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Plant Physiol. (1998) 117: 455-464
Identification of the Gene Encoding the Tryptophan Synthase
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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We
report the isolation of a Chlamydomonas reinhardtii cDNA
that encodes the 
-subunit of tryptophan synthase (TSB). This cDNA
was cloned by functional complementation of a
trp-operon-deleted strain of Escherichia
coli. Hybridization analysis indicated that the gene exists in
a single copy. The predicted amino acid sequence showed the greatest
identity to TSB polypeptides from other photosynthetic organisms. With
the goal of identifying mutations in the gene encoding this enzyme, we
isolated 11 recessive and 1 dominant single-gene mutation that
conferred resistance to 5-fluoroindole. These mutations fell into three
complementation groups, MAA2, MAA7, and
TAR1. In vitro assays showed that
mutations at each of these loci affected TSB activity. Restriction
fragment-length polymorphism analysis suggested that
MAA7 encodes TSB. MAA2 and TAR1 may act to regulate the activity of
MAA7 or its protein product.
In the unicellular green alga Chlamydomonas
reinhardtii, there is a paucity of auxotrophic mutations. Classic
genetic screens have led to the isolation of only Arg-requiring strains
(for review, see Harris, 1989 Given these observations, we reasoned that an alternative strategy for
investigating amino acid metabolism in C. reinhardtii would be to isolate the DNA encoding a biosynthetic enzyme that could
be used to characterize restriction fragment-length polymorphisms in
mutant strains that are resistant to amino acid analogs. We chose to
study TSB. Trp synthase performs two enzymatic functions and is
composed of two subunits, Using a C. reinhardtii cDNA library, we complemented an
Escherichia coli strain lacking TSB activity. The
complementing cDNA was sequenced and found to be similar to genes
encoding TSB from other organisms. The predicted protein product showed
highest identity to homologs from other photosynthetic organisms.
Hybridization analysis of genomic DNA indicated that TSB is encoded by
a single-copy gene in C. reinhardtii.
With the goal of identifying mutations in TSB, we examined 14 prototrophic strains that were resistant to 5-FI. These mutations fell
into three complementation groups, MAA2, MAA7,
and TAR1. Hybridization analysis of wild-type and mutant DNA
probed with the TSB-encoding cDNA revealed a restriction
fragment-length polymorphism associated with the maa7-8
mutation. We conclude that MAA7 is likely to encode
TSB.
Strains and Culture Conditions
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
). An attempt to isolate Trp auxotrophs on
the basis of resistance to the Trp precursor analog 5-MA was
unsuccessful, although strains with altered Trp biosynthetic enzyme
activity were recovered (Dutcher et al., 1992). The failure to isolate amino acid auxotrophs has been attributed to the inability to effectively import amino acids other than Arg (Kirk and Kirk, 1978).
and , which together catalyze the
conversion of indole-3-glycerol phosphate to Trp. The -subunit catalyzes the conversion of indole-3-glycerol phosphate to indole and
glyceraldehyde-3-phosphate, whereas the -subunit catalyzes the
condensation of Ser and indole into Trp. TSB was particularly attractive because of its conservation in many organisms (Crawford, 1989; Zhao et al., 1994), and the fact that both auxotrophic and nonauxotrophic mutations affecting TSB in Arabidopsis
thaliana have been isolated on the basis of resistance to the TSB
substrate analog 5-FI (Barczak et al., 1995).
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
trpEA2
(r
m+leuthi)
(Yanofsky and Horn, 1995), which was a kind gift of Dr. Charles
Yanofsky (Stanford University, CA), was used as the host strain for
functional identification of a Chlamydomonas reinhardtii
cDNA encoding TSB. This strain was maintained in 2× yeast extract
tryptone liquid or on Luria-Bertani solid medium (Sambrook et al.,
1989). The medium used for selection of Trp prototrophs (minimal indole
medium) contained the E salt mixture of Vogel and Bonner (1956)
supplemented with indole, biotin, thiamine, Glc, acid-hydrolyzed casein
(Zhao et al., 1994), and Leu (40 g mL1).
supplemented with 8 mM sodium
acetate (Holmes and Dutcher, 1989) under constant illumination (150 µmol photons m2 s1)
at 21°C. Growth on solid medium took place at 21°C (for most purposes) or at 25°C (when scoring drug resistance) under constant illumination (150 µmol photons m2
s1).
