JOURNAL OF BACTERIOLOGY, Aug. 1975, p. 637-641 Copyight 0 1975 American Society for Microbiology
Vol. 123, No. 2 Printed in U.S.A.
Derepression in Saccharomyces cerevisiae Can Be Dissociated from Cellular Proliferation and Deoxyribonucleic Acid Synthesis' HENRY R. MAHLER,* KATHERINE ASSIMOS, AND CHI CHUNG LIN Mitochondrial Biogenesis Group, Chemical Laboratories, Indiana University, Bloomington, Indiana 47401
Received for publication 31 March 1975
A method has been developed that permits precise control of release from catabolite repression in Saccharomyces cerevisiae. It consists of transferring cells growing exponentially on 5% glucose to a derepression medium at high cell density. Derepression then proceeds with reproducible kinetics and is complete within 6 to 7.5 h for various intra- and extramitochondrial markers, in the absence of any substantial increase in cellular dry weight or protein. Nuclear (and mitochondrial) deoxyribonucleic acid synthesis can be interrupted in certain thermosensitive (cdc) mutants at the nonpermissive temperature; a shift to this temperature before the onset of derepression has no effect on its outcome.
Catabolite repression and its release in yeasts, such as Saccharomyces cerevisiae, represents one of the more striking examples of a cellular regulatory regime, involving a multiplicity of functions of these unicellular eukaryotes (reviewed in 12, 14, 19). We (9, 10, 15, 17, 18, 20) have been interested in the phenomenon, particularly from the point of view of its effect on the elaboration of fully functional mitochondria, capable of efficient respiration and energy transduction-processes known to require the presence of a nonfermentable carbon source and to be repressible by high glucose in the medium. One of the major unanswered questions in this area concerns the mechanism by which a large number of genes, located both in the mitochondria and in the nucleus, are first turned on over a short period of time at the onset of derepression, maintained at that level over a period of several hours, and eventually turned down or off to terminate the process. One plausible set of models suggests that transcription of the genes capable of derepression is directly dependent on the prior synthesis of nuclear deoxyribonucleic acid (DNA), either because of gene dosage effects or because previously formed DNA cannot be transcribed due to its interaction with a resident repressor (1, 2, 13, 21). We have now tested this model explicitly by the use of appropriate thermosensitive mutants (4) incapable of either initiating the synthesis or continuing the elongation of nuclear DNA at the nonpermissive temperature (5-8). To do so, we have developed 'Publication no. 2690 from the Chemical Laboratories, Indiana University, Bloomington.
a derepression paradigm which, unlike those generally in use, employs cells collected in the exponential phase, permits precise timing of the initiation of derepression, and allows it to occur in the virtually complete absence of cellular proliferation. MATERIALS AND METHODS Yeast strains and media. The following strains, provided by L. H. Hartwell, were used: A364A (wild type); H185.3.4 (cdc 28); 135.1.1 (cdc 4-3; 314 (cdc 4-1); 146.2.3 (cdc 21-1); and 13052 (cdc 8-3). They were maintained on agar slants containing yeast extract- (1%)-peptone (2%)-glucose (2%) medium (16,18) and precultured in the same medium. Derepression experiments. Cells were grown on either yeast extract-peptone-glucose or YM-1 medium (4) containing 5% glucose to an absorbancy at 600 nm of 0.40, corresponding to a cell density of 3 x 107/ml. They were harvested by centrifugation, washed, and resuspended at an absorbancy at 600 nm c 2.0 in a derepression medium consisting of either complete YM- 1 or the same medium without peptone, with 2.5 g of glucose plus 30 g of ethanol per liter as carbon source. Samples were withdrawn at the times indicated in Results, and cells were sedimented and broken for enzyme assays. Cell breakage, enzyme localization, and assays. These procedures have been described in (15, 17, 18), as has the rationale for the selection and the localization of the enzymes used. The enzymes assayed were reduced nicotinamide adenine dinucleotide (NADH) oxidase; cytochrome c oxidase (EC 1.9.3.3); NADH: cytochrome c (oxido) reductase (EC 1.6.2.1); succinate:cytochrome c (oxido) reductase (EC 1.3.99.1); Li-isocitrate dehydrogenase (EC 1.1.1.42; IDH); Lmalate dehydrogenase (EC 1.1.1.37; MDH); and Lglutamate dehydrogenase (EC 1.4.1.2; GDH). Of 637
