Download Two Anthranilate Synthase Genes in Arabidopsis

The Plant Cell, Vol. 4, 721-733, June 1992 O 1992 American Society of Plant Physiologists
Two Anthranilate Synthase Genes in Arabidopsis:
Defense-Related Regulation of the Tryptophan Pathway
Krishna K. Niyogi and Gerald R. Finkl
Department of Biology and Whitehead lnstitute for Biomedical Research, Massachusetts lnstitute of Technology,
Nine Cambridge Center, Cambridge, Massachusetts 02142
Arabidopsis thaliana has two genes, ASA7 and ASA2, encoding the a subunit of anthranilate synthase, the enzyme catalyzing
the first reaction i n the tryptophan biosynthetic pathway. As a branchpoint enzyme i n aromatic amino acid biosynthesis,
anthranilate synthase has an important regulatory role. The sequences of the plant genes are homologous to their microbial
counterparts. Both predicted proteins have putative chloroplast transit peptides at their amino termini and conserved
amino acids involved in feedback inhibition by tryptophan. ASA7 and ASA2 cDNAs complement anthranilate synthase
a subunit mutations i n the yeast Saccharomyces cerevisiae and i n Escherichia coli, confirming that both genes encode
functional anthranilate synthase proteins. The distributions of ASA7 and ASA2 mRNAs i n various parts of Arabidopsis
plants are overlapping but nonidentical, and ASA7 mRNA is approximately 10times more abundant in whole plants. Whereas
ASA2 i s expressed at a constitutive basal level, ASA7 is induced by wounding and bacterial pathogen infiltration, suggesting a nove1 role for ASA7 i n the production of tryptophan pathway metabolites as part of an Arabidopsis defense
response. Regulation of key steps i n aromatic amino acid biosynthesis i n Arabidopsis appears to involve differential expression of duplicated genes.
INTRODUCTION
The enzyme anthranilatesynthase (AS; EC 4.1.3.27) catalyzes
the first reaction branching from the aromatic amino acid pathway toward the biosynthesisof tryptophan in plants, fungi, and
bacteria. Tryptophan is required primarily for protein synthesis in bacteria and fungi, whereas in plants the tryptophan
pathway also provides precursorsfor the synthesis of key “secondary” metabolites such as the major endogenous auxin,
indole-3-acetic acid (IAA), and other molecules that may help
protect plants against pathogens and herbivores.
Because AS is a branchpoint enzyme in aromatic amino acid
biosynthesis, regulation of AS is critical for controlling the flux
of intermediates in the pathway. In plants, fungi, and bacteria,
AS enzyme activity is feedback inhibited by tryptophan. In
microorganisms, the genes encoding AS and other tryptophan
biosynthetic enzymes are regulated in a manner that reflects
the functional importance of the amino acid as the end product of the pathway. For example, in Escherichia coli the
synthesis of tryptophan pathway enzymes is regulated by tryptophan via transcription repression and attenuation (for review,
see Yanofsky and Crawford, 1987). As a result, the enzymes
are synthesized only under conditions of tryptophan starvation. In the yeast Saccharomyces cerevisiae and other fungi,
transcription of the genes encoding tryptophan biosynthetic
enzymes is under general amino acid control rather than
pathway-specific control (for review, see Hinnebusch, 1988).
The genes are expressed ata constitutive basal level, and increased transcription occurs not only in response to tryptophan
starvation, but also upon starvation for a number of other amino
acids. The regulation of tryptophan biosynthetic genes in plants
is undefined.
Microbial AS is composed of two nonidentical subunits, an
a subunit (also called Component I), which binds the substrate
chorismate and carries out its aromatization, and a subunit
(also called Component II), which transfers an amino group
from the other substrate glutamine (for reviews, see Zalkin,
1980; Hütter et al., 1986; Yanofsky and Crawford, 1987;
Crawford, 1989). This glutamine-dependentAS reaction (1) requires both a and p subunits. The a subunit alone can
synthesize anthranilate from chorismate using ammonia as
the amino donor rather than glutamine, but only if the ammonia is present at a sufficiently high concentration. This reaction
is termed the ammonia-dependent AS reaction (2). Both AS
reactions require Mg2+ as a cofactor.
Chorismate
1
To whom correspondence should be addressed.
+ glutamine -. anthranilate +
Chorismate
+
glutamate
NH3 -. anthranilate
+
pyruvate
pyruvate
+
(11
(2)
722
The Plant Cell
The a subunit is usually a monofunctional polypeptide,whereas
the p subunit is often part of a multifunctional polypeptide in
which one domain contains the glutamine amidotransferase
activity, and the others catalyze subsequent reactions in the
tryptophan biosynthetic pathway. For example, in Neurospora
crassa the TRPl gene encodes a single polypeptide containing the AS p subunit, phosphoribosylanthranilateisomerase,
and indole-3-glycerolphosphate synthase (Hütter et al., 1986).
The a and p subunits associate to form a heteromeric AS enzyme, i.e., an a$;! heterotetramer in E. coli (Ito and Yanofsky,
1969) and N. crassa (Hulett and DeMoss, 1975) and an ap heterodimer in S. cerevisiae (Prantl et al., 1985). Recent studies
of AS from Salmonella typhimurium (Caligiuri and Bauerle, 1991)
and Brevibacferium lacfofermenfum (Matsui et al., 1987) have
pinpointed particular amino acids in the a subunit that are important for feedback inhibition by tryptophan. Microbial AS
genes have homology to genes encoding at least two other
chorismate-utilizingenzymes, para-aminobenzoicacid (PABA)
synthase (Goncharoff and Nichols, 1984; Crawford, 1989) and
isochorismate synthase (Ozenberger et al., 1989).
Relatively little is known about the biochemistry and regulation of AS from plants (for reviews, see Gilchrist and Kosuge,
1980; Poulsen and Verpoorte, 1991; Singh et al., 1991). Crude
fractionation studies have shown that AS is separable from
other enzymes in the pathway (Hankins et al., 1976), but the
subunit composition of plant AS has not been determined. The
role of feedback inhibition of AS enzyme activity by tryptophan
(Belser et al., 1971) has been investigated using plant cell cultures resistant to 5-methyltryptophan, a false feedback inhibitor
of AS. The mutant cells contain a feedback-resistant AS, and
as a result the cultures accumulate tryptophan (Widholm,
1972a. 1972b; Carlson and Widholm, 1978; Ranch et al., 1983)
and, in some cases, no longer require exogenous auxin for
growth (Widholm, 1977; Sung, 1979). Plant cell cultures fed
with anthranilate or indole show apparently unregulated accumulation of tryptophan (Widholm, 1974). Together, these
results imply that tryptophan levels in cultured cells are controlled mainly by regulation of AS. However, besides feedback
inhibition of AS, control of tryptophan biosynthesis in plants
is largely uncharacterized.The synthesis of pathway enzymes,
including AS, is not repressed by tryptophan in plant tissue
cultures (Widholm, 197l). Subcellular fractionation experiments
(Mousdale and Coggins, 1986) have led to the conclusion that
AS, like the rest of the aromatic amino acid pathway (SchulzeSiebert and Schultz, 1989), is sequestered in plastids, although
there has been a report of a cytosolic AS isozyme (Brotherton
et al., 1986).
