Differential Regulation of the Duplicated Isocytochrome C Genes in Yeast (Yeast Regulation/Heme Deficiency/Glucose Repression/Cytochrome C Regulation) THOMAS M

Proc. NatI. Acad. Sci. USA Vol. 81, pp. 4475-4479, July 1984 Genetics

Differential regulation of the duplicated isocytochrome c genes in yeast (yeast regulation/heme deficiency/glucose repression/cytochrome c regulation) THOMAS M. LAZ, DENNIS F. PIETRAS, AND FRED SHERMAN Departments of Biochemistry and of Radiation Biology and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642 Communicated by Herschel L. Roman, March 26, 1984

ABSTRACT The two unlinked genes CYCI and CYC7 en- acid (ALA) synthetase (10, 12); since ALA is an intermedi- code iso-l-cytochrome c and iso-2-cytochrome c, respectively, ate in the biosynthesis of heme, hemi mutants are deficient in the yeast Saccharomyces cerevisiae. An examination of the in heme and all heme-containing proteins, including the iso- steady-state level of CYCI and CYC7 mRNAs in normal and cytochromes c. In contrast, the CYC2 and CYC3 genes ap- mutant strains grown under different conditions, along with pear to affect only the isocytochromes c. These mutants may previous results of apoprotein levels, demonstrate that CYCI be involved in some aspect of the enzymatic attachment of a and CYC7 have similar and different modes of regulation. heme to the apoisocytochromes c or the transport of the apo- Both CYCI and CYC7 mRNAs are diminished after anaerobic cytochromes c into the mitochondria (9). Whereas hemi and growth. In contrast, CYCI mRNA but not CYC7 mRNA is de- cyc3 mutations can completely block the isocytochrome c creased by heme deficiency in hem) mutants. Although both production, the cyc2 mutation can block at most only 90% of CYCI and CYC7 mRNAs are substantially lowered after the isocytochromes c. Immunological studies of apocyto- growth in glucose medium, there is a difference in the kinetics chromes c demonstrate that hemi, cyc2, and cyc3 mutants of glucose derepression. CYCI mRNA levels rise in the early lack apoiso-1-cytochrome c but contain substantial amounts logarithmic phase of growth before complete exhaustion of of apoiso-2-cytochrome c (9). The absence of apoiso-1-cyto- glucose, whereas CYC7 mRNA levels rise in the late logarith- chrome c and the presence of apoiso-2-cytochrome c in these mic phase when the level of CYCI mRNA has plateaued. For a mutants may be explained by differences in the rates of syn- brief period before cessation of growth, the level of CYC7 thesis or degradation of either apoisocytochromes c or their mRNA attains a level corresponding to the high derepressed respective mRNAs. level of CYCJ mRNA. The high amount of CYC7 mRNA is As part of a systematic investigation of the regulation of surprising because iso-2-cytochrome c constitutes only 5% of the isocytochromes c and to explain the differences in the the total cytochrome c complement in derepressed cells. We accumulation of the apoisocytochromes c, we have exam- suggest that iso-2-cytochrome c has the potential to comprise a ined the steady-state mRNA levels of both isocytochromes c major proportion of cytochrome c under certain physiologic under conditions that have been shown to decrease cyto- conditions that have not been experimentally defined. The chrome c levels. These conditions include the cyc3 and hem] cyc3 mutant, which lacks the ability to attach heme groups to mutations, anaerobic growth, and glucose repression. Al- apocytochromes c, contains both CYCI and CYC7 mRNAs in though some aspects of transcriptional regulation of both normal amounts. Yet, cyc3 mutants contain only apoiso-2-cy- genes have been investigated previously (13-20), we have tochrome c and not apoiso-1-cytochrome c. The lack of accu- chosen to systematically compare the regulation ofthe CYCJ mulation of apoiso-1-cytochrome c in cyc3 