J. BACTERIOL.
MAHLER, ASSIMOS, AND LIN
638
these, the first five are of mitochondrial localization, the attendant changes in cell number, turbidthe first four in the inneir membrane and subject to ity, dry weight, and homogenate protein, and strong, the last in the m Latrix and subject to weak, the initial, repressed, and final, derepressed catabolite repression. Th e first two require a mito- levels of a more complete battery of enzymes. chondrial contribution fior their biogenesis during short-term derepression e) xperiments. IDH is localized All these experiments were performed with cells in the mitochondrial mattrix; MDH is found in both of A364A, the wild type, at 30 C, the usual the mitochondrial maxtri: x and in the cytosol; GDH is temperature for such studies. It can be seen that, found only in the latter. A 1l these activities except for in spite of the absence of any substantial IDH increase substantiallIy during derepression. changes (< 20%) in cell mass or protein after the
RESULTS AN]D DISCUSSION Derepression doesn iot require growth. Earlier experiments, in w hich growing cells of S. cerevisiae had been r eleased from catabolite repression as a result o f the exhaustion of their supply of glucose and the resulting transition from glycolytic to resp iratory metabolism, had indicated that such der epression did not require any extensive increase in cellular number or mass (1-3, 10, 18). H lowever, a more precise answer to this question, particularly as concerns its timing and kinetics had to await the development of a technique Ithat permits close experimental control of its initiation. The simple procedure outlined abo ye does just that. Representative data are summarized in Fig. 1 and 2 and the first co lumn of Table 1, which depict, respectively, tihe kinetics of derepression of four represental tive enzymes, as well as '°°r-
100- MDH (cyto)
MDH (Mito)
p
cty
F
2nd h, all the various marker activities exhibited
the expected increases. (The initial increase could be prevented by performing the derepression experiment at 36 C- see below.) Increases became detectable within 90 min and were complete by 6 to 7.5 h. The markers selected included enzymes localized exclusively in the cytosol such as GDH, and one found in both the cytosol and the mitochondrial matrix (MDH), as well as a number of activities characteristic of the mitochondrial inner membrane, some with an obligatory intramitochondrial contribution (cytochrome c oxidase and NADH oxidase) and some without (succinate dehydrogenase and succinate cytochrome c reductase). The inhibition patterns in response to site-specific inhibitors of protein synthesis agreed with this expectation and with previous studies (11, 12, 18, 19). The derepression of all activities was completely blocked by cycloheximide at concentrations > 10 the results for mitochondrial MDH are shown as an example. In contrast, chloramphenicol blocked the synthesis of cytochrome c oxidase completely but had only a partial effect on the synthesis of either MDH or c reductase. Since the former is located in the matrix, these inhibitions are probably not due to a block in the biogenesis of the enzyme proteins per se, but instead reflect the formation of incompetent mitochondria, deficient either in certain structural parameters or in their capacity to generate sufficient adenosine triphosphate for biosynthetic purposes (11, 18). Derepression does not require DNA ability of a synthesis. We then tested the derepression number of cdc mutants to undergo at a nonpermissive temperature, where their defined lesions at different stages of nuclear DNA synthesis should become manifest. Results are shown in Table 1 experiments of these with the essential control, which show together that derepression in the wild type was unaffected by the temperature shift. Detailed kinetics for the derepression of four representative enzymes are shown in Fig. 3. The results are relatively unambiguous: all the enzymatic activities tested in all mutants became dere-
,g/ml;
NADH:cytochrome
5C
50-
/
4
cyt c. 02 (Mo)
">
A*
ol (-)
.O -0
NADH: c (Mito)
4-
2
I
2-
b
*
01~~~~~~~~~~~
2
-
4
0
6
_______x_ 4 2 6
erttransfer (h)
time aft(er FIG. at 30
C.
