Current Genetics 2,103 107 (1980)
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© by Springer-Verlag 1980
Measure of Asymmetrical Transcription of the Yeast OMP Decarboxylase Gene Expressed in Yeast or in E. coli J. C. Hubert, M. L. Bach and F. Lacroute Laboratoire de G6n6tique Physiologique, I.B.M.C. du C.N.R.S., 15 rue R. Descartes, F-67084 Strasbourg Cedex, France
Summary. The yeast OMP decarboxylase gene has been cloned in the phage fd in both orientations to obtain pure DNA of each strand. With these probes the transcription of each strand of the yeast gene was measured in wild type yeast, either repressed or induced, in a yeast strain transformed by an hybrid plasmid containing the OMPdecase gene, and in E. coli strains bearing the yeast gene cloned in different plasmids. In yeast it appears that in all conditions tested transcription was asymmetrical, 95% being transcribed from one strand. In E. eoli, transcription of the yeast gene occurred on both strands. Key words: Single strand DNA - Transcription - fd cloning - Gene regulation.
Introduction Highly asymmetrical transcription in vivo is a general rule for higher eukaryotic genes, stable RNA products being transcribed from one of the two strands (Martin et al., 1967). It appeared of interest, therefore, to see if this asymmetrical transcription would be true for the yeast OMP decarboxylase gene either in its normal chromosomal location or in different plasmids situated in yeast, its normal host, or in E. coli. Moreover it is important to establish the effect of the transcriptional regulation of this gene (Bach et al., 1979) on the degree of asymmetry. For this purpose we have cloned in both orientations a 1.1 kb HindlII DNA fragment bearing the ura3 gene in a bacteriophage fd vector constructed by Herrmann et al. (Herrmann et al., 1980). This gave us DNA probes specific for each ura3 gene strand. This paper describes the cloning of the ura3 gene and results obtained concerning the symmetry of its transcription.
Materials and Methods Yeast Strains. Yeast strains were all derived from a wild type strain FL 100 (a) ATCC 28383. ural (21 50) is an isogenic strain derepressed for dihydroorotic dehydrogenase, ura3- pFL 2 is a strain constructed by Chevallier et al. (unpublished results) from an a ura3- strain carrying two mutations at the ura3 locus (~ ura3-251-373) and transformed by a chimeric plasmid containing the E. coli plasmid pBR 322, the EcoRI-D fragment of the 2#m yeast plasmid and a 1.1 kb HindlII fragment bearing the ura3 gene. Bacterial strains. The bacterial strains were derivatives ofE. eoli K12. Strain MB 1000 pyrF:: mu trp LaeZ hsdR: this strain was also described as strain DB 6656 elsewhere (Bach et al., 1979). Clone 1 and clone 2 are the E. coli MB 1000 strain transformed by plasmid pMB 9 (Tet R) bearing DNA fragments specifying OMPdecase (Bach et al., 1979). Clone 6 and clone 8 are the same E. coli MB 1000 strain transformed by the plasmid pBR 322 bearing a HindIII 1.1 kb fragment containing the ura3 gene. The two clones differ in the orientation of the inserted fragment (Botstein D., personal communication). Strain E. coli MB 1000 pyrF F'Lac receptor of the phage fd was obtained from E. coli pyrF by crossing with E. coli F'Fac. Bacteriophage Vectors. Bacteriophage fd wild type and fd 106 (single stranded DNA vector) was a generous gift from R. Herrmann Conditions for Growth, Cell Labeling and Hybridization. Yeast cells were grown in Yeast Nitrogen Base (Difco) medium with or without uracil at 30 °C. E. coli cells were grown in M9 medium containing either excess uracil (40 /~g/ml) or limiting uracil (1 #g/ml) at 37 °C. Mid-exponential phase cells were labeled with 3H-adenine, 20 ~zCi/ml having a specific activity of 1.7 Ci/mmole. To stop labeling after 2 rain for E. eoli cells and after 5 rain for yeast ceils, one volume of cell suspension was pipetted into two volumes of cold ethanol. The cells were centrifuged, washed once with the RNA extraction buffer (acetate buffer containing i mM acetate, 5 mM NaC1 and 0.1 mM magnesium acetate, pH 5.1). RNA was extracted from yeast ceils according to the procedure of Waldron and Lacroute (1975) as modified by Losson and Lacroute (1979). RNA was extracted from E. coli cells by the
0172-8083/80/0002/0103/SO 1.00
104 same method except that E. coli crude extracts were obtained by sonication. Nitrocellulose filters with probe DNA were prepared by filtration as described by Gillespie and Spiegelman (1965). Hybridization conditions were those given by Losson and Lacroute (1979). Specific "hybridization to each strand of the ura3 gene was measured in quadruplicate in the following way: radioactive RNAs were incubated in a small glass vial with one filter loaded with 1 ~g fd ura3 minus strand DNA, one filter loaded with 1 ~tg fd ura3 plus strand DNA, and one filter loaded with 1 #g fd 106 DNA. The plus strand was named arbitrarily as the strand which hybridized the most RNA. To evaluate hybridization to the ura3-minus or-plus strand, cpm retained on an fd DNA filter were substracted from cpm on an fd ura3 DNA filter. The amount of hybridization was expressed as a fraction of the input.
