies.

Acknowledgements. The research was supported by grants fl'om the Fonds voor Funda- menteel Wetenschappelijk Onderzoek. E.R. obtained a fellowship from the Nationaal Fonds voor Wetenschappelijk Onderzoek. We thank the firms Ciba (Basel) and Lepetit (Milan) for gifts of rifampicin. Dr. K. Nozawa kindly send us the drug-sensitive strain F l l .

References

Altman, S.: Isolation of tyrosine tRNA precursor molecules. Nature (Lond.) New Biol. 229, 19-21 (1971).

Argetsinger, J. E., Gussin, G. N. : Intact ribonueleic acid from defective particles of bacteriophage R17. J. molec. Biol. 21, 421-434 (1966).

Chrambach, A., Reisfeld, R. A., Wyekoff, M., Zaecari, J. : A procedure for rapid and sensitive staining of protein fractionated by polyacrylamide gel electrophoresis. Analyt. Biochem. 20, 150-154 (1967).

Dunker, A. K., Rueckert, R. R. : Observations on molecular weight determinations on polyacrylamide gel. J. biol. Chem. 244, 5074-5080 (1969).

Fiers, W., Lepoutre, L., Vandendriessche, L. : Studies on the bacteriophage MS2. I. Distribu- tion of purine sequences in the viral RNA and in yeast RNA. J. molec. Biol. 18, 432-450 (1965).

Fiers, W., Montagu, M. van, De Wachter, R., Haegeman, G., Win Jou, W., Messens, E., Remaut, E., Vandenberghe, A., Styvendaele, B. van: Studies on the primary structure and the replication mechanism of bacteriophage RNA. Cold Spr. Harb. Symp. quant. Biol. 34, 697-705 (1969).

Goldschmidt, R. : In vivo degradation of nonsense fragments in E. coll. Nature (Lond.) 228, 1151-1154 (1970).

Hartmann, G., IIonikel, K. 0., Kniisel, F., Nuesch, J. : The specific inhibition of the DNA- directed RNA synthesis by rifampicin. Biochim. biophys. Acta (Amst.) 145, 843-844 (1967).

Heisenberg, M.: Formation of defective bacteriophage particles by fr amber mutants. J. molec. Biol. 17, 136-144 (1966)o

Lin, J., Tsung, C. M., Fraenkel-Conrat, H. : The coat protein of the RNA bacteriophage MS2. J. molec. Biol. 24, 1-14 (1967).

Lodish, tI. F., Horiuchi, K., Zinder, N. D.: Mutants of the bacteriophage f2. V. On the production of noninfectious phage particles. Virology 27, 139-155 (1965).

Lodish, H. F., Robertson, H. D. : Cell-free synthesis of bacteriophage f2 maturation protein. J. molec. Biol. 4,9, 9-22 (1969).

Morrison, S. L., Zipser, D.: Folypeptide products of nonsense mutations. I. Termination fragments from nonsense mutations in the z gene of the lac operon of Eseherichia coll. J. molec. Biol. 50, 359-371 (1970).

Nathans, D., Oeschger, M. P., Eggen, K., Shimura, Y.: Bacteriophage-specific proteins in E. coli infected with an RNA bacteriophage. Proc. nat. Acad. Sci. (Wash.) .56, 1844-1851 (1966).

Nathans, D., Oeschger, M. P., Polmar, S. K., Eggen, K.: Regulation of protein synthesis directed by coliphage MS2 RNA. I. Phage protein and RNA syathesis in cells infected with suppressible mutants. J. molec. Biol. 39, 279-292 (1969).

Nozawa, R., Mizuno, D.: Isolation and properties of an actinomycin sensitive mutant of Escherichia coli. J. Biochem. 63, 795-797 (1968).

Platt, T., Hiller, J. I-I., Weber, K.: In vivo degradation of mutant lac repressor. Nature (Lond.) 228, 1154-1156 (1970).

228 E. Vandamme et al. : Nonsense Fragments of Amber Mutants of Bacteriophage MS2

Remaut, E., Fiers, W.: Studies on the bacteriophage MS2. XVI. The termination signal of the A protein cistron. Submitted for publication.

Sarabhai, A. S., Stretton, A. O. W., Brenner, S., Bolle, A.: Co-linearity of the gene with the polypeptide chain. Nature (Lond.) 201, 13-17 (1964).

Sinsheimer, R. L., Starman, B., Nagler, C., Guthrie, S.: The process of infection with bacteriophage qiX174. I. Evidence for a "replicative form". J. molec. Biol. 4, 142-160 (1962).

Steitz, J. A. : Isolation of the A protein from bacteriophage R17. J. molec. Biol. 83, 937-945 (1968).

Sugiyama, T., Stone, H. 0., Jr., Nakada, D. : Protein synthesis directed by an amber coat- protein mutant of the RNA phage MS2. J. molec. Biol. 42, 97-117 (1969).

Susuki, T., Garen, A. : Fragments of alkaline phosphatase from nonsense mutants. I. Isolation and characterization of fragments from amber and ochre mutants. J. molec. Biol. 45, 549-566 (1969).

Vifiuela, E., Algranati, I. D., Feix, G., Garwes, D., Weissmann, C., Ochoa, S. : Virus-specific proteins in Escheriehia coli infected with some amber mutants of phage MS2. Biochim. biophys. Aeta (Amst.) 155, 558-565 (1968).

Vifiuela, E., Algranati, I. D., Ochoa, S. : Synthesis of virus-specific proteins in Eseheriehia coli infected with the RNA bacteriophage MS2. Europ. J. Biochem. 1, 3-11 (1967).

Weber, K., Osborn, M. : The reliability of molecular weight determinations by dodecyl sulphato- polyacrylamide gel electrophoresis. J. biol. Chem. 244, 4406-4412 (1969).

Communica t ed b y P. S ta r l inger

E. Vandamme E. Remaut M. van Montagu W. Fiers Laboratory of Molecular Biology Ledeganckstraat, 35 Ghent 9000 Belgium

studies on the bacteriophage ms 2

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