600 nm
1.
Derepression of marker activities
Repressed cells =
0.4 in
yeast
w ere ex
in A364A
grown to absorbancy at con-
ctract-peptone-glucose eplusn3%aethanol Symbols
taining 2% glucose before to YM-Iplus 0.25% glucose
Ioramphenicol (4 mg/m); .g/ml). Abbreviations are
(0) Control; (0) Plus ch (A) plus cycloheximide (2( 9 as in
Table 1.
pressed to the same (or a greater) extent as in
DEREPRESSION IN S. CEREVISIAE
VOL. 123, 1975
639
1.8
_fi 1.6
0
0
0
0
4-o
0
'0
@i 1.4 1.2
01
D
2.. 100
1.-
2.9
o 2.7 2 < 2.5
>~
~
~
F-
*
0
0
~~~
- ;2.5
S
0
0=5 80 -6
I
2.3(F
2.0
I
t, ^ .10-
3.0
o
a0
', 2.2
L0
.0 n°
2.5
1.8
u ._i
O)0NJ
a
2
0
Co
0
)-
o
a
o
L
*
*0..
.
0ii
0
0
6
4
2
4
6
time after transfer (h)
8
time after transfer (h)
FIG. 2. Effect of derepression on various cellular parameters of A364A: the data symbolized with 0 and 0 obtained at 30 C, those with at 36 C. (0) Conditions as in Fig. 1; (0, 0) cells were grown to absorbancy at
were 600
nm
(A.,,)
0.4 in YM-1
plus
5%
glucose before concentration and derepression
on
YM-1 minus peptone
plus 0.25% glucose plus 3% ethanol.
TABLE 1. Repressed and derepressed levels of enzymes of cdc mutants at 36 C Strain and gene, Activitya
NADH:O0 cyt C:02 NADH:c succ:c
GDH MDH
IDH
A364 (wild type) 36 C 30 C
1.9 9.2 1.6 3.0 0.80 4.4 0.33 2.3 0.50 13.8 62 240
1.2 6.7 2.5 6.9 0.50 4.5 0.19 2.4 1.7
12.3 54 177 3.2 3.8
1853.4 cdc 28
13052 cdc 8-3
146.2.3 cdc 21-1
1351.1 cdc 4-3
0.65 7.8 5.8 13.9 0.44 2.8 0.10 2.3 0.75 7.5 20 159 2.0 3.6
0.64
0.79 5.0
1.5
6.9 1.3
4.5
2.5
8.6 0.30 6.3 0.10 1.7 0.40 10.5 35 247 3.8 6.0
7.8
7.5
6.2
0.27
0.48
1.70 0.25 1.2
3.8 0.10 1.4
49 141 3.4 2.3
1.5 5.4 17
73 5.3 7.8
a Abbreviations: NADH: 0, NADH oxidase; cyt c:02, cytochrome c oxidase; succ:c, succinate:cytochrome c (oxido) reductase; GDH, .-glutamate dehydrogenase; MDH, L-malate dehydrogenase; IDH, Li-isocitrate dehydrogenase. The first (roman) number indicates specific activity at time of transfer (repressed); the second (italic) represents the final (derepressed) value after 7.5 or 10 h in derepression medium. c Tested at 38 C.
the wild type. Thus derepression does not depend (5-7) on a normal level of either the initiation (cdc genes 28 and 4) or elongation of progeny DNA (cdc genes 8 and 21) in the nucleus. These results therefore rule out any model for the regulation of derepression that postulates the synthesis of such new, "naked" (i.e., unoccupied by a regulatory element, such
as a repressor) genes as a required precondition. Actually the conclusion can be stated in even stronger terms. Recent studies by C. S. Newlon and W. L. Fangman (private communication) with the same strain have shown that the cdc mutants that are incapable of elongating nuclear DNA after a shift-up to 36 C are equally deficient in performing this function for mito-
