and the accumulation of GS2 mRNA may in part be due to a phytochrome-mediated response [14].
Photorespiratory production of ammonia also reg- ulates pea GS2 expression [13].
In the work on bacteria it has been shown that there is a single gene whose expression is con-
trolled by two regulatory proteins in a manner which is sensitive to the nitrogen status of the cell.
In addition the enzyme itself is regulated by a reversible activationdeactivation mechanism sen-
sitive to the same cellular stimulus [15]. However, the work in higher plants has shown that the
regulation of GS activity is quite different, involv- ing multiple GS genes which are individually con-
trolled [6,7,10]. This complexity may reflect the greater compartmentalization and differentiation
of higher plant cells andor organs. But, at present, very little is known of the components
that regulate GS activity unlike the situation in prokaryotes. In leaves there are the two forms of
GS: GS1, cytosolic; GS2, chloroplastic. GS2 con- tains additional cysteine residues per subunit,
which may account for the specific susceptibility of this isozyme to sulfhydryl reagents [16 – 18].
Cana6alia lineata, used in this study, contains 10 of canavanine in its seed dry weight [19].
Moreover, this
compound, an
analogue of
arginine, is known to have roles in chemical de- fense and nitrogen storage. It is catabolised to
canaline and urea by arginase. The former com- pound is degraded to homoserine and ammonia by
canaline reductase. The latter compound is de- graded to carbon dioxide and ammonia by urease.
Ammonia is then reassimilated by the GS GOGAT cycle to glutamate [20,21]. Thus, the
understanding of the nitrogen metabolism in this plant, C. lineata, is important in the study of
canavanine metabolism.
We previously reported the study of the bio- chemical
and immunological
aspects of
GS isozymes in C. lineata [22]. The three organs each
have a predominant form which is different from the others in their molecular sizes. In the present
work we set out to clone cDNAs encoding cytoso- lic and chloroplastic GSs and to establish the
molecular basis for the different regulation of the isozyme activities. We also confirmed that the two
additional cysteine residues in GS2 are responsible for the specific susceptibility of this isozyme to
sulfhydryl reagents.
2. Materials and methods
2
.
1
. Plant material and chemicals The leaves of 4 – 5-week-old plants of C. lineata
were used for RNA extraction. Restriction en- zymes,
DNA modifying
enzymes and
PCR reagents
were purchased
from Boehringer
Mannheim Germany. Medium components for the bacterial culture were obtained from DIFCO
USA and
Sigma USA.
Radioisotope [a-
35
S]dATP was obtained from Amersham USA. All other chemicals were purchased from Sigma-
Aldrich.
2
.
2
. Bacterial strains, media, and recombinant DNA techniques
Bacterial strains, JM109, XLOLR and BL21 DE3, were cultured in Luria – Bertani broth. The
XL1Blue-MRF’ bacteria were grown in NZYP medium. When required, kanamycin was added at
50 mgl. Plasmid DNA was purified with the Mini-Prep kit Promega, USA and DNA frag-
ments were isolated by restriction endonuclease digestion, electrophoresis and gel elution using a
DNA purification system Jet-Sorb, GENOMED, USA. Ligation and transformation were done as
described by Sambrook et al. [23]. All sequences were determined on both strands. The nucleotide
and deduced amino acid sequences were analyzed with the DNASIS and PROSIS programs. Multi-
ple sequence alignments of GS1 the cytosolic isoform and GS2 the chloroplastic isoform
copied from the GenBank database were per- formed using the Clustal W 1.7 multiple se-
quence alignment program.
Leaves of C. lineata for nucleic acid isolation were harvested and ground to a fine powder in
liquid nitrogen. Total RNA was isolated using the guanidium isothiocyanate methods [24]. PolyA
+
RNA was affinity-purified on oligodT-cellulose Sigma-Aldrich, USA according to Sambrook et
al. [23] and used for cDNA library preparation.
2
.
3
. Construction of a lZAP expression cDNA library and isolation of chloroplast GS cDNA
clones The polyA
+
RNA from the leaves was used to construct a cDNA expression library using the
lZAP cDNA synthesis kit Stratagene, USA. The recombinants were packaged using Gigapack III
gold packaging extracts Stratagene, USA. After infection of XL1-blue MRF’ cells and amplifica-
tion, the recombinant phages were plated onto NZYP – agar plates. After incubation at 42°C for
the small plaques to become visible, IPTG-treated nitrocellulose membranes Amersham were ap-
plied onto the agar plates to express the cDNAs and to lift the expressed proteins. After another
incubation of 3.5 h at 37°C, the membranes were hybridized with an antibody raised against the
GS2 1:8000 dilution and visualized using horse radish peroxidase-conjugated antirabbit IgG. The
immunopositive clones were selected and charac- terized further. Using properties of the ZAP Ex-
press™ vector, the positive clones were excised with Exassist helper phage in XL1-blue MRF’
cells and the resulting phagemid-packaged phage particles were used to infect XLOLR cells. The
transformed cells were plated on LB-kanamycin agar plates and colonies were selected for plasmid
preparations and sequencing.