We are studying AS in Arabidopsis fhaliana to gain further
insight into the structure and regulation of the tryptophan biosynthetic pathway in plants. We find that Arabidopsis has two
genes encoding the a subunit of AS. The two genes are regulated differently in response to wounding and bacterial
pathogen infiltration, providing molecular genetic evidence that
the tryptophan branch of the aromatic pathway has a role in
plant defense responses.
RESULTS
lsolation of Two Genes Encoding Arabidopsis
Anthranilate Synthase
A fragment of the yeast TRP2 gene (Zalkin et al., 1984) containing the region most highly conserved between yeast and
bacterial AS a subunit genes was used as a DNA hybridization probe to isolate homologous cDNAs from Arabidopsis. A
cDNA with 500/0 nucleotide and 39% amino acid identity to
the yeast probe was subsequently used to identify two classes
of additional Arabidopsis cDNA clones. Restriction mapping
and further sequence analysis revealed that one class was
identicalto the initial Arabidopsis clone, whereas the other class
represented a second gene that also had significant similarity
to known AS a subunit genes. Genomic clones corresponding to each cDNA were isolated and sequenced.
These two Arabidopsis AS genes, designated ASAl (GenBank accession no. M92353) and ASA2 (GenBank accession
no. M92354), share 63% nucleotide identity within proteincoding exons. The structures of the ASAl and ASA2 genes are
summarized schematically in Figure 1.
ASAl:
XSHd PHd SHd P
Hc
Hc
HdHdP
EXHc
PHc
Hd C
HcHd
ASA2:
,
t o
Ikb
Figure 1. Schematic Structures of ASAl and ASA2 Genes.
The boxes represent exons, and dashed lines connect homologous exons between ASAl and ASAP. The 5' to 3' transcriptional orientation of
both genes is from left to right. The restriction sites shown are A, Accl; C, Clal; E, EcoRI; Hc, Hincll; Hd, Hindlll; P, Pstl; S, Sacl; and X, Xbal.
Arabidopsis Anthranilate Syntheses
B
RNA
RNA
!-*-*
GATC - +
723
two genes cross-hybridized weakly, but there was no evidence
of additional hybridizing sequences (data not shown). Restriction fragment length polymorphism (RFLP) linkage analysis
(S. Hanley and H. Goodman, personal communication) placed
kb
-9.5-
-7.5-4.4H*
«. -2.4-
it
UJ X
-1.4-
X
zi
_
o
^
S
8
U UJ
—23130—
—9416 —
— 6557 —
-0.24-
—4361 —
ASA1
ASA2
ASA1
ASA2
Figure 2. Analysis of /AS/47 and ASA2 mRNAs.
(A) RNA gel blot analysis. Ten micrograms of Arabidopsis total RNA
and 1 ng of poly(A)+ RNA were fractionated by electrophoresis on a
1% agarose gel containing formaldehyde (Ausubel et al., 1989), transferred to nitrocellulose, and hybridized with a 32P-labeled 2-kb Xhol
fragment of pKN41 (ASA1) and a 1.8-kb BamHI fragment of pKN108A
(ASA2). The sizes of the RNA markers (Bethesda Research Laboratories) are shown in kilobases.
(B) Primer extension analysis. For ASA1, an antisense oligonucleotide complementary to nucleotides 2391 to 2418 in the ASA1 sequence
(GenBank accession no. M92353) was hybridized to 25 ng of Arabidopsis total RNA at 55°C, and cDNA was synthesized using reverse
transcriptase (Ausubel et al., 1989). Products were analyzed by denaturing polyacrylamide gel electrophoresis. The ASA1 sequencing ladder
was generated using the same primer and pKN211A template DNA.
For ASA2, an antisense oligonucleotide complementary to nucleotides
2846 to 2873 in the ASA2 sequence (GenBank accession no. M92354)
was hybridized to 50 ng of Arabidopsis total RNA at 55°C. The ASA2
sequencing ladder was generated using the same primer and pKN143C
template DNA.
— 564—
ASA1
ASA2
B
2
4553 - - 0.0
Cp-2
24.2
er
39.2
ASA2
Both genes are transcribed, resulting in mRNAs approximately 2200 nucleotides in length, as shown by RNA gel blot
analysis in Figure 2A. The 5' ends of the mRNAs encoded by
the two genes were determined by primer extension (Figure
2B) and confirmed by S1 nuclease protection (data not shown).
For ASA1 mRNA, there are two major 5' ends, resulting in 5'
untranslated leaders of 90 and 93 nucleotides. The mRNA corresponding to ASA2 begins at a single site 41 nucleotides
upstream of the first AUG codon. As determined by cDNA sequencing, the ASA1 and ASA2 genes have 3' transcribed but
untranslated regions of 156 nucleotides and 162 nucleotides,
respectively. As is the case for many plant genes (Joshi, 1987),
no sequences were found in these 3' regions that exactly match
the AAUAAA consensus sequence for animal poly(A) addition signals.
Arabidopsis ASA1 and ASA2 are duplicated, but unlinked,
nuclear genes. The cloned genes hybridized to different sets
of fragments on a genomic DNA gel blot, as shown in Figure
3A. Under conditions of reduced hybridization stringency, the
TSB1
125.3
AB5-13
160.6
-
47.2
21502
49.9
cer-8
71.9
Figure 3. Analysis of ASA1 and ASA2 Genomic Loci.
(A) Genomic DNA gel blot analysis. Two micrograms of Arabidopsis
genomic DNA was digested with the indicated restriction enzymes,
fractionated by electrophoresis on a 0.8% agarose gel, transferred to
a Zeta-Probe membrane (Bio-Rad), and hybridized with a ^P-labeled
1-kb EcoRI fragment of pKNSA (ASA1) and a 1.2-kb EcoRI fragment
of pKN1A (ASA2). Sizes of DNA markers (Hindlll-digested X DNA) are
shown in base pairs.