640
J. BACTERIOL.
MAHLER, ASSIMOS, AND LIN
o00o NADH:
loor
A0
02 0
75-
o
*
I
A
NADH: c 75F
U
A
0
50k
50k
0
H .? .>
-
22I5
0
251
a
0 0
E 01 Ex 10)o w Succinate: c0 FE
10 o
GDH
75F
0 7r5
a 0
0
50o
5
0
2
25
*8 0 -os
C
2
4
6
8
u-0O
A 0
U 0
0
0
A
0
8
A
2
4
6
8
time after transfer (h) FIG. 3. Derepression of thermosensitive cdc mutants, blocked in DNA synthesis, at a nonpermissive was studied at 38 C). Symbols: (A) A364A (wild type); (0) cdc 8; (0) cdc 21; (U) cdc 4-3; (0) cdc 28. Abbreviations are as in Table 1. temperature (36 C, except for cdc 28, which
chondrial DNA. Thus derepression can proceed in the absence of a substantial level of all cellular DNA synthesis, whether nuclear or mitochondrial. This statement probably rules out a requirement for synthesis of new DNA as a prerequisite for the transcription of the numerous structural genes that must participate in the process of derepression, but it cannot exclude, given the present level of detection, a possible synthesis of a small number of regulatory genes, particularly if this process is independent of the products specified by cdc genes 8 and 21. Conversely, the results with the initiation mutants, which have been shown to lead to dissociation of mitochondrial from nuclear DNA synthesis with a resultant overproduction of the latter (3, 22), appear to rule out any substantital effect of mitochondrial gene dosage in the regulation of derepression. ACKNOWLEDGMENTS We are greatly indebted to L. Hartwell for providing us with all of the strains used in this investigation and for many useful suggestions, as well as to C. S. Newlon for communicating to us her results before publication and for her permission to cite them in this paper. This investigation was supported by Public Health Service grant GM 12228 from the Institute of General Medical
Sciences. H. R. M. is recipient of Public Health Service career award KO 05060 from the same institute.
LITERATURE CITED 1. Barath, S., and H. Kuntzel. 1972. Cooperation of mitochondrial and nuclear genes specifying the mitochondrial genetic apparatus in Neurospora crassa. Proc. Natl. Acad. Sci. U.S.A. 69:1371-1374. 2. Barath, S., and H. Kiintzel. 1972. Induction of mitochondrial RNA polymerase in Neurospora crassa. Nature (London) New Biol. 240:195-197. 3. Cottrell, S., M. Rabinowitz, and G. S. Getz. 1974. Mitochondrial deoxyribonucleic acid synthesis in a temperature-sensitive mutant of deoxyribonucleic acid replication of S. cerevisiae. Biochemistry 12:4374-4378. 4. Hartwell, L. H. 1967. Macromolecule synthesis in temperature-sensitive mutants of yeast. J. Bacteriol. 93:1662-1670. 5. Hartwell, L. H. 1971. Genetic control of the cell division cycle in yeast. II. Genes controlling DNA replication and its initiation. J. Mol. Biol. 59:183-194. 6. Hartwell, L. H. 1973. Three additional genes required for DNA synthesis in S. cerevisiae. J. Bacteriol. 115: 966-974. 7. Hartwell, L. H. 1974. Saccharomyces cerevisiae cell cycle. Bacteriol. Rev. 38:164-198. 8. Hartwell, L. H., J. Culotti, J. R. Pringle, and B. J. Rein. 1974. Genetic control of the cell division cycle in yeast: a model. Science 183:46-51. 9. Henson, C. P., C. N. Weber, and H. R. Mahler. 1968. Formation of yeast mitochondria. II. Effects of antibiotics of enzyme activity during derepression. Biochemistry 7:4445-4454.
