Gene, 105 (1991) 129-134 0 1991 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119/91/$03.50 129 GENE 06042
`
`Glucose-responsive and oleic acid-responsive elements in the gene encoding the peroxisomal trifunctional
`enzyme of
`
`Candida tropicalis (Gene regulation; b-oxidation; oleic acid; heterologous expression; yeast; hydratase-dehydrogenase-epimerase; recombinant DNA;
`
`Saccharomyces cerevisiae)
`
`Department of Biochemistry, Mckfaster University, Hamilton, Ontario, LSN 325 (Canada)
`
`Candida
`
`tropicalis.
`
`HDE
`cerevisiae
`Saccharomyces
`gene has allowed for determination of regions responsible for the control of expression of the
`HDE
`
`tropicalis
`has identified possible consensus nt sequences for glucose- and oleic acid-responsive upstream elements in these genes. The regulation of the
`HDE
`cerevisiae
`closely resembles that found in C.
`tropicalis,
`
`encoding HDE; kb, kilobase or 1000 bp; nt, nucleotide(s); SDS, sodium dodecyl sulfate; u, unit(s); YNE, see Fig. I legend. tion enzymes when these yeasts are grown in carbon sources such as oleic acid (reviewed by Lazarow and Fujiki, 1985; Borst, 1986). We are interested in the mechanisms of regulation of the gene encoding the peroxisomal trifunc- tional /?-oxidation enzyme of C.
`
`tropicalis
`and S.
`cerevisiae
`are excellent model systems for the study of peroxisome biogenesis. Peroxisomes proliferate and there is a marked increase in the specific activities of the /Soxida-
`
`Correspondence 10:
`
`Dr. R.A. Rachubinski, Department of Biochemistry, McMaster University, 1200 Main Street West, Hamilton, Ontario, L8N 325 (Canada) Tel. (416)525-9140, ext. 2428; Fax (416)522-9033. Abbreviations: AOX, acyl-CoA oxidase;
`
`AOX,
`gene encoding AOX; bp, base pair(s); C.,
`CATL, gene
`Cundida;
`HDE, gene
`encoding CATL; HDE, trifunctional enzyme;
`
`Received by G.C. Shore: 10 June 1991 Accepted: 18 June 1991 Received at publishers: 11 July 1991 SUMMARY We have investigated the regulation of expression of the gene (HDE), encoding the peroxisomal trifunctional enzyme hydratase-dehydrogenase-epimerase (HDE), of the diploid yeast
`gene. Expression was monitored by immunoblot analysis of yeast lysates with anti-HDE serum. Regions have been identified that are responsible for both repression by glucose and induction by oleic acid. A glucose-responsive region lies between nucleotides (nt) -526 and -393. An oleic acid-responsive region lies between nt -393 and -341. An additional region controlling derepression by nonfermentable carbon sources is located downstream from nt -341. Comparison of the nt sequences of these regions to upstream regions of other oleic acid-responsive genes of C.
`suggesting that similar mechanisms of transcriptional control operate in both yeasts. INTRODUCTION Peroxisomes have many metabolic functions, notably the /?-oxidation of fatty acids. Yeasts such as C.
`has been instrumental in studying peroxisome biogenesis and function, genetic work with this species has been hampered by the lack of a transformation system and by the asexual, diploid nature of the yeast (Rachubinski, 1990). The amenability of S.
`
`tropicalis,
`HDE. Although C.
`tropicalis
`
`cerevisiae
`
`to genetic manipula- tion and the fact that peroxisomes proliferate when S.
`cerevisiae
`is grown on oleic acid (Veenhuis et al., 1987) suggested that S.
`cerevisiae
`could act as an heterologous expression system for studying the regulation of the
`HDE
`gene. In our experimental system, the HDE polypeptide could be detected immunologically by a polyclonal anti- serum that did not cross-react with the endogenous tri-
`
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`
`James A. Sloots, John D. Aitchison and Richard A. Rachuhinski
`Heterologous expression in
`of constructs containing deletions in the upstream region of the
`gene in S.
`CATL, catalase;
`

`

`130 functional enzyme of S.