mutants, which con- and CYC7 genes. tain CYCI mRNA, suggests that apoiso-1-cytochrome c is ex- Our studies demonstrate that both CYCI and CYC7 tensively regulated by a post-transcriptional process. steady-state mRNA levels were diminished in cells grown anaerobically or under glucose repression, whereas only The total complement of cytochrome c in derepressed cells CYCI mRNA is diminished in hemi strains. However, the of the yeast Saccharomyces cerevisiae is typically composed cyc3 mutation has no effect on either CYCI or CYC7 mRNA of 95% iso-1-cytochrome c and 5% iso-2-cytochrome c (1). levels. Thus, the basis of the differential regulation of the Both iso-1-cytochrome c and iso-2-cytochrome c carry out two apoisocytochromes c in cyc3 strains must be at a post- identical or similar functions in mitochondrial electron trans- transcriptional step. Lastly, glucose derepressions of CYCI port (2). Iso-1-cytochrome c and iso-2-cytochrome c are en- and CYC7 mRNAs are different; the level of CYCI mRNA coded by the unlinked nuclear genes CYCJ and CYC7 (3-6), plateaus by the midlogarithmic growth phase, whereas CYC7 respectively, that are -=80% homologous in their DNA se- mRNA accumulation is initiated and peaks in the late loga- quence (7) and that appear to be evolutionarily related (8). rithmic phase. The biosynthesis of the two isocytochromes c, similar to the biosynthesis of other heme proteins, is decreased by growth MATERIALS AND METHODS in glucose media (catabolite repression) and by growth with Yeast Strains. The following mutant strains were obtained anaerobic conditions (reviewed in ref. 1). However, the for- either from our stock collections or constructed by standard mation of the isocytochromes c from glucose-repressed and genetic methods: DP44-5C (MATa CYCIP CYC7+ hem] lysi anaerobically grown cells shows different kinetics (1). In ad- metl4); DP39-2D (MATa cycl-J CYC7+ hemi lys2 trp2 dition, recent work (9) has shown that the expression of the his-); M15-4A (MATa CYCI+ CYC7+ cyc3-10 lys2); and CYCI and CYC7 genes is affected differently by mutations at M15-4D (MA Ta cycl-J CYC7+ lys2 his] trp2). Normal the following three other loci: hem] (ref. 10), cyc2 (ref. 11), strains included D311-3A (MATa CYCI+ CYC7+ lys2 his] and cyc3 (ref. 11). The hemi locus encodes 8-aminolevulinic trp2), M15-4B (MATa CYCI+ CYC7+ lys2), and D273-10B (MATa CYCJ+ CYC7+). The cycl-J mutation is a deletion the locus and The publication costs of this article were defrayed in part by page charge encompassing entire CYCI adjacent regions payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: ALA, 8-aminolevulinic acid.

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Cells for the glucose derepression study were obtained by overnight growth in 1 liter of YPD medium at 30'C and were harvested at appropriate intervals during the growth cycle. RNA Analysis. Total RNA was isolated, electrophoresed on 1.5% agarose denaturing gels, and transferred to nitrocel- Nick translation lulose paper essentially as described (22). Single-stranded Heat denaturation M13 mp9 phage DNAs with CYC) and CYC7 inserts were used for hybridization probes as schematically illustrated in Fig. 1. The CYCJ probe, denoted mCYC1, contains a 345- * Immobilized specific mRNA base-pair EcoRI to Taq I insert in the M13 mp9, whereas the fO" +( probe + (CYC1 mRNA) CYC7 probe, mCYC7, contains a 349-base-pair Sau3A to (mCYCl) t \- 5'-AM 3' Hpa II insert in the M13 mp9. With the A of the initiating N ATG codon corresponding to nucleotide position 1, the