(B) Schematic representation of the locations of ASA1 and ASA2 on
Arabidopsis chromosomes 2 and 5. Selected visible and RFLP genetic
markers are shown to the left of each vertical line, and map distances
in centimorgans (S. Hanley and H. Goodman, personal communication) are to the right.
724
The Plant Cell
Plurality
ASA1
ASA2
TRP2 (S.C.)
TrpE (E.c. )
TrpE ( B . s . )
PabB (E.c )
.
......... I . ........ I ......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .I .
10
20
30
50
40
60
70
80
90
100
170
180
190
200
***
Plural ity
ASAl
ASA2
TRP2(S.c.)
TrpE (E.c. )
TrpE ( B . s . )
PabB (E.c.)
......... I .........l.........I.........I.........I.........I.........l............l.........l.........
110
Plurality
ASAl
ASA2
TRP2(S.c.)
TrpE (E.c. )
TrpE(E3.s.)
PabB (E.c.)
120
140
160
150
I
......... I ......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
210
Plurality
ASAl
ASA2
TRP2 ( S . c. )
TrpE (E.c. )
TrpE (E3.s.)
PabB (E.c.)
130
220
230
240
250
.........I . . ....... l ......... I..... .... I.........I.
310
320
tt
330
340
2 60
270
280
2 90
I
300
........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .I
350
**
360
370
3 90
380
400
t
t
Plurality
ASAl
ASA2
TRPZ(5.c.)
TrpE (E.c.)
TrpE(B.s.)
PabB (E.c. )
......... I ......... I . . . . . . . . . I . .
410
420
.......I.... ..... I..... .... I ......... I . ........ (.........I.........
430
440
450
t
Plurality
4 60
470
480
t
I
4 90
500
t
...GSV.V..I ... I..FSHVMH..S.V.G.L...L...L)ALRA..P.GT.SGAPKVW\MELI.ELE..RRG.\l.G..G..SF.G.-MD..I..RT...--
ASAl
ASA2
TRPZ(S.c.)
TrpE (E.c. )
TrpE ( B . s . )
PabB (E.c.)
......... I ......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
510
520
..
530
t
-_-----_----____
Plurality
G.A..QAGAGIVRDS.P
ASA1
ASA2
TRP2 ( S .c. )
TrpE(E.c.1
TrpE (F3.s.)
PabB (E.c )
I
. . . . . . . . . . . . . . .
.
......... .........
610
620
630
540
550
. .- . . .
-%
--.
570
580
5 90
I
600
t
..E ..E..NKA......I..A.......
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. . .
640
650
Figure 4. Alignment of Amino Acid Sequences of AS a Subunit Proteins.
, ..E... -
5 60
660
670
680
6 90
700
Arabidopsis Anthranilate Synthases
ASAl and ASA2 on different chromosomes on an Arabidopsis
RFLP genetic map (Nam et al., 1989). The ASAl gene is located on chromosome 5 at 14.8 centimorgans, whereasASA2
is located on chromosome 2 at 47.2 centimorgans (Figure 38).
Sequence analysis of 5908 bp of ASAl and 6661 bp of ASA2
genomic DNA did not reveal any sequences related to microbial
AS p subunit genes or any other tryptophan biosyntheticgenes.
Based on available mapping data, there does not appear to
be significant clustering of the cloned Arabidopsis tryptophan
biosynthetic genes in any particular region of the genome (Figure 38; Last et al., 1991).
Comparison of the ASA1 and ASA2 Deduced Proteins
The amino acid sequences of deduced proteins encoded by
ASAl and ASA2 are shown in Figure 4. For each gene, protein
translation was assumed to begin at the first AUG codon downstream of the transcription start site(s). The ASAl gene is
capable of encoding a protein of 595 amino acids with calculated molecular mass of 66212 D; ASA2 can encode a 69711-D
protein composed of 621 amino acids.
The ASAl and ASA2 predicted amino acid sequences were
aligned with the AS a subunits from yeast, E. coli, and Bacillus subtilis, along with the PABA synthase a subunit from E.
coli (Figure 4). As seen in Table 1, ASAl and ASA2 are 67%
identical to each other and 30 to 36% identical to microbial
AS a subunit sequences. The ASAl and ASA2 predicted proteins contain conserved amino acids involved in feedback
inhibition of AS by tryptophan (Figure 4).
60th ASA1 and ASA2 proteins are predicted to have aminoterminal extensions not present in the corresponding microbial
proteins (Figure 4). The amino acid compositions and secondary structures of these extensions are characteristic of plant
chloroplast transit peptides (von Heijne et al., 1989). The aminoterminal amino acid sequences of the mature, processed
Arabidopsis ASA1 and ASA2 proteins are not known, but the
sequences IKCV in ASAl and IKCS in ASA2 are near matches
to a proposed cleavage site consensus [(V/I)X(A/C).1A] (Gavel
and von Heijne, 1990). The putative chloroplast transit peptides are the most dissimilar regions of the aligned proteins,
as evidenced by the fact that there is only 20% amino acid
725
identity between the peptides encoded by the first exons of
ASAl and ASA2. There is no apparent homology between the
ASA1 and ASA2 transit peptides and the transit peptides of
other Arabidopsis aromatic amino acid biosynthetic enzymes
(Klee et al., 1987; Berlyn et al., 1989; Keith et al., 1991; Last
et al., 1991).
ASA7 and ASA2 Encode Functional AS a Subunits
Arabidopsis ASAl and ASA2 complemented AS mutations in
yeast and E. coli, verifying the assertion, based on sequence
analysis, that ASAl and ASA2 are AS a subunit genes. To test
complementation in yeast, we made a deletionlinsertion mutation of the TRP2 gene encoding the AS a subunit in yeast.
A strain (KNY1) containing this mutation (frp2A9O::HlS3)requires tryptophan supplementation of minimal medium for
growth. As shown in Figure 5, ASAl and ASA2 cDNAs under
the control of the yeast GALl promoter complemented the auxotrophic phenotype of the frp2 mutation, confirming that both
ASAl and ASA2 encode functional anthranilatesynthases. Consistent with the control of expression by the inducible GALl
promoter, the KNYl strain containing the ASAl cDNA (pKN41)
or the ASA2 cDNA (pKNlO9A-1) grew on galactose medium
Table 1. Pairwise Amino Acid ldentity between Plant AS a
Subunit Sequences and Microbial AS a and PABA Synthase a
Subunit Sequences
ASAl
ASA2
TRP2a
ASA2
TRP2a
TrpEb
TrpEC
PabBb
(04
(O4
(04
( 0 4
(O4
67
33
30
31
31
36
35
35
31
32
33
TrpEb
TrpEC
30
27
26
29
Percent identity was calculated for the sequences as aligned in
Figure 4.
a
Derived from S. cerevisiae.