`
`cerevisiue.
`
`HDE
`
`tropicalis HDE
`
`cerevisiae
`
`tropicalis
`
`expression (35fold and 2%fold, respectively) and repressed, although to a lesser extent,
`
`CATL
`(7.6-fold and 4.9-fold, respectively) and
`AUX
`
`HUE
`
`HDE, CATL
`
`AUX
`
`tr~p~i~aiis.
`
`AOX
`
`gene was expressed under the control of its own promoter and functioned as its own reporter. The aim of the present study was to dissect the regulatory region of a gene encoding a yeast peroxisomal protein and to show that C.
`and that its expression is subject to induction by growth in oleic acid. EXPERIMENTAL AND DISCUSSION (a) Expression of genes encoding HDE, acyl-CoA oxidase and catalase in C~~~i~~ ~~o~icu~is Lysates prepared from C.
`cells grown on differ- ent carbon sources were analyzed by immunoblotting to assess the effects of carbon source on expression of the genes encoding HDE, AOX and CATL. Although Tween-40 was only needed for the solubili~ation of oleic acid, it was nevertheless added as an invariant component to all media. The addition of glucose to both high and low concentrations in YNE greatly repressed
`(IO-fold and 7%fold, respectively) expression (Fig. 1, compare lanes - with lanes GS and GU.2, respec- tively). These results suggest that glucose repression and derepression play an important role in the regulation of expression of
`4.5fold (Fig. 1, compare lanes G0.20”.2 with lanes GO.Z), demonstrating an induction of the expression of these genes by oieic acid. High levels of expression were seen for all three genes in YNE (contains only Tween-40) (Fig. 1, A and B, lanes -), indicating that Tween-40 can be metabo- lized by the @oxidation pathway and used as a carbon source in C.
`expres- sion 1.4-fold, and AOX expression 1.3-fold (Fig. 1, com- pare lanes O* with lanes -). Taken together, the results indicate that oleic acid and Tween-40 probably act via different mechanisms.
`(1. l-fold) and A OX (1.2-fold) expression was higher when cells were grown in gfycerol-ethanol as opposed to growth in low glucose (Fig. 1, compare Ianes GY2E2 with G0.2), demon- strating that a nonfermentable carbon source can influence the level of expression of these genes, The addition of 0.2:; oleic acid to nonfermentable glycerol-ethanol further increased expression of
`
`The addition of 0.27; oleic acid to low glucose increased expres- sion of
`
`HDE
`
`CATL
`
`tropicalis.
`The addition of 2% oleic acid to YNE increased HDE expression 1.3-fold,
`CATL
`
`HDE
`
`TL
`
`HDE
`
`CATL
`
`2.6-fold and of AOX 4.7-fold (Fig. 1, compare lanes GY’E’O”.’ with lanes GY”E2), again showing an induction of expres- sion of these genes by oleic acid. Reduction of
`
`HDE
`
`G5
`
`6”’
`
`0.2 0.z G 0 GY2$ GY’; 6’“- GY*
`
`EZ 0” HDE
`
`B
`
`G5 Go ’ o’20’2 G 0 GY2i GY'i 6 ‘- GY' E2 0’ CATL AOX Fig. 1. Effects of various carbon sources on expression of HDE, AUX and CATL. in C. ~ru~~ca~~~. The yeast C. rr~p~caljs Berkhout strain pK233 (ATCC20336) (Tanabe et al., 1966) was grown i? minimal medium (YNE; 0.67% yeast nitrogen base/0.05% yeast extract/OS”/, (w/v) Tween-40 (Erdmann et al., 1989) containing one of the following as a carbon source: So/, glucose (high glucose); 0.2% glucose (low glucose); 0.2% glucose/0.2?b (w/v) oleic acid (glucose-oleic acid); 2”/, (v/v) gly- cerot; 2y, (v/v) ethanol; 27.; (v/v) glyceroI/2)?b (v/v) ethanol (glycerol- ethanol); ?