YEAST
VOL.
7: 78 1-803 ( I99 I )
Four Major Transcriptional Responses in the Methionine/Threonine Biosynthetic Pathway of Saccharomyces cerevisiae HARRY A. MOUNTAIN*, ANDERS S. BYSTROM, JORGEN TANG LARSEN AND CHRISTOPHER K O R C H t
Depurtment of Microbiology. University of Umed, S-90187 Umed. Sweden Received 12 February 1991; accepted 4 June 1991
Genes encoding enzymcs in the threonine/methioninc biosynthetic pathway were cloned and used to investigate their transcriptional response to signals known to affect gene expression on the basis of enzyme specific-activities. Four major responses were evident: strong repression by methionine of M E T 3 , MET5 and METIQ. as previously described for MET3. M E T 2 and MET2.5; weak repression by methioninc of MET6; weak stimulation by methionine but no rcsponsc to threonine was seen for T H R I , H O M 2 and H O M 3 ; no response to any of the signals tested. for HOM6 and M E S I . In a BOR3 mutant. T H R I , HOMZ and H O M 3 mRNA levels were increased slightly. The stimulation of transcription by mcthioninc for H O M 2 , H O M 3 and THRI is mediated by the CCN4 gene product and hencc these genes are under the general amino acid control. In addition to the strong repression by methionine, MET5 is also regulated by the general control. KEY WORDS
-
Methioninejthreoninc biosynthesis; transcriptional regulation; general amino acid control; yeast.
INTRODUCTION Amino acid biosynthesis in Saccharomyces cerevisiae is regulated at the enzyme level, at the level of gene expression, or both (Jones and Fink, 1982; Messenguy, 1987). Pathway-specific gene regulation and an interlinked regulatory system called the general amino acid control (general control) have been found (reviewed by Hinnebusch, 1988). Pathway-specific controls have been observed for genes in the arginine, lysine. methionine, leucine, isoleucine and valine biosynthetic pathways and controls are mediated by cis- and rrans-regulatory elements at the transcriptional level. In contrast, the C P A l gene is regulated at the translational level (Werner er ul., 1987). In the general control system, starvation for any of tcn amino acids leads to thederepression ofabout 30 amino acid biosynthetic enzymes in nine different biosynthetic pathways (Hinnebusch, 1988). To date, the amino acid biosynthetic genes under this control have all been shown to have all or part of the *To whom enquiries should be addressed. tPresent address: Imperial Holly Research and Development Centre. 5320 Mark Dabling Boulevard, Colorado Springs, Colorado 80918, U.S.A.
0749 503X:91/080781-23 $1 1.50 0 1991 by John Wiley &Sons Ltd
sequence A T G G T C A T in the 5' non-coding region (Hopeand Struhl, 1985; Arndt and Fink, 1986; Hill el al., 1986a). This cis- regulatory sequence specifically binds the GCN4 regulatory protein which thereby activates gene expression at the transcriptional level (Struhl, 1982; Donahue er al., 1983; Arndt and Fink, 1986; Hope and Struhl, 1985). The GCN4 gene product is itself under translational regulatory control such that the protein is only produced upon amino acid starvation (Hinnebusch, 1984; Thireos er al.. 1984). Fourteen regulators of GCN4 mRNA translation have been defined (reviewed by Hinnebusch, 1988). One of these is the GCDl gene product which is a negative regulator of GCN4 mRNA translation. A gcdl mutant is therefore constitutively derepressed for genes under the general control (Wolfner er al.. 1975). The methionineithreonine biosynthetic pathway is diagrammed in Figure 1 and metabolic signals affecting expression of genes in this pathway are summarized in Table I . For most of these genes, the data are based o n measurements of the specific activities ofcorresponding enzymes isolated from cells grown in different media. Only the MET2, MET3, METl7i25, SAM1 and very recently MET16 genes
782
H.A. MOUNTAIN E T A L .
acids on the mRNA levels, the HOMand THR gene transcript levels were also examined in borrelidinresistant mutants which are potentially defective in the regulation of the methionine/threonine biosynthetic pathway. Borrelidin is a threonine analogue which specifically inhibits threonyl-tRNA synthetase. Of the four mutations conferring resistance to the antibiotic, BORI is an allele of HOM3 which encodes a feedback-resistant mutant of aspartokinase (Seibold et al., 1981), BOR3 encodes an altered threonyl-tRNA synthetase (Nass and Poralla, 1976), and the others are uncharacterized. Furthermore, to determine whether these methionine/threonine genes are under the general control we have investigated their regulatory responses in strains carrying the gcdl-101 allele or the gcn4-101 allele.
-=/
pzEYJ
ABBREVIATIONS
The designations of the genotypes of the wildtype and mutant alleles and their gene products are as follows: HOM2 and hom2, aspartate semialdehyde dehydrogenase (L-aspartate-p-semialdeMOt Mel-1RNA S-.dsn-xylmuhionme hyde:NADP oxidoreductase (phosphorylating), ( AdoMcI ) EC 1.2.1.11); HOM3 and hom3, aspartate kinase Figure I . Methionineithreonine biosynthesis in Saccharomyces (L-aspartate 4-phosphotransferase, EC 2.7.2.4); cmvisiae. Based on the figure of Jones and Fink (1982). and HOM6 and hom6, homoserine dehydrogenase updated. (L-homoserine:NAD oxidoreductase, EC 1. I . 1.3); MESl and mesl, methionyl-tRNA synthetase (Lhave been shown to be repressed at the transcrip- methionine:tRNAMc' ligase, EC 6 . I . 1.10); MET3 tional level by the addition of methionine or and met3, ATP sulphurylase (ATP:sulphate AdoMet (Baroni et al., 1986; Cherest et al., 1985; adenylyltransferase, EC 2.7.7.4); MET5 and met5, Sangsoda er al., 1985; Thomas and Surdin-Kerjan, part of the sulphite reductase (hydrogen sulphide: 1987; Thomas et al., 1990). SAM2 mRNA is NADP oxidoreductase, EC 1.8.1.2); MET6 and induced by methionine (Thomas er al., 1988). met6, homocysteine methyltransferase (5-methyl Recently, HOM2 has been shown to be under the tetrahydropteroyl-tn-L-glutamatex-homocysteine general amino acid control (Thomas and Surdin- S-methyltransferase, EC 2.1.1.14); MET14 and met14 APS kinase (ATP: adenylyl sulphate-3'Kerjan, 1989). In this paper we describe the cloning and physical phosphotransferase, EC 2.7.1.25); THRI and characterization of genomic DNAs encoding the rhrl, homoserine kinase (ATP:L-homoserine-0HOM2. HOM3. HOM6. THRI, MET3. MET5 and phosphotransferase, EC 2.7.1.39). Corresponding MET6 amino acid biosynthetic genes. A MET14 phenotypes are Horn+ and Hom- (a requirement clone (Fitzgerald-Hayes et a[., 1982) obtained from for threonine and methionine), Thr+ and Thr-, and J . Carbon was also physically characterized. We Met+ and Met-. BORI, BOR2 and BOR3 are obtained a MESl clone (Meussdoerffer and Fink, dominant borrelidin-resistant mutants and hor4 is a 1983) from G. R. Fink. Using these nine genes as recessive borrelidin-resistant mutant. Other general abbreviations are: bp, base pairs; probes in Northern blot hybridizations we investigated their transcriptional regulation. The levels of kb, kilobase pairs; nt, nucleotides; cM, centimRNAs were examined in total RNA isolated from morgans; Tris, tris(hydroxymethy1)aminomethane; cells growing in repressed and derepressed con- DTT, dithiothreitol; SDS, sodium dodecyl ditions according to the different metabolic signals sulphate; NaAc, sodium acetate; EDTA, ethylenelisted in Table 1. In addition to the effects of amino diaminetetra-acetate disodium salt; AdoMet, SMotbanyllRNA
.Usthiomnc
S -adenorylhomosynci-c
TRANSCRIPTION OF METHlONlNE AND THREONINE BIOSYNTHETIC GENES
adenosylmethionine; 3AT, 3-amino- 1,2,4-triazole. YEP, YCp and YIP vectors refer to yeast episomal, yeast centromere and yeast integrative plasmids, respectively . MATERIALS AND METHODS Materials
All the amino acids and 3AT were obtained from the Sigma Chemical Company. Other media components were obtained from Difco. Northern blot hybridizations were carried out on Gene Screen Plus@ hybridization transfer membranes from DuPont. Autoradiography was carried out on Kodak X-Omat AR5 films or Amersham Hyper Film-MP. In most cases the film was pre-flashed to give an optical density of 0.1 above the film base. Cronex intensifying screens from DuPont were used during autoradiography with exposures being made at - 70°C. Probes for Northern blot analysis were labelled using a Oligolabelling@ kit (purchased from Pharmacia) incorporating [a-”P]dCTP (3000 Ciimmol) from New England Nuclear. Restriction enzymes were purchased from New England Biolabs, Pharmacia, or Boehringer Mannheim. T4 DNA ligase and RNase-free DNase were purchased from Pharmacia. St ru ins
The source and genotypes of yeast and bacterial strains used in this study are listed in Table 2. Mediu
Complete (YEPD), minimal (MM) and synthetic complete (AA, containing all amino acids, uracil and adenine) media have been described previously by Sherman et al. (1986). Unless otherwise indicated, amino acid supplements were the same concentrations as for AA medium. 3AT was added at 10 mM. Genetic procedures
Yeast genetic manipulations were performed as described by Sherman et al. (1986). Difficulties encountered in sporulating certain diploids following Sherman et al. (1986) were overcome by following a procedure by Bilinski er al. (1987). Yeast cells were transformed by using 0.1 M-lithium acetate according to Ito et ul. (1983). Bacterial transformations followed the procedure of Hanahan (l983), as modified by Korch er ul. (1991).
