Vol. 138, No. 1

JOURNAL OF BACTERIOLOGY, Apr. 1979, p. 122-125 0021-9193/79/04-0122/04$02.00/0

Suppression of Polar Effects of Nonsense Mutations by Ultraviolet Irradiation THOMAS J. POLLOCK,t ETHEL S. TESSMAN, AND IRWIN TESSMAN* Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907 Received for publication 28 November 1978

Nonsense mutations in capsid genes F and G of phages S13 and 4X174 decreased the expression of genes downstream. These polar effects were suppressed by ultraviolet irradiation of the host before infection. Activities of the downstream genes were restored to between 30 and 95% of their normal levels, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the viral proteins. The effect of ultraviolet irradiation may be to suppress rhodependent termination of transcription. The polarity of one mutation was poorly suppressed by ultraviolet irradiation.

Polar mutations not only eliminate the expression of the mutated gene itself but, by definition, also reduce expression of genes in the distal part of the operon (14). Studies of the trp operon (1113) have shown that polar mutations result in marked shortening of trp mRNA. Shortening of 4OX174 mRNA has also been observed (9). Shortened mRNA molecules produced by polar mutations in the trp and gal operons are due primarily to termination of transcription rather than to degradation of mRNA (trp in vivo [10, 11]; gal in vitro [6]). Escherichia coli mutants that suppress the polarity of nonsense and frameshift mutations (4, 15, 19) possess an altered rho factor (17, 18). The discovery that rho mutations suppress polarity provides an important clue to the understanding of transcription termination (reviewed in reference 1). We report an analogous phenomenon: suppression of polarity by UV irradiation. The polar mutations studied here were in the capsid genes of the small DNA phages S13 and 4)X174, and suppression was achieved by irradiating the rho' host cell before infection. MATERIALS AND METHODS Preparation of labeled extracts of phage-infected cells, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fluorography, and densitometry have been described previously (16). The only significant difference was in the gel composition, which was, in the order of solution: 18.5% (wt/vol) acrylamide, 0.15% (wt/vol) N,N'-methylenebisacrylamide, 0.11% (vol/ vol) N,N,N',N'-tetramethylethylenediamine, 0.11% (vol/vol) 2-mercaptoethanol, 28% (vol/vol) denaturing buffer (0.4 M Tris-acetate [pH 9.1], 4 M urea, 0.8% sodium dodecyl sulfate), and 0.19% (wt/vol) ammonium persulfate. t Present address: Laboratory of Neurochemistry, National Institutes of Health, Bethesda, MD 20014.

122

Phage mutants were from the collections of M. Hayashi and M. N. Hayashi (4XamF57), R. L. Sinsheimer (OXamG9), E. S. Tessman (Sl3amF28, S13amG83), and I. Tessman (S13amE15,

4XamF1005). E. coli AP1 is a UV-sensitive (Hcr-)

derivative of E. coli C isolated by A. Puga and was the host for all tests of phage polarity.

RESULTS Three adjacent capsid genes, F, G, and H, of S13 and 4X174 are involved in polar effects. The genes are arranged in the order F-G-H (3) and are transcribed and translated from left to right (20, 22). Strongly polar mutations have been isolated in genes F and G (7, 9, 20, 21); polar mutations in G affect H and polar mutations in F affect both G and H. The effect of UV was studied on two polar mutants of S13, the gene F mutant amF28 and the gene G mutant amG83, and on two polar mutants of 4X174, the gene F mutant amFI005 and the gene G mutant amG9. Extracts of infected cells were subjected to electrophoresis in a sodium dodecyl sulfate-polyacrylamide gel. A fluorogram (Fig. 1) shows the phage-coded proteins. The proteins in the amG83 extract from unirradiated cells revealed a polar effect on the expression of gene H. Simple inspection showed that the H band was restored by irradiation of the cells. The nonpolar amE15 mutant provided a standard for comparison. In OX, one can see the polar effect of amF1005 on the expression of genes G and H and the polar effect of amG9 on the expression of gene H. In both cases, UV suppressed the polar effect. The nonpolar amF57 mutant was the standard. For a quantitative analysis of the gels shown in Fig. 1, the relative amount of radioactive label in each protein band was determined by scan-

OcoLO

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1 S

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that appreciable H protein was then made.