Growth of Bacteriophage and DNA Extraction. Bacteriophages were grown on E. eoli strain MB 1000 F'Lae in the condidtions described by Herrmann et al. (in press). The phages were isolated from the culture supernatant by the technique of Yamamoto et al. (1970). DNA was extracted from the phages by hot phenol treatment followed by ethanol precipitation.
Determination of OMP Decarboxylase Activity. The assay of Wolcott and Ross (1966) was used and enzyme activity was determined relative to protein concentration (Lowry et al., 1951).
J.C. Hubert et al.: Asymmetrical Transcription in Yeast infected by phage fd 106 carrying two resistance markers ( c h l o r a m p h e n i c o l and k a n a m y c i n ) at a m.i. o f 0.05 as described by H e r r m a n n et al. (1980) in n u t r i e n t m e d i u m containing c h l o r a m p h e n i c o l ( 2 5 / J g / m l ) . Phage fd replicatire f o r m D N A was digested in the k a n a m y c i n gene by endonuclease HindIII, treated with alkaline phosphatase to p r e v e n t recircularization and ligated by T4 ligase with the ura3 fragment as described in Methods.
Transformation and Harvesting o f Phages with Opposite Direction DNA Inserts E. coli pyrF F'Lac cells were transformed directly with the ligation m i x t u r e according to Seeburg and Schaller (1975). The ura3 D N A fragment has thus been inserted in the phage R F in b o t h orientations giving rise to single stranded D N A phages with each strand o f the ura3 gene. Bactoria which gave resistant colonies on minimal m e d i u m plus c h l o r a m p h e n i c o l and were sensitive to k a n a m y c i n were tested for phage p r o d u c t i o n as described by H e r r m a n n et al. (1980).
DNA Extraction from Agarose Gels. DNA extraction from agarose gels followed by ligation was done as follows. DNA-containing agarose, about 0.2 ml was cut out from the get and put in a conical "Eppendorff' centrifuge tube with 0.15 ml 5 M ammonium acetate and 0.4 ml phenol saturated with T.E. buffer (10 mM Tris HC1 pH 8.0, 1 mM EDTA). The tube was then heated in a water bath with frequent shaking until a single liquid phase was obtained (this is generally between 72 ° and 78 °C). The tube was then cooled in melting ice and centrifuged. The supernatant aqueous phase containing the DNA was separated from the interphase containing the denaturated agarose and the phenol phase containing ethidium bromide. The DNA was then concentrated from approximatively 0.3 ml to 20 ~1 by dialysis with air pressure against T.E. buffer. Phosphatased vector (which can also be added at the first extraction step), and an equal volume of double concentration ligation buffer were added directly to the residual volume of DNA for ligation. To the 40 ~zl was added 2 g l o f 1 0 2 M A T P a n d 0 . 2 u n i t o f T 4 ligase. After one day's incubation in the refrigerator ( 6 - 8 °C) the ligation mixture was used for E. eoli transformation (15 ttl for 0.3 ml competent E. coli cell suspension).
Test for Orientation o f the Integrated ura3 Fragment Analysis to determine the orientation o f ura3 D N A inserted in fd D N A (single stranded) was carried o u t with
Results Cloning o f the ura3 Gene in Phage f d 106 The chimeric plasmid p B R 322 2/~D ura3 was digested by HindIII endonuclease (Biolabs). The ura3 1.1 kb D N A was separated by electrophoresis o f the cleaved D N A on an 0.8% agarose gel.
Vector Preparation Bacteriophage fd 106 replicative f o r m ( R F ) doublestranded D N A was o b t a i n e d f r o m E. coli Hfr 3 3 0 0 cells
Fig. 1. About 5 x 1010phages from each of the two clones to be tested were mixed and adjusted to 1% SDS (for disintegration of phage capsid), 50 mM Tris HC1 pH 8.0, 200 mM NaC1 in a final volume of 20 gl. The sample was incubated 2 min at 100 °C and cooled in an isolated water bath to room temperature over a period of 2 h. In slot 1: fd 106 vector; 2: recombinant phage 1; 3: recombinant phage 2; 4: a mixture of phage 1 and phage 2. Hybridized molecules in slot 4 give rise to several bands. This could be due to a different extent of annealing between two DNA molecules or by the formation of aggregates (Herrmann et al., 1980)
J. C. Hubert et al.: Asymmetrical Transcription in Yeast crude phage preparations following the technique of Herrmann et al. (1980). Phages with identical inserts in opposite orientations form DNA/DNA hybrids which can be detected by agarose gel electrophoresis by their molecular weight. The results of such an experiment are shown in Fig. 1. It is clear that the two phages tested hybridize; therefore each possesses one strand of the ura3 DNA.