2
.
4
. Cloning of cytosolic GS cDNA After massive in vivo excision of the l bacterio-
phage library, the resulting phagemid-packaged phage particles were used to infect XLOLR cells.
The transformed cells were cultured in LB- kanamycin media. The resulting plasmid library
was used to transform an Escherichia coli DglnA mutant FDB213 [25,26]. FDB213 cells, generously
provided by Professor Ausubel Massachusetts General Hospital, Boston, USA, were trans-
formed by the CaCl
2
procedure [23] and screened by functional rescue[27].
2
.
5
. Construction of expression 6ector The open reading frame of the Clgln
1
cDNA in pBK-CMV vector at EcoRI and XhoI sites was
cloned into pET30 + Novagen: An NcoI – XhoI fragment containing the bases from 429 to 1345 of
Clgln
1
cDNA was cloned into pET30 + to obtain pETGS1nx. The remaining open reading frame
bases from 63 to 428 was prepared by PCR amplification using a 5 NcoI linker-primer 5-
CCACATGTCTTTAGTCTCAGATCTC-3 and a
3 T7
primer 5-GTAATACGACTCAC-
TATAGGGC-3. The PCR product was digested with NcoI and cloned into pETGS1nx to obtain
pETGS1. The open reading frame of Clgln
2
cDNA, except for the transit peptide region, was cloned into
pET30 + : an NcoI – SacI fragment containing bases 241 – 414 was prepared by PCR amplification
using a 5 NcoI linker-primer 5-CCACATGC- CATGG
CAACTAAGTCTGAAAA TG-
GCACC-3 and a 3 T7 primer 5-GTAATACG ACTCACTATAGGGC-3. The PCR product
was digested with NcoI and SacI and cloned into the expression vector pET30a + Novagen, USA.
Subsequently, the remaining portion of the GS cDNA was inserted by using the internal SacI and
XhoI fragment to obtain pETGS2.
2
.
6
. Site-directed mutagenesis Site-directed mutagenesis was performed by
PCR extension of the following complementary primers to the opposite site strands of pClgln
2
[18]. Cys306 Ser C306S, 5-GG AAT GGT GCA GGA TCC CAC ACT AAT TAT AG-3
and 5-CT ATA ATT AGT GTG GGA TCC TGC ACC ATT CC-3; Cys371 Ser C371S,
5-GCT AAC CGC GGG AGC TCA ATC CGT GTG-3 and 5-CAC ACG GAT TGA GCT CCC
GCG GTT AGC-3 mismatched bases are shown in and restriction enzyme sites underlined; muta-
genic oligonucleotides were designed to generate new restriction sites to facilitate the confirmation
of the mutation. The oligonucleotide primers were extended during thermal cycling 93°C for 30
s, 55°C for 60 s, 72°C for 12 min; 12 cycles using Pow DNA polymerase Boehringer Mannheim,
Germany. The mutated plasmid containing stag- gered nicks was generated. Following the thermal
cycling, the product was treated with DpnI target sequence: 5-G
m6
ATC-3 to digest parental DNA template and to select for mutation-containing
synthesized DNA. The nicked DNA having the desired mutations was transformed into E. coli
strain JM109. The mutated DNA fragments 835 bp were cut out by treating with PstI and XbaI
and substituted for the corresponding fragment of pETGS2. The 853 bp fragment was sequenced to
confirm the mutation.
2
.
7
. Expression and purification of recombinant proteins
E. coli strain BL21DE3 Novagen, USA har- boring the recombinant plasmids were grown at
37°C in Luria – Bertani broth containing 50 mgml kanamycin
[23]. Expression
of recombinant
proteins was induced with 1 mM IPTG when the cell density reached an A
600
of 0.4 – 0.8. The cells were allowed to grow either for 3 h at 37°C or for
16 h at 25°C. The recombinant protein was purified
by the
procedure recommended
by Novagen.
A typical enzyme assay was performed as fol- lows: to a 450 ml reaction mixture containing all
components 80 mM glutamic acid, 6 mM NH
2
OH, 2 mM DTT, 20 mM MgCl
2,
150 mM, Tris – HCl, pH7.8, variable concentrations of the
sulfhydryl reagents and enzyme, incubated for 5 min at 35°C, was added with 50 ml 80 mM ATP.
Other details about the enzyme and protein assays performed are described by Choi and Kwon [22]
and in Ref. [28].
3. Results and discussion