Isolation of a TSB-Encoding cDNA from C. reinhardtii
A wild-type C. reinhardtii cDNA library (a generous gift of Dr. Andy Wang, Iowa State University, Ames) in the vector
ZAP II (Stratagene) was converted into a phagemid library
using the "mass excision of pBluescript from a lambda ZAP library"
protocol from Life Technologies. The phagemids were converted into a
double-stranded plasmid library using the host strain XLOLR
(Stratagene) according to the supplier's instructions. The final
isolation of the plasmid library was performed using a Plasmid Maxi Kit
(Qiagen, Chatsworth, CA).
trpEA2,
which lacks the entire coding region of the trp operon
(Yanofsky and Horn, 1995). The cells were plated onto minimal indole
medium and incubated at 37°C. Plasmids were isolated from
trp+ transformants and sequenced at the DNA
Sequencing Facility at Iowa State University. A single plasmid,
pCU-100, was chosen for further study.
Computer Analysis of cDNA
The sequence of the C. reinhardtii cDNA encoding TSB was compared with DNA and protein sequence databases using the BLASTN (Altschul et al., 1990) and BLASTX (Altschul et al., 1990; Gish and
States, 1993) programs, respectively. The predicted translation product
of the pCU-100 insert was compared with protein sequence databases
using the BLASTP program (Altschul et al., 1990). An alignment of 23 TSB protein sequences obtained from GenBank (Benson et al., 1997) was
made using the PILEUP (Devereux et al., 1984) or CLUSTAL-W (Thompson et
al., 1994) programs. Phylogenetic trees were inferred by
Fitch-Margoliash analysis using a Dayhoff PAM250 substitution matrix
with PROTDIST, NEIGHBOR, and FITCH programs from the PHYLIP
package, version 3.5 (Felsenstein, 1993, 1996). Unweighted parsimony
trees were inferred using the PROTPARS program from the PHYLIP package
(Felsenstein, 1993). Trees generated with different alignment programs
or with UGMPA, neighbor-joining, and parsimony methods were
qualitatively similar.
DNA-Hybridization Analysis
Total C. reinhardtii DNA was isolated as described by Newman et al. (1990). Conditions for DNA blotting and hybridization of
pCU-100 DNA to genomic DNA were performed as described by Johnson and
Dutcher (1991), except that the hybridization temperature was 55°C.
Filters were washed in 15 mM Na citrate, 150 mM
NaCl, pH 7.0, 0.2% SDS once at room temperature (5 min) and twice at 63°C (20 min each wash). The probe was
32P-labeled using the Multiprime DNA Labeling
System (Amersham).
Genetic Analyses
Standard techniques used to determine segregation of genetic markers were performed essentially as described by Harris (1989). Diploid strains were constructed by mating nit2-1
AC17 strains to NIT2 ac17-1 strains and selecting for
growth on solid medium lacking acetate and containing 2 mM
NaNO3 as the sole source of N (Fernández et al.,
1989). Because mitotic recombination and chromosome-loss events
sometimes confused scoring for analog resistance in complementation
tests, at least eight independent diploid strains of a given genotype
were examined. For the same reason, potential diploid strains were kept
under selective conditions until transfer onto other diagnostic media.
The effectiveness of the diploid-strain-selection technique was
confirmed by DNA hybridization using the mating-type-specific probe
from plasmid pThi4.9 (Ferris, 1995).
Selections for 5-FI Resistance
Each culture used in the selections was derived from an independent single colony. For UV-radiation mutagenesis, 20 cultures of 0.5 × 106 to 1.0 × 106 cells each were plated onto acetate medium and exposed to UV radiation from 20-W sunlamps (model FS20, Westinghouse, Baltimore, MD) for 45 s. This mutagenesis procedure results in approximately 50% lethality. The plates were immediately wrapped in foil and allowed to recover overnight at 21°C. Mutagenized cells were replica plated (RepliPlate, FMC, Inc., Rockland, ME) onto acetate medium supplemented with 25 µM 5-FI (Sigma) and 1.5 mM L-Trp (Sigma) (FIT medium), wrapped in a paper towel to inhibit photodegradation of Trp and 5-FI, and incubated at 25°C under constant illumination (30 µmol photons m2 s1). For
-radiation mutagenesis, 35 cultures of 0.5 × 106 to 1.0 × 106
cells were plated onto solid FIT medium. Each was exposed to 10,000 rad, which resulted in approximately 50% lethality, from a
137Cs irradiator (model 143, J.L. Shepherd and
Associates, Glendale, CA). Plates of mutagenized cultures were wrapped
in a paper towel and incubated under constant illumination (30 µmol
photons m2 s1) at
25°C. Potential 5-FI-resistant colonies were collected for 2 weeks
and rescored for growth on FIT medium. Only one 5-FI-resistant colony
was collected from a given plate. Each 5-FI-resistant isolate was
tested for growth in the presence and in the absence of both Trp and
5-FI.