II mCYC1 insert encompasses nucleotide positions 7 through Hybridization 352 and the mCYC7 insert encompasses nucleotide positions 72 through 277. In all cases, RNA preparations were checked quantitatively by ethidium bromide fluorescence after agarose gel electrophoresis and absorbance at 260 nm. Prehybridization and hybridization were carried out as described (22), with the following modifications. Typically, the probes for the RNA analysis consisted of 0.2-0.3 ,g of unla- 5' AAMAA 3' beled phage mCYC1 or mCYC7 DNA and -0.3 ,tg of dena- tured M13 mp9 replicative form that had been nick-translat- FIG. 1. Schematic representation of the hybridization of a single- stranded M13 with an insert, a nick-translated replicative form (RF) ed to a specific activity of 4 x 107 cpm/,ug with [32P]idCTP. of M13 without an insert, and a mRNA transcribed from a region After overnight hybridization, the filters were washed with encompassing the insert. gentle shaking; four washes were carried out for 5 min each at 55°C in the first wash solution (0.3 M sodium chloride/0.03 (21). The hem) and cyc3-10 mutations used in this study M sodium citrate, pH 7/0.1% NaDodSO4) and six washes were obtained from the mutants GT38-7A (10) and B-614 (8, were carried out for 1 hr each at 55°C in the second wash 9), respectively. solution (15 mM sodium chloride/1.5 mM sodium citrate, pH Culture Media. YPD medium contains 1% yeast extract 7/0.1% NaDodSO4). Under these conditions, there was no (Difco), 2% peptone (Difco), and 2% glucose and is used for significant cross-hybridization between the mCYC1 probe growth of normal strains. YPD + ET medium is YPD medi- and CYC7 mRNA and between the mCYC7 probe and CYC1 um containing ergosterol and Tween 80 (Sigma) as described mRNA. Because the mCYC1 and mCYC7 probes contain (9). YPD + ALA medium is YPD mediurt supplemented inserts of almost identical length, the relative amounts of with ALA (Sigma) as described (9). YP3%D medium is a CYC1 and CYC7 mRNAs can be compared when the same high-glucose medium containing 1% yeast extract, 2% pep- preparation of labeled M13 mp9 is used for hybridization. tone, and 3% glucose. Glucose Determination. The glucose concentration of the Growth Conditions. Glucose-derepressed cells were ob- medium at the time of cell harvesting was determined with tained by overnight growth of yeast in 15 ml of YPD medium the Statzyme Glucose 500 kit from Worthington. at 30°C with vigorous shaking. Strains containing the hem) mutation were grown under the following conditions: YPD + RESULTS ET medium, which allows growth of heme-deficient yeast by The effects of deficiency in heme concentration and in heme providing essential metabolites but does not allow the syn- attachment on CYCJ and CYC7 steady-state mRNA levels thesis of heme; and YPD + ALA medium, which allows were investigated by hybridizing RNA from hem) and cyc3 growth by bypassing the' HEMI mutation and allowing the strains with mCYC1 and mCYC7 probes. These results (Fig. synthesis of heme. Cells were harvested at 5 x 107 cells per 2) demonstrate that the hem) block diminishes CYCI mRNA ml. Glucose-repressed cells were obtained by first growing but not CYC7 mRNA levels and that the cyc3 block does not yeast at 300C in YPD medium to 5 x 107 cells per ml, fol- affect either CYCI or CYC7 mRNA levels. The extent of the lowed by transfer to a 10-fold larger volume of YP3%D medi- CYC1 mRNA deficiency during heme deprivation was fur- um; cells were harvested after 10 min of incubation in the ther investigated by examining a longer exposure of an auto- YP3%D medium at 300C. Yeast were grown anaerobically at radiogram of the gel shown in Fig. 2, which also contained 300C in YPD + ET medium under nitrogen atmosphere. dilutions 'of the wild-type RNA (data not shown). The results Cells were harvested at -5 x 106 cells per ml after the addi- demonstrate that CYCJ mRNA from the hem) mutant grown tion of cycloheximide to a final concentration of0. 