Derived from E. coli.
Derived from 6. subfilis.
Figure 4. (continued).
The predicted amino acid sequences of ASAl and ASA2 proteins were fit to the alignment of bacterial and yeast AS a subunit sequences (Crawford,
1989), with minor modifications. The sequences shown are Arabidopsis ASAl and ASA2, S. cerevisiae TRP2 [TRPP S.C.)](Zalkin et al., 1984),
E. coli TrpE [TrpE (E.c.)] (Nichols et ai., 1981), 6. subtilis TrpE [TrpE (B.s.)](Band et al., 1984), and E. coli PabB [PabB (E.c.)] (Goncharoff and
Nichols, 1984), the a subunit of PABA synthase. The TRP2 sequence was modified by the introduction of frameshifts at amino acid positions
622 and 638, as described by Crawford (1989). The plurality shows amino acids that are conserved in two-thirds of the aligned sequences at
each position. Hyphens indicate introduced gaps. Amino acid positions showing identity to the ASA1 sequence are shaded. Conserved amino
acids that are affected in inactivating missense mutants of S. typhimurium WpE (Bauerle et al., 1987) are designated with daggers. Five of six
of these amino acids are invariant in the AS a subunit sequences shown. Amino acid positions affected in strongly feedback-resistant mutants
of S.typhimurium trp€ (Caligiuri and Bauerle, 1991) are noted with asterisks. Five of nine of these amino acids are identically conserved in ASAl
and ASA2, and two of nine are replaced by similar amino acids.
726
The Plant Cell
Regulation of ASA1 and ASA2 mRNA Levels
ra
!
1
a s
I K l
undiluted
glc - ura
glc - ura - trp gal - ura - trp
Figure 5. Complementation of a Yeast trp2 Deletion Mutation by
Arabidopsis ASA1 and ASA2.
The yeast strain KNY1 (MATa trp2A90::HIS3 ura3-52 his3A200 leu2A2
Gal+) was transformed with the plasmids pKN10 (TRP2), pSE936*
(vector), pKN41 (ASA1), pKN109C (antisense ASA2), and pKN109A-1
(ASA2), and Ura+ transformants were grown to saturation at 30°C in
liquid SC medium containing 2% glucose lacking uracil. Serial dilutions were made with water, and 2 nL of each were spotted on the
indicated agar plates: SC medium with 2% glucose lacking uracil (glcura), SC medium with 2% glucose lacking uracil and tryptophan (glcura-trp), and SC medium with 2% galactose lacking uracil and tryptophan (gal-ura-trp). Growth after 4 days at 30°C is shown.
lacking tryptophan but not on glucose medium without tryptophan. The URA3 ARS1 CEN4 vector alone (pSE936*) or
antisense ASA2 in the URA3, 2-um vector (pKN109C) did not
allow growth of KNY1 on either galactose or glucose medium
lacking tryptophan. As a positive control, the yeast TRP2 gene
under the control of its own promoter (pKN10) complemented
the trp2 mutation in KNY1 for growth on both glucose and galactose medium without tryptophan.
We also tested the ability of ASA1 cDNAs to complement
trpE mutations in £ coli. As shown in Figure 6, expression of
an ASA1 cDNA allowed growth of E. coli strains containing either a deletion mutation in trpE (trpAES) or a nonsense mutation
in trpE (trpE5972) on M9 glycerol minimal medium lacking tryptophan. The vector alone was unable to complement either
trpAES or trpE5972. Complementation of trpAES by ASA1
(pKN37) was also observed on modified M9 glycerol minimal
medium without tryptophan containing only 1 mM NH4CI
(data not shown), conditions under which E. coli requires
glutamine-dependent AS activity (1) in order to grow (Paluh
et al., 1985). Expression of ASA1 in an £ coli strain lacking
both endogenous AS subunits (trpAED27; Jackson and
Yanofsky, 1974) allowed growth on M9 medium lacking tryptophan (data not shown), conditions under which the ammoniadependent AS activity (2) provided by the AS a subunit is
sufficient. However, this strain did not grow on modified M9
(1 mM NH4CI) lacking tryptophan (data not shown), suggesting that ASA1 encodes an AS a subunit that can interact
with the E. coli AS p subunil in vivo and that such interaction
is necessary for catalyzing the glutamine-dependent AS
reaction (1).
We observed a quantitative difference in the steady state levels of ASA1 and ASA2 mRNAs in Arabidopsis plants (Figure
2A). The ASA1 mRNA was the major AS a subunit transcript,
present at a level about 10-fold greater than that of ASA2, as
determined by RNA gel blot analysis using probes of approximately equal specific activity.
RNA gel blot analysis of RNAs isolated from different parts
of Arabidopsis plants revealed a qualitative difference in the
patterns of ASA1 and ASA2 expression, as shown in Figure
7A. Roots and rosette leaves of 5-week-old plants contained
approximately equal amounts of ASA1 mRNA. The level of ASA1
mRNA was lower in stem tissue and undetectable in flowers,
developing (green) siliques, and mature dry seeds. In contrast,
ASA2 mRNA was present at low, but detectable, levels in roots,
leaves, and developing siliques and at slightly lower levels in
stem tissue. There was no detectable ASA2 mRNA in flowers
or dry seeds. The absence of detectable ASA1 orASA2 mRNA
in certain tissues was not due to any general problem with
the RNA from those tissues, because the mRNA corresponding to the ALS gene encoding acetolactate synthase
(acetohydroxyacid synthase) (Mazur et al., 1987), the first enzyme of the isoleucine-valine biosynthetic pathway, was
detected in all Arabidopsis tissues examined (Figure 7A).
Addition of tryptophan or auxin did not affect the expression of ASA1 or ASA2 RNAs in wild-type plants. Arabidopsis
plants grown on synthetic medium without tryptophan had
steady state levels of ASA1 and ASA2 mRNAs equivalent to
plants grown with 50 uM tryptophan (data not shown). Because
trpAES
trpE5972
vector
ASA1
+trp
Figure 6. Complementation of E. coli trpE Mutations by Arabidopsis
ASA1.