783
D N A methods Large-scale isolation ofplasmid DNA, restriction enzyme analysis, and agarose gel electrophoresis were performed according to Maniatis et al. (1982). Isolation ofplasmid DNA from yeast was according to Hoffman and Winston ( 1 987). Mini-preparations of plasmid DNA from Escherichia coli were made according to Holmes and Quigley ( 1 98 I), except that we used 5% instead of 0.5% Triton X-100 in the STET solution and boiling was for 3 min instead of 45s. Probes for Northern blot analysis were purified on Sephadex G-50 Nick@ columns from Pharmacia. Northern blots Total RNA was prepared from S288C cells in mid-log phase (OD, -0.6 estimated with an LKB Ultrospec I 1 spectrophotometer) growing at 30°C in MM supplemented with methionine, threonine or lysine at 0, 0.2, 2 and 20 mM. Also, RNA was isolated from MM-grown strains containing the following mutations: B O R l , BOR2. BOR3, hor4. These represent steady-state levels of RNA. In addition, total RNA was also prepared from cells after shifting to the appropriate medium. In this case strains were pre-grown to about OD, ‘v 0.5 and then harvested, washed, re-suspended three times in 40 ml of sterile distilled water, and then suspended in the medium of interest (at OD, -0.5) and grown for 4 h (Penn et al., 1983). All RNAs for the tests for general control were isolated from shifted cells. For the gcdl-101 and gcnl-101 strains (9043-7D and L869, respectively), histidine or leucine, respectively, were added to MM to avoid the general control being induced because of starvation for these amino acids in these auxotrophic strains. The 9043-7D strain with the temperaturesensitive gcdl-I01 allele was grown at 25°C. Total RNA was prepared from 20 ml of cells in the appropriate medium following the procedure, suitably scaled down, described in Kelly et al. (1988). Contaminating DNA was removed by resuspending the washed and dried pellet in 20mMNaAc, pH 4.5, 10mM-MgCI, and 10mM-NaCI, and digesting with 30 units of RNase-free DNase (Pharmacia) at room temperature for 30 min. After extracting the solution twice with phenol/chloroform, followed by a chloroform extraction, the nucleic acids were re-precipitated and centrifuged. The pellet was resuspended in 20 mM-sodium phosphate (pH 6.5), 2 mM-EDTA, debris removed by centrifugation, and the nucleic acid concentration
Adenylsulphate kinase PAPS reductase
MET14 METI6, METI7*, MET22, METI, MET8 MET5, MET10 METI8, MET8 MET20, M E T I , MET2
MET6
MET25
ATP sulphurylase
MET3
0-acetylhomoserine/ 0-acetylserine sulphydrylase Homocysteine methyltransferase
Homoserine acetyltransferase
Sulphite reductase
Sulphate permeases
Enzyme
C H R I . sell
Gene
+ +
+
+
+met +met - tRNA'" AdoMet +met +met - tRNAM" AdoMet +met +met -tRNAMet +met
+ +AdoMet
AdoMet +met AdoMet +met +met - tRNAM" +met +met 1 mM-AdoMet
Low enzyme levels
AdoMet -met -met -tRNA'"' -AdoMet -met -met - tRNAM" - AdoMet -met - met-tRNAM" -met -
+
AdoMet -met - AdoMet -met - tRNAM" -met -met 0.05 mM-AdoMet -
High enzyme levels
Regulatory signals for:
3.5 (met)
31 (met)
5-19 (met)
25 (met)
> 1 (met) 4.5 11
11&140 (met)
+
Ratio of steady-state enzyme levels -amino acid/ amino acid
Table 1. Genes and enzymes of methionine and threonine biosynthesis. Known signals and regulatory responses of enzyme levels
References
Probable transcriptional activator for thc M E T genes Unknown functions
MET4
M E T 7 . M E T 13. M E T I S , MET21. M E T 2 3 , MET24. N I I S I . PHSI
- mct Not known
+met +met
- mct
0.5 (met)
3 (1YS) 0.5 ( + bor)" 0.5 ( + mct)a > 1 (thr) 2--5 (met)
4 7(thr)
I . 3 4 (met)
2.0 (met) 2.7 (AdoMct) j. k
"Note that ratios less than I signify induction by the indicated compound. "Breton and Surdin-Kerjan (1977): 'Cherest er a/. (1969); Therest C I a/. (1973a); 'Ferro and Spence (1973): 'Cherest rr a/. (1971); 8 Antoniewski and de Robichon-Szulmajster (1973); 'dde Robichon-Szulmajster and Cherest (1967); 'Baroni er a/.( 19x6);'de Robichon-Szulmajstrr and Corrivaux (1963); 'de Robichon-Szulmajster and Corrivaux (1964); 'Nass and Hascnbank (1970); "'de Robichon-S7ulmajster 6'1 01. (1973): "Surdin (1967); PLor and Cossins (1972); T h o m a s 1.r a/. (19YO); Thomas CI a/. (199 I). *A complementing allele of the ME7'2.7gene. mcr17, affects PAPS reductax.
THRl THH4
HOM6
Aspartate semialdehyde deh ydrogcnase Homoscrinc dehydrogenasc Homoserine kinasc Threoninc kinase
HOM2
tRNAM"'! - thr. +met
met-t RNAMCL ? - mct
+ lys,
+ borrelidin
- thr.
-met
+ homoserinc
+ thr, +met
Aspartokinase
HOM3
+
+met AdoMet
Glucose-6-phospha te dchydrogenasc
MET19
U
*Z
786
H. A. MOUNTAIN ET AL.
Table 2. List o f yeast and bacterial strains used in this study Strain Yeast strains F337 F350 F352 F483 F615 F762 F763 L869 S288C 6080-1 1C 6040- 1 A 6460-8D 9043-7D H21012A H2121B H2 179d H2 17AB 1 S2206D S2614C STX272-4D D286-2A D286-2A BORl D286-2A BOR2 D286-2A BOR3 D286-2A b o d Bacterial strain DH5a
Genotype a ade9 ura3 horn3 serl a met5 leu2 a met6 a cdc6 his4-519 adel ura3-52 lys2 thrl a ilvl arg6 ura3 horn3 trp2 hisl adel met1 gal2 a ura3-52 t r p l d l a ura3-52 trpl-A1 a gcn4-I01 leu2-3,112 a ma12 gal2 a met14 trpl-A1 lysl-1 a adel arg4 his2 lys7 ura3-52 a met3 a gcdl-I01 his4-519 a met6 ura3-52 a horn6 ura3 a ura3-52 leu2-3,112 a ura3-52 a ura3 horn3 gal2 his2 his6 Ieul a horn2 arol C lysl adel trpl arg4-1 gaI2 a horn6 adel leu1 ura3-52 his2 his4 ade6 ural a adel hisl a adel hisl BORI a adel hisl BOR2 a adel hisl BOR3 a adel hisl bor4
F - endAl hsdRl7(ri, m:)supE44 thi- 1 h recA 1 gyrA96 relA 1 A(1acZYA- argF)U 169, cp80dlacZAM 15
Source G.R.F. G.R.F. G.R.F. G.R.F. G.R.F. G.R.F. G.R.F. G.R.F. G.R.F. G.R.F. G.R.F. G.R.F. G.R.F. This work This work This work This work YGSC YGSC YGSC
K.P. K.P. K.P. K.P. K.P. BRL
G.R.F.,Dr G. R. Fink; YGSC, Yeast Genetic Stock Center; K.P., Dr K. Poralla, BRL, Bethesda Research Laboratories.
determined spectrophotometrically. The DuPont manual on the running of denaturing formaldehyde agarose gels and RNA transfer to Gene Screen Plus@ membranes was followed. Total RNA (10 pg) was applied to each lane of the Northern blot gels. Probes were labelled to 3-5 x lo9 cpm/pg DNA and lo8cpm were added to each hybridization. All Northern blots were probed for ACTI mRNA as an internal loading control. The probe was a 314 bp BglII internal fragment of the ACTI gene (Ng and Abelson, 1980) cloned into M13tg131. Purified replicative form was used as a probe.