OX 174

S13 No UV

UV rf OD u) aD LL LLJ

UV

UV INo No

LUV

0

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0 ° cn co

° IL

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F'--- H-H

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-D-

To

obtain

an

estimate

of

the

amount

of

H

protein made by the polar G mutant in comparison with its normal production, the amG83 peak heights were compared with the corresponding ones for the amE15 extracts. The amG83/ amE15 ratios for all of the bands would be the same if the proteins were made in the same proportions in the amG infection as in the control. For D and F, the ratios were indeed api==proximately equal, with and without UV, firming the expectation that the amG mutation would not affect those products. UV restored the H ratio to about 0.8 ± 20% of the ratios observed for the D and F bands. Thus, UV suppressed about 65 to 95% of the polar effect of the amG mutation. For the 4X174 analysis (Table 2), the mutant 4XamF57 provided the standard proportions of the phage products. This is a nonpolar amber mutation at the 3' end of the F gene and makes a large amount of F fragment (F') that is only slightly shorter than the original F protein. A polar F mutant, amF1 005, inhibited expression of two genes, G and H, almost completely con-

-B- 1.

123

UV SUPPRESSION OF POLARITY

VOL. 138, 1979

U_

** *

^

&@

the absence of UV. Here too the

polar

effect

was suppressed by UV so that expression of both G and H was largely restored. The G mutant,

FIG. 1. Fluorogram showing UV suppression of polarity in nonsense mutants of phages S13 and amG9, behaved qualitatively like the S13 amber G mutant. For the two polar 4X mutants, UV 4X1 74. E. coli API, an Hcr- d'erivative of strain C, was grown at 37°C to 2 x 10' cells per ml in M9 restored expression of the distal genes to about inorganic salts (2) supplemented with 0.4% (wt/vol) TABLE 1. UV suppression ofpolarity of an S13 glucose and 10-5 M FeCl3. Part of the culture was amber G mutant irradiated with UV (150 J/m2). Irradiated and unirradiated cells were made 10-2 M in MgSO4 and Optical densities of infected with phage at a multiplicity of 10 plaqueelectropherogram forming units per cell. Irradiated cells were labeled bands Phage gene product with 25 ,iCi of [3H]lysine (60 mCi/mol, 500 ,iCi/ml) +UV -UV from 15 to 45 min after infection; unirradiated cells were labeled with 10 ACi from 23 to 26 min after S13amE15 infection. Extracts were subjectd to electrophoresis in 12.5 9.3 D a sodium dodecyl sulfate-polyacrylamide gel. The 12.9 15.4 F polar G mutants eliminated the H proteins, and the 2.6 2.9 G H G and the proteins. F mutant eliminated polar 8.4 10.8 H S13amG83 ning the electropherogram with a Joyce-Loebl 19.0 9.4 D Mark III microdensitometer. The peak heights 20.0 20.0 F for the products of genes D, F, G, and H are c0.2 G c0.2 shown in Table 1 for S13 and in Table 2 for 9.7 0.7 H 4X174 extracts. The gene B product was not used in the analysis because its amount was low amG83/amE15 ratio and variable in amber F extracts. 1.5 1.0 D In Table 1 the first block of data is for 1.5 1.3 F S13amE15, which served as a control, providing G s0.08 c0.07 the standard proportions of each of the phage 1.2 0.06 H products with and without preirradiation of the Fraction of H cells with UV. The second block of data shows protein restored by that the amber G mutant was strongly polar in UV the absence of UV, as indicated by the lack of 0.8 0.06 H/D of Preirradiation H protein peak. any detectable 0.8 0.05 H/F the cells, however, suppressed the polar effect so

124

J. BACTERIOL.

POLLOCK, TESSMAN, AND TESSMAN

TABLE 2. UV suppression ofpolarity of 0.X1 74 amber F and G mutants Phage gene product

D F' G H D F G H D F G H

D F G H

ity was not observed (s20%). A longer film exposure revealed that G and H were present at about 10% of their standard amounts (data not Optical densities of electropherogram shown). bands A dose-response curve (Fig. 2) shows that suppression of polarity increased with UV dose. +UV -UV Polarity was measured in 4XamF1005; the conoXamF57 trol was provided by 4XamF57. The large errors 5.9 10.0 undoubtedly obscure the fact that the curve 17.0 8.8 must begin to level off by a dose of 150 J/m2, 3.2 1.7 judging from Tables 1 and 2. 5.5 11.3 The tables show another effect of UV irradia0XamF1005 tion. Viral protein synthesis is often easier to 9.2 17.3 study if the cells are preirradiated with UV to SO.2 -

Suppression of polar effects of nonsense mutations by ultraviolet irradiation.

Vol. 138, No. 1 JOURNAL OF BACTERIOLOGY, Apr. 1979, p. 122-125 0021-9193/79/04-0122/04$02.00/0 Suppression of Polar Effects of Nonsense Mutations by...
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