The E. coli strains trpAES tnaA2 and trpE5972 were transformed with
pSE936* (vector) or pKN37 (ASAT), and ampicillin-resistant transformants were grown to saturation at 37°C in liquid Luria-Bertani medium
containing 100 ng/mL ampicillm and then streaked onto agar plates
containing M9 minimal medium plus 0.2% glycerol, 100 ng/mL ampicillin, 200 uM L-tryptophan (+trp) or M9 minimal medium plus 0.2%
glycerol, 100 |ig/mL ampicillin, 100 uM isopropylthiogalactoside
(-trp). Growth after 4 days at 37°C is shown.
Arabidopsis Anthranilate Synthases
B
hours after Infection
hours after wounding
« £
r
«
8
0
727
1
3
6
controlI065(avlrulent)
9 121518
4326 (virulent)
0 1.53.5 612.524 50 0 1.53.5 612.524 50 0 1.53.5 612.524 50
ASA1
•*• -99•*»*?* f» ASA1
ASA2
ASA2
ALS
ALS
•«•-.«•-* m-1f.-m If ^ » w ^ 1F» » VW ASA1
ASA2
*' "» »*
•-*»«•• ALS
<•}>•«*• **> «•• •«• — -i"
Figure 7. RNA Gel Blot Analysis of ASA1 and AS/42 Expression.
Total RNA was fractionated by electrophoresis on 1% agarose gels containing formaldehyde (Ausubel et al., 1989), transferred to nitrocellulose,
and hybridized with 32P-labeled probes.
(A) Tissue specificity. Five micrograms of total RNA per lane from rosette leaves (leaf), roots (root), stems of flowering stalks (stem), immature
and fully opened flowers (flower), developing green seed pods (silique), and mature dry seeds (seed) of 5-week-old Arabidopsis pgm plants were
hybridized with a 32P-labeled 1-kb EcoRI fragment of pKNSA (ASA1), a 1.2-kb EcoRI fragment of pKN1A (ASA2), and a 3.3-kb Ncol-Xbal fragment
of pGH1 (ALS) (kindly provided by G. Haughn, University of Saskatchewan). Equal loading of RNA in each gel lane was confirmed by ethidium
bromide staining.
(B) Wounding. Ten micrograms of total RNA per lane from wounded Arabidopsis leaves were hybridized with a 32P-labeled 2-kb Xhol fragment
of pKN41 (ASA1), a 1.8-kb BamHI fragment of pKN108A (ASA2), a 3.3-kb Ncol-Xbal fragment of pGH1 (ALS), and a 1-kb Kpnl-BamHI fragment
of pABT4 (ABT4) (Marks et al., 1987).
(C) Pathogen infiltration. Ten micrograms of total RNA per lane from leaves of 3-week-old Arabidopsis plants inoculated as described previously
(Dong et al., 1991) with 10 mM MgSO4 (control), P. s. pv tomato MM1065 (1065 [avirulent]), and P. s. pv maculicola ES4326 (4326 [virulent]) were
hybridized with the same probes as given in (B). The liter of the inoculated bacterial strains was 1 x 105 colony-forming units per milliliter. Bacterial growth was followed as described by Dong et al. (1991). Disease symptoms caused by ES4326 were readily apparent 48 hr after inoculation.
the tryptophan pathway provides the precursors for IAA biosynthesis in plants, we also examined the effect of exogenous
IAA application on the steady state levels of ASA1 and ASA2
mRNAs. Spraying 3-week-old wild-type Arabidopsis plants with
100 uM IAA did not discernibly affect ASA1 and ASA2 mRNAs
relative to water-treated control plants (data not shown),
whereas levels of mRNAs corresponding to known auxininduced genes were increased (J. Normanly and G. R. Fink,
unpublished results).
Expression of ASA1 mRNA, but not ASA2 mRNA, was
strongly induced by wounding of Arabidopsis leaf tissue. As
shown in Figure 7B, the steady state level of ASA1 mRNA increased dramatically 3 hr after wounding and then increased
again after 12 hr, reaching a maximum at 18 hr. In striking contrast to ASA1, the level of ASA2 mRNA declined gradually after
wounding of Arabidopsis leaves. A similar decrease was observed for ALS and (5-tubulin (ABT4) mRNAs (Figure 7B).
Infiltration of Arabidopsis leaves with virulent and avirulent
strains of Pseudomonas syringae (Dong et al., 1991) specifically induced ASA1 mRNA. The avirulent strain P. s. pv tomato
MM1065 causes a resistance response when inoculated into
Arabidopsis ecotype Columbia, whereas infiltration of the virulent strain P. s. pv maculicola ES1065 results in disease (Dong
etal., 1991). Figure 7C shows that the steady state level of ASA1
mRNA increased transiently 6 hr after inoculation with the avirulent pathogen R s. pv tomato MM1065. Infiltration of the virulent
strain P. s. pv maculicola ES4326 resulted in a strong induction of ASA1 mRNA, beginning at 12.5 hr and persisting until
the end of the time course. Mock inoculation of Arabidopsis
leaves caused a slight, transient increase in the level of ASA1
mRNA at 1.5 hr that may be due to wounding of the plants
during the inoculation procedure; similar increases were observed 1.5 hr after bacterial inoculations (Figure 7C). The levels
of ASA2 and ALS mRNAs were relatively unaffected by bacterial infiltration, whereas (3-tubulin (ABT4) mRNA increased
slightly 12 to 48 hr after infiltration of ES4326 (Figure 7C).
DISCUSSION
Arabidopsis AS Resembles Microbial AS
We have isolated and characterized two Arabidopsis genes,
ASA1 and ASA2, encoding the a subunit of AS. The initial clone
was obtained using part of the yeast TRP2 gene (Zalkin et al.,
728
The Plant Cell
1984) as a heterologous probe. Two lines of evidence have led
to the conclusion that ASAl and ASA2 encode Arabidopsis AS.
First, the sequences of both genes are homologous to AS a
subunit genes from bacteria and yeast (Figure 4 and Table
1). Second, ASA7 and ASA2 cDNAs complemented mutations
in yeast TRP2 and E. coli frpE (Figures 5 and 6).
The structure of plant AS appears to resemble that of
microbial AS. With the exception of the putative chloroplast
transit peptides at their amino termini, the Arabidopsis ASAl
and ASA2 proteins align well with AS a subunit proteins from
yeast and bacteria (Figure 4). We found no evidence for fused
a and p subunits as in Rhizobium meliloti (Bae et al., 1989)
and the photosynthetic eukaryote Euglena gracilis (Hankins
and Mills, 1976). When expressed in E. coli, the Arabidopsis
a subunit encoded by ASA7 was apparently able to interact
with the E. coli p subunit (Figure 6 and data not shown). Although there is no information about the subunit structure of
plant AS, our data suggest that in Arabidopsis the ASA1 and
ASA2 proteins interact with an AS p subunit, presumably encoded by a distinct gene(s).