All Northern hybridization data in Figures 2-1 1 for the tests for the general control (+3AT, +Met+Val) in S288C and the gcdl-101 and gcn4-101 strains are from shift experiments, as are the amino acid omission tests (AA-Met, AA-Thr). For MM+Met data, except for +0.2mM-Met where only steady-state experiments were done, both steady-state and shift data showed the same patterns. Shift data are shown for MET3, MET14, MET6, THRl and the HOM genes. Steady-state data are shown for MET5 and MESI. All data for MM + Thr and MM + Lys and for the borrelidinresistant strains are steady-state data.
787
TRANSCRIPTION OF METHIONINE AND THREONINE BIOSYNTHETIC GENES
__
ne r >n P h
+ I
z
)
*
2
O
+
?
N
e
+
?
0
+
d
+
3
> m
r
z z z u
+
I B F ?
d
%
u
%
,
+
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N
i
t
u D
Y
z
%
, , ? ? ?
W U z F 1
z
u
0 O
N
*
I
B
Z
?
?
3 s : +
-
u O
X
+
+
P P E S I I S E E S I S I I E E E E
Figure 2. HIS4. Northern hybridization. The probe for HIS4 and URA3 was pNKY48 (Alani and Kleckner, 1987), which contains no other yeast sequences. The Northern blot shows transcript levels in 10 pg of total RNA, per track (see Materials and Methods for details).
a
b Y
Y
D z
D z
2
2
%
O z
U
Figure 3. MET3. Restriction map and subclones of the genomic regions containing the cloned gene (a) and Northern hybridization (b) of the probe to investigate transcriptional regulation. (a) Restriction maps of the DNA complementing the mutation and the complementing activities of derived subclones are shown. All probes were based on the YIP vector carrying URA3, pRG412-1. An asterisk indicates the cloned segment used as the probe in the Northern hybridizations. Abbreviations for restriction enzyme sites in this and subsequent figures (Figures 4a-9a, 1 la) are B1, BamHI; B2, BglII; C, ClaI; E, EcoRI; H, HindIII; K, KpnI; N, NdeI; P1, PstI; P2 PvuII; S1, SacI; S2, SacII; S3, SalI; S4, SsiI; XI, XbaI; X2, XhoI; X3, XmnI. (b) The Northern blot shows transcript levels in 10 pg of total RNA, per track (see Materials and Methods for details). S288C was the source of RNA except for the lanes indicated gcdl or gcn4 where the strains 9043-7D and L869 were the respective sources. MM is minimal synthetic medium and the supplements are indicated. AA is MM with all amino acids, adenine and uracil added as in Sherman ei al. (1986); appropriate omissions and additions are indicated.
Densitometry
This was carried out using a Bio-Rad Laboratories Video Densitometer model 620 and the data were analysed with the Bio-Rad Laboratories program 1D Analyst I1 for Microsoft Windows. Densitometry was only carried out on autoradiograms made using pre-flashed film. Only those exposures which gave
signals in the linear optical density range of the film (0.1-1.5 above the pre-flashed film base level) were measured. The photographs presented in Figures 2-1 1 are of autoradiograms which reproduced the best and are not necessarily those which were scanned densitometrically. The probes all contained, in addition to the gene of interest, the URA3 gene to serve as an internal
788
H. A. MOUNTAIN ETAL.
b
a MET5
Met5 Phew
Region
1Kb
P1
S2 S3
E S1
type
* m
+ U
W
U
B P Z 5 5 %
pCK129
I I
II
\
; !
"
I
I I
+
O
9
N
+
9
: +
,
+
4.
e"
x
I
f
8 f
0
9
r< . .N
8
:
U
Y
I
I
N
z; +
+
+
pCK13O
MET5
I pCK143
I
I
--
pCK144
I I
I
uRA3
pCK133'
ACT1
+
0
€.
pCKlO6
0
U
pCKlD8 pCK109
2
I I
Y
5
+
+
I I I I I I
- +
control for differences in the amount of RNA loaded. The approximately 900 nt long URA3 transcript is known to be expressed constitutively under the conditions we tested (Penn et al., 1983) and its expression does not alter in response to the general control (Silverman et al., 1982). However, variation in the level of the URA3 mRNA and its frequently
2 f
% ; +
E I
H I 3 5 2 2 I
0
I
N
I pCK119'
+
" 1 9 0 O N ;
pCK112 pCK113
P
8 2 h
pCK111
-n
+
pCK107
0
0
"
C 4
N.
N
*
*
Y
2 f 2 ;m m 0
+
+
I H I I E I 2 Z S E urn3 MET14
ACT1
weak signal (Figures 2-1 1) prompted us to reprobe the blots for ACTZ mRNA, again a control for RNA loading. A stronger, and hence more reliable signal for comparison was obtained. Some variation was still seen. To compensate for this and to allow for comparisons of the extent of repression or induction of transcript levels, first we normalized all
789
TRANSCRIPTION OF METHIONINE AND THREONINE BIOSYNTHETIC GENES
-
a
MET6
Region B,
1Kb
Pl
El
X1
54 B 2 y P 2
Met6 Pheno.
b
type
-3
pHAM35'
E.
m Y
w
T
2
a
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l
5
? ? 9 9
P
v c
m
m
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%
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m
m
0
O
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0
u C I
"
+
*
+
,
*
)
c4
Y
0
?
.
N
N
T
e
;; +
-
+
' TZ I E 1 1 P I i E E 2 I I E T
I I I I
pHAM39 pHAM40
MET6
URA3
pHAM41 ACT1
pTL22^
Figure 6. MET6. Restriction map, probe (a) and Northern hybridization (b). Legend as for Figure 3.
a HOM2 Region
b .
1Kb
I pBY165 pTL6 pTL5 pTL14 pTL13 pTL12 pTL17 pTL18'
ACTl
Figure 7. HOM2. Restriction map, probe (a) and Northern hybridization (b). Legend as for Figure 3. The bor mutants were compared to their parental strain D286.
790
H. A. MOUNTAIN ETAL.
a HOM3-HlSl
Region
c
1Kb
h
i
- >- ->
P2 Y
pBY158
U
m
d
a
o
El P2
P
e 3
HIS1
o
I
%
?
0
N i
+
L
4
T
*
U
I
m
r
Z
Y
Y
P
P
E .
Y
Y
r " P
E
f
0
0
0
,
N
O
N
s z s
.
-
.
+
*
?