13 mg/ml. in YPD + ET medium (lane 3) was decreased by a factor of Probe: mCYC I mCYC7 FIG. 2. RNA blot analysis of CYCI mRNA (lanes 1-4) and CYC7 mRNA (lanes 5-8) in normal and mu- Pertinent genotype: WT cvc3 hem! WT (vc3 /wml8Z1 tant strains. The pertinent genotypes of the following strains were: lane 1, D311-3A (CYCIJ CYC7+ HEMI+ CYC3+); lane 2, M15-4A (CYC1+ CYC7+ 1 2 3 4 5 6 7 8 HEMI+ cyc3-10); lanes 3 and 4, DP44-5C (CYCI+ CYC7+ heml CYC3+Q; lane 5, M15-4D (cycl-l Ac^ CYC7+ HEM)+ CYC3 ); lane 6, DP39-2B (cycl-91 f CYC7+ HEM) + cyc3-10); and lanes 7 and 8, DP39-2D - r (cyci-l CYC7+ hem) CYC3+). ALA and ET (ergosterol and Tween 80) are supplements added to YPD medium to support growth of hem] strains; YPD + Growth medium: YPD YPD YPD YPD YPD YPD YPD YPD ALA medium allows'the formation of heme proteins, +E A + + whereas YPD + ET medium does not (see text). WT, ET AL A ET ALA wild type. Downloaded by guest on October 1, 2021 Genetics: Laz et al. Proc. NatL. Acad. Sci. USA 81 (1984) 4477

Experiment I Experiment 2

k,- r- a -3 raN ("l v_ r Dry weight: - fI N \ - "-t _N N (mg/ml) C- etI e. -

1 2 3 4 5 6 7 8 9 10 11

CYCI mRNA

FIG. 3. RNA blot analysis of CYCJ and CYC7 mRNAs CYC7 mRNA during the growth of strain D273-1OB (CYCI + CYC7+) in 9... YPD medium. Experiments 1 and 2 represent two independent experiments. -200 compared to mRNA from the normal strain (lane 1) or measurements showed that the CYCI mRNA level was di- from hemi mutants grown in YPD + ALA medium (lane 4), minished by a factor of =10, whereas the CYC7 mRNA level which overcomes the block caused by the hemi mutation. was lowered by a factor of -15 after glucose repression (data Fig. 2 (lane 7) demonstrates that heme is not required for not shown). Similarly, the CYCI and CYC7 mRNAs were CYC7 mRNA production. The hemi mutant strains grown in diminished in cells grown anaerobically (Fig. 5, lanes 2 and YPD + ET medium are not diminished in CYC7 mRNA. The 4) when compared to derepressed cells (lanes 1 and 3). The addition of ALA to the growth medium has no effect on the decrease was found to be -1/10th for the CYCJ mRNA and CYC7 mRNA levels in hemi strains (lane 8). Although the -1/20th for the CYC7 mRNA. In addition to the lower level of mRNA in lane 7 is even higher than that in the con- mRNA levels, anaerobically grown cells yielded smaller mo- trol (lane 5), the cells were harvested at a slightly higher cell lecular weight transcripts (lane 5 compared to control lane 4) density and, as described below, there is a dependency of due to degradation during extractions with anaerobically the CYC7 mRNA level on the cell density. Although both grown cells. iso-1-cytochrome c and iso-2-cytochrome c proteins are diminished up to 90% by the cyc3 mutation, this mutation has 100

no significant effect on the mRNA levels of either CYCI or I-- CYC7 (compare lanes 2 and 6 to the WT lanes 1 and 5 in Fig. 2). Thus, in contrast to the results with hemi mutants, the 4 Ai lack of apoiso-1-cytochrome c in cyc3 mutants is not due to 6 C4 z the lack of CYCI mRNA. In addition, the cyc2 mutation has 04 z no effect on CYCI or CYC7 mRNA levels (data not shown). tN Determination of the amounts of CYCI and CYC7 mRNAs I- during the growth of cells in glucose medium (Figs. 3 and 4) and determination of the exhaustion of glucose (Fig. 4) demonstrate that the mRNA levels increase as the glucose concentration diminishes and that both mRNA levels decline after cessation of growth. However, the kinetics of formation and induction of the two mRNAs differ. CYCI mRNA begins to be formed in the early logarithmic phase of growth when glucose is present; the level plateaus in the midloga- I- rithmic growth phase and declines during the stationary 4 growth phase. In contrast, CYC7 mRNA is still repressed to when the CYCJ mRNA level plateaus; the level of CYC7 0 mRNA begins to increase dramatically only when the glu- 04 cose concentration is nearly exhausted during the late logarithmic growth phase. Formation of CYC7 mRNA is com- pleted in about 1 hr when its level is approximately equiva- lent to the level of CYCJ mRNA. The CYC7 mRNA level declines rapidly thereafter in the stationary phase of growth. 