) and other compounds.
TSB Activity Assays
Cultures for TSB activity assays were grown in acetate medium supplemented with K2HPO4 to a final concentration of 1 mM (Holmes and Dutcher, 1989). Cells
were grown to mid-log phase at 21°C and harvested by centrifugation.
Cell pellets were resuspended in breaking buffer (Dutcher et al., 1992)
to a final concentration of approximately 25% (v/v) and transferred to
a 30-mL centrifuge bottle. The resuspended cells were frozen in liquid
N2 and stored at 
70°C. Cell extracts were
prepared by sonicating thawed cells on ice, followed by passage through
a 38.1-mm, 22-gauge syringe needle at 4°C. Debris were removed by
centrifugation at 12,000g for 10 min at 4°C. Each
supernatant was aliquoted into microcentrifuge tubes and stored at
70°C. The protein content of each extract was determined using the
Bio-Rad protein assay kit and compared with BSA standards according to
the manufacturer's instructions.
with the following modifications. Reactions (7 mL) were preincubated
for 10 min at 30°C with all components except indole. At several time
points after the addition of indole, 1-mL aliquots were removed and the
reaction stopped by mixing with 4 mL of toluene. The indole content of
each toluene phase was measured colorimetrically using Ehrlich's
reagent prepared as described by Yanofsky (1955), except that the
reagent was freshly made for each set of reactions.
. Specific activities are presented in
units per milligram of total protein.
Drug-Resistance Phenotypes
5-FI-resistant strains were tested for growth on solid acetate medium supplemented with 7 µM 5-FI, 1.6 mM 5-MA, 1.39 mM 6-MA, 1.29 mM 5-MT, 60 µM anisomycin, 2.8 mM canavanine, 4 mM colchicine, 64 µM cycloheximide, or 5 µM oryzalin. Stocks of these compounds were prepared as described by Dutcher et al. (1992) and James and Lefebvre (1989).
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Cloning a TSB cDNA from C. reinhardtii
Because of the high degree of conservation among TSB proteins from different organisms, we sought to identify a TSB-encoding gene from C. reinhardtii by rescuing an E. coli strain lacking TSB activity. A plasmid library of wild-type C. reinhardtii cDNA was transformed into the trp-operon-deleted E. coli strain JMB9trpEA2. Approximately 1010
transformants were plated onto minimal indole medium and grown for
5 d. Five transformants were isolated that survived when
restreaked onto selective medium. Plasmids isolated from these
transformants were sequenced. Four of the plasmid preparations were not
homogeneous, presumably because of some instability of the plasmids. A
preparation of the fifth plasmid, pCU-100, was homogeneous and
sequenced in its entirety. The 1840-bp cDNA contained a single large
open reading frame of 1332 bp. The predicted translation product of 444 amino acids (Fig. 1) displayed strong
identity to known and predicted TSB sequences (Fig.
2). The highest scoring matches from a
BLASTP search (Altschul et al., 1990) were to TSB sequences from other photosynthetic organisms. For example, the C. reinhardtii
predicted translation product was 75% identical and 86% similar to
the TRP2 precursor protein from maize (Zea mays) (P = 2.0216). Identity and similarity to TSB from
the cyanobacterium Synechocystis sp. were 71 and 83%,
respectively (P = 1.3206). Matches to TSB
proteins from nonphotosynthetic organisms had slightly lower scores.