Transcription o f the ura3 gene in Yeast Labeled RNA was hybridized to plus and minus strand DNA probes. Table 1 presents the percentage of ura3 mRNA hybridized on each strand of the ura3 gene. The data show that the transcription is very asymmetric. In all cases (wild type, derepressed mutant and nonchromosomally integrated strain) the same strand was transcribed with the greatest efficiency (more than 96%). The ura3 messenger levels accord well with the data found for hybridization with total ura3 DNA by Chevallier et al. (unpublished results) and by Losson et al. (1979). It appears in our experiments that the level of mRNA transcribed on the ura3 plus strand in ura3- pFL 2 strain is about 60 times higher than the level in wild type FL 100 and 15 times higher than in the derepressed mutant ural ( 2 1 - 5 0 ) . These values vary coordinately with the observed enzyme activity measured by Chevallier et al. (unpublished results) and confirm that the ura3 gene is under transcriptional control.
105 Transcription o f the ura3 Gene in E. coli Table 2 presents the hybridization results obtained for the transcription in E. coli of the yeast ura3 gene cloned into different E. coli plasmids. The four plasmids used were pMB 9-1 and pMB 9-2 with, respectiviely, a 5.2 kb and 3.3 kb yeast DNA insert, including the ura3 gene and pBR 3 22-6 and pBR 3 22-8 containing a HindIII 1.1 kb ura3 DNA fragment with opposite orientations. It appears that, contrary to the situation in yeast, transcription takes place on both strands. Nevertheless, the transcription on the plus strand was always significantly higher than on the minus strand. Previous results had shown that the OMPdecase levels found in E. coli strains with pMB 9-1 or pMB 9-2 were much higher when strains were grown on a pyrimidine limiting medium than when grown on a medium fully supplemented by pyrimidine (Bach et al., 1979). One interpretation of this result was that some transcriptional regulation was retained, even in E. coli for the ura3 gene. For this reason the transcription on each ura3 strand was measured in these two different physiological conditions. Table 3 shows no significant difference in the transcriptional regulation inE. eoli in the different physiological conditions. This result is in accordance with preliminary results obtained by G. Loison (in our laboratory), who found from quantitative immunological precipitation of the OMPdecase protein in these same conditions of growth, that the amount of enzyme protein was the
Table 1. Transcription of the ura3 gene in Yeast. Cells were labeled with tritiated adenine for 5 min, RNAs were extracted, and hybridization was carried out as described in Material and Methods. The amount of hybridization is expressed as the fraction of radioactivity specifically retained versus the input. The radioactivity specifically retained was calculated by substracting the counts on the fd DNA filters from the counts on the plus or minus fd ura3 DNA filter. Each value is an average of four independent determinations Yeast strain
DNA fd ura3 minus strand
DNA fd ura3 plus strand
Amount of hybrid- % of total ura3 ization x 10-5 transcription on the minus strand
Amount of hybridization x 10-5
% of total ura3 transcription on the plus strand
Exp. no. 1
Wild type FL 100 ura1-21-50 ura3- pFL 2
0.18 0.52 5.9
3 6 5
2.68 7.94 123
97 94 95
Exp. no. 2
Wild type FL 100 ura1-21.50 ura3- pFL 2
0 0.6 4.12
0 1 4
1.19 6.66 106
100 99 96
Exp. no. 3
Wild type FL 100 ura1-21-50 ura3- pF1 2
0.09 0.08 4.2
3 1 3
2.5 8.1 132
97 99 97
106
J.C. Hubert et al.: Asymmetrical Transcription in Yeast
Table 2. Transcription of the Yeast ura3 Gene in E. coli, Cells were labeled with 3H-adenine for 2 min. The amount of transcription is expressed as in Table 1 DNA fd ura3 minus strand
DNA fd ura3 plus strand
Amount of hybrid- % of total ura3 transization x 10- 5 cription on the minus strand
Amount of hybrid- % of total ura3 transization x 10- 5 cription on the plus strand
1 2 3
69 97 119
17 / 21 / 22
20
343 370 429
83 79 78
80
1 2 3
84 102 120
32 ] 351 40
36
177 187 179
68 65 60
64
1 2 3 4
49 60 67 186
43 t 43 39 37
40.5
66 80 107 312
57 57 61 63
59.5
1 2 3 4
44 67 78 113
31 35 / 31 35
33
96 124 113 208
69 65 69 65
67.25
Strains
MB 1000 clone 6
MB 1000 clone 8
MB 1000 clone 1
MB 1000 clone 2
Table 3. Effect of Growth Conditions on Transcription of the Yeast ura3 Gene in E. coli. Cells were grown as described in Material and Methods, OMPdecase specific activities are expressed as miUimoles of OMP transformed/mg protein. The amount of hybridization is expressed as in Table 1 Strains
Excess uracil Enzymatic activity
E. colipyrF clone 1 E. coli pyrF clone 2
0.10 0.04
Limiting conditions Amount of hybridization x 10-5 plus strand
minus strand
186 113
312 208
same in the two cases. Differences in activity seem therefore to be due to an increase in stability o f the protein during growth on pyrimidine limiting medium; the accumulation o f the substrate (OMP) in this condition could be involved.