6 8 The levels of CYCJ and CYC7 mRNAs were examined in a Time, hr variety of strains grown in glucose medium and grown anaerobically. As shown in Fig. 5, the CYCJ and CYC7 mRNA FIG. 4. Glucose derepression of CYCI and CYC7 mRNA during levels are significantly diminished by glucose repression the growth of strain D273-1OB in YPD medium. The open symbols (lanes 3 and 6), when compared to the derepressed state (a, A) denote data points from experiment 1 and the filled symbols (lanes 1 and 4). Glucose repression of CYC7 mRNA was (e, *) denote data points from experiment 2. (Upper) The percent found in the strain M15-4D in the two CYCI mRNA (0, *) and percent CYC7 mRNA (A, ,) are based on cycle CYC7' (Fig. 5), densitometry scans of the autoradiograms presented in Fig. 3. A sol- CYC1+ CYC7' strains D273-10B (Fig. 5) and D311-3A (data id line has been drawn to reveal the peak in experiment 2. (Lower) not shown), and in the wild-type strain "Fleischmann's The growth of cells is presented as mg of dry weight per ml of the Bakers' yeast" (data not shown). A series of dilutions of culture (o, *). Also shown is the percent glucose in the medium derepressed mRNA were used to estimate the levels of during the growth cycle (A, A). Initially there was 2% glucose in mRNAs after glucose repression and anaerobiosis. These YPD medium. Downloaded by guest on October 1, 2021 4478 Gerietics: Laz et aL Proc. NatL Acad. Sci. USA 81 (1984)

Probe: mCYC I mCYC7

Physiologic condition: 0l No Glc 0 N, GIc FIG. 5. RNA blot analysis of CYCI and CYC7 mRNAs from cells grown under glucose 1 2 3 4 5 6 repression and anaerobiosis. The strains used were D273-10B (CYCI+) (lanes 1-3) and M15- 4D (cycl-1 CYC7+) (lanes 4-6). RNA was iso- _ lated from derepressed cells grown aerobically (02) (lanes 1 and 4), from cells grown anaerobically (N2) (lanes 2 and 5), and from glucose- repressed cells (Glc) (lanes 3 and 6). The blots were probed with mCYC1 (lanes 1-3) or mCYC7 (lanes 4-6). The multiple bands seen in lane 5 are due to degradation that occurs with anaerobically grown cells (see text). DISCUSSION synthesis of catalase T. The precise relationship between glucose, oxygen, and heme in regulating heme proteins still This study, along with previous work (9), describes the ef- remains to be determined. fects of the unlinked genetic mutations hem] and cyc3 and In addition to the differences in the regulation by heme, the physiologic states ofglucose repression and anaerobiosis other heme proteins respond differently to glucose repres- on the levels of mRNAs and apoproteins and holoproteins of sion and anoxia. Unlike iso-1-cytochrome c and iso-2-cyto- iso-1-cytochrome c and iso-2-cytochrome c. The results in- chrome c, the apoproteins of cytochrome b (31), cytochrome dicate that growth in glucose medium and growth with an- c peroxidase, and cytochrome c1 (24) are not diminished in aerobic conditions diminish the steady-state level of CYCI anaerobically grown cells. Also unlike iso-1-cytochrome c and CYC7 mRNAs. For hem] mutants, the level of CYCI and iso-2-cytochrome c, cytochrome P-450 is present in glu- mRNA, but not CYC7 mRNA, is diminished. In the cyc3 cose-grown cells but absent in derepressed respiring cells mutants, the level of CYCJ and CYC7 mRNAs is unaffected, (32). Thus, various heme proteins respond differently to the even though cyc3 strains lack apoiso-1-cytochrome c. Last- induction by heme, the induction by oxygen, and the repres- ly, the formation of CYCJ and CYC7 mRNAs show different sion by glucose. kinetics during glucose derepression. In general, our results Additional differences in the expression of iso-1-cyto- are consistent with the results of others who have also ob- chrome c and iso-2-cytochrome c were revealed with the