“;, (v/v) glycerol/2”/, (v/v) ethanolj0.2”, (w/v) oleic acid; or 2”” (w/v) oleic acid. 50-ml cultures of cells grown to late exponential phase (A,,,,, = 0.4) were pelleted, washed in sterile HaO, and resuspended in an equal volume of 50 mM Tris HCI pH &O/SO mM NaCl/O. 1 mM EDTA/O.l mM ZnCl,/lS mM phenylmethylsulfonylfluoride. The cells were disrupted by vortexing with glass beads. The crude lysates were clarified by cejltrjfugation. Protein was determined by the method of Bradford (1976) using ovalbumin as a standard. Proteins were analyzed by 0.12, SDS-polyacrylamide gel electrophoresis (Fujiki et al., 1984) followed by immunoblotting using antisera against: (panel A) HDE and (panel B) CATL and AOX. [ ‘*‘I]Protein A (Amersham, Oakville, ON) was used to detect antigen-antibudy complexes (Burnette, 1981). Auto- radiography was performed at -70°C with prellashed (Laskey and Mills, 1977) XAR-5 film (Eastman Kodak Co., Rochester, NY). The films were analyzed with a GS-300 scanning densitometer and the GS-350 software (Hoefer Scientific Instruments, San Francisco, CA). Quantitative anal- yses were the average of at least two independent observations, except for C. tropicah grown in YNE alone or YNE plus one of 2y10 (v/v) glycerol, 27; (v/v) ethanol, or 2$,; (w/v) oleic acid. G, glucose; GY, glycerol; E, ethanol; 0, oleic acid; I - 1, YNE. Superscripts refer to 9, of a particular carbon source. Equai amounts of protein were run in each lane. (5. l-fold),
`
`CATL
`
`AUX
`
`(4.7-fold) expression when ethanol is added to gIyceroI-cont~ning medium (Fig. 1, compare lanes GY2E2 to GY’) indicates that ethanol represses the expression of these genes. This argues that derepression, and not induction by a carbon source (glycerol) in the absence of glucose, is the principal mecha- nism resulting in increased expression of these genes. It appears that when C.
`
`trop~~a~i~~
`
`is grown on carbon sources not metabolized by the /&oxidation pathway, a general negative control mechanism involving repression and derepression effects the expression of
`
`CATL.
`CATL
`in different carbon sources suggests these genes share common regulatory elements.
`
`HDE, AOX
`HDE, AOX
`
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`Page 2 of 6
`
`Therefore, the
`is expressed in S.
`and
`in C.
`6.2-fold, of
`2.6-fold and of
`(3.7-fold), CA
`3.3-fold, of
`A
`(4.5fold) and
`and
`The coordinate expression of
`and
`

`

`cerevisiue.
`
`The YCpSO vector assures a stable average gene dosage of one copy per cell (Struhl, 1983). Deletions (Fig. 2B) in the upstream region of
`
`131
`
`HDE
`
`(Fig. 3) were constructed to determine the 5’ elements mediating the responses detailed above.
`HDE
`expression in S.
`cerevisiue
`
`was assayed by immunoblotting of total lysates with anti-HDE serum which is specific for the heter- ologously synthesized HDE. The removal of sequences from nt -775 (5WT; see Fig. 2B) to nt -613 (5R) had essentially no effect on the levels of
`expression in either low glucose or glucose- oleic acid (Fig. 4, compare lanes 5WT and 5R). Deletion of the next 53 nt to -560 (5E) showed a threefold and twofold increase in
`expression when cells were grown in low glucose and glucose-oleic acid, respectively, suggesting the presence of a repressive control element between nt -613 and -560 (Fig. 4, compare lanes 5E with lanes 5R). Sequences between nt -560 (5E) and -526 (5B) positively regulate
`expression decreased to approx. 40% of 5WT in glucose and glucose-oleic acid (Fig. 4, compare lanes 5B with lanes 5E). Removal of the next 133 nt to -393 caused a 6.6-fold increase and a 36-fold increase in
`expres- sion relative to 5WT in glucose and glucose-oleic acid, respectively (Fig. 4, compare lanes 5P with lanes 5WT). Therefore, the region between nt -526 and -393 contains a negative regulatory element. There are two sequences within this region that are conserved in a number of oleic acid-responsive genes of C.