VOL. 123, 1975 10. Jayaraman, J., C. Cotman, H. R. Mahler, and C. W. Sharp. 1966. Biochemical correlates of respiratory deficiency. VII. Glucose repression. Arch. Biochem. Biophys. 116:224-251. 11. Kim, I-C., and D. S. Beattie. 1973. Formation of the yeast mitochondrial membrane. I. Effects of inhibitors of protein synthesis on the kinetics of enzyme appearance during glucose derepression. Eur. J. Biochem. 36:509-518. 12. Linnane, A. W., J. M. Haslam, H. B. Lukins, and P. Nagley. 1972. The biogenesis of mitochondria in microorganisms. Annu. Rev. Microbiol. 26:163-198. 13. Lloyd, D., G. Turner, R. K. Poole, W. G. Nichole, and G. I. Roach. 1971. A hypothesis of nuclear-mitochondrial interactions for the control of mitochondrial biogenesis based on experiments with Tetrahymena pyriformis. Subcell. Biochem. 1:91-95. 14. Mahler, H. R. 1973. Biogenetic autonomy of mitochondria. CRC Crit. Rev. Biochem. 1:381-460. 15. Mahler, H. R., and C. C. Lin. 1971. The derepression of A-amino-levulinate synthetase in yeast. Biochem. Biophys. Res. Commun. 61:963-970. 16. Mahler, H. R., P. Perlman, C. Henson, and C. Weber. 1968. Selective effects of chloramphenicol, cyclohexi-
DEREPRESSION IN S. CEREVISIAE
641
mide and nalidixic acid on the biosynthesis of respiraenzymes in yeast. Biochem. Biophys. Res. Commun. 31:474-480. 17. Perlman, P. S., and H. R. Mahler. 1970. Intracellular localization of enzymes in yeast. Arch. Biochem. Biophys. 136:245-259. 18. Perlman, P. S., and H. R. Mahler. 1974. Derepression of mitochondi¶a and their enzymes in yeast: regulatory aspects. Arch. Biochem. Biophys. 162: tory
248-271. 19. Sager, R. 1972. Cytoplasmic genes and organelles. Academic Press Inc., New York. 20. South, D. J., and H. R. Mahler. 1968. RNA synthesis in yeast mitochondria: a derepressible activity. Nature (London) 218:1226-1232. 21. Williamson, D. H. 1970. The effect of environmental and genetic factors on the replication of mitochondrial DNA in yeast. p. 247-276. In P. L. Miller (ed.), Control of organelle development. Academic Press Inc., New York. 22. Wintersberger, U., J. Hirsch, and A. M. Fink. 1974. Studies on nuclear and mitochondrial DNA-replication in a temperature-sensitive mutant of Saccharomyces cerevisiae. Mol. Gen. Genet. 131:291-299.
Vol. 123, No. 2 Printed in U.S.A.
Derepression in Saccharomyces cerevisiae Can Be Dissociated from Cellular Proliferation and Deoxyribonucleic Acid Synthesis' HENRY R. MAHLER,* KATHERINE ASSIMOS, AND CHI CHUNG LIN Mitochondrial Biogenesis Group, Chemical Laboratories, Indiana University, Bloomington, Indiana 47401
Received for publication 31 March 1975
A method has been developed that permits precise control of release from catabolite repression in Saccharomyces cerevisiae. It consists of transferring cells growing exponentially on 5% glucose to a derepression medium at high cell density. Derepression then proceeds with reproducible kinetics and is complete within 6 to 7.5 h for various intra- and extramitochondrial markers, in the absence of any substantial increase in cellular dry weight or protein. Nuclear (and mitochondrial) deoxyribonucleic acid synthesis can be interrupted in certain thermosensitive (cdc) mutants at the nonpermissive temperature; a shift to this temperature before the onset of derepression has no effect on its outcome.
Catabolite repression and its release in yeasts, such as Saccharomyces cerevisiae, represents one of the more striking examples of a cellular regulatory regime, involving a multiplicity of functions of these unicellular eukaryotes (reviewed in 12, 14, 19). We (9, 10, 15, 17, 18, 20) have been interested in the phenomenon, particularly from the point of view of its effect on the elaboration of fully functional mitochondria, capable of efficient respiration and energy transduction-processes known to require the presence of a nonfermentable carbon source and to be repressible by high glucose in the medium. One of the major unanswered questions in this area concerns the mechanism by which a large number of genes, located both in the mitochondria and in the nucleus, are first turned on over a short period of time at the onset of derepression, maintained at that level over a period of several hours, and eventually turned down or off to terminate the process. One plausible set of models suggests that transcription of the genes capable of derepression is directly dependent on the prior synthesis of nuclear deoxyribonucleic acid (DNA), either because of gene dosage effects or because previously formed DNA cannot be transcribed due to its interaction with a resident repressor (1, 2, 13, 21). We have now tested this model explicitly by the use of appropriate thermosensitive mutants (4) incapable of either initiating the synthesis or continuing the elongation of nuclear DNA at the nonpermissive temperature (5-8). To do so, we have developed 'Publication no. 2690 from the Chemical Laboratories, Indiana University, Bloomington.