For example, the predicted C. reinhardtii translation
product was 61% identical and 76% similar to the TrpB protein from
E. coli (P = 6.9152).
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end of the cDNA
was missing from pCU-100. The observation that the truncated cDNA
produced a functional protein in E. coli indicated that the missing sequence is not essential for function in this bacterium. TSB
sequences from other photosynthetic eukaryotes contain
chloroplast-localization peptides at their amino terminus (Berlyn et
al., 1989; Wright et al., 1992; Zhao and Last, 1995). Stromal-targeting
domains have no amino acid consensus, but they are generally enriched in hydroxylated amino acids and deficient in acidic amino acids. The
amino terminus of the transit peptide is generally devoid of Gly and
Pro and charged amino acids; the middle region is generally enriched in
Ser, Thr, Arg, and Lys; and the carboxy terminus contains a possible
processing site of Val-X-Ala (Gavel and von Heijne, 1990). It is
therefore likely that the amino-terminal domain of C. reinhardtii TSB is a chloroplast-localization peptide.
) and the phylogeny was constructed using the method of Fitch and
Margoliash (1967). We observed clustering of the C. reinhardtii TSB with the enzymes from other photosynthetic organisms, including the cyanobacterium Synechocystis sp.
Trees generated with and without the chloroplast transit peptides of Arabidopsis, maize, rice, and C. reinhardtii were
qualitatively similar.
Copy Number in C. reinhardtii
In A. thaliana and maize, the gene encoding TSB exists in two copies (Berlyn et al., 1989; Last et al., 1991; Wright et
al., 1992). We used the C. reinhardtii cDNA from pCU-100 as the probe of genomic DNA blots to determine the copy number of this
gene. When genomic DNA was digested with the restriction enzyme
SalI, which does not cut within the cDNA, a single
TSB-specific hybridization band was found (Fig.
3, lanes 1 and 2). Digests with
AccI and SstI (either alone or in a double
digest), which cut near the 5 end of the cDNA, also yielded a single
hybridization band (data not shown). Presumably, the sequence 5 of the
restriction sites had insufficient homology to allow hybridization or
was contained within a fragment too small to be retained under our experimental conditions. Likewise, when genomic DNA was digested with
NcoI, a restriction enzyme that cuts once in the middle of the cDNA, two hybridization bands were observed (Fig. 3, lanes 3 and
4). Although we cannot exclude the possibility of a highly divergent
copy of the gene, we conclude that a single gene encodes TSB in
C. reinhardtii.
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Isolation of Mutant Strains with Altered TSB Activity
In many Trp-producing organisms, TSB converts the indole analog 5-FI into the toxic Trp analog 5fluorotryptophan (Miozzari et al., 1977; Barczak et al., 1995; for review, see Somerville, 1983). In
A. thaliana, plants with defects in TSB activity have been
isolated by virtue of resistance to 5-FI (Barczak et al., 1995). With
the intention of obtaining mutations in the gene encoding C. reinhardtii TSB, we isolated a collection of 5-FI-resistant strains. Five independent 5-FI-resistant strains were isolated from
approximately 1.5 × 107 cells mutagenized
with UV light, and seven independent strains were isolated from
approximately 2.5 × 107 cells mutagenized
with radiation (Table I). In an
attempt to isolate Trp auxotrophs, the selection medium contained 1.5 mM Trp. However, each resistant strain grew in the absence
of exogenous Trp. Incubation at 15, 21, 25, and 30°C had no
apparent effect on Trp prototrophy.
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Genetic Analysis of 5-FI-Resistance Mutations
Crosses were performed to determine the genetic location of the 5-FI-resistance mutations. Because these mutations might have represented new alleles of previously mapped 5-FI-resistance mutations (Dutcher et al., 1992), each mutation was mapped with respect to
maa2-1, maa7-2, or other loci on linkage group
III. As shown in Table I, eight of the mutations were linked to
maa2-1 and maa7-2, and map approximately 20 map
units from the NIT2 locus. The other four mutations were
found to be linked to each other on linkage group VI (Table I). A
genetic map of these loci is shown in Figure
4.
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Slow-Growth Phenotypes in 5-FI-Resistant Strains
In addition to its analog-resistance phenotype, the maa2-1 mutation confers a slow-growth phenotype (Dutcher et al., 1992). Likewise, two of the 5-FI-resistant strains isolated in
this study, maa2-8 and maa7-3, displayed a
slow-growth phenotype after 9 d of growth. This phenotype was most
noticeable in meiotic progeny of crosses between these strains, and a
strain without growth defects in which single colonies could be viewed
side by side under identical conditions (see Fig. 6). Because each of
these strains has a similar plating efficiency, it is likely that the observed growth defects are the result of an increased doubling time.