`(Table I; Fig. 3). One of these sequences is similar to the consensus sequence [ATTTCFF] for regulation of SUC2 by glucose in S.
`(Sarokin and Carlson, 1986). It is interesting to speculate that these sequences may be similarly involved in the glucose response of all these genes.
`
`HDE
`
`HDE
`
`HDE
`
`HDE
`
`l
`
`1
`
`cerevisiue
`
`HDE
`
`tropicalis
`
`(b) Expression of HDE deletion constructs in Saccharo- myces cerevisiae Determination of the upstream elements involved in the regulation of the
`
`HDE
`gene necessitated its introduction into the genetically manipulable yeast S.
`cerevisiue.
`HDE
`
`gene was inserted into the CEN-containing plasmid YCpSO producing plasmid YCp505H (Fig. 2A), which was , EcoRI/Hindlll
`
`B
`
`5WT 5R 5E 5B 5P 5T 50 5V ATG I II I \/ I I ! -7
`
`-6
`
`1
`3
`
`-5-5
`6
`0
`
`2
`6
`
`-3-3-3
`9
`3
`
`43
`19
`
`-2
`0
`5
`
`7
`5
`
`HDE gene.
`A)
`Subcloning of the
`cerevisiae.
`HDE
`A 4.2-kb Sal1 fragment containing the
`HDE
`gene (Aitchison et al., 1990) was ligated into SalI- digested pGEMSZf( + ) to produce recombinant plasmid 3T. YCp505H was constructed by ligating the ScaI-Sac1 fragment containing the
`
`HDE
`
`gene from 3T into the Sal1 site of YCp50. Both the fragment and the vector were treated with T4 DNA polymerase to produce blunt ends. 1.8 kb of pGEMSZf( + ) (blackened box) flanked the 5’ end of the
`
`HDE
`
`gene to act as a buffer for subsequent BAL 31 digestion. The thin arrow indicates the direction of transcription of the
`E. coli
`HDE
`
`DHS-a cells were transformed as described by Bethesda Research Laboratories (Burlington, ON) and grown in Luria broth supplemented with 100 pg ampicillin/ml. Recombinants were selected by colony hybridization (Grunstein and Hogness, 1975) and characterized by restriction endo- nuclease digestion. (Panel B) Generation of 5’ deletion constructs. BAL 31 exonuclease was used to produce deletions in the upstream region of the
`
`HDE
`
`gene. Digestions were performed in K’glutamate buffer (Hanish and McClelland, 1988) at 37°C with 1 u of BAL 3l/pmol of XbaI-linearized YCp505H, which removed approx. 35 bp/min from each end. The digested DNA ends were made blunt using T4 DNA polymerase, and BumHI linkers (GGGATCCC) were added. The plasmids were then digested with BamHI and recircularized. BAL 31 deletion end points were determined by dideoxy sequencing (Sanger et al., 1977). Numbering (all minus) is as in Fig. 3. used to transform S.
`
`HDE
`
`expression in 5P (nt -393) and 5T (nt -341) is 6.6-fold and 2. l-fold that of 5WT, respectively, in low glu- cose, and 36-fold and 4-fold that of 5WT, respectively, in glucose-oleic acid (Fig. 4, compare lanes 5P, 5T, and 5WT). This region contains an oleic acid-responsive element, because the ratios of 5P/5T (relative to 5WT) is threefold higher in glucose-oleic acid than in low glucose. Therefore, sequences unique to 5P, i.e., nt -393 to -341, respond positively to oleic acid. A nonanucleotide sequence [consensus = zGGTT$TTA] between nt -393 and -341 of
`
`HDE
`is conserved in oleic acid-responsive genes of C.
`tropic&is
`
`(Table I; Fig. 3). This sequence may be a bind- ing site for an oleic acid-responsive factor. Removal of nt downstream from nt -341 (5T) to give 5Q and 5V progressively reduced
`expression to the limits of detection in both glucose and glucose-oleic acid (Fig. 4) suggesting that the minimal length for a functional
`
`HDE
`
`HDE
`
`promoter lies between nt -341 and -205. To extend the above results, 5P and 5T, which delineate
`
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`Page 3 of 6
`
`The
`Fig. 2. Constructions for analysis ofthe
`(Panel
`gene for expression in S.