ex- served decreases in the CYCJ mRNA level by glucose re- amination of cyc3 mutants. Unlike hem] mutants that lack pression (13, 17), anaerobiosis (16), and heme deficiency (16, all heme proteins, cyc3 mutants specifically lack only iso-1- 18) and decreases in the CYC7 mRNA levels by glucose re- cytochrome c and iso-2-cytochrome c. Hemi mutants con- pression (20). tain the CYC7 mRNA and the corresponding apoprotein and Iso-2-cytochrome c and iso-1-cytochrome c represent two lack the CYCI mRNA and the corresponding apoprotein, classes of heme proteins, those whose apoproteins are either whereas cyc3 mutants also contain the CYC7 mRNA and the present or absent, respectively, in heml mutants. The apo- corresponding apoproteins but contain the CYCI mRNA and proteins unaffected by heme deficiency in addition to iso-2- lack the corresponding apoprotein. Therefore, under condi- cytochrome c include the following: cytochrome c peroxi- tions in which the apoprotein is not processed, either CYC) dase (23); cytochrome cl (24); cytochrome b (23); cyto- mRNA is not translated or apoiso-1-cytochrome c is selec- chrome oxidase subunits II, III, and VII (23); and several tively degraded. proteins of complex III (25). On the other hand, apoproteins Determination of the levels of CYCI and CYC7 mRNAs and the corresponding mRNAs of iso-1-cytochrome c, cata- during the growth cycle demonstrated that transcription of lase T, and catalase A (16, 26, 27) are absent in hemi mu- CYC7 mRNA is more sensitive to glucose repression than tants. There is a relationship between glucose repression, transcription of CYCJ mRNA (Figs. 4 and 5). It remains to anaerobiosis, and heme deficiency, the three parameters be determined whether this difference is due to two similar that play a role in the regulation of the heme proteins repre- regulatory regions with different sensitivities or to entirely sented by iso-1-cytochrome c. The heme biosynthetic path- different regulatory mechanisms. These measurements also way contains enzymes whose synthesis is sensitive to glu- reveal that the steady-state levels of CYC) and CYC7 mRNA cose repression and anoxia (28, 29). Also, anaerobic experi- were approximately equal during a short period in the late ments are usually conducted with cells grown in glucose logarithmic phase of growth. This brief increase of CYC7 medium. Thus, the glucose regulation and anaerobic regula- mRNA has been consistently observed in a variety of normal tion of iso-1-cytochrome c and certain other heme proteins strains if numerous measurements are taken from the late could be reduced to the single parameter of heme concentra- logarithmic phase of growth through the stationary phase of tions. In fact, Guarente and Mason (18) have uncovered a growth. The similarity in the amount of CYCJ and CYC7 site upstream from the CYC1 gene that mediates heme regu- mRNAs was unexpected because iso-2-cytochrome c consti- lation on CYCJ transcription. However, it is not altogether tutes only -5% of the total cytochrome c complement in de- clear that the heme deficiencies can explain the lack of heme repressed cells, even when measurements are made through- proteins in cells grown in glucose medium or grown anaero- out the growth cycle (1). Apparently the high level of CYC7 bically. Heme biosynthesis is lowered but not eliminated mRNA is not reflected in a corresponding high level of iso-2- during glucose repression and anaerobiosis (28, 29). In addi- cytochrome c because the period may be too brief for com- tion, regulation by glucose and oxygen may be independent plete translational expression. This high level of CYC7 processes because cells grown anaerobically with derepress- mRNA suggests a possible functional role for iso-2-cyto- ing conditions of limiting glucose are still deficient in heme chrome c. While CYC7 mRNA and the corresponding iso-2- proteins (30). Also, Hortner et al. (16) have presented evi- cytochrome c are low in repressed and anaerobically grown dence indicating that heme deficiency, glucose repression, cells and are relatively low in completely derepressed cells, and anoxia may be separate regulatory conditions for the it is possible that prolonged growth in the physiologic condi- Downloaded by guest on October 1, 2021 Genetics: Laz et aL Proc. NatL Acad Sci. USA 81 (1984) 4479