The strain maa2-1 also had an increased doubling time
(Dutcher et al., 1992).
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TSB Activity in Wild-Type and 5-FI-Resistant Strains
Crude cell extracts of wild-type and mutant cells were assayed for TSB activity (Fig. 5). In each assay that had measurable TSB activity, the reaction rate was linear with time and protein concentration. Extracts from strains with the maa2-1 or maa2-8 mutation had approximately 65 and 75% of wild-type activity, respectively (Fig. 5). Assays of maa2-1 extracts for anthranilate synthase activity, and combined anthranilate phosphosphoribosyl transferase/phosphoribosyl anthranilate isomerase/indoleglycerol phosphate synthase assays showed no reduction in these enzymatic activities (Dutcher et al., 1992). Taken together,
these results indicated that mutations at the MAA2 locus
affect Trp synthase activity specifically. Because the activity of Trp
synthase was not assayed, it is not known whether both Trp synthase
activities were affected.
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). Thus, it appears that mutations at the MAA7 locus
specifically affect Trp synthase activity.
Other Drug-Resistance Phenotypes
Because mutations at MAA2, MAA7, and TAR1 each affect TSB activity in vitro, other means were necessary to determine which of these loci encodes TSB. Mutations in TSB would be expected to display a predictable range of cross-resistance phenotypes (for review, see Sommerville, 1983). Mutations in TSB (a) should not confer resistance to toxins unrelated to Trp biosynthesis, (b) may confer resistance to analogs of other intermediates of Trp biosynthesis, and (c) should not confer resistance to 5-MT, which is an analog of Trp rather than of a biosynthetic intermediate of Trp.
TSB Hybridization Analysis of maa7-8
; Li and Last, 1996). In C. reinhardtii the mutations maa2-1 and maa7-2
were isolated based on resistance to 5-MA (Dutcher et al., 1992). We
tested each 5-FI-resistant strain for cross-resistance to 5-MA and
6-MA. Strains with any of the maa2 or tar1
alleles were fully resistant to 1.6 mM 5-MA and 1.39 mM 6-MA (Table II). Mutations
at the MAA7 locus displayed a range of phenotypes (Table
II). Six maa7 mutations conferred resistance to both 5-MA
and 6-MA. In contrast, the maa7-5 mutation failed to confer
resistance to either analog. Strains with the maa7-6
mutation were resistant to 5-MA and sensitive to 6-MA. Combined with
the range of biochemical phenotypes (Fig. 5), these data indicated that
a series of nonidentical mutations at the MAA7 locus was
recovered. Resistance to 5-FI, 5-MA, and 6-MA conferred by the
maa2, maa7, and tar1 mutations may
result from reduced or eliminated conversion of the intermediate analog
into a Trp analog (Tilby, 1978).
View this table:
Table II.
Cross-resistance phenotypes of 5-FI-resistant
strains
Strains were spotted onto solid acetate medium supplemented with 7 µM 5-FI, 1.6 mM 5-MA, 1.39 mM
6-MA, or 1.29 mM 5-MT. The presence or absence of growth
after 9 d is indicated by a + or , respectively.
). These mechanisms of toxicity suggest that a mutant
form of TSB would not confer resistance to 5-MT. As shown in Table II,
each maa2 and tar1 mutation conferred resistance
to 5-MT, whereas no maa7 mutation conferred resistance.
These data are consistent with the hypothesis that MAA7
encodes TSB. MAA2 and TAR1 may encode other
products involved in Trp metabolism.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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We report the isolation of a C. reinhardtii cDNA that
encodes TSB. The lack of a 5 untranslated region and start codon
indicate that the cDNA is incomplete. Despite this, the cDNA
complements a trpB mutation in E. coli,
suggesting that the missing sequence is not essential for function in
the organism. All but approximately the amino-terminal 46 amino acids
encoded by the cDNA showed significant identity to other TSB proteins.