Two Genes, Two Pathways
The presence of two Arabidopsis genes encoding the AS a
subunit is consistent with the suggestion that there may be
two aromatic amino acid pathways in plants (Jensen, 1986;
Keith et al., 1991; Last et al., 1991). Duplication of genes has
also been observed for three other Arabidopsis aromatic amino
acid biosynthetic enzymes: 3-deoxy-o-arabino-heptulosonate
bphosphate (DAHP) synthase (Keith et al., 1991), 5enolpyruvylshikimate-3phosphate(EPSP) synthase (Klee et al., 1987), and
tryptophan synthase p subunit (Berlyn et al., 1989; Last et al.,
1991). The available evidence suggests that duplication of
genes encoding certain enzymes of this pathway may be a
general feature of plants (Last et al., 1991).
Two aromatic amino acid biosynthetic pathways could be
separated within the plant cell due to different compartmentation (Jensen, 1986). Cytosolic and plastidic isozymes of AS
have been described in 5-methyltryptophan-resistant tobacco
cell cultures (Brothertonet al., 1986). However, both Arabidopsis AS a subunit genes encode proteins with putative
amino-terminal chloroplast transit peptides, suggesting that
the products of both genes may reside within plastids. Like
ASA7 and ASA2, the gene pairs encoding tryptophan synthase
p (Berlyn et al., 1989; Last et al., 1991) and DAHP synthase
(Keith et al., 1991) and the one published EPSP synthase gene
(Klee et al., 1987) encode putative chloroplast transit peptides
at their amino termini. Although we have not proven that ASAl
and ASA2 proteins are plastid-localized,preliminary subcellular fractionation experiments indicated that there is AS activity
in Arabidopsis chloroplasts, whereas none was detectable in
a cytosolic fraction (K. K. Niyogi and G. R. Fink, unpublished
results). It is possible that ASAl and ASA2 are sequestered
in different compartments within the plastid. If our results can
be extrapolated to tobacco, the cytosolic AS isozyme, which
was detectable only in tissue culture cells and not in regenerated plants (Brotherton et al., 1986), might be due to aberrant
processing or localization of a plastidic AS protein in cultured
cells (Singh et al., 1991). Of course, there may be additional,
distantly related AS genes in Arabidopsis that encode cytosolic isozymes.
60th aromatic amino acid pathways could be present in
plastids, but the two pathways might be expressed in distinct
cell types. Our results showing expression of ASA7 and ASA2
in different parts of Arabidopsis plants revealed subtle qualitative differences in ASAl and ASA2 tissue specificity (Figure
7A). Transgenic plants containing fusions of ASA7 and ASA2
promoters to the reporter gene 0-glucuronidase (Jefferson et
al., 1987) should be useful for determining precisely in which
cell types the two genes are expressed.
Two Genes, Two Functions
Expression of the ASAl gene is regulated by increased demand for tryptophan pathway metabolites, whereas theASA2
gene appears to be constitutivelyexpressed. Addition of tryptophan or auxin to Arabidopsis plants had no effect on ASA7
orASA2 mRNA levels (data not shown), suggestingthat repression below the basal levels of expression does not occur in
response to exogenous primary and secondary products of
the pathway.
The dramatic increases in ASAl steady state mRNA levels
following wounding (Figure 76) and bacterial pathogen infiltration (Figure 7C) suggest a role for ASAl in production of
secondary metabolites as part of an Arabidopsis defense response. Because ALS mRNA was unaffected by these
treatments, the induction of ASA7 is probably not due to a
general stress-relatedrequirement for amino acids for protein
synthesis. Tryptophan pathway metabolites that may be
involved in plant defense responses include indole-3-methylglucosinolates and indole phytoalexins. lndole glucosinolates,
which are found in Arabidopsis (Hogge et al., 1988) and which
have a number of toxic hydrolysis products (for review, see
Fenwick et al., 1983), accumulate following insect infestation
and wounding of Brassica species (Koritsas et al., 1989).
Production of indole phytoalexins by cruciferous plants has
been observed following elicitation with bacterial and funga1
pathogens and UV light (Takasugi et al., 1986; Devys et al.,
1988; Browne et al., 1991). In Arabidopsis, the phytoalexin produced in response to ?
I s. pv syringae infection is
3-thiazol-2'-yl-indole(Tsuji et al., 1992). lnductionof ASA1 mRNA
may therefore be necessary for increased synthesis of indole
phytoalexins.
The second AS gene, ASA2, behaves constitutively under
conditions that induce ASA1. The different regulation of ASAl
and ASA2 may reflect different functions. Duplication of AS
genes to perform distinct functions has a precedent in the bacterium I? aeruginosa, which has one AS gene for tryptophan
biosynthesis and another involved in pyocyanin pigment synthesis (Essar et al., 1990).
Arabidopsis Anthranilate Synthases
In Arabidopsis, the constitutiveASA2 gene may function primarily to maintain a basal leve1 of tryptophan pathway
metabolites. The regulated ASA7 gene may also have a role
in basal biosynthetic activity, so the two genes may have some
functional redundancy, but the induction of ASAl in situations
requiring increased synthesis of secondary products derived
from the tryptophan pathway suggests that ASA1 has additional
roles. Arabidopsis mutants affected in AS activity should be
useful for testing this hypothesis.
The different regulation of ASAl and ASA2 parallels the situation for the duplicated genes encoding Arabidopsis DAHP
synthase (Keith et al., 1991), the first enzyme in the aromatic
amino acid pathway. The DAHP synthase gene DHSl, like
ASAl, respondsto wounding and pathogen infection, whereas
the second DAHP synthase gene DHS2, like ASAP, is unaffected. These results suggest that for two canonical control
points in the aromatic amino acid pathway, AS and DAHP synthase, regulation in plants involves differential expression of
duplicated genes.
METHODS
Plant Material
Arabidopsis thaliana plants derived from the Columbia ecotype were
grown in soil under continuous illumination at 22OC as described by
Last and Fink (1988). Total DNA from rosette leaves of 3-week-oldwildtype plantsand RNAfrom 3-week-oldwhole pgm (Caspar et al., 1985)
plants were isolated as described by Ausubel et al. (1989). Total RNA
was isolatedfrom small amounts of plant materialbya miniprep method
(Nagy et al., 1988).