E
0
N
+
? i
f
E E I E I E I H I I I I I I I E
pBY16Y
Y
I- I
Figure 8. HOM3. Restriction map, probe (a) and Northern hybridization (b). Legend as for Figure 3. The bor mutants were compared to their parental strain D286.
values to the same ACT1 level and then for each individual gene the mRNA signal in MM was taken as 1 and all other levels were expressed relative to this. These data are shown in Tables 4 and 5. Relative to the loading control of ACTl mRNA, the URA3 transcript level (Tables 4 and 5) can be seen to be generally unresponsive to the conditions. This is as expected. Because of the variation in loading that did occur, the frequently weak URA3 mRNA did not make as reliable a control as that of ACTl mRNA. Isolation and confirmation of clones containing methioninelthreonine biosynthetic genes
Hybrid plasmids containing the T H R l , HOM2, HOM3, HOM6, MET3 and MET6 genes were isolated from yeast genomic libraries, based on the YCpSO vector (Rose et al., 1987), by complementation of the corresponding phenotypes on synthetic plates. Complementation was always made in com-
bination with selection for Ura', the selectable marker of the vector being URA3. MET5 was also cloned from a YEp24-based library (Carlson and Botstein, 1982) by complementation of met5 in strain F350. Hybrid plasmids were re-isolated in E. coli from positive yeast clones. These plasmids were re-transformed into yeast and Ura' transformants were selected and the complementation of the mutant phenotype, by the plasmid, confirmed. A set of plasmids from each cloning that passed these tests above, were isolated as minipreps, digested with restriction enzymes, and analysed by agarose gel electrophoresis. For complementation of a given mutation, the plasmids obtained in all cases contained identical restriction patterns over part of the insert map. MET3, MET5, MET14 and HOM3 fragments were subcloned into pCKlOl, a YEp vector based on pCH100 (Hadfield et al., 1986) but with the pBR322 segment replaced by pUC19 (YanishPerron et al., 1985; C . Korch, in preparation).
79 1
TRANSCRIPTION OF METHIONINE AND THREONINE BIOSYNTHETIC GENES
a THRl
Region
pBY16a
pTL4 Pn3 pTL2 pTL23'
-
Thrl Pheno-
1 Kb D
I
b 3
D
P C
b,
m
-* E
c o
.
3
m >
1
3
> 0
m >
+ U
Y
B B
P 5
U
$
l
1
9
9
0
0
0
0
I!
0
4
N
N
N
4
0
0
I
I I
N
::
.
O
+
+
+
*
*
+
*
+
+
+
I
I
I
~n
*
.
.
.
?
?
5
I
I1
w
?
O
?
N
?
O N
N
O
?
N
?
O
N
Figure 9. THR1. Restriction map, probe (a) and Northern hybridization (b). Legend as for Figure 3. The bor mutants were compared to their parental strain D286.
HESl
uRA3 ACT1
Figure 10. MESI. Northern hybridization. The probe for MESl was a 948 bp XbaI internal fragment (Walter et al., 1983) cloned into the URA3-based YIP vector, pRG412-1 and designated pBY164. The Northern blot shows transcript levels in 10 pg of total RNA, per track (see Materials and Methods for details).
792
H. A. MOUNTAIN ETAL.
b
a HOM6 Region
Horn6
1Kb
PI PI
pTLl
53
IH
B1
H \ H X2
Phenox1 type
HI
.
,
I I
pTL7 pTLl6'
+
Figure 11. HOM6. Restriction map, probe (a) and Northern hybridization (b). Legend as for Figure 3. The bor mutants were compared to their parental strain D286
THRI, HOM2 and HOM6 were subcloned in YEp352 (Hill et al., 1986b). HOM2 fragments were subcloned in pRG4 16, a low copy YCp vector carrying URA3, aspTL12, pTL13 andpTL14(seeFigure 7a), as was MET6. The restriction map of the MET3 clone (Figure 3a) overlaps that obtained for the MET3 gene (Cherest et al., 1985, 1987) and subsequent sequencing of the region confirmed that it represented the structural gene. Two met5 complementing clones (Figure 4) were isolated and denoted pMET5-43 and pK4. pK4 was originally isolated as a horn6 complementing plasmid and its large insert (about 17 kb) was subcloned to separate the hom6 and met5 complementing abilities. MET5 and HOM6 are closely linked on the right side of chromosome X (Mortimer et al., 1989) and the isolation of both complementing activities on the same piece ofgenomic DNA strongly suggests that both structural genes have been cloned. MET14 is tightly linked to the centromere of chromosome XI and was isolated in association
with the centromeric DNA of this chromosome (Fitzgerald-Hayes et al., 1982). Its nucleotide sequence has been determined (Korch et al., 1991). The restriction map of the DNA complementing horn3 agrees with that previously described (Rafalski and Falco, 1988, 1990) and the original plasmid also complemented the tightly linked his1 mutation (Mortimer et al., 1989) and showed a restriction pattern matching that of the HIS1 region (Hinnebusch and Fink, 1983)and hence the plasmid contains the structural HOM3 gene. The MET6, HOM2 and THRI complementing clones were shown to carry the appropriate genes, and not to represent extra-genic suppressors, by mapping the site of integration of the cloned DNA when targeted into the genome on a YIP plasmid. MET6 complementing DNA integrated 1.2 cM from trp2 on chromosome V and showed the expected linkages to arg6, hom3 and his1 (Table 3) and hence, the cloned DNA contains the MET6 structural gene. The restriction map of the clone
793
TRANSCRIPTION OF METHIONINE AND THREONINE BIOSYNTHETIC GENES
Table 3. Genetic analysis of the integration of plasmids carrying the MET6, HOM2 and THRl genes. In all cases the plasmid was cut at a site in the subcloned DNA and used to transform H217d (a ura3-52, leu2-3, 112) to Ura+. The resultant strains were crossed to the a strains indicated and after sporulation the tetrads were analysed. The plasmids pTL22, pTLl5 and pTL18 potentially carry MET6, THRI and HOM2 DNA, respectively
a Strain H2179d::pTL22 XbaI H2179d::pTL22 XbaI
H2179d::pTL15 XbaI
H2179d::pTLlS Pst 1
a Strain
x F352
x F615
x 6040-1A
x S2614C
Gene pair met6::URA3x met6 met6::URA3 x arg6 met6::URA3 x hom3 met6::URA3x his1 met6::URA3x trp2 THRI::URA3 x arg4 THRI::URA3x adel THR1::URASx leu2 leu2 x adel hom2::URA3 x arol hom2::URA3 x hom2
Tetrads PD 127 54 36 41 82 65 23 22 32 98 121
NPD 0 0 2 1 0 0 25 25 46 1
0
Total TT 0 30 46 42 2 26 43b
127 84
91
Map distance” (CM)
0 17.9 34.5 28.6 1.2 14.3
44b
13b 22‘ 0
121
11.6 0
All of the markers segregated randomly. In all cases ‘:: URA3’ is the gene introduced on the pTL plasmid. Talculated from map distances = 100/2 (TT + 6NPD)/(PD+NPD +‘IT) (Perkins, 1949). hBoth leu2 and adel show centromere linkage; these data allow the position of ::URA3 relative to its centromere to be determined (reviews by Fincham and Day, 1963). ‘For this cross, the URA3 gene introduced on the plasmid can be distinguished from the one in S2614C because the plasmid ‘pops out’ readily if a selection is made for Met+ or Thr+,and the strains carrying the plasmid become Ura-.
agreed with that obtained previously (Csaikl and Csaikl, 1986). THRl complementing DNA integrated 14 cM from arg4 and 23cM from the centromere of chromosome VIII, which is appropriate for THRI DNA (Table 3). For HOM2, complementing DNA integrated 11.6cM from AROI on chromosome IV, which indicates that the plasmid contains HOM2 DNA (Table 3). RESULTS To obtain an overall picture of the regulation of the methionine/threonine biosynthetic pathway at the transcriptional level, a number of genes encoding enzymes in this pathway were cloned. These, along with additional cloned genes generously given to us, were used to study the transcriptional responses to conditions known to influence the specific activities of the corresponding enzymes (Table 1). Furthermore, their regulatory responses were examined in strains with either a gcdl or a gcn4 mutation to determine whether the genes were under the general amino acid control. Figures 3a-9a and 1l a show the restriction maps for the genomic regions containing the genes iso-
lated and the derivation of the probes used in the Northern hybridizations, the results of which are also shown in these figures (Figures 2, 3 b 9 b , 10, 1 1b). Densitometric estimates of the relative levels of all the transcripts are presented in Tables 4 and 5. There were variations in the loading of samples as evidenced by the variations in the internal controls of ACT1 and URA3 signals. We therefore estimated the levels of all transcripts relative to the stronger and more reliable ACT1 transcript (see Materials and Methods). The values in Tables 4 and 5 must be treated with a degree of caution because of the inherent difficulties in accurately quantifying transcripts by densitometric measurements of autoradiograms (Swillenset al., 1989). Care was taken to ensure that the autoradiograms were suitable for densitometry (Materials and Methods), but even so the procedure is at best semi-quantitative. Using the 18s and 23s rRNAs and the known sizes of the HIS4 and URA3 transcripts as molecular size standards, the approximate sizes of the mRNAs of the cloned genes are MET3 1.7-1.8 kb (in agreement with Cherest et al., 1985), MET5 5.5 kb, MET6 2.5 kb (in agreement with Csaikl and Csaikl, 1986), MET14 0.7 kb, HOM2 1.3 kb, HOM3 1.8 kb (in agreement with Rafalski and Falco, 1988, 1990), HOM6 1.2 kband THRI 1.2 kb.