Discussion Transcription of the ura3 gene in yeast is clearly very asymmetrical. The question is whether the small amount o f RNA hybridized to the minus strand is significant. If one considers the results obtained with the chromosomally located ura3 gene expressed either at its induced or basal level, RNA hybridized to the minus strand was at the limit of sensitivity o f the technique. This could be seen by the variation in the experimental values. In the case o f the nonchromosomally transformed strain the
Enzymatic activity
3.2 1.1
Amount of hybridization × 10 - 5 plus strand
minus strand
108 78
267 204
amount o f RNA hybridized to the minus strand was clearly significant. Two possibilities could be invoked: either some RNA is indeed transcribed from this strand or the counts came from radioactive ura3 DNA complementary to the minus strand. This last possibility is raised by the fact that the nonchromosomally transformed strain has a large number o f copies (~60) of the ura3 gene so that even during a short pulse o f radioactivity significant labeling of ura3 DNA sequences was obtained. It would be possible to choose between these possibilities if one could obtain an increased sensitivity of the hybridization technique by reduction of background. In the case of the yeast gene cloned in E. coli, transcription occurred on both strands. Similar results were obtained by Chang et al. (1975) with mouse mitochondrial DNA cloned in theE. colipSC 101 plasmid and by Miller et al. (1977) with a Drosophila DNA fragment cloned in the same plasmid. If transcription o f the ura3 DNA fragment in
J. C. Hubert et al.: Asymmetrical Transcription in Yeast
E. coli was completely due to E. coli promotors, one would expect to reverse the direction of transcription when the orientation of the insert is inverted. This is clearly n o t the case since the plus strand was always transcribed more. On the other hand, if the transcription of the ura3 DNA fragment was completely due to internal promotors (either the normal yeast p r o m o t o r or some different yeast sequence recognized as a p r o m o t o r by the E. coli RNA polymerase) the amount of transcription on each strand would n o t be changed at all by the inversion of the ura3 DNA insert. This is also clearly n o t the case. One must therefore conclude that there is participation of both external and internal promotors in the transcription observed. Knowledge o f the size o f the transcripts on each strand by Northern hybridization (Alwine et al., 1977) could clarify the problem. Recently Berg et al. (1979) have shown that anEcoRI fragment of yeast mitochondrial DNA cloned in E. coli was transcribed on both strands in contrast to the asymmetrical transcription found in mitochondria by Rabinowitz (1976). Our results lead to the same conclusion in the case o f yeast nuclear DNA transcribed in E. coli. "Biohazards associated with the experiments described in this publication have been examined previously hy the French National Control Comittee"
Acknowledgements. We aregreatly indebted to Dr. Richard Herrmann for helpful advice and for his generous gift of different fd vectors and grateful to B. Winsor for reading the manuscript. This work was supported in part by grants from the C.N.R.S. (ATP "Microbiologie" No. 3070) and the I.N.S.E.R.M. (Institut National de la Sant6 et de la Recherche M6dicale, contrat No. 77.1.073.1. and ATP I.N.S.E.R.M. No. 79.104).
