Current Genetics (1984) 8:45-48

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© Springer-Verlag 1984

Regulation of transcription of the Saccharomyces cerevisiae CYC1 gene: Identification of a DNA region involved in heme control Rosmarie Gudenusl, Andrew Spence2, Andreas Hartig 1, Michael Smith 2 , and Helmut Ruisl 1 Institut fiir Allgemeine Biochemie der Universit~it Wien, Ludwig Boltzmann-Forschungsstelle ftir Biochemie, W~ihringer Str. 38, A-1090 Wien, Austria 2 Department of Biochemistry, Faculty of Medicine, The University of British Columbia, 2146 Health Sciences Mall, Vancouver, B.C., Canada V6T 1W5

Summary. A Saccharornyces cerevisiae mutant (hem1 cycl-1) was transformed with plasmids bearing a chromosomal centromer (CEN3) and a 2 /lm DNA replication origin. In one of the plasmids a functional CYC1 gene was present, in a second plasmid an XhoI fragment located between bases -245 and -678 upstream from the translation initiation codon had been deleted, in a third plasmid this region had been inverted. Results of hybridization experiments carried out with mRNA isolated from heme-deficient and heme-containing transformants indicated that heme controls transcription of the CYC1 gene and that DNA sequences located within the upstream XhoI fragment are involved in activation of the gene by heme. Key words: Iso-l-cytochrome c - S a c c h a r o m y c e s cerevisiae - Heme - Transcription

Introduction Regulation of gene expression in the yeastSaccharomyces cerevisiae is currently being widely studied since it is considered a model system especially suitable for the investigation of general principles of eukaryotic gene regulation. One reason for the great interest in this system is that it can be analyzed more easily than other eukaryotes. Yeast genes can be isolated by yeast transformation (Hinnen et al. 1978), they can be subjected to mutagenesis in vitro and reintroduced into yeast cells where any effects of mutations can then be tested in vivo (see e.g. Botstein and Davis 1982). This kind of approach is used to analyze the regulation of expression

Offprint requests to." H. Ruis

of individual genes or of groups of genes that are under coordinate control. Among the genes suitable for this type of analysis are those coding for hemoproteins of S. cerevisiae. It has been demonstrated that at least some hemoprotein genes, those coding for iso-l-cytochrome c, catalase T and catalase A, are coordinately controlled by glucose, oxygen and heme (H6rtner et al. 1982). Comparison of regulatory elements of these genes should therefore contribute to our understanding of mechanisms involved in coordinate control of eukaryotic genes. The CYC1 gene, which codes for the main cytochrome c protein of S. cerevisiae, iso-l-cytochrome c, has already been studied more extensively than other hemoprotein genes of yeast. It has been shown to be under transcriptional control by glucose (Zitomer et al. 1979), and first attempts have been reported to identify upstream regulatory regions involved in glucose control (Guarente and Ptashne 1981). More recently, regulation of the gene by heme and oxygen has been demonstrated (H6rtner et al. 1982), and the existence of an upstream region involved in oxygen control has been reported (Lowry et al. 1983). The experiments described in this paper have been carried out to investigate the control of the CYC1 gene by heme. The results obtained indicate that a DNA region upstream from the transcription start of the CYC1 gene is necessary for its activation by heine. While this work was in progress, results, which are in line with the findings reported in this paper, have been published by Guarente and Mason (1983).