`gene.
`expression, because when removed, levels of
`

`

`-690
`-700
`-710
`-720
`-730
`-740
`-750
`-760
`-770
`GTCGA CGTTTTGCCG CCACTTGTGA AGGGAAGMG AGTCGTTGAG TTGATGTAAT TAAGCTGGCA CGTAGATACC AGAAGGTTCT AGAGTAGAGC
`-670
`-660
`-650
`-640
`-630
`-620
`-610
`-600
`-590
`-680
`TTGGGTGGTG TTTGGCCCTG TTTGGACCAC GGATAGAGAT GGAGAATCCC TTGGTTAGAG CGGAGAGGAA AAAATTGAAA CTTTGCATAT CCCACTTCAT
`-580
`-570
`-560
`-550
`-540
`-530
`-520
`-510
`-500
`-490
`TATCCTTGAT GTAACCGTTT TATGGGGTAA TTAAAGTGTG GAAAAATMT CAGGGAGACA TATTCCCGAT CAATTGGGTG GTGGTCGCTC AATTTCTGTG
`-480
`-470
`-460
`-450
`-440
`-43D
`-420
`-410
`-400
`-390
`&TAGTAGGC TCAGTGGTGT GTATTGGGAT TGGTAGTAGT CTGTATMGC AGTGTTATAT AACCCATTGC TTGTTGATTC CTATTTTGCT GGCAAAAGTG
`-380
`-370
`-360
`-350
`-340
`-330
`-320
`-310
`-300
`-290
`ACAACTGTAG TTGTGAGATA ATCCTCGGTT ATTACGCCTG GGGGGGCAGA CAGCCAAAGT TGTGCCCGTG CGACAATGGC ATCAGMGAA ACAGAAAAAA
`-280
`-270
`-260
`-250
`-240
`-230
`-220
`-210
`-200
`-190
`AAAACACAGG CATTTTTATC CACATGCACA CTACCCCCAC TATTCCTGTC TGCAGTGTGC TTGTGTGTGG CCCCCCGCAG MTCAACAGG GCAAACTCTG
`-180
`-170
`-160
`-150
`-140
`-130
`-120
`-110
`-100
`-90
`GAGCCTGMT CTTTATATAA ACTTCAGGCA TTGGCCCCCC TTTTCACAAT TCTTCACATC CACCATTTTT TTTCTTCTTT CCTACCATAT TAGTTTTTTT
`-40
`+l
`-80
`-70
`-60
`-50
`-30
`-20
`-10
`TTATTCTTTT CCTACCTATC TGATTATTAT CAAACATCTG GTCATCCTCA AAAGAAAGM AGAAACTATA ACMTCMTC
`ATG
`HDE gene.