107
References Alwine, J., Kemp, D. J., Stark, G. R.: Proc. Natl. Acad. Sci. USA 74, 5350-5354 (1977) Bach, M. L., Laeroute, F., Botstein, D.: Proc. Natl. Acad. Sci. USA 76,386-390 (1979) Berg, P. E., Lewin, A., Christianson, T., Rabinowitz, M.: Nucleic Acids Res. 6, 2133-2150 (1979) Chang, A. C. Y., Lansman, R. A., Clayton, D. A., Cohen, S. N.: Cell 6, 231-244 (1975) Gillespie, D., Spiegelman, S.: J. Mol. Biol. 12, 829-842 (1965) Herrmann, R., Neugzbauer, K., Pirkl, E., Zentgraf, H., Schaller, H.: Mol. Gen. Genet. 177, 231-242 (1980) Losson, R., Lacroute, F.: Proc. Nail. Acad. Sci. USA 76, 51345137 (1979) Lowry, O. H., Rosenbrough, N. J., Farr, A. L., Randall, R. J.: J. Biol. Chem. 193,265-275 (1951) Martin, G. S., Tochini-Valentini, G. P.: Genetic transcription and RNA polymerase. In: Regulation of nucleic acid and protein biosynthesis. Koningberger, V. V., Bosch, L. (eds.), pp. 9 1 97. Amsterdam, London, New York Elsevier 1967 Miller, D. L., Gubbins, E. J., Pegg III, E. W., Donelson, J. E.: Biochemistry 16, 1031 1038 (1977) Rabinowitz, M., Jacovcic, S., Martin, N., Hendler, F., Halbreich, A., Lewin, A., Morimoto, R.: In: The genetic function of mitochondrial DNA. Saccone, DL., Kroon, A. M. (eds.), pp. 219-230. Amsterdam: North Holland, Elsevier 1976 Seeburg, P. H., Sehaller, H.: J. Mol. Biol. 92, 261-277 (1975) Waldron, C., Lacroute, F.: J. Bacteriol. 122, 855-865 (1975) Wolcott, J. H., Ross, C.: Biochim. Biophys. Acta 122, 532-534 (1966) Yamamoto, K. R., Alberts, B. M., Benzinger, R., Lawhorne, L., Treiber, G. : Virology 40, 734-744 (1970)
Communicated by F. Kaudewitz Received February 7, 1980
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© by Springer-Verlag 1980
Measure of Asymmetrical Transcription of the Yeast OMP Decarboxylase Gene Expressed in Yeast or in E. coli J. C. Hubert, M. L. Bach and F. Lacroute Laboratoire de G6n6tique Physiologique, I.B.M.C. du C.N.R.S., 15 rue R. Descartes, F-67084 Strasbourg Cedex, France
Summary. The yeast OMP decarboxylase gene has been cloned in the phage fd in both orientations to obtain pure DNA of each strand. With these probes the transcription of each strand of the yeast gene was measured in wild type yeast, either repressed or induced, in a yeast strain transformed by an hybrid plasmid containing the OMPdecase gene, and in E. coli strains bearing the yeast gene cloned in different plasmids. In yeast it appears that in all conditions tested transcription was asymmetrical, 95% being transcribed from one strand. In E. eoli, transcription of the yeast gene occurred on both strands. Key words: Single strand DNA - Transcription - fd cloning - Gene regulation.
Introduction Highly asymmetrical transcription in vivo is a general rule for higher eukaryotic genes, stable RNA products being transcribed from one of the two strands (Martin et al., 1967). It appeared of interest, therefore, to see if this asymmetrical transcription would be true for the yeast OMP decarboxylase gene either in its normal chromosomal location or in different plasmids situated in yeast, its normal host, or in E. coli. Moreover it is important to establish the effect of the transcriptional regulation of this gene (Bach et al., 1979) on the degree of asymmetry. For this purpose we have cloned in both orientations a 1.1 kb HindlII DNA fragment bearing the ura3 gene in a bacteriophage fd vector constructed by Herrmann et al. (Herrmann et al., 1980). This gave us DNA probes specific for each ura3 gene strand. This paper describes the cloning of the ura3 gene and results obtained concerning the symmetry of its transcription.
Materials and Methods Yeast Strains. Yeast strains were all derived from a wild type strain FL 100 (a) ATCC 28383. ural (21 50) is an isogenic strain derepressed for dihydroorotic dehydrogenase, ura3- pFL 2 is a strain constructed by Chevallier et al. (unpublished results) from an a ura3- strain carrying two mutations at the ura3 locus (~ ura3-251-373) and transformed by a chimeric plasmid containing the E. coli plasmid pBR 322, the EcoRI-D fragment of the 2#m yeast plasmid and a 1.1 kb HindlII fragment bearing the ura3 gene. Bacterial strains. The bacterial strains were derivatives ofE. eoli K12. Strain MB 1000 pyrF:: mu trp LaeZ hsdR: this strain was also described as strain DB 6656 elsewhere (Bach et al., 1979). Clone 1 and clone 2 are the E. coli MB 1000 strain transformed by plasmid pMB 9 (Tet R) bearing DNA fragments specifying OMPdecase (Bach et al., 1979). Clone 6 and clone 8 are the same E. coli MB 1000 strain transformed by the plasmid pBR 322 bearing a HindIII 1.1 kb fragment containing the ura3 gene. The two clones differ in the orientation of the inserted fragment (Botstein D., personal communication). Strain E. coli MB 1000 pyrF F'Lac receptor of the phage fd was obtained from E. coli pyrF by crossing with E. coli F'Fac. Bacteriophage Vectors. Bacteriophage fd wild type and fd 106 (single stranded DNA vector) was a generous gift from R. Herrmann Conditions for Growth, Cell Labeling and Hybridization. Yeast cells were grown in Yeast Nitrogen Base (Difco) medium with or without uracil at 30 °C. E. coli cells were grown in M9 medium containing either excess uracil (40 /~g/ml) or limiting uracil (1 #g/ml) at 37 °C. Mid-exponential phase cells were labeled with 3H-adenine, 20 ~zCi/ml having a specific activity of 1.7 Ci/mmole. To stop labeling after 2 rain for E. eoli cells and after 5 rain for yeast ceils, one volume of cell suspension was pipetted into two volumes of cold ethanol. The cells were centrifuged, washed once with the RNA extraction buffer (acetate buffer containing i mM acetate, 5 mM NaC1 and 0.1 mM magnesium acetate, pH 5.1). RNA was extracted from yeast ceils according to the procedure of Waldron and Lacroute (1975) as modified by Losson and Lacroute (1979). RNA was extracted from E. coli cells by the
0172-8083/80/0002/0103/SO 1.00
104 same method except that E. coli crude extracts were obtained by sonication. Nitrocellulose filters with probe DNA were prepared by filtration as described by Gillespie and Spiegelman (1965). Hybridization conditions were those given by Losson and Lacroute (1979). Specific "hybridization to each strand of the ura3 gene was measured in quadruplicate in the following way: radioactive RNAs were incubated in a small glass vial with one filter loaded with 1 ~g fd ura3 minus strand DNA, one filter loaded with 1 ~tg fd ura3 plus strand DNA, and one filter loaded with 1 #g fd 106 DNA. The plus strand was named arbitrarily as the strand which hybridized the most RNA. To evaluate hybridization to the ura3-minus or-plus strand, cpm retained on an fd DNA filter were substracted from cpm on an fd ura3 DNA filter. The amount of hybridization was expressed as a fraction of the input.
Growth of Bacteriophage and DNA Extraction. Bacteriophages were grown on E. eoli strain MB 1000 F'Lae in the condidtions described by Herrmann et al. (in press). The phages were isolated from the culture supernatant by the technique of Yamamoto et al. (1970). DNA was extracted from the phages by hot phenol treatment followed by ethanol precipitation.
Determination of OMP Decarboxylase Activity. The assay of Wolcott and Ross (1966) was used and enzyme activity was determined relative to protein concentration (Lowry et al., 1951).
J.C. Hubert et al.: Asymmetrical Transcription in Yeast infected by phage fd 106 carrying two resistance markers ( c h l o r a m p h e n i c o l and k a n a m y c i n ) at a m.i. o f 0.05 as described by H e r r m a n n et al. (1980) in n u t r i e n t m e d i u m containing c h l o r a m p h e n i c o l ( 2 5 / J g / m l ) . Phage fd replicatire f o r m D N A was digested in the k a n a m y c i n gene by endonuclease HindIII, treated with alkaline phosphatase to p r e v e n t recircularization and ligated by T4 ligase with the ura3 fragment as described in Methods.
Transformation and Harvesting o f Phages with Opposite Direction DNA Inserts E. coli pyrF F'Lac cells were transformed directly with the ligation m i x t u r e according to Seeburg and Schaller (1975). The ura3 D N A fragment has thus been inserted in the phage R F in b o t h orientations giving rise to single stranded D N A phages with each strand o f the ura3 gene. Bactoria which gave resistant colonies on minimal m e d i u m plus c h l o r a m p h e n i c o l and were sensitive to k a n a m y c i n were tested for phage p r o d u c t i o n as described by H e r r m a n n et al. (1980).
DNA Extraction from Agarose Gels. DNA extraction from agarose gels followed by ligation was done as follows. DNA-containing agarose, about 0.2 ml was cut out from the get and put in a conical "Eppendorff' centrifuge tube with 0.15 ml 5 M ammonium acetate and 0.4 ml phenol saturated with T.E. buffer (10 mM Tris HC1 pH 8.0, 1 mM EDTA). The tube was then heated in a water bath with frequent shaking until a single liquid phase was obtained (this is generally between 72 ° and 78 °C). The tube was then cooled in melting ice and centrifuged. The supernatant aqueous phase containing the DNA was separated from the interphase containing the denaturated agarose and the phenol phase containing ethidium bromide. The DNA was then concentrated from approximatively 0.3 ml to 20 ~1 by dialysis with air pressure against T.E. buffer. Phosphatased vector (which can also be added at the first extraction step), and an equal volume of double concentration ligation buffer were added directly to the residual volume of DNA for ligation. To the 40 ~zl was added 2 g l o f 1 0 2 M A T P a n d 0 . 2 u n i t o f T 4 ligase. After one day's incubation in the refrigerator ( 6 - 8 °C) the ligation mixture was used for E. eoli transformation (15 ttl for 0.3 ml competent E. coli cell suspension).
Test for Orientation o f the Integrated ura3 Fragment Analysis to determine the orientation o f ura3 D N A inserted in fd D N A (single stranded) was carried o u t with
Results Cloning o f the ura3 Gene in Phage f d 106 The chimeric plasmid p B R 322 2/~D ura3 was digested by HindIII endonuclease (Biolabs). The ura3 1.1 kb D N A was separated by electrophoresis o f the cleaved D N A on an 0.8% agarose gel.