MET3 MET5 MET14 MET6 HOM2 HOM3 THRI HOM6 MESl HIS4 URA3
Table 4.
1
1 1 1 1 1 1 1 1 1 1
E E 1.1 0 0.8
VOL.
7: 78 1-803 ( I99 I )
Four Major Transcriptional Responses in the Methionine/Threonine Biosynthetic Pathway of Saccharomyces cerevisiae HARRY A. MOUNTAIN*, ANDERS S. BYSTROM, JORGEN TANG LARSEN AND CHRISTOPHER K O R C H t
Depurtment of Microbiology. University of Umed, S-90187 Umed. Sweden Received 12 February 1991; accepted 4 June 1991
Genes encoding enzymcs in the threonine/methioninc biosynthetic pathway were cloned and used to investigate their transcriptional response to signals known to affect gene expression on the basis of enzyme specific-activities. Four major responses were evident: strong repression by methionine of M E T 3 , MET5 and METIQ. as previously described for MET3. M E T 2 and MET2.5; weak repression by methioninc of MET6; weak stimulation by methionine but no rcsponsc to threonine was seen for T H R I , H O M 2 and H O M 3 ; no response to any of the signals tested. for HOM6 and M E S I . In a BOR3 mutant. T H R I , HOMZ and H O M 3 mRNA levels were increased slightly. The stimulation of transcription by mcthioninc for H O M 2 , H O M 3 and THRI is mediated by the CCN4 gene product and hencc these genes are under the general amino acid control. In addition to the strong repression by methionine, MET5 is also regulated by the general control. KEY WORDS
-
Methioninejthreoninc biosynthesis; transcriptional regulation; general amino acid control; yeast.
INTRODUCTION Amino acid biosynthesis in Saccharomyces cerevisiae is regulated at the enzyme level, at the level of gene expression, or both (Jones and Fink, 1982; Messenguy, 1987). Pathway-specific gene regulation and an interlinked regulatory system called the general amino acid control (general control) have been found (reviewed by Hinnebusch, 1988). Pathway-specific controls have been observed for genes in the arginine, lysine. methionine, leucine, isoleucine and valine biosynthetic pathways and controls are mediated by cis- and rrans-regulatory elements at the transcriptional level. In contrast, the C P A l gene is regulated at the translational level (Werner er ul., 1987). In the general control system, starvation for any of tcn amino acids leads to thederepression ofabout 30 amino acid biosynthetic enzymes in nine different biosynthetic pathways (Hinnebusch, 1988). To date, the amino acid biosynthetic genes under this control have all been shown to have all or part of the *To whom enquiries should be addressed. tPresent address: Imperial Holly Research and Development Centre. 5320 Mark Dabling Boulevard, Colorado Springs, Colorado 80918, U.S.A.
0749 503X:91/080781-23 $1 1.50 0 1991 by John Wiley &Sons Ltd
sequence A T G G T C A T in the 5' non-coding region (Hopeand Struhl, 1985; Arndt and Fink, 1986; Hill el al., 1986a). This cis- regulatory sequence specifically binds the GCN4 regulatory protein which thereby activates gene expression at the transcriptional level (Struhl, 1982; Donahue er al., 1983; Arndt and Fink, 1986; Hope and Struhl, 1985). The GCN4 gene product is itself under translational regulatory control such that the protein is only produced upon amino acid starvation (Hinnebusch, 1984; Thireos er al.. 1984). Fourteen regulators of GCN4 mRNA translation have been defined (reviewed by Hinnebusch, 1988). One of these is the GCDl gene product which is a negative regulator of GCN4 mRNA translation. A gcdl mutant is therefore constitutively derepressed for genes under the general control (Wolfner er al.. 1975). The methionineithreonine biosynthetic pathway is diagrammed in Figure 1 and metabolic signals affecting expression of genes in this pathway are summarized in Table I . For most of these genes, the data are based o n measurements of the specific activities ofcorresponding enzymes isolated from cells grown in different media. Only the MET2, MET3, METl7i25, SAM1 and very recently MET16 genes
782
H.A. MOUNTAIN E T A L .
acids on the mRNA levels, the HOMand THR gene transcript levels were also examined in borrelidinresistant mutants which are potentially defective in the regulation of the methionine/threonine biosynthetic pathway. Borrelidin is a threonine analogue which specifically inhibits threonyl-tRNA synthetase. Of the four mutations conferring resistance to the antibiotic, BORI is an allele of HOM3 which encodes a feedback-resistant mutant of aspartokinase (Seibold et al., 1981), BOR3 encodes an altered threonyl-tRNA synthetase (Nass and Poralla, 1976), and the others are uncharacterized. Furthermore, to determine whether these methionine/threonine genes are under the general control we have investigated their regulatory responses in strains carrying the gcdl-101 allele or the gcn4-101 allele.
-=/
pzEYJ
ABBREVIATIONS
The designations of the genotypes of the wildtype and mutant alleles and their gene products are as follows: HOM2 and hom2, aspartate semialdehyde dehydrogenase (L-aspartate-p-semialdeMOt Mel-1RNA S-.dsn-xylmuhionme hyde:NADP oxidoreductase (phosphorylating), ( AdoMcI ) EC 1.2.1.11); HOM3 and hom3, aspartate kinase Figure I . Methionineithreonine biosynthesis in Saccharomyces (L-aspartate 4-phosphotransferase, EC 2.7.2.4); cmvisiae. Based on the figure of Jones and Fink (1982). and HOM6 and hom6, homoserine dehydrogenase updated. (L-homoserine:NAD oxidoreductase, EC 1. I . 1.3); MESl and mesl, methionyl-tRNA synthetase (Lhave been shown to be repressed at the transcrip- methionine:tRNAMc' ligase, EC 6 . I . 1.10); MET3 tional level by the addition of methionine or and met3, ATP sulphurylase (ATP:sulphate AdoMet (Baroni et al., 1986; Cherest et al., 1985; adenylyltransferase, EC 2.7.7.4); MET5 and met5, Sangsoda er al., 1985; Thomas and Surdin-Kerjan, part of the sulphite reductase (hydrogen sulphide: 1987; Thomas et al., 1990). SAM2 mRNA is NADP oxidoreductase, EC 1.8.1.2); MET6 and induced by methionine (Thomas er al., 1988). met6, homocysteine methyltransferase (5-methyl Recently, HOM2 has been shown to be under the tetrahydropteroyl-tn-L-glutamatex-homocysteine general amino acid control (Thomas and Surdin- S-methyltransferase, EC 2.1.1.14); MET14 and met14 APS kinase (ATP: adenylyl sulphate-3'Kerjan, 1989). In this paper we describe the cloning and physical phosphotransferase, EC 2.7.1.25); THRI and characterization of genomic DNAs encoding the rhrl, homoserine kinase (ATP:L-homoserine-0HOM2. HOM3. HOM6. THRI, MET3. MET5 and phosphotransferase, EC 2.7.1.39). Corresponding MET6 amino acid biosynthetic genes. A MET14 phenotypes are Horn+ and Hom- (a requirement clone (Fitzgerald-Hayes et a[., 1982) obtained from for threonine and methionine), Thr+ and Thr-, and J . Carbon was also physically characterized. We Met+ and Met-. BORI, BOR2 and BOR3 are obtained a MESl clone (Meussdoerffer and Fink, dominant borrelidin-resistant mutants and hor4 is a 1983) from G. R. Fink. Using these nine genes as recessive borrelidin-resistant mutant. Other general abbreviations are: bp, base pairs; probes in Northern blot hybridizations we investigated their transcriptional regulation. The levels of kb, kilobase pairs; nt, nucleotides; cM, centimRNAs were examined in total RNA isolated from morgans; Tris, tris(hydroxymethy1)aminomethane; cells growing in repressed and derepressed con- DTT, dithiothreitol; SDS, sodium dodecyl ditions according to the different metabolic signals sulphate; NaAc, sodium acetate; EDTA, ethylenelisted in Table 1. In addition to the effects of amino diaminetetra-acetate disodium salt; AdoMet, SMotbanyllRNA
.Usthiomnc
S -adenorylhomosynci-c
TRANSCRIPTION OF METHlONlNE AND THREONINE BIOSYNTHETIC GENES
adenosylmethionine; 3AT, 3-amino- 1,2,4-triazole. YEP, YCp and YIP vectors refer to yeast episomal, yeast centromere and yeast integrative plasmids, respectively . MATERIALS AND METHODS Materials
All the amino acids and 3AT were obtained from the Sigma Chemical Company. Other media components were obtained from Difco. Northern blot hybridizations were carried out on Gene Screen Plus@ hybridization transfer membranes from DuPont. Autoradiography was carried out on Kodak X-Omat AR5 films or Amersham Hyper Film-MP. In most cases the film was pre-flashed to give an optical density of 0.1 above the film base. Cronex intensifying screens from DuPont were used during autoradiography with exposures being made at - 70°C. Probes for Northern blot analysis were labelled using a Oligolabelling@ kit (purchased from Pharmacia) incorporating [a-”P]dCTP (3000 Ciimmol) from New England Nuclear. Restriction enzymes were purchased from New England Biolabs, Pharmacia, or Boehringer Mannheim. T4 DNA ligase and RNase-free DNase were purchased from Pharmacia. St ru ins
The source and genotypes of yeast and bacterial strains used in this study are listed in Table 2. Mediu
Complete (YEPD), minimal (MM) and synthetic complete (AA, containing all amino acids, uracil and adenine) media have been described previously by Sherman et al. (1986). Unless otherwise indicated, amino acid supplements were the same concentrations as for AA medium. 3AT was added at 10 mM. Genetic procedures
Yeast genetic manipulations were performed as described by Sherman et al. (1986). Difficulties encountered in sporulating certain diploids following Sherman et al. (1986) were overcome by following a procedure by Bilinski er al. (1987). Yeast cells were transformed by using 0.1 M-lithium acetate according to Ito et ul. (1983). Bacterial transformations followed the procedure of Hanahan (l983), as modified by Korch er ul. (1991).