`HDE
`
`expression seen with 5T, the positive elements responsible for the expression seen with 5P are located between nt -393 and -341 (Fig. 5, panel G5). Low glucose increased
`
`HDE
`
`HDE
`
`HDE
`
`Candida tropicalis
`
`(.5WT), were chosen to investigate the effects of var- ious carbon sources. Since only 5P showed detectable levels of
`
`has been determined previously (Aitchison et al., 1991). The first nt of the start codon is designated + 1. The first digits of numerals are aligned with the corresponding nt. Nucleotides 5’ to this nt are designated by negative values. The double-underlined nt sequence corresponds to a consensus nt sequence for response to glucose. The sequence in bold type and underlined is similar to the glucose-responsive element of SUC2. The single-underlined sequence corresponds to a consensus nt sequence for response to oleic acid. The sequence in bold type corresponds to a TATA consensus sequence. the oleic acid-responsive region, along with the full-length
`expression in high glucose (Fig. 5, panel G’), sequences upstream from nt -393 contain elements involved in glucose repression, in agreement with results presented above showing a negative regulatory region between nt -526 and -393. As there is no detectable
`expression due to derepression in both 5P (3.9-fold) and 5T (Fig. 5, compare lanes Go.2 with G5), indicating that ele- ments responsible for derepression are located downstream TABLE I Sequences of the responsive regions of the
`genes encoding peroxisomal proteins il Genes b Glucose’ Oleated (distance)” (distance)r A B
`GGGAGACATA -528 -519 GAGAGAGAGA -482 -413 GGGAGAGAGA -396 -387 GGTAGACTTA -580 -571 GGGAAACAGA -422 -43 I Consensus GGGAGAgAbA TTTCTGTGAGT -488 -478 ATTGTCTGAGT -330 -320 TTTTTGTATGT -194 -184 TTCATGTGAAA -431 -421 TTTGTGTGAGG -600 -590 TTTNTGTGAG: G CGGTTATTA -355 -341 CGGTTATTC -359 -351 CGGTAGTTA -223 -215 TGGTTGTTG -253 -261 TGGTTATTA -695 -687 :GGTT;TT~ G ,’ The sequences of the glucose-responsive (nt -526 to -393) and the oleic acid-responsive (nt -393 to -341) regions of the
`
`HDE
`
`HDE
`
`HDE
`
`POX18
`
`P450alk
`
`CA TL
`
`HDE
`gene were compared to the sequences of the upstream regions of other oleic acid-responsive genes of C.
`tropicalis.
`h HDE,
`POX4,
`POXZ8,
`
`P450aik,
`
`CATL,
`catalase (Murray and Rachubinski, 1989). ’ Sequences in the glucose-responsive region of
`HDE
`tropicalis.
`Column A, conserved sequence A; column B, conserved sequence B similar to the SUC2 glucose-responsive sequence of S.
`cerevisiae.
`’
`HDE
`tropicalis.
`’
`Distance in nt upstream from the A ( + 1) of the start codon. Last digits of numbers are aligned with corresponding nt. Nucleotides denoted in bold type deviate from the derived consensus sequences.
`
`LCY Biotechnology Holding, Inc.
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`Page 4 of 6
`
`132
`Fig. 3. Nucleotide sequence of the upstream region of the
`The nt sequence of
`gene conserved in other
`POX4
`trifunctional enzyme;
`fatty acyl-CoA oxidase (Okazaki et al., 1986);
`peroxisomal 18-kDa protein (Szabo et al., 1989);
`alkane-inducible cytochrome P450 (Sanglard and Loper, 1989);
`conserved in other oleic acid-responsive genes of C.
`Sequence in the oleic acid-responsive region of
`conserved in other oleic acid-responsive genes of C.
`

`

`cerevisiae
`is regulated in a fashion similar to that seen in C.
`tr~picul~s,
`except that the extent of repression, derepression and activation differs, The
`HDE
`
`gene is sub- ject to greater repression and derepression but to lower activation by oleic acid when expressed in S.
`
`cerevisiue,
`cerevisiae
`
`0.2 OS2
`
`v&his C.
`
`GO Fig. 4. Expression of deletion constructs in S.
`
`cerevisiue. S. cerevisiae
`
`~ro~jcul~s to metabolize fatty acids. Removal of sequences upstream from 5P (nt -393) results in a resto- ration of high levels of expression of
`
`HDE
`
`cerevisiae,
`
`HDE
`
`G 0 GY?E2 G?; 0”‘”
`
`DL-1 (Van Loon et al., 1983) was transformed by the method of Ito et al. (1983). Transformants harboring various deletion constructs were grown in YNE supplemented with leucine (30 pg/ml) and histidine (20 rg/ml) containing either low glucose (upper panel, G”.2) or glucose-oleic acid (lower panel, GO 20”.2; see Fig. 1 for details of media composition, gel, and abbreviations), lysed and analyzed for
`expression by immuno- blotting (see Fig. 1 for details). Lanes of each panel contain equal amounts of protein. 0.2 0.2 0.2 G
`,-1_ 5WT 5P 1 ,_-_L__, ,-__ -1..___, i__l_.,, _._ _I_--_, 5T 5WT 5P 5T 5WT 5P 5T 5WT’5WT SF 5T kDa - 110 - 84 Fig. 5. Effects of various carbon sources on
`
`expression 133 in S.