Materials and methods Strain RG3-13B (a trpl ura3 leu2.3 leu2-!12 heml (formerly ole3) cycl.1) was derived from a cross of strains GM-3C-2

R. Gudenus et al.: Heme control of transcription of yeast CYC1 gene

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(~ trpl his4 leu2-3 leu2-112 cycl-1 cycT) (Faye et al. 1981) and Mcx11-M3 (a hem1 lysl), which had been obtained as a segregant from a cross of strains DczHI-IB (a leul hem1) (Richter et al. 1980) and D585-11C (a lysl). Cells were grown on synthetic complete medium (Sherman et al. 1971) containing 1% glucose, Tween 80 (2.6 g/l) and ergosterol (12 rag/l) in the presence or absence of 6-aminolevulinate (50 rag/l). Medium deficient in leucine was used for growth of transformed strains. Cells were ahvays harvested when the glucose concentration in the medium had decreased to 0.2%. Glucose concentration was monitored with Diastix (Ames Laboratories) test strips (Cross and Ruis 1978). Yeast strains were transformed with plasmids essentially as described by Beggs (1978). Total yeast RNA was isolated as previously described (Richter et al. 1980). Poly A+-RNA was prepared from total RNA by oligo(dT)cellulose chromatography (Aviv and Leder 1972). Agarose gel etectrophoresis of RNAs on gels containing formaldehyde was carried out as described by Maniatis et al. (1982). Transfer of RNAs to nitrocellulose and hybridization with DNA 32p-labelled by nick translation (Maniatis et al. 1975) was carried out according to Thomas (1980). Plasmids pYeCYCI(2.5) (Faye et al. 1981) and pYeCYC1 (0.60) (Smith et al. 1979) were kindly provided by Dr. B. D. Hall. Plasmid pYeCEN3(41) (Clarke and Carbon 1980) was generously donated by Dr. J. Carbon. Plasmid pYA301 (Langford et al. t983) constructed by inserting a 3.4 kb BamHI-EcoRI fragment containing the yeast actin gene into plasmid pBR322 was kindly given to us by Dr. D. Gallwitz. Plasmid DNAs were amplified in Escheriehia coli strains HB101 or RR1 and were isolated by the quick method of Ferguson et al. (1981) or over CsC1 gradients as described by Ish-Horowicz and Burke (1981). Total yeast DNA to be used for hybridization according to Southern (1975) was isolated by the procedure of Wagner and Fangman (in preparation). Restriction enzymes were purchased from BRL, Neu-Isenburg, Germany. Restriction endonuclease digestions were carried out as recommended by the supplier. Eleetrophoresis of DNA fragments was carried out as described by Montgomery et al. (1978). DNA fragments were isolated from agarose gels according to Dretzen et al. (1981) or by the method of Langridge et al. (1980). Cytochrome spectra of yeast cells frozen in liquid nitrogen were recorded in a Beckman DU-8 spectrophotometer. EP

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Results and discussion The plasmids used in this study were derived from pYeCYCI(2.5), which carries the CYC1 gene on a 2.5 kb BamHI-HindlII fragment (Faye et al. 1981). Digestion of this plasmid with XhoI produced two fragments of 6 kb and 433 bp, which were separated on agarose gels. Plasmid pYeCYC1A245/678 was constructed by religating the 6 kb fragment. To construct pYeCYC1i245/678, which carries the small XhoI fragment in inverted orientation, the 6 kb fragment was ligated in the presence of a 5-fold molar excess of the 433 bp fragment. Both ligation mixes were used to transform E. coli RR1. The parental plasmid and each of its derivatives were digested with BamHI and HindlII to release a 2.1 or 2.5 kb fragment carrying CYC1. Each of the three fragments was inserted into the yeast-E, coli shuttle vector Y E p l 3 (Broach et al. 1979). To allow the resulting plasmids to be maintained in yeast at a low copy number, a 2.2 kb BamHI-BgllI fragment carrying the yeast centromere CEN3 was inserted at the BamHI site of each plasmid. The centromere fragment was purified from plasmid pYeCEN3(41) (Clarke and Carbon 1980). Plasmids were isolated from several ampicillin resistant transformants of E. coli strain RR1, and those carrying CEN3 were identified by restriction analysis. The relative orientation of CEN3 and CYC1 was the same in each of the plasmids chosen for this study: YC13CYCI(2.5), YC13CYCl(A245/678) and YC13CYCl(i245/678). The structures of these plasmids are illustrated in Fig. 1. Strain RG3-13B was used as a recipient for the three plasmids containing CYC1 gene sequences. This strain carries the hem1 mutation and a large deletion in the chromosomal CYC1 region. It accumulates no transcripts hybridizing to a CYC1 probe (Fig. 2, Table 1). The h e m l mutation causes deficiency in the first enzyme of the heine biosynthetic pathway, 6-aminolevulinate