783
D N A methods Large-scale isolation ofplasmid DNA, restriction enzyme analysis, and agarose gel electrophoresis were performed according to Maniatis et al. (1982). Isolation ofplasmid DNA from yeast was according to Hoffman and Winston ( 1 987). Mini-preparations of plasmid DNA from Escherichia coli were made according to Holmes and Quigley ( 1 98 I), except that we used 5% instead of 0.5% Triton X-100 in the STET solution and boiling was for 3 min instead of 45s. Probes for Northern blot analysis were purified on Sephadex G-50 Nick@ columns from Pharmacia. Northern blots Total RNA was prepared from S288C cells in mid-log phase (OD, -0.6 estimated with an LKB Ultrospec I 1 spectrophotometer) growing at 30°C in MM supplemented with methionine, threonine or lysine at 0, 0.2, 2 and 20 mM. Also, RNA was isolated from MM-grown strains containing the following mutations: B O R l , BOR2. BOR3, hor4. These represent steady-state levels of RNA. In addition, total RNA was also prepared from cells after shifting to the appropriate medium. In this case strains were pre-grown to about OD, ‘v 0.5 and then harvested, washed, re-suspended three times in 40 ml of sterile distilled water, and then suspended in the medium of interest (at OD, -0.5) and grown for 4 h (Penn et al., 1983). All RNAs for the tests for general control were isolated from shifted cells. For the gcdl-101 and gcnl-101 strains (9043-7D and L869, respectively), histidine or leucine, respectively, were added to MM to avoid the general control being induced because of starvation for these amino acids in these auxotrophic strains. The 9043-7D strain with the temperaturesensitive gcdl-I01 allele was grown at 25°C. Total RNA was prepared from 20 ml of cells in the appropriate medium following the procedure, suitably scaled down, described in Kelly et al. (1988). Contaminating DNA was removed by resuspending the washed and dried pellet in 20mMNaAc, pH 4.5, 10mM-MgCI, and 10mM-NaCI, and digesting with 30 units of RNase-free DNase (Pharmacia) at room temperature for 30 min. After extracting the solution twice with phenol/chloroform, followed by a chloroform extraction, the nucleic acids were re-precipitated and centrifuged. The pellet was resuspended in 20 mM-sodium phosphate (pH 6.5), 2 mM-EDTA, debris removed by centrifugation, and the nucleic acid concentration
Adenylsulphate kinase PAPS reductase
MET14 METI6, METI7*, MET22, METI, MET8 MET5, MET10 METI8, MET8 MET20, M E T I , MET2
MET6
MET25
ATP sulphurylase
MET3
0-acetylhomoserine/ 0-acetylserine sulphydrylase Homocysteine methyltransferase
Homoserine acetyltransferase
Sulphite reductase
Sulphate permeases
Enzyme
C H R I . sell
Gene
+ +
+
+
+met +met - tRNA'" AdoMet +met +met - tRNAM" AdoMet +met +met -tRNAMet +met
+ +AdoMet
AdoMet +met AdoMet +met +met - tRNAM" +met +met 1 mM-AdoMet
Low enzyme levels
AdoMet -met -met -tRNA'"' -AdoMet -met -met - tRNAM" - AdoMet -met - met-tRNAM" -met -
+
AdoMet -met - AdoMet -met - tRNAM" -met -met 0.05 mM-AdoMet -
High enzyme levels
Regulatory signals for:
3.5 (met)
31 (met)
5-19 (met)
25 (met)
> 1 (met) 4.5 11
11&140 (met)
+
Ratio of steady-state enzyme levels -amino acid/ amino acid
Table 1. Genes and enzymes of methionine and threonine biosynthesis. Known signals and regulatory responses of enzyme levels
References
Probable transcriptional activator for thc M E T genes Unknown functions
MET4
M E T 7 . M E T 13. M E T I S , MET21. M E T 2 3 , MET24. N I I S I . PHSI
- mct Not known
+met +met
- mct
0.5 (met)
3 (1YS) 0.5 ( + bor)" 0.5 ( + mct)a > 1 (thr) 2--5 (met)
4 7(thr)
I . 3 4 (met)
2.0 (met) 2.7 (AdoMct) j. k
"Note that ratios less than I signify induction by the indicated compound. "Breton and Surdin-Kerjan (1977): 'Cherest er a/. (1969); Therest C I a/. (1973a); 'Ferro and Spence (1973): 'Cherest rr a/. (1971); 8 Antoniewski and de Robichon-Szulmajster (1973); 'dde Robichon-Szulmajster and Cherest (1967); 'Baroni er a/.( 19x6);'de Robichon-Szulmajstrr and Corrivaux (1963); 'de Robichon-Szulmajster and Corrivaux (1964); 'Nass and Hascnbank (1970); "'de Robichon-S7ulmajster 6'1 01. (1973): "Surdin (1967); PLor and Cossins (1972); T h o m a s 1.r a/. (19YO); Thomas CI a/. (199 I). *A complementing allele of the ME7'2.7gene. mcr17, affects PAPS reductax.
THRl THH4
HOM6
Aspartate semialdehyde deh ydrogcnase Homoscrinc dehydrogenasc Homoserine kinasc Threoninc kinase
HOM2
tRNAM"'! - thr. +met
met-t RNAMCL ? - mct
+ lys,
+ borrelidin
- thr.