a derepression paradigm which, unlike those generally in use, employs cells collected in the exponential phase, permits precise timing of the initiation of derepression, and allows it to occur in the virtually complete absence of cellular proliferation. MATERIALS AND METHODS Yeast strains and media. The following strains, provided by L. H. Hartwell, were used: A364A (wild type); H185.3.4 (cdc 28); 135.1.1 (cdc 4-3; 314 (cdc 4-1); 146.2.3 (cdc 21-1); and 13052 (cdc 8-3). They were maintained on agar slants containing yeast extract- (1%)-peptone (2%)-glucose (2%) medium (16,18) and precultured in the same medium. Derepression experiments. Cells were grown on either yeast extract-peptone-glucose or YM-1 medium (4) containing 5% glucose to an absorbancy at 600 nm of 0.40, corresponding to a cell density of 3 x 107/ml. They were harvested by centrifugation, washed, and resuspended at an absorbancy at 600 nm c 2.0 in a derepression medium consisting of either complete YM- 1 or the same medium without peptone, with 2.5 g of glucose plus 30 g of ethanol per liter as carbon source. Samples were withdrawn at the times indicated in Results, and cells were sedimented and broken for enzyme assays. Cell breakage, enzyme localization, and assays. These procedures have been described in (15, 17, 18), as has the rationale for the selection and the localization of the enzymes used. The enzymes assayed were reduced nicotinamide adenine dinucleotide (NADH) oxidase; cytochrome c oxidase (EC 1.9.3.3); NADH: cytochrome c (oxido) reductase (EC 1.6.2.1); succinate:cytochrome c (oxido) reductase (EC 1.3.99.1); Li-isocitrate dehydrogenase (EC 1.1.1.42; IDH); Lmalate dehydrogenase (EC 1.1.1.37; MDH); and Lglutamate dehydrogenase (EC 1.4.1.2; GDH). Of 637
J. BACTERIOL.
MAHLER, ASSIMOS, AND LIN
638
these, the first five are of mitochondrial localization, the attendant changes in cell number, turbidthe first four in the inneir membrane and subject to ity, dry weight, and homogenate protein, and strong, the last in the m Latrix and subject to weak, the initial, repressed, and final, derepressed catabolite repression. Th e first two require a mito- levels of a more complete battery of enzymes. chondrial contribution fior their biogenesis during short-term derepression e) xperiments. IDH is localized All these experiments were performed with cells in the mitochondrial mattrix; MDH is found in both of A364A, the wild type, at 30 C, the usual the mitochondrial maxtri: x and in the cytosol; GDH is temperature for such studies. It can be seen that, found only in the latter. A 1l these activities except for in spite of the absence of any substantial IDH increase substantiallIy during derepression. changes (< 20%) in cell mass or protein after the
RESULTS AN]D DISCUSSION Derepression doesn iot require growth. Earlier experiments, in w hich growing cells of S. cerevisiae had been r eleased from catabolite repression as a result o f the exhaustion of their supply of glucose and the resulting transition from glycolytic to resp iratory metabolism, had indicated that such der epression did not require any extensive increase in cellular number or mass (1-3, 10, 18). H lowever, a more precise answer to this question, particularly as concerns its timing and kinetics had to await the development of a technique Ithat permits close experimental control of its initiation. The simple procedure outlined abo ye does just that. Representative data are summarized in Fig. 1 and 2 and the first co lumn of Table 1, which depict, respectively, tihe kinetics of derepression of four represental tive enzymes, as well as '°°r-