X E

(B/Bg)H

P

YC13CYC1(A 2 4 5 1678)

YC13CYC1(i2451678) B

kb 0

X F 1

H 2

B kb 0

XXmX E 1

2

H

Fig. 1. Plasmid constructions containing CYC1 gene sequences. The plasmids shown were derived from pYeCYCI(2.5), YEP13 and pYeCEN3(41). B: BamHI, Bg: BglII, E: EeoRI, H: HindIII, X: XhoI, Xm: XmaI, P: PstI

R. Gudenus et al.: Heme control of transcription of yeast CYC1 gene

47

J

4 5 6

?

8

9

Fig. 2. Hybridization of poly A+-enriched RNA with CYC1 DNA. Poly A+-enriched RNAs from yeast ceils were separated by agarose gel electrophoresis (1.1% gels), transferred to nitrocellulose and hybridized with a nicktranslated CYC1 fragment isolated from plasmid pYeCYCI(0.60). RNA from: lanes 1, 2: Strain RG3-13B transformed with plasmid YC13CYCI(2.5); 3, 4: RG3-13B transformed with YC13CYCl(~245/678); 5, 6: RG3-13B transformed with YC13CYC1 (i245/678); 7, 8: untransformed RG3-13B; 9, 10: strain DczHI-IB. Lanes 1, 3, 5, 7, 9: cells grown in the presence of 6-aminolevulinate (50 rag/l); lanes 2, 4, 6, 8, 10: cells grown in the absence of 8-aminolevulinate. Arrow: position of iso-l-cytochrome c mRNA Table 1. Levels of iso-l-cytochrome c mRNA in strains transformed with CYCl-plasmids. Poly A+-en!:iched RNAs were separated by agarose gel electrophoresis, transferred to nitrocellulose and probed with the nick-translated CYC1 fragment isolated from plasmid pYeCYC1 (0.60). To correct for differences in amounts of total mRNAs in the preparations, control hybridizations were carried out with plasmid pYA301 and values obtained by scanning of autoradiograms were corrected assuming that equal amounts of actin mRNA are present in all transformants Strain

Plasmid

6-amino- Relative levulin- level of ate iso-l-cytochrome c mRNA

RG3-13B RG3-13B RG3-13B RG3-13B RG3-13B RG3-13B RG3-13B RG3-13B DczHI-IB DczHI-IB

YC13CYC1 (2.5) YC13CYC1 (2.5) YC13CYC1 (/,245/678) YC13CYC1 (zx245/678) YCl 3CYCl (i245/678) YC13CYCl (i245/678)

+

a Arbitrarily set to 100

-

+ -

+ +

100 a 2.1 3.6 10.9 1.3 25.7 0

-

0

+

155.2

-

2.1

10

530

570 nm

Fig. 3. Cytochrome spectia of yeast cells used for RNA isolation. Aliquots of cells used for RNA isolation were frozen in liquid nitrogen and cytochrome spectra were recorded in a Beckman DU-8 spectrophotometer. 1, 2: Strain RG3-13B transformed with YC13CYCI(2.5); 3, 4: RG313B transformed with YC13CYC1 (•245/678); 5, 6: RG3-13B transformed with YC13CYC1(i245/ 678); 7, 8: untransformed RG313B; 9, 10: strain DczHI-IB. Lanes 1, 3, 5, 7, 9: cells grown in the presence of 8-animolevulinate (50 mg/liter); lanes 2, 4, 6, 8, 10: cells grown in the absence of 6aminolevulinate