`
`ACKNOWLEDGEMENTS This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada. J.D.A. is a recipient of an Ontario Graduate Studentship. R.A.R. is a recipient of a Medical Research Council of Canada Scholarship. We thank Gillian Regoeczi for expert technical assistance. REFERENCES Aitchison, J.D. and Rachubinski, R.A.: In vivo import of
`
`indicating that negative control involving repression and derepression is involved in the regulation of the
`HDE
`gene. Since the expression of the
`HDE
`gene is similar in C.
`tropicaiis
`cerevisiae,
`
`these two yeasts probably possess similar mechanisms for the control of expression of this gene. (c) Conclusions (I) Carbon sources regulate the expression of
`
`HDE,
`
`AOX
`
`CATL
`
`tropicalis
`
`cerevisiae
`
`by induc- tive, repressive and derepressive mechanisms. (2) The mechanism of regulation of expression of
`
`HDE
`
`tropicalis
`
`cerevisiae.
`
`(3)
`A giucose-responsive region lies between nt -526 and -393 of
`HDE,
`
`and two sequences within this region are conserved in a number of oleic acid-responsive genes of C.
`
`~r~~~calis.
`(4)
`An oleic acid-responsive region lies between nt -393 and -341 of
`HDE,
`
`and a sequence within this region is conserved in a number of oteic acid-responsive genes of C.
`
`tropicalis.
`
`Candida gropi-
`
`Candida nlbicans.
`
`Curr. Genet. 17 (1990) 48 I-486. Aitchison, J.D., Sloots, J.A., Nuttley, W.M. and Rachubinski, R.A.: Sequence of the gene encoding
`
`Candida tropicalis
`
`peroxisomal tri- functional enzyme. Gene 105 (1991) 135-136. Borst, P.: How proteins get into microbodies (peroxisomes, glyoxysomes, glycosomes). Biochim. Biophys. Acta 866 (1986) 179-203. Bradford, M.M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72 (1976) 248-254.
`
`HDE
`
`expression by con- structs 5WT, 5P, and ST. Transformants harboring constructs 5WT, 5P and ST were grown in various carbon sources. Yeast lysates were pre- pared and analyzed for
`
`HDE
`
`expression by immunoblotting. Carbon sources, gel, and methods are as in Fig. 1. Equal amounts ofprotein were run in each lane. from nt -341. Addition of oleic acid to low glucose increased expression of 5WT to detectable levels, SP ICfold and 5T 1.3-fold (Fig. 5, compare lanes G”.200.2 with lanes G0.2), providing further evidence for an oleic acid- responsive element between nt -393 and -341. Substitution of nonfermentable glycerol-ethanol for low glucose in media containing oleic acid resulted in increased expression for 5WT (9.3-fold), 5P (3.4-fold) and ST (45-fold) (Fig. 5, compare lanes GY2E200.2 with G”.200.2), indicating that sequences responsive to glycerol-ethanol (or the absence of glucose) are downstream from nt -341 (5T) (Fig. 5, compare lanes GY2E200.2 with G0,20a.2). The addition of 0.2% oteic acid to glycerol-ethanol increased
`
`HDE
`
`expression 1.4-fold (Fig. .5, 5WT, compare lanes GY2E200.* with GY2E2). This increase is approx. 40% ofthat seen in C. trop~cu~is(3.3-fold; Fig. 1). Switching from a fermentable to a nonfermentable carbon source increased
`
`HDE
`
`expression of 5WT at least eightfold (Fig. 5, compare lanes GY2E2 with lanes Go,‘), (about twice that seen under the same conditions in C.
`
`tropicalis;
`
`HDE
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`5WT 5R 5E 5B 5P 5T 58 5V
`3.5-fold; Fig. 1). These results suggest that
`which is consistent with the reduced ability of S.
`in S.
`and in S.
`and
`in C.
`and S.
`is similar in C.
`and S.