Wounding of plants was performed by cutting rosette leaves of
3-week-oldpgm plants into a damp glass beaker that was then covered and incubated in the dark at room temperature. At various time
points, 2 to 3 g of tissue was frozen in liquid nitrogen and stored at
-7OOC.
lnoculation of 3-week-old wild-type Arabidopsis plants with Pseudomonas syringae pv tomato MM1065 and P s.pv maculicola ES4326
was performed as described by Dong et al. (1991).
DNA and RNA Methods
Standard techniques of DNA analysis and cloning were performed
as described by Ausubel et al. (1989). DNA fragments were purified
from agarose gels using GeneClean(Bio 101, La Jolla, CA). DNA hybridization probes were labeled using a-32P-dATP(Amersham) and
random hexamer primers (Prime Time, lnternationalBiotechnologies,
New Haven, CT). For DNA gel blot analysis of genomic DNA, total DNA
was digestedwith restrictionenzymes(New England Biolabs, Beverly,
MA), separated on 0.8% agarose gels, and transferred to Zeta-Probe
(Bio-Rad) in 0.4 M NaOH. Genomic DNA blots were hybridized in 1.5
x SSPE (1 x SSPE is 0.15 M NaCI, 10 mM sodium phosphate, 1 mM
EWA, pH 7.4), 05% Blotlo, 1%SDS, O5 mg/mL heat-denatured sheared
herring sperm DNA, and 10% dextran sulfate at 65OC overnight and
washed in 0.1 x SSC (1 x SSC is 0.15 M NaCI, 15 mM sodium citrate), 0.1% SDS at 5OOC.
729
RNA gel blot analysiswas done as describedby Ausubel et al. (1989).
except that 0.1 mg/mL heat-denatured sheared herring sperm DNA
and 5% dextran sulfate were used in the hybridizationsolution. Nitrocellulose filters were washed in 0.1 x SSC, 0.1% SDS at 65OC. An RNA
ladder (Bethesda Research Laboratories) was used to estimate sizes
on RNA gels. Densitometric scanning of RNA blot autoradiograms
was done using a computing densitometer (Molecular Dynamics,
Sunnyvale, CA).
Primer extensionanalysisof RNA was done as described by Ausubel
et al. (1989)with minor modifications. Yeast tRNA (10 pg) was included
in the hybridization. Moloney murine leukemiavirus (M-MLV) reverse
transcriptase(400 units; Bethesda Research Laboratories) was used
insteadof avian myeloblastosisvirus reversetranscriptase,and alkaline hydrolysis was substituted for RNase A treatment.
Screening of cDNA and Genomic Libraries
The Saccharomyces cerevisiae TRP2 gene (Zalkin et al., 1984) and
the Escherichiacoli trpE gene (Nichols et al., 1981) were compared
to identify conservedamino acid sequences, and a 91Sbp EcoRV fragment (nucleotides 1271 to 2189) of the yeast TRP2 gene in pME514
(Braus et al., 1985) was chosen as a DNA hybridization probe. An
Arabidopsis cDNA library in IgtlO (Clontech, Palo Alto, CA) was grown
in E. coli host strain C600, and 50,000 plaqueswere transferredto Biotrans nylon filters (ICN Biomedicals, Irvine, CA) in duplicate and
hybridized to the 32P-labeledprobe in 25% formamide, 5 x SSPE,
5 x Denhardt's solution (1 x Denhardfssolution is 0.02% Ficoll,0.02%
polyvinylpyrrolidone, 0.02% BSA), 0.1 mg/mL heat-denaturedsheared
herringsperm DNA, 5% dextran sulfate at 42OC overnight. Filterswere
washed four times for 15 min each in 25% formamide, 5 x SSC, 0.1%
SDS at 42OC and then exposedto x-rayfilm (Kodak)with an intensifying screenat 70% for 7 days. Phageclones yieldingduplicated positive
hybridization signals were purified by two more rounds of plaque hybridization. The 1.2-kb cDNA insert of I K N l was subcloned in both
orientations into the EcoRl site of pUC118 (Vieira and Messing, 1987)
to create pKNlA and pKNlC.
The EcoRl fragment containing the 1.2-kb cDNA and pKNlA was
then used as a hybridization probe to isolate additional cDNAs from
a randomhexamer-primedArabidopsis cDNA library in IgtlO (Learned
and Fink, 1989). Hybridizationwas done in 5 x SSPE, 5 x Denhardt's
solution, 0.2% SDS, 0.1 mg/mL heat-denaturedsheared herringsperm
DNA, 5% dextran sulfate at 65OC overnight, and the most stringent
wash was in 0.25 x SSC, 0.1% SDS at room temperature. The 1.0-kb
cDNA insert from purified phage IKN8 was subcloned in both orientations into the EcoRl site of pUC118, yielding pKN8A and pKN8C.
The cDNAs from pKN8A and pKNlA were then used to isolate
genomic clones corresponding toASA7 and ASA2, respectively, from
an Arabidopsis genomic library in IEMBL3 (Clontech). The ASA7
genomic clone IKN21was subcloned as follows: a 3.2-kb Hindlll partia1digest fragment was subcloned in both orientations into the Hindlll
site of pUC119 to make pKN212A and pKN212C; an overlapping 4.2kb EcoRl fragment was subcloned into EcoRlcut pUC118 to make
pKN211A; a 3.2-kb EcoRl-Xbalfragmentthat is a subset of the pKN211A
insertwas ligated in the opposite orientation into pUC118 cut with EcoRl
and Xbal to make pKN214. Two adjacent Hindlll fragments from ASA2
genomic clone IKN12 were each ligated into the Hindlll site of pUCll9
in both orientations; pKN140A and pKN140C contain a 3.1-kb fragment,
and pKN143A and pKN143C contain a 3.6-kb fragment.
Additional ASA7 and ASA2 cDNAs were isolatedfrom an Arabidopsis cDNA library in IYES-R (Elledge et al., 1991). A 533-bp EcoRl
fragment of pKN212Awasusedto isolate kKN37and XKN41, from which
730
The Plant Cell
pKN37 and pKN41were looped out in vivo by site-specific recombination using JM107lhKC as host (Elledge et al., 1991). A 612-bp Sacl
fragment of pKN143A was used to isolate IKN34 and IKN35 and
thereby pKN34and pKN35.The cDNA in pKN35 lacked 198 bp at the
5'end and 438 bp at the 3'end of the coding region, so a nearly fulllengthASA2 cDNA was constructed as follows. The Xhol fragmentcontaining the cDNA from pKN35was ligated into the Sal1site of pUC119
to make pKN104, and then the 844-bp Bglll-Pstl fragment of pKNlC
was ligated into Bglll- and Pstlcut pKN104 to generate pKNlO8A.