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synthase. Mutant cells grown in the absence of heme or heme precursor do n o t produce any mature cytochromes. A c c u m u l a t i o n of cytochromes is, however, observed when culture media are supplemented with ~-aminolevulinate (Fig. 3). As demonstrated in Fig. 3, all strains used in this investigation lacked cytochromes completely when grown in the absence of heme precursor. When they were supplemented with ~-aminolevulinate, significant a c c u m u l a t i o n of cytochrome c was observed in strain D c z H I - I B , a hem1 m u t a n t carrying a wild type CYC1 gene at its normal chromosomal location, and in strain RG3-13B transformed with a plasmid c o n t a i n i n g the complete CYC1 gene, b u t n o t in u n t r a n s f o r m e d RG3-13B or in cells transformed with plasmids bearing a deletion or an inversion of the 5'-upstream 433 basepair XhoI fragment. Cytochrome spectra shown in Fig. 3 were recorded using aliquots of cells used for RNA isolation. Results o b t a i n e d b y " N o r t h e r n " h y b r i d i z a t i o n of poly A+-enriched RNAs isolated from strain D c z H I - I B , from strain RG3-13B and from RG3-13B transformed with different plasmids (Fig. 1), are summarized in Fig. 2 and Table 1. Transformants bearing the CYC1 gene with u n m o d i f i e d upstream sequences have levels of iso1-cytochrome c m R N A very similar to those o f cells bearing the chromosomal wild type allele of the gene. Compared to heme-deficient cells, an approximately 50 fold stimulation of iso-l-cytochrome c m R N A levels

48 was observed in such cells when they are supplemented with heme precursor. When the XhoI fragment located in the 5'-upstream region of the gene is deleted, heme stimulation of transcription is completely lost. In the experiment illustrated in Fig. 2 and Table 1, the level of CYC1 transcript was even somewhat higher in cells grown in the absence of heme. Since this effect was not observed in other experiments its significance is questionable. Cells transformed with the plasmid containing an inverted XhoI fragment also have a low level of iso-l-cytochrome c m R N A when they are grown in the presence of ~-aminolevulinate. The amount of this transcript is, however, reproducibly increased when these cells are cultivated in the absence of heme. Similar results were obtained in hybridization experiments with total RNA instead o f poly A+-enriched RNA (data not shown). The data obtained indicate that the upstream XhoI fragment is involved in transcriptional control by heme. To exclude the possibility that instability of the CEN3 or CYC1 regions o f the plasmids during cultivation of the transformants might have caused differences in expression of the CYC1 gene a control experiment was carried out. Total DNA was isolated from aliquots of the cells used for RNA isolation, the DNA preparations were digested with HindlII and with HindIII plus BamHI and Southern hybridizations were carried out with a labelled CYC1 probe. No indication of any alteration within the CYC1 Or CEN3 regions of the plasmids was found (results not shown). In summary, the results of our investigation are strong evidence for transcriptional control of the CYC1 gene b y heme. They further indicate that a DNA region located between the two XhoI sites 245 and 678 basepairs upstream from the translation initiation codon of the gene is necessary for activation of the gene b y heme. Using a similar approach, Lowry et al. ( 1 9 8 3 ) h a v e recently obtained evidence for involvement of t h e region bounded b y the two XhoI sites in oxygen regulation of the gene. Further studies are required to clarify whether heme and oxygen control are mediated b y a single DNA element or whether separate regulatory regions exist. It must be emphasized in this context that the possibility cannot be ruled out from the data presently available that oxygen control is exclusively caused b y low heme concentrations in yeast cells grown anaerobically (Lukaskiewicz and Biliriski 1979). Since yeast genes coding for two other hemoproteins controlled b y heme, catalase T and catalase A, have recently been cloned (Spevak et al. 1983; G. Cohen and H. Ruis, unpublished results) it will be of particular interest to identify DNA elements involved in heme control of these genes and to compare the sequences and positions of control regions of the three heme-regulated genes.