100- MDH (cyto)
MDH (Mito)
p
cty
F
2nd h, all the various marker activities exhibited
the expected increases. (The initial increase could be prevented by performing the derepression experiment at 36 C- see below.) Increases became detectable within 90 min and were complete by 6 to 7.5 h. The markers selected included enzymes localized exclusively in the cytosol such as GDH, and one found in both the cytosol and the mitochondrial matrix (MDH), as well as a number of activities characteristic of the mitochondrial inner membrane, some with an obligatory intramitochondrial contribution (cytochrome c oxidase and NADH oxidase) and some without (succinate dehydrogenase and succinate cytochrome c reductase). The inhibition patterns in response to site-specific inhibitors of protein synthesis agreed with this expectation and with previous studies (11, 12, 18, 19). The derepression of all activities was completely blocked by cycloheximide at concentrations > 10 the results for mitochondrial MDH are shown as an example. In contrast, chloramphenicol blocked the synthesis of cytochrome c oxidase completely but had only a partial effect on the synthesis of either MDH or c reductase. Since the former is located in the matrix, these inhibitions are probably not due to a block in the biogenesis of the enzyme proteins per se, but instead reflect the formation of incompetent mitochondria, deficient either in certain structural parameters or in their capacity to generate sufficient adenosine triphosphate for biosynthetic purposes (11, 18). Derepression does not require DNA ability of a synthesis. We then tested the derepression number of cdc mutants to undergo at a nonpermissive temperature, where their defined lesions at different stages of nuclear DNA synthesis should become manifest. Results are shown in Table 1 experiments of these with the essential control, which show together that derepression in the wild type was unaffected by the temperature shift. Detailed kinetics for the derepression of four representative enzymes are shown in Fig. 3. The results are relatively unambiguous: all the enzymatic activities tested in all mutants became dere-
,g/ml;
NADH:cytochrome
5C
50-
/
4
cyt c. 02 (Mo)
">
A*
ol (-)
.O -0
NADH: c (Mito)
4-
2
I
2-
b
*
01~~~~~~~~~~~
2
-
4
0
6
_______x_ 4 2 6
erttransfer (h)
time aft(er FIG. at 30
C.
600 nm
1.
Derepression of marker activities
Repressed cells =
0.4 in
yeast
w ere ex
in A364A
grown to absorbancy at con-
ctract-peptone-glucose eplusn3%aethanol Symbols
taining 2% glucose before to YM-Iplus 0.25% glucose
Ioramphenicol (4 mg/m); .g/ml). Abbreviations are
(0) Control; (0) Plus ch (A) plus cycloheximide (2( 9 as in
Table 1.
pressed to the same (or a greater) extent as in
DEREPRESSION IN S. CEREVISIAE
VOL. 123, 1975
639
1.8
_fi 1.6
0
0
0
0
4-o
0
'0
@i 1.4 1.2
01
D
2.. 100
1.-
2.9
o 2.7 2 < 2.5
>~
~
~
F-
*
0
0
~~~
- ;2.5
S
0
0=5 80 -6
I
2.3(F
2.0
I
t, ^ .10-
3.0
o
a0
', 2.2
L0
.0 n°
2.5
1.8
u ._i
O)0NJ
a
2
0
Co
0
)-
o
a
o
L
*
*0..
.
0ii
0
0
6
4
2
4
6
time after transfer (h)
8
time after transfer (h)
FIG. 2. Effect of derepression on various cellular parameters of A364A: the data symbolized with 0 and 0 obtained at 30 C, those with at 36 C. (0) Conditions as in Fig. 1; (0, 0) cells were grown to absorbancy at
were 600
nm
(A.,,)
0.4 in YM-1
plus
5%
glucose before concentration and derepression
on
YM-1 minus peptone
plus 0.25% glucose plus 3% ethanol.