-met
+ homoserinc
+ thr, +met
Aspartokinase
HOM3
+
+met AdoMet
Glucose-6-phospha te dchydrogenasc
MET19
U
*Z
786
H. A. MOUNTAIN ET AL.
Table 2. List o f yeast and bacterial strains used in this study Strain Yeast strains F337 F350 F352 F483 F615 F762 F763 L869 S288C 6080-1 1C 6040- 1 A 6460-8D 9043-7D H21012A H2121B H2 179d H2 17AB 1 S2206D S2614C STX272-4D D286-2A D286-2A BORl D286-2A BOR2 D286-2A BOR3 D286-2A b o d Bacterial strain DH5a
Genotype a ade9 ura3 horn3 serl a met5 leu2 a met6 a cdc6 his4-519 adel ura3-52 lys2 thrl a ilvl arg6 ura3 horn3 trp2 hisl adel met1 gal2 a ura3-52 t r p l d l a ura3-52 trpl-A1 a gcn4-I01 leu2-3,112 a ma12 gal2 a met14 trpl-A1 lysl-1 a adel arg4 his2 lys7 ura3-52 a met3 a gcdl-I01 his4-519 a met6 ura3-52 a horn6 ura3 a ura3-52 leu2-3,112 a ura3-52 a ura3 horn3 gal2 his2 his6 Ieul a horn2 arol C lysl adel trpl arg4-1 gaI2 a horn6 adel leu1 ura3-52 his2 his4 ade6 ural a adel hisl a adel hisl BORI a adel hisl BOR2 a adel hisl BOR3 a adel hisl bor4
F - endAl hsdRl7(ri, m:)supE44 thi- 1 h recA 1 gyrA96 relA 1 A(1acZYA- argF)U 169, cp80dlacZAM 15
Source G.R.F. G.R.F. G.R.F. G.R.F. G.R.F. G.R.F. G.R.F. G.R.F. G.R.F. G.R.F. G.R.F. G.R.F. G.R.F. This work This work This work This work YGSC YGSC YGSC
K.P. K.P. K.P. K.P. K.P. BRL
G.R.F.,Dr G. R. Fink; YGSC, Yeast Genetic Stock Center; K.P., Dr K. Poralla, BRL, Bethesda Research Laboratories.
determined spectrophotometrically. The DuPont manual on the running of denaturing formaldehyde agarose gels and RNA transfer to Gene Screen Plus@ membranes was followed. Total RNA (10 pg) was applied to each lane of the Northern blot gels. Probes were labelled to 3-5 x lo9 cpm/pg DNA and lo8cpm were added to each hybridization. All Northern blots were probed for ACTI mRNA as an internal loading control. The probe was a 314 bp BglII internal fragment of the ACTI gene (Ng and Abelson, 1980) cloned into M13tg131. Purified replicative form was used as a probe.
All Northern hybridization data in Figures 2-1 1 for the tests for the general control (+3AT, +Met+Val) in S288C and the gcdl-101 and gcn4-101 strains are from shift experiments, as are the amino acid omission tests (AA-Met, AA-Thr). For MM+Met data, except for +0.2mM-Met where only steady-state experiments were done, both steady-state and shift data showed the same patterns. Shift data are shown for MET3, MET14, MET6, THRl and the HOM genes. Steady-state data are shown for MET5 and MESI. All data for MM + Thr and MM + Lys and for the borrelidinresistant strains are steady-state data.
787
TRANSCRIPTION OF METHIONINE AND THREONINE BIOSYNTHETIC GENES
__
ne r >n P h
+ I
z
)
*
2
O
+
?
N
e
+
?
0
+
d
+
3
> m
r
z z z u
+
I B F ?
d
%
u
%
,
+
N
N
i
t
u D
Y
z
%
, , ? ? ?
W U z F 1
z
u
0 O
N
*
I
B
Z
?
?
3 s : +
-
u O
X
+
+
P P E S I I S E E S I S I I E E E E
Figure 2. HIS4. Northern hybridization. The probe for HIS4 and URA3 was pNKY48 (Alani and Kleckner, 1987), which contains no other yeast sequences. The Northern blot shows transcript levels in 10 pg of total RNA, per track (see Materials and Methods for details).
a
b Y
Y
D z
D z
2
2
%
O z
U
Figure 3. MET3. Restriction map and subclones of the genomic regions containing the cloned gene (a) and Northern hybridization (b) of the probe to investigate transcriptional regulation. (a) Restriction maps of the DNA complementing the mutation and the complementing activities of derived subclones are shown. All probes were based on the YIP vector carrying URA3, pRG412-1. An asterisk indicates the cloned segment used as the probe in the Northern hybridizations. Abbreviations for restriction enzyme sites in this and subsequent figures (Figures 4a-9a, 1 la) are B1, BamHI; B2, BglII; C, ClaI; E, EcoRI; H, HindIII; K, KpnI; N, NdeI; P1, PstI; P2 PvuII; S1, SacI; S2, SacII; S3, SalI; S4, SsiI; XI, XbaI; X2, XhoI; X3, XmnI. (b) The Northern blot shows transcript levels in 10 pg of total RNA, per track (see Materials and Methods for details). S288C was the source of RNA except for the lanes indicated gcdl or gcn4 where the strains 9043-7D and L869 were the respective sources. MM is minimal synthetic medium and the supplements are indicated. AA is MM with all amino acids, adenine and uracil added as in Sherman ei al. (1986); appropriate omissions and additions are indicated.
Densitometry
This was carried out using a Bio-Rad Laboratories Video Densitometer model 620 and the data were analysed with the Bio-Rad Laboratories program 1D Analyst I1 for Microsoft Windows. Densitometry was only carried out on autoradiograms made using pre-flashed film. Only those exposures which gave
signals in the linear optical density range of the film (0.1-1.5 above the pre-flashed film base level) were measured. The photographs presented in Figures 2-1 1 are of autoradiograms which reproduced the best and are not necessarily those which were scanned densitometrically. The probes all contained, in addition to the gene of interest, the URA3 gene to serve as an internal
788
H. A. MOUNTAIN ETAL.
b
a MET5
Met5 Phew
Region
1Kb
P1
S2 S3
E S1
type
* m
+ U
W
U
B P Z 5 5 %
pCK129
I I
II
\
; !
"
I
I I
+
O
9
N
+
9
: +
,
+
4.
e"
x
I
f
8 f
0
9
r< . .N
8
:
U
Y
I
I
N
z; +
+
+
pCK13O
MET5
I pCK143
I
I
--
pCK144
I I
I
uRA3
pCK133'
ACT1
+
0
€.
pCKlO6
0
U
pCKlD8 pCK109
2
I I
Y
5
+
+
I I I I I I
- +
control for differences in the amount of RNA loaded. The approximately 900 nt long URA3 transcript is known to be expressed constitutively under the conditions we tested (Penn et al., 1983) and its expression does not alter in response to the general control (Silverman et al., 1982). However, variation in the level of the URA3 mRNA and its frequently
2 f
% ; +
E I
H I 3 5 2 2 I
0
I
N
I pCK119'
+
" 1 9 0 O N ;
pCK112 pCK113
P
8 2 h
pCK111
-n
+
pCK107
0
0
"
C 4
N.
N
*
*
Y
2 f 2 ;m m 0
+
+
I H I I E I 2 Z S E urn3 MET14
ACT1
weak signal (Figures 2-1 1) prompted us to reprobe the blots for ACTZ mRNA, again a control for RNA loading. A stronger, and hence more reliable signal for comparison was obtained. Some variation was still seen. To compensate for this and to allow for comparisons of the extent of repression or induction of transcript levels, first we normalized all
789
TRANSCRIPTION OF METHIONINE AND THREONINE BIOSYNTHETIC GENES
-
a
MET6
Region B,
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-3
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I I I I
pHAM39 pHAM40
MET6
URA3
pHAM41 ACT1
pTL22^
Figure 6. MET6. Restriction map, probe (a) and Northern hybridization (b). Legend as for Figure 3.
a HOM2 Region
b .
1Kb
I pBY165 pTL6 pTL5 pTL14 pTL13 pTL12 pTL17 pTL18'
ACTl
Figure 7. HOM2. Restriction map, probe (a) and Northern hybridization (b). Legend as for Figure 3. The bor mutants were compared to their parental strain D286.
790
H. A. MOUNTAIN ETAL.
a HOM3-HlSl
Region
c
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pBY16Y
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Figure 8. HOM3. Restriction map, probe (a) and Northern hybridization (b). Legend as for Figure 3. The bor mutants were compared to their parental strain D286.
values to the same ACT1 level and then for each individual gene the mRNA signal in MM was taken as 1 and all other levels were expressed relative to this. These data are shown in Tables 4 and 5. Relative to the loading control of ACTl mRNA, the URA3 transcript level (Tables 4 and 5) can be seen to be generally unresponsive to the conditions. This is as expected. Because of the variation in loading that did occur, the frequently weak URA3 mRNA did not make as reliable a control as that of ACTl mRNA. Isolation and confirmation of clones containing methioninelthreonine biosynthetic genes
Hybrid plasmids containing the T H R l , HOM2, HOM3, HOM6, MET3 and MET6 genes were isolated from yeast genomic libraries, based on the YCpSO vector (Rose et al., 1987), by complementation of the corresponding phenotypes on synthetic plates. Complementation was always made in com-
bination with selection for Ura', the selectable marker of the vector being URA3. MET5 was also cloned from a YEp24-based library (Carlson and Botstein, 1982) by complementation of met5 in strain F350. Hybrid plasmids were re-isolated in E. coli from positive yeast clones. These plasmids were re-transformed into yeast and Ura' transformants were selected and the complementation of the mutant phenotype, by the plasmid, confirmed. A set of plasmids from each cloning that passed these tests above, were isolated as minipreps, digested with restriction enzymes, and analysed by agarose gel electrophoresis. For complementation of a given mutation, the plasmids obtained in all cases contained identical restriction patterns over part of the insert map. MET3, MET5, MET14 and HOM3 fragments were subcloned into pCKlOl, a YEp vector based on pCH100 (Hadfield et al., 1986) but with the pBR322 segment replaced by pUC19 (YanishPerron et al., 1985; C . Korch, in preparation).