Vector Preparation Bacteriophage fd 106 replicative f o r m ( R F ) doublestranded D N A was o b t a i n e d f r o m E. coli Hfr 3 3 0 0 cells
Fig. 1. About 5 x 1010phages from each of the two clones to be tested were mixed and adjusted to 1% SDS (for disintegration of phage capsid), 50 mM Tris HC1 pH 8.0, 200 mM NaC1 in a final volume of 20 gl. The sample was incubated 2 min at 100 °C and cooled in an isolated water bath to room temperature over a period of 2 h. In slot 1: fd 106 vector; 2: recombinant phage 1; 3: recombinant phage 2; 4: a mixture of phage 1 and phage 2. Hybridized molecules in slot 4 give rise to several bands. This could be due to a different extent of annealing between two DNA molecules or by the formation of aggregates (Herrmann et al., 1980)
J. C. Hubert et al.: Asymmetrical Transcription in Yeast crude phage preparations following the technique of Herrmann et al. (1980). Phages with identical inserts in opposite orientations form DNA/DNA hybrids which can be detected by agarose gel electrophoresis by their molecular weight. The results of such an experiment are shown in Fig. 1. It is clear that the two phages tested hybridize; therefore each possesses one strand of the ura3 DNA.
Transcription o f the ura3 gene in Yeast Labeled RNA was hybridized to plus and minus strand DNA probes. Table 1 presents the percentage of ura3 mRNA hybridized on each strand of the ura3 gene. The data show that the transcription is very asymmetric. In all cases (wild type, derepressed mutant and nonchromosomally integrated strain) the same strand was transcribed with the greatest efficiency (more than 96%). The ura3 messenger levels accord well with the data found for hybridization with total ura3 DNA by Chevallier et al. (unpublished results) and by Losson et al. (1979). It appears in our experiments that the level of mRNA transcribed on the ura3 plus strand in ura3- pFL 2 strain is about 60 times higher than the level in wild type FL 100 and 15 times higher than in the derepressed mutant ural ( 2 1 - 5 0 ) . These values vary coordinately with the observed enzyme activity measured by Chevallier et al. (unpublished results) and confirm that the ura3 gene is under transcriptional control.
105 Transcription o f the ura3 Gene in E. coli Table 2 presents the hybridization results obtained for the transcription in E. coli of the yeast ura3 gene cloned into different E. coli plasmids. The four plasmids used were pMB 9-1 and pMB 9-2 with, respectiviely, a 5.2 kb and 3.3 kb yeast DNA insert, including the ura3 gene and pBR 3 22-6 and pBR 3 22-8 containing a HindIII 1.1 kb ura3 DNA fragment with opposite orientations. It appears that, contrary to the situation in yeast, transcription takes place on both strands. Nevertheless, the transcription on the plus strand was always significantly higher than on the minus strand. Previous results had shown that the OMPdecase levels found in E. coli strains with pMB 9-1 or pMB 9-2 were much higher when strains were grown on a pyrimidine limiting medium than when grown on a medium fully supplemented by pyrimidine (Bach et al., 1979). One interpretation of this result was that some transcriptional regulation was retained, even in E. coli for the ura3 gene. For this reason the transcription on each ura3 strand was measured in these two different physiological conditions. Table 3 shows no significant difference in the transcriptional regulation inE. eoli in the different physiological conditions. This result is in accordance with preliminary results obtained by G. Loison (in our laboratory), who found from quantitative immunological precipitation of the OMPdecase protein in these same conditions of growth, that the amount of enzyme protein was the
Table 1. Transcription of the ura3 gene in Yeast. Cells were labeled with tritiated adenine for 5 min, RNAs were extracted, and hybridization was carried out as described in Material and Methods. The amount of hybridization is expressed as the fraction of radioactivity specifically retained versus the input. The radioactivity specifically retained was calculated by substracting the counts on the fd DNA filters from the counts on the plus or minus fd ura3 DNA filter. Each value is an average of four independent determinations Yeast strain
DNA fd ura3 minus strand
DNA fd ura3 plus strand
Amount of hybrid- % of total ura3 ization x 10-5 transcription on the minus strand
Amount of hybridization x 10-5
% of total ura3 transcription on the plus strand
Exp. no. 1
Wild type FL 100 ura1-21-50 ura3- pFL 2
0.18 0.52 5.9
3 6 5
2.68 7.94 123
97 94 95
Exp. no. 2
Wild type FL 100 ura1-21.50 ura3- pFL 2
0 0.6 4.12
0 1 4
1.19 6.66 106
100 99 96
Exp. no. 3
Wild type FL 100 ura1-21-50 ura3- pF1 2
0.09 0.08 4.2
3 1 3
2.5 8.1 132
97 99 97
106
J.C. Hubert et al.: Asymmetrical Transcription in Yeast