tions represented by the brief period in late logarithmic 4. Lawrence, C. W., Sherman, F., Jackson, M. & Gilmore, R. A. phase of growth could result in cells with high amounts of (1975) Genetics 81, 615-629. iso-2-cytochrome c. In fact, we previously reported that cer- 5. Downie, J. A., Stewart, J. W., Brockman, N., Schweingruber, tain batches of commercially grown Fleischmann's Bakers' A. M. & Sherman, F. (1977) J. Mol. Biol. 113, 369-384. 6. Sherman, F., Stewart, J. W., Helms, C. & Downie, J. A. yeast contained large amounts of iso-2-cytochrome c, even (1978) Proc. Nati. Acad. Sci. USA 75, 1437-1441. though this strain behaved identically to defined laboratory 7. Montgomery, D. L., Leung, D. W., Smith, M., Shalit, P., strains when carefully examined after various growth condi- Faye, G. & Hall, B. D. (1980) Proc. Nati. Acad. Sci. USA 77, tions (1). Although high amounts of iso-2-cytochrome were 541-545. not observed in Fleischmann's Bakers' yeast grown with 8. McKnight, G. L., Cardillo, T. & Sherman, F. (1981) Cell 25, various laboratory conditions and although we were unable 409-419. to mimic the conditions of the commercially grown yeast, 9. Matner, R. R. & Sherman, F. (1982) J. Biol. Chem. 257, 9811- there appears to be a certain physiologic state that results in 9821. high levels of iso-2-cytochrome c; this state may reflect the 10. Woods, R. A., Sanders, H. K., Briquet, M., Foury, F., Drys- dale, B.-E. & Mattoon, J. R. (1975) J. Biol. Chem. 250, 9090- high levels of CYC7 mRNA observed for a brief period in 9098. growth cycle, a transitory state of incomplete glucose dere- 11. Rothstein, R. J. & Sherman, F. (1980) Genetics 94, 871-889. pression. 12. Gollub, E. G., Liu, K., Dayan, J., Aldersberg, M. & Sprinson, In addition, this transitory high level of CYC7 mRNA re- D. B. (1977) J. Biol. Chem. 252, 2846-2854. sembles the high level of CYC7 mRNA in certain CYC7 mu- 13. Zitomer, R. S. & Hall, B. D. (1976) J. Biol. Chem. 251, 6320- tants that overproduce iso-2-cytochrome c. The dominant 6326. CYC7-H2 and CYC7-H3 mutants and certain recessive cyc8 14. Zitomer, R. S., Montgomery, D. L., Nichols, D. L. & Hall, cyclO, cyc9 cyclO, and cyclO cycli double mutants contain B. D. (1979) Proc. Natl. Acad. Sci. USA 76, 3627-3631. high levels of CYC7 mRNA and iso-2-cytochrome c that are 15. Guarente, L. & Ptashne, M. (1981) Proc. Natl. Acad. Sci. USA 78, 2199-2203. similar to the normal levels of CYCI mRNA and iso-1-cyto- 16. Hortner, H., Ammerer, G., Hartter, E., Hamilton, B., Rytka, chrome c, respectively (33). These CYC7-H2 and CYC7-H3 J., Bilinski, T. & Ruis, H. (1982) Eur. J. Biochem. 128, 179- mutants contain gross alterations in the 5'-noncoding region 184. of the CYC7 locus but apparently have CYC7 transcripts 17. Montgomery, D. L., Boss, J. M., McAndrew, S. J., Marr, L., with normal start sites. It is tempting to suggest that these Walthall, D. A. & Zitomer, R. S. (1982) J. Biol. Chem. 257, CYC7 overproducers are continuously producing a maximal 7756-7761. rate of transcription similar to the rate of transcription ob- 18. Guarente, L. & Mason, T. (1983) Cell 32, 1279-1286. served with the normal CYC7' strains during a brief transi- 19. Lowry, C. V., Weiss, J. L., Walthall, D. A. & Zitomer, R. S. tory state. (1983) Proc. NatI. Acad. Sci. USA 80, 151-155. 20. Wright, C. F., Walthall, D. A., Boss, J. M. & Zitomer, R. S. The CYCJ and CYC7 genes are reminiscent of several oth- (1983) Curr. Genet. 