79 1
TRANSCRIPTION OF METHIONINE AND THREONINE BIOSYNTHETIC GENES
a THRl
Region
pBY16a
pTL4 Pn3 pTL2 pTL23'
-
Thrl Pheno-
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Figure 9. THR1. Restriction map, probe (a) and Northern hybridization (b). Legend as for Figure 3. The bor mutants were compared to their parental strain D286.
HESl
uRA3 ACT1
Figure 10. MESI. Northern hybridization. The probe for MESl was a 948 bp XbaI internal fragment (Walter et al., 1983) cloned into the URA3-based YIP vector, pRG412-1 and designated pBY164. The Northern blot shows transcript levels in 10 pg of total RNA, per track (see Materials and Methods for details).
792
H. A. MOUNTAIN ETAL.
b
a HOM6 Region
Horn6
1Kb
PI PI
pTLl
53
IH
B1
H \ H X2
Phenox1 type
HI
.
,
I I
pTL7 pTLl6'
+
Figure 11. HOM6. Restriction map, probe (a) and Northern hybridization (b). Legend as for Figure 3. The bor mutants were compared to their parental strain D286
THRI, HOM2 and HOM6 were subcloned in YEp352 (Hill et al., 1986b). HOM2 fragments were subcloned in pRG4 16, a low copy YCp vector carrying URA3, aspTL12, pTL13 andpTL14(seeFigure 7a), as was MET6. The restriction map of the MET3 clone (Figure 3a) overlaps that obtained for the MET3 gene (Cherest et al., 1985, 1987) and subsequent sequencing of the region confirmed that it represented the structural gene. Two met5 complementing clones (Figure 4) were isolated and denoted pMET5-43 and pK4. pK4 was originally isolated as a horn6 complementing plasmid and its large insert (about 17 kb) was subcloned to separate the hom6 and met5 complementing abilities. MET5 and HOM6 are closely linked on the right side of chromosome X (Mortimer et al., 1989) and the isolation of both complementing activities on the same piece ofgenomic DNA strongly suggests that both structural genes have been cloned. MET14 is tightly linked to the centromere of chromosome XI and was isolated in association
with the centromeric DNA of this chromosome (Fitzgerald-Hayes et al., 1982). Its nucleotide sequence has been determined (Korch et al., 1991). The restriction map of the DNA complementing horn3 agrees with that previously described (Rafalski and Falco, 1988, 1990) and the original plasmid also complemented the tightly linked his1 mutation (Mortimer et al., 1989) and showed a restriction pattern matching that of the HIS1 region (Hinnebusch and Fink, 1983)and hence the plasmid contains the structural HOM3 gene. The MET6, HOM2 and THRI complementing clones were shown to carry the appropriate genes, and not to represent extra-genic suppressors, by mapping the site of integration of the cloned DNA when targeted into the genome on a YIP plasmid. MET6 complementing DNA integrated 1.2 cM from trp2 on chromosome V and showed the expected linkages to arg6, hom3 and his1 (Table 3) and hence, the cloned DNA contains the MET6 structural gene. The restriction map of the clone
793
TRANSCRIPTION OF METHIONINE AND THREONINE BIOSYNTHETIC GENES
Table 3. Genetic analysis of the integration of plasmids carrying the MET6, HOM2 and THRl genes. In all cases the plasmid was cut at a site in the subcloned DNA and used to transform H217d (a ura3-52, leu2-3, 112) to Ura+. The resultant strains were crossed to the a strains indicated and after sporulation the tetrads were analysed. The plasmids pTL22, pTLl5 and pTL18 potentially carry MET6, THRI and HOM2 DNA, respectively
a Strain H2179d::pTL22 XbaI H2179d::pTL22 XbaI
H2179d::pTL15 XbaI
H2179d::pTLlS Pst 1
a Strain
x F352
x F615
x 6040-1A
x S2614C
Gene pair met6::URA3x met6 met6::URA3 x arg6 met6::URA3 x hom3 met6::URA3x his1 met6::URA3x trp2 THRI::URA3 x arg4 THRI::URA3x adel THR1::URASx leu2 leu2 x adel hom2::URA3 x arol hom2::URA3 x hom2
Tetrads PD 127 54 36 41 82 65 23 22 32 98 121
NPD 0 0 2 1 0 0 25 25 46 1
0
Total TT 0 30 46 42 2 26 43b
127 84
91
Map distance” (CM)
0 17.9 34.5 28.6 1.2 14.3
44b
13b 22‘ 0
121
11.6 0
All of the markers segregated randomly. In all cases ‘:: URA3’ is the gene introduced on the pTL plasmid. Talculated from map distances = 100/2 (TT + 6NPD)/(PD+NPD +‘IT) (Perkins, 1949). hBoth leu2 and adel show centromere linkage; these data allow the position of ::URA3 relative to its centromere to be determined (reviews by Fincham and Day, 1963). ‘For this cross, the URA3 gene introduced on the plasmid can be distinguished from the one in S2614C because the plasmid ‘pops out’ readily if a selection is made for Met+ or Thr+,and the strains carrying the plasmid become Ura-.
agreed with that obtained previously (Csaikl and Csaikl, 1986). THRl complementing DNA integrated 14 cM from arg4 and 23cM from the centromere of chromosome VIII, which is appropriate for THRI DNA (Table 3). For HOM2, complementing DNA integrated 11.6cM from AROI on chromosome IV, which indicates that the plasmid contains HOM2 DNA (Table 3). RESULTS To obtain an overall picture of the regulation of the methionine/threonine biosynthetic pathway at the transcriptional level, a number of genes encoding enzymes in this pathway were cloned. These, along with additional cloned genes generously given to us, were used to study the transcriptional responses to conditions known to influence the specific activities of the corresponding enzymes (Table 1). Furthermore, their regulatory responses were examined in strains with either a gcdl or a gcn4 mutation to determine whether the genes were under the general amino acid control. Figures 3a-9a and 1l a show the restriction maps for the genomic regions containing the genes iso-
lated and the derivation of the probes used in the Northern hybridizations, the results of which are also shown in these figures (Figures 2, 3 b 9 b , 10, 1 1b). Densitometric estimates of the relative levels of all the transcripts are presented in Tables 4 and 5. There were variations in the loading of samples as evidenced by the variations in the internal controls of ACT1 and URA3 signals. We therefore estimated the levels of all transcripts relative to the stronger and more reliable ACT1 transcript (see Materials and Methods). The values in Tables 4 and 5 must be treated with a degree of caution because of the inherent difficulties in accurately quantifying transcripts by densitometric measurements of autoradiograms (Swillenset al., 1989). Care was taken to ensure that the autoradiograms were suitable for densitometry (Materials and Methods), but even so the procedure is at best semi-quantitative. Using the 18s and 23s rRNAs and the known sizes of the HIS4 and URA3 transcripts as molecular size standards, the approximate sizes of the mRNAs of the cloned genes are MET3 1.7-1.8 kb (in agreement with Cherest et al., 1985), MET5 5.5 kb, MET6 2.5 kb (in agreement with Csaikl and Csaikl, 1986), MET14 0.7 kb, HOM2 1.3 kb, HOM3 1.8 kb (in agreement with Rafalski and Falco, 1988, 1990), HOM6 1.2 kband THRI 1.2 kb.
MET3 MET5 MET14 MET6 HOM2 HOM3 THRI HOM6 MESl HIS4 URA3
Table 4.
1
1 1 1 1 1 1 1 1 1 1
E E 1.1 0 0.8