R. Gudenus et al.: Heme control of transcription of yeast CYC1 gene

Acknowledgements. This work was supported by grants from the Fonds zur F6rderung der wissenschaftlichen Forschung (Vienna), the ~sterreicbische Forschungsgemeinschaft (Vienna) and by the Medical Research Council of Canada. M. S. is a Career Investigator of the Medical Research Council of Canada. A. S. acknowledges support by the Natural Science and Engineering Research Council of Canada.

References Aviv H, Leder P (1972) Proc Natl Acad Sci USA 69:1408-1412 Beggs JD (1978) Nature 275:104-109 Botstein D, Davis RW (1982) Principles and practice of recombinant DNA research with yeast. In: The molecular biology of the yeast Saccharomyces, metabolism and gene expression. Cold Spring Harbor Laboratory, p 607 Broach JR, Strathern JN, Hicks JB (1979) Gene 8:121-133 Clarke L, Carbon J (1980) Nature 287:504-509 Cross HS, Ruis H (1978) Mol Gen Genet 166:37-43 Dretzen G, Bellard M, Sassone-Corsi P, Chambon P (1981) Anal Biochem 112:295-298 Faye G, Leung DW, Tatchell K, Hall BD, Smith M (1981) Proc Natl Acad Sci USA 78:2258-2262 Ferguson J, Groppe JC, Reed SI (1981) Gene 16:191-197 Langford C, Nellen W, Niessing J, Gallwitz D (1983) Proc Natl Acad Sci USA 80:1496-1500 Guarente L, Mason T (1983) Cell 32:1279-1286 Guarente L, Ptashne M (1981) Proc Natl Acad Sci USA 78:21992203 Hinnen A, Hicks JB, Fink GR (1978) Proc Natl Acad Sci USA 75:1929-1933 H6rtner H, Ammeter G, Hartter E, Hamilton B, Rytka R, Bilifiski T, Ruis H (1982) Eur J Biochem 128:179-184 Hsiao CL, Carbon J (1981) Gene 15:157-166 Ish-Horowicz D, Burke JF (1981) Nucl Acid Res 9:2989-2998 Langridge J, Langridge P, Bergquist PL (1980) Anal Biochem 103:264-271 Lowry CV, Weiss JL, Walthall DA, Zitomer RS (1983) Proc Natl Acad Sci USA 80:151-155 Lukaskiewicz J, Bitifiski T (1979) Acta Biochim Pol 26:161169 Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, p. 202 Maniatis T, Jeffrey A, Kleid DG (1975) Proc Natl Acad Sci USA 72:1184-1188 Montgomery DL, Hall BD, Gillam S, Smith M (1978) Cell 14: 673-680 Richter K, Ammerer G, Hartter E, Ruis H (1980) J Biol Chem 255:8019-8022 Sherman F, Fink GR, Lawrence CW (1971) Methods in yeast genetics. Cold Spring Harbor Laboratory Smith M, Leung D, Gillam S, Astell C, Montgomery D, Hall B (1979) Cell 16:753-761 Southern EM (1975) J Mol Biol 98:503-517 Spevak W, Fessl F, Rytka J, Traczyk A, Skoneczny M, Ruis H (1983) Mol Celt Biol (in press) Thomas PS (1980) Proc Natl Acad Sci USA 77:5201-5205 Zitomer RS, Montgomery DL, Nichols DL, Hall BD (1979) Proc Natl Acad Sci USA 76:3627-3631 Communicated by R. J. Schweyen Received July 12, 1983

Regulation of transcription of the Saccharomyces cerevisiae CYC1 gene: Identification of a DNA region involved in heme control.

A Saccharomyces cerevisiae mutant (hem1 cycl-1) was transformed with plasmids bearing a chromosomal centromer (CEN3) and a 2 μm DNA replication origin...
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