Table 2. Transcription of the Yeast ura3 Gene in E. coli, Cells were labeled with 3H-adenine for 2 min. The amount of transcription is expressed as in Table 1 DNA fd ura3 minus strand
DNA fd ura3 plus strand
Amount of hybrid- % of total ura3 transization x 10- 5 cription on the minus strand
Amount of hybrid- % of total ura3 transization x 10- 5 cription on the plus strand
1 2 3
69 97 119
17 / 21 / 22
20
343 370 429
83 79 78
80
1 2 3
84 102 120
32 ] 351 40
36
177 187 179
68 65 60
64
1 2 3 4
49 60 67 186
43 t 43 39 37
40.5
66 80 107 312
57 57 61 63
59.5
1 2 3 4
44 67 78 113
31 35 / 31 35
33
96 124 113 208
69 65 69 65
67.25
Strains
MB 1000 clone 6
MB 1000 clone 8
MB 1000 clone 1
MB 1000 clone 2
Table 3. Effect of Growth Conditions on Transcription of the Yeast ura3 Gene in E. coli. Cells were grown as described in Material and Methods, OMPdecase specific activities are expressed as miUimoles of OMP transformed/mg protein. The amount of hybridization is expressed as in Table 1 Strains
Excess uracil Enzymatic activity
E. colipyrF clone 1 E. coli pyrF clone 2
0.10 0.04
Limiting conditions Amount of hybridization x 10-5 plus strand
minus strand
186 113
312 208
same in the two cases. Differences in activity seem therefore to be due to an increase in stability o f the protein during growth on pyrimidine limiting medium; the accumulation o f the substrate (OMP) in this condition could be involved.
Discussion Transcription of the ura3 gene in yeast is clearly very asymmetrical. The question is whether the small amount o f RNA hybridized to the minus strand is significant. If one considers the results obtained with the chromosomally located ura3 gene expressed either at its induced or basal level, RNA hybridized to the minus strand was at the limit of sensitivity o f the technique. This could be seen by the variation in the experimental values. In the case o f the nonchromosomally transformed strain the
Enzymatic activity
3.2 1.1
Amount of hybridization × 10 - 5 plus strand
minus strand
108 78
267 204
amount o f RNA hybridized to the minus strand was clearly significant. Two possibilities could be invoked: either some RNA is indeed transcribed from this strand or the counts came from radioactive ura3 DNA complementary to the minus strand. This last possibility is raised by the fact that the nonchromosomally transformed strain has a large number o f copies (~60) of the ura3 gene so that even during a short pulse o f radioactivity significant labeling of ura3 DNA sequences was obtained. It would be possible to choose between these possibilities if one could obtain an increased sensitivity of the hybridization technique by reduction of background. In the case of the yeast gene cloned in E. coli, transcription occurred on both strands. Similar results were obtained by Chang et al. (1975) with mouse mitochondrial DNA cloned in theE. colipSC 101 plasmid and by Miller et al. (1977) with a Drosophila DNA fragment cloned in the same plasmid. If transcription o f the ura3 DNA fragment in
J. C. Hubert et al.: Asymmetrical Transcription in Yeast
E. coli was completely due to E. coli promotors, one would expect to reverse the direction of transcription when the orientation of the insert is inverted. This is clearly n o t the case since the plus strand was always transcribed more. On the other hand, if the transcription of the ura3 DNA fragment was completely due to internal promotors (either the normal yeast p r o m o t o r or some different yeast sequence recognized as a p r o m o t o r by the E. coli RNA polymerase) the amount of transcription on each strand would n o t be changed at all by the inversion of the ura3 DNA insert. This is also clearly n o t the case. One must therefore conclude that there is participation of both external and internal promotors in the transcription observed. Knowledge o f the size o f the transcripts on each strand by Northern hybridization (Alwine et al., 1977) could clarify the problem. Recently Berg et al. (1979) have shown that anEcoRI fragment of yeast mitochondrial DNA cloned in E. coli was transcribed on both strands in contrast to the asymmetrical transcription found in mitochondria by Rabinowitz (1976). Our results lead to the same conclusion in the case o f yeast nuclear DNA transcribed in E. coli. "Biohazards associated with the experiments described in this publication have been examined previously hy the French National Control Comittee"
Acknowledgements. We aregreatly indebted to Dr. Richard Herrmann for helpful advice and for his generous gift of different fd vectors and grateful to B. Winsor for reading the manuscript. This work was supported in part by grants from the C.N.R.S. (ATP "Microbiologie" No. 3070) and the I.N.S.E.R.M. (Institut National de la Sant6 et de la Recherche M6dicale, contrat No. 77.1.073.1. and ATP I.N.S.E.R.M. No. 79.104).
107
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Communicated by F. Kaudewitz Received February 7, 1980