`CQfis hydratase-dehydrogenase-epimerase into peroxisomes of
`

`

`Candidu tropicalis:
`
`primary structures deduced from genomic DNA sequence. Proc. Natl. Acad. Sci. USA. 83 (1986) 1232-1236. Rachubinski, R.A.: Genetic methods for and gene structure in other
`
`Cundidu
`
`species. In: Kirsch, D.R., Kelly, R. and Kurtz, M.B. (Eds.), The Genetics of Candida. CRC Press, Boca Raton, FL, 1990, pp. 177-186. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chain- terminating inhibitors. Proc. Natl. Acad. Sci. USA. 74 (1977) 5463-5467. Sanglard, D. and Loper, J.C.: Characterization of the alkane-inducible cytochrome P450 (P450alk) gene from the yeast
`
`Cundidu tropic&:
`
`134 Burnette, W.N.: Western blotting: electrophoretic transfer of proteins from SDS-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112 (1981) 195-203. Erdmann, R., Veenhuis, M., Mertens, D. and Kunau, W.-H.: Isolation of peroxisome-deficient mutants of
`
`identiti- cation of an upstream BamHI site polymorphism. Nucleic Acids Res. 17 (1989) 3600. Okazaki, K., Takechi, T., Kambara, N., Fukui, S., Kubota, I. and Kamiryo, T.: Two acyl-coenzyme A oxidases in peroxisomes of the yeast
`Nat]. Acad. Sci. USA. 86 (1989) 5419-5423. Fujiki, Y., Rachubinski, R.A. and Lazarow, P.B.: Synthesis of a major integral membrane polypeptide of rat liver peroxisomes on free poly- somes. Proc. Natl. Acad. Sci. USA. 81 (1984) 7127-7131. Grunstein, M. and Hogness, D.S.: Colony hybridization: amethod for the isolation of cloned DNA’s that contain a specific gene. Proc. Nat]. Acad. Sci. USA. 72 (1975) 3961-3965. Hanish, J. and McClelland, M.: Activity ofDNA modification and restric- tion enzymes in KGB, a potassium glutamate buffer. Gene Analyt. Techn. 5 (1988) 105-107. Ito, H., Fukuda, Y., Murata, K. and Kimura, A.: Transformation ofintact yeast cells treated with alkali cations. J. Bacterial. 153 (1983) 163-168. Laskey, R.A. and Mills, A.D.: Enhanced autoradiographic detection of ‘*P and “‘1 using intensifying screens and hypersensitized film. FEBS Lett. 82 (1977) 314-316. Lazarow, P.B. and Fujiki, Y.: Biogenesis ofperoxisomes. Annu. Rev. Cell Biol. 1 (1985) 489-530. Murray, W.W. and Rachubinski, R.A.: Nucleotide sequence of per- oxisomal catalase from the yeast
`
`Saccharomyces cerevisiue. Proc.
`
`identification of a new P450 family. Gene 76 (1989) 121-136. Sarokin, L. and Carlson, M.: Short repeated elements in the upstream regulatory region of the SUC2 gene of
`Mol. Cell. Biol. 6 (1986) 2324-2333. Struhl, K.: The new yeast genetics. Nature 303 (1983) 391-396. Szabo, L.J., Small, G.M. and Lazarow, P.B.: The nucleotide sequence of
`
`Sacchuromyces cerevisiue.
`
`POXI??,
`a gene encoding a small oleate-inducible peroxisomal protein from
`Candidu tropicalis.
`Gene 75 (1989) 119-126. Tanabe, I., Okada, J. and Ono, H.: Isolation and determination
`of
`
`yeasts utilizing kerosene as a sole source of carbon. Agr. Biol. Chem. 30 (1966) 1175-1182. Van Loon, A.P., Van Eijk, E. and Grivell, L.A.: Biosynthesis of the ubiquinol-cytochrome c reductase complex in yeast. Discoordinate synthesis of the 1 I-kd subunit in response to increased gene copy number. EMBO J. 2 (1983) 1765-1770. Veenhuis, M., Mateblowski, M., Kunau, W.-H. and Harder, W.: Proliferation of microbodies in
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`Succhuromyces cerevisiae.
`Yeast 3 (1987) 77-84.
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`Candidu tropicalis pK233:
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