7, 117-122. er duplicated genes that encode similar proteins with pre- 21. Singh, A. & Sherman, F. (1978) Genetics 89, 653-665. sumably identical functions but that exhibit different regula- 22. Zaret, K. & Sherman, F. (1982) Cell 28, 563-573. tory responses. For example, the two alcohol dehydrogen- 23. Saltzgaber-Muller, J. & Schatz, G. (1978) J. Biol. Chem. 253, ases, ADH I and ADH II, are encoded by the two genes 305-310. ADCJ and ADR2, respectively, which are 90% homologous 24. Ross, E. & Schatz, G. (1976) J. Biol. Chem. 251, 1997-2004. (34, 35). ADR2 mRNA is repressed by glucose, whereas 25. Lin, C. P., Gollub, E. & Beattie, D. (1982) Eur. J. Biochem. ADCJ mRNA is induced in an opposite manner (36). Similar- 128, 309-313. ly the two enolase polypeptides, enolase 1 and enolase 2, are 26. Woloszczuk, W., Sprinson, D. B. & Ruis, H. (1980) J. Biol. encoded by the two genes and respectively, Chem. 255, 2624-2627. ENO] ENO2, 27. Richter, K., Ammerer, G., Hartter, E. & Ruis, H. (1980) J. which are 95% homologous (37). It appears as if ENO] Biol. Chem. 255, 8019-8022. expression is unaffected by growth in various media, where- 28. Labbe-Bois, R. & Labbe, P. (1978) in Biochemistry and Genet- as EN02 is induced by growth in glucose medium (38). Also, ics of Yeasts, eds. Bacila, M., Horecker, B. & Stoppani, A. PHOS codes for a phosphate repressible acid phosphatase (Academic, New York), pp. 97-117. and PH03 codes for a constitutive acid phosphatase (39). 29. Lukaskiewicz, J. & Bilinski, T. (1979) Acta Biochim. Pol. 26, Thus, iso-1-cytochrome c and iso-2-cytochrome c, as well as 161-169. other pairs of duplicated genes in yeast, may have evolved 30. Lowdon, M. S., Gordon, P. A. & Stewart, P. R. (1972) Arch. different regulatory properties in addition to different struc- Mikrobiol. 85, 355-361. 31. Clejan, L., Beattie, D. S., Gollub, E. G., Liu, B. K. P. & tural characteristics in response to nutritional variations. Sprinson, D. B. (1980) J. Biol. Chem. 255, 1312-1316. The underlying selection pressures could have results not 32. Karenlampi, S. O., Martin, E. & Hanninen, 0. 0. P. (1981) only in subtle differences in enzymic properties but also in Biochem. J. 194, 407-413. differences in the pattern of regulation that could not be 33. Kosiba, B. E., Errede, B., Cardillo, T. S. & Sherman, F. readily evolved from a single gene with a single regulatory (1982) Recent Adv. Yeast Biol. 1, 156-172. region. 34. Bennetzen, J. L. & Hall, B. D. (1982) J. Biol. Chem. 257, 3018-3025. This investigation was supported in part by Public Health Service 35. Young, T., Williamson, V., Taguchi, A., Smith, M., Sled- Grant R01 GM12702 and Training Grants T32 GM07102 and T32 ziewski, A., Russell, D., Osterman, J., Denis, C., Cox, D. & GM07098 from the National Institutes of Health and in part by U.S. Beier, D. (1982) in Genetic Engineering ofMicroorganisms for Department of Energy Contract DE-AC02-76EV03490 at the Uni- Chemicals, eds. Hollaender, A., DeMoss, R. D., Kaplan, S., versity of Rochester, Department of Radiation Biology and Bio- Konisky, J., Savage, D. & Wolf, R. S. (Plenum, New York), physics. This paper has been designated report no. UR-3490-2356. pp. 335-361. 36. Denis, C. L., Ferguson, J. & Young, E. T. (1983) J. Biol. 1. Sherman, F. & Stewart, J. W. (1971) Annu. Rev. Genet. 5, Chem. 258, 1165-1171. 257-296. 37. Holland, M. J., Holland, J. P., Thill, G. P. & Jackson, K. A. 2. Mattoon, J. R. & Sherman, F. (1966) J. Biol. Chem. 241, 409- (1981) J. Biol. Chem. 256, 1385-1395. 419. 38. McAlister, L. & Holland, M. J. (1982) J. Biol. Chem. 257, 3. Sherman, F., Stewart, J. W., Margoliash, E., Parker, J. & 7181-7188. Campbell, W. (1966) Proc. Nati. Acad. Sci. USA 55, 1498- 39. Rogers, D. T., Lemire, J. M. & Bostian, K. A. (1982) Proc. 1504. Natl. Acad. Sci. USA 79, 2157-2161. Downloaded by guest on October 1, 2021