It is likely that these 46 amino acids are part of a
chloroplast-transit peptide that is present in TSB from other
photosynthetic eukaryotes. The presence of a chloroplast-localization
sequence at the 5 end of the cDNA suggests that TSB is a
monofunctional polypeptide in C. reinhardtii. This finding
is consistent with findings in bacteria and plants (Berlyn et al.,
1989; Crawford, 1989; Wright et al., 1992). In contrast, Trp synthase
and TSB activities are contained within a single polypeptide in
fungi (Zalkin and Yanofsky, 1982; Burns and Yanofsky, 1989), and TSB is
part of a pentafunctional Trp biosynthetic polypeptide in Euglena
gracilis (Hankins and Mills, 1977). A phylogenetic analysis of the
predicted translation product and 23 other TSB proteins showed that
C. reinhardtii TSB is most closely related to TSB enzymes
from other photosynthetic organisms. This relationship is maintained
even when chloroplast transit peptides are excluded from phylogenetic
analyses. Hybridization analysis of C. reinhardtii genomic
DNA probed with the cDNA from pCU-100 suggested that the gene exists in
a single copy. This is based on observing single bands in digests with
enzymes that cut once or not at all in the cDNA (Fig. 3). Although both
A. thaliana and maize have two copies of TSB
(Berlyn et al., 1989; Last et al., 1991; Wright et al., 1992), C. reinhardtii is likely to have only a single gene.
;
Last and Fink, 1988; Li and Last, 1996). Among the C. reinhardtii strains examined, all maa2 and
tar1 alleles conferred resistance to both 5-MA and 6-MA. The
maa7 mutations examined showed allele-specific variation in
cross-resistance phenotypes. This variation was compelling evidence
that a series of nonidentical mutations at MAA7 was
recovered.
), fungi (Miozzari et al., 1977), and
cultured mammalian tissue (Taub, 1977), part or all of Trp analog
toxicity is based on incorporation of these analogs into protein.
). In E. coli, fluorotryptophans and methyltryptophans also act by false
corepression of the trp operon and false-feedback inhibition
of anthranilate synthase and Trp-specific
deoxy-D-arabino-D-heptulosonatephosphate
synthase (for review, see Somerville, 1983). False-feedback inhibition of anthranilate synthase is also observed in S. cerevisiae
(Miozzari et al., 1977) and A. thaliana (Li and Last, 1996).
In A. thaliana, a feedback-resistant form of anthranilate
synthase confers resistance to a wide range of analogs of anthranilate
and Trp (Li and Last, 1996). If MAA2 and TAR1
encode enzymes that are normally feedback inhibited by Trp, the
observed alterations in TSB activity would likely be an indirect effect
of the mutations. An indirect effect on TSB activity would also be
likely if either locus encoded a mutant tryptophanyl-tRNA synthetase or
tRNATrp. In addition, Trp synthase physically
interacts with TSB in other organisms, either as independently
translated subunits (for review, see Hyde and Miles, 1990; Radwanski et
al., 1995) or as a multifunctional polypeptide (Hankins and Mills,
1977; Zalkin and Yanofsky, 1982; Burns and Yanofsky, 1989; Schwarz et
al., 1997).
could therefore
affect TSB activity. The lethality phenotype of the maa2-8
MAA7-9 double-mutant strains is consistent with any of these
models. Regardless of the nature of the maa2-8 mutation, the
indirect reduction in TSB activity was lethal in combination with the
mutant form of TSB encoded by MAA7-9. It is worth noting that the maa7-1 mutation, not included in this study,
displayed a conditional lethal phenotype. Strains with this mutation
failed to grow at 16°C (Dutcher et al., 1992).
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FOOTNOTES |
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Received December 11, 1997;
accepted February 24, 1998.
The accession number for the TSB sequence reported in this article is
AF047024.
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ABBREVIATIONS |
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Abbreviations:
5-FI, 5-fluoroindole.
5-MA, 5-methylanthranilate.
5-MT, 5-methyltryptophan.
6-MA, 6-methylanthranilate.
TSB, -subunit of Trp synthase.
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ACKNOWLEDGMENTS |
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We thank Dr. Charles Yanofsky for providing E. coli strain JMB9, Dr. Andy Wang for providing the cDNA library used in these experiments, and Dr. Rob Last for sharing unpublished results. We also thank Drs. Shelley Copley and Ron Somerville for invaluable discussions of experimental design, and Dr. Sylvia Fromherz and Andrea Preble for helpful discussions and critical review of the manuscript.
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LITERATURE CITED |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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