DNA Sequencing
DNA sequence was determined using the dideoxy chain termination
method (Sanger et al., 1977)with modified T7 DNA polymerase(Tabor
and Richardson, 1987) and either single-stranded templates generated using M13KW (Vieira and Messing, 1987) or alkaline-denatured
double-stranded templates (Chen and Seeburg, 1985). Overlapping
unidirectional deletion series were made using exonuclease 111
(Henikoff, 1984) with exonuclease VI1 instead of S1 nuclease. Deletion series of fragments subcloned in both orientationswere made so
that both strands were completely sequenced. Sequencing reactions
(Sequenase, United States Biochemical) were analyzed on denaturing gradient polyacrylamide gels (Biggin et al., 1983).
The overlapping deletions in combination with synthetic oligonucleotideswere used to sequence the entire genomic or cDNA clones
in pKN212A, pKN212C, pKN211A,pKN214, pKNBA, pKNBC, pKN140A,
pKN140C, pKN143A, pKN143C,pKNlA, and pKN1C. Partia1sequences
were obtained for the inserts in pKN34, pKN35, pKN36, pKN37, and
pKN41 to determine the 5' ends of these cDNAs and to confirm the
positions of introns in the genomic clones. The 5'ends of the cDNAs
in pKN37 and pKN41 are nucleotides2508 and 2386, respectively, in
the ASA7 sequence (GenBankaccession no. M92353). The 5'end of
the pKN35 cDNA is nucleotide3023 in theASA2 sequence (GenBank
accession no. M92354). In addition, the cDNA in pKN35 has a single
silent T- to -C transition mutation (nucleotide3753 in theASA2sequence)
relative to the sequence of pKN34 and of the genomic clone, and the
cDNA in pKN34 has an unspliced fourth intron.
Sequence analysis was done using UWGCG programs (Devereux
et al., 1984). Data base searcheswere performedusing FastA (Pearson
and Lipman, 1988). Protein secondary structure predictionswere done
using PREDICT89 (Finer-Moore and Stroud, 1984).
Expression ln Yeast and E. coli
Yeast media were prepared as described by Sherman et al. (1986).
The yeaststrainKNYl (MATa ttp2A90::HlS3ura3-52his3A2OOleu2A7
Gal+) was constructed as follows. The ends of the 1.77-kb BamHl fragment of pJH-H1(kindly provided by J. Hill, Carnegie Mellon University,
Pittsburgh, PA) wntaining the HlS3gene were made blunt using Klenow
fragment of DNA polymerase I,and the resultingfragment was ligated
into Stul- and Hpal-cutpME514to create pKNtrpPAB, a constructcontaining a deletionof nucleotides 1106to 1794 in the TRP2 coding region
(Zalkinet al., 1984)as well as an insertion of the HlS3 gene. The plasmid pKNtrp2ABwas digestedwith EwRl and Sal1and usedto transform
yeast strain L4242 (MATa ura3-52 his3A200leu2A 7 Gal+) (6. Ruskin
and G. R. Fink, unpublished results) by the lithium acetate method
(Ausubel et al., 1989). His+transformants were selected on synthetic
complete (SC) medium lacking histidine, and His+Trp- clones were
identifiedby replica plating. Genomic DNA gel blot analysisconfirmed
the replacement of the wild-type TRP2 gene by the deletionlinsertion
mutation trp2A 90::HIS3.
The 4.2-kb BamHI-Sal1fragment of pME514 was subcloned into
BamHI- and Sall-cut YEp24 (Botstein et al., 1979) to create pKN10,
which contains the wild-type yeast TRP2 gene under the control of
its own promoter in a high-copy 2-pm, URA3 vector. The plasmid
pSE936*, the URA3 ARS7 CEN4 empty vector counterpart of pKN37
and pKN41, was generated by site-specificrecombination betweenlox
sites on IYES-R in host JMlWlIKC (Elledge et al., 1991); pSE936'
differs from pSE936 (Elledge et al., 1991) because it lacks the Notl
site and has only a single lox site. The BamHl fragment containing
the ASA2 cDNA from pKNlO8A was subcloned in both orientations
into the yeast expression vector pDAD2 (URA3, 2 Wm) to generate
pKNlO9A (sense orientation) and pKNlO9C (antisense).The plasmid
pDAD2(D. Miller, D. Pellman, and G. R. Fink, unpublished results)was
created by inserting a polylinker and PH05 terminator downstream
of the GAL7 promoter in pCGSlO9 (a kind gift of J. Schaum and J.
Mao, Collaborative Research, Bedford, MA). Yeast strain KNYl was
transformed with pKNlOSA, grown to saturation in SC containing2%
galactose and lacking uracil, and cells were plated on SC containing
2% galactose without uracil and tryptophan. Trp+ colonies arose at
a frequency of approximately 5 x 10-8. The plasmid was rescued
from one of these Trp+clones (pKNlO9A-I), and DNA sequencing revealed that a single base pair substitution had occurred to generate
an in-frameATG in the adaptor region three codons upstream of the
ASA2 reading frame.
E. coli mediawere prepared as described by Ausubel et al. (1989).
E. coli strains were transformed by the CaCI, method (Ausubel et al.,
1989).
ACKNOWLEDGMENTS
We gratefully acknowledgethe work of Paula L. Grisafi in sequencing
the 5'half of theASA7 gene, the RFLP mappingdata provided by Susan
Hanley and HowardGoodman (MassachusettsGeneral Hospital), and
the help of Brian Keith with the pathogen infection experiment. We
thank Charles Yanofsky for bacterial strains and Stephen Elledge for
providing the IYES-R library prior to publication. We thank Bonnie
Bartel, Judy Bender, Brian Keith, and Jennifer Normanly for critical
reading of the manuscript. K.K.N. thanks past and present members
of the lab, especiallyRob Last and Brian Keith, for helpful discussions.
This work was supportedby the National Science Foundation. K.K.N.
was supported by a National Science Foundation Graduate Fellowship. G.R.F. is an American Cancer Society Professor of Genetics.
Received March 25, 1992; accepted April 22, 1992.
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Plant Cell 1992;4;721-733
DOI 10.1105/tpc.4.6.721
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