TABLE 1. Repressed and derepressed levels of enzymes of cdc mutants at 36 C Strain and gene, Activitya
NADH:O0 cyt C:02 NADH:c succ:c
GDH MDH
IDH
A364 (wild type) 36 C 30 C
1.9 9.2 1.6 3.0 0.80 4.4 0.33 2.3 0.50 13.8 62 240
1.2 6.7 2.5 6.9 0.50 4.5 0.19 2.4 1.7
12.3 54 177 3.2 3.8
1853.4 cdc 28
13052 cdc 8-3
146.2.3 cdc 21-1
1351.1 cdc 4-3
0.65 7.8 5.8 13.9 0.44 2.8 0.10 2.3 0.75 7.5 20 159 2.0 3.6
0.64
0.79 5.0
1.5
6.9 1.3
4.5
2.5
8.6 0.30 6.3 0.10 1.7 0.40 10.5 35 247 3.8 6.0
7.8
7.5
6.2
0.27
0.48
1.70 0.25 1.2
3.8 0.10 1.4
49 141 3.4 2.3
1.5 5.4 17
73 5.3 7.8
a Abbreviations: NADH: 0, NADH oxidase; cyt c:02, cytochrome c oxidase; succ:c, succinate:cytochrome c (oxido) reductase; GDH, .-glutamate dehydrogenase; MDH, L-malate dehydrogenase; IDH, Li-isocitrate dehydrogenase. The first (roman) number indicates specific activity at time of transfer (repressed); the second (italic) represents the final (derepressed) value after 7.5 or 10 h in derepression medium. c Tested at 38 C.
the wild type. Thus derepression does not depend (5-7) on a normal level of either the initiation (cdc genes 28 and 4) or elongation of progeny DNA (cdc genes 8 and 21) in the nucleus. These results therefore rule out any model for the regulation of derepression that postulates the synthesis of such new, "naked" (i.e., unoccupied by a regulatory element, such
as a repressor) genes as a required precondition. Actually the conclusion can be stated in even stronger terms. Recent studies by C. S. Newlon and W. L. Fangman (private communication) with the same strain have shown that the cdc mutants that are incapable of elongating nuclear DNA after a shift-up to 36 C are equally deficient in performing this function for mito-
640
J. BACTERIOL.
MAHLER, ASSIMOS, AND LIN
o00o NADH:
loor
A0
02 0
75-
o
*
I
A
NADH: c 75F
U
A
0
50k
50k
0
H .? .>
-
22I5
0
251
a
0 0
E 01 Ex 10)o w Succinate: c0 FE
10 o
GDH
75F
0 7r5
a 0
0
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5
0
2
25
*8 0 -os
C
2
4
6
8
u-0O
A 0
U 0
0
0
A
0
8
A
2
4
6
8
time after transfer (h) FIG. 3. Derepression of thermosensitive cdc mutants, blocked in DNA synthesis, at a nonpermissive was studied at 38 C). Symbols: (A) A364A (wild type); (0) cdc 8; (0) cdc 21; (U) cdc 4-3; (0) cdc 28. Abbreviations are as in Table 1. temperature (36 C, except for cdc 28, which
chondrial DNA. Thus derepression can proceed in the absence of a substantial level of all cellular DNA synthesis, whether nuclear or mitochondrial. This statement probably rules out a requirement for synthesis of new DNA as a prerequisite for the transcription of the numerous structural genes that must participate in the process of derepression, but it cannot exclude, given the present level of detection, a possible synthesis of a small number of regulatory genes, particularly if this process is independent of the products specified by cdc genes 8 and 21. Conversely, the results with the initiation mutants, which have been shown to lead to dissociation of mitochondrial from nuclear DNA synthesis with a resultant overproduction of the latter (3, 22), appear to rule out any substantital effect of mitochondrial gene dosage in the regulation of derepression. ACKNOWLEDGMENTS We are greatly indebted to L. Hartwell for providing us with all of the strains used in this investigation and for many useful suggestions, as well as to C. S. Newlon for communicating to us her results before publication and for her permission to cite them in this paper. This investigation was supported by Public Health Service grant GM 12228 from the Institute of General Medical
Sciences. H. R. M. is recipient of Public Health Service career award KO 05060 from the same institute.
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