The differential expression of AAT isoforms has provided workers with a model system that can be used to investigate the basis of gene expression in plants.
Plant Physiol. (1994) 104: 417-423
Cloning and Characterization of a cDNA Encoding Aspartate Aminotransferase-P1 f rom f upinus angustifoli'us Root Tips' Christopher S. Winefield, Brett D. Reddington, William T. Jones, Paul H. S. Reynolds*, and Kevin J. F. Farnden Biochemistry Department, University of Otago, Dunedin, New Zealand (C.S.W., B.D.R., K.J.F.F.); and Plant lmprovement Division, Hort Research, Private Bag 1 1030, Palmerston North, New Zealand (W.T.]., P.H.S.R.).
nodule development (Reynolds and Famden, 1979). Isoformspecific monoclonal antibodies have been raised against L. angustifolius AAT-Pl and AAT-P2proteins (Jones et al., 1990, 1994). The differential expression of AAT isoforms has provided workers with a model system that can be used to investigate the basis of gene expression in plants. Plant AAT cDNAs have been cloned recently from L. angustifolius (Reynolds et al., 1992), Medicago sativa (Udvardi and Kahn, 1991; Gantt et al., 1992), Daucus carota (Turano et al., 1992), Glycine max (Wadsworth et al., 1993), and Panicum miliaceum (Taniguchi et al., 1992). In this paper we describe the isolation and characterization of a cDNA clone encoding the cytosolic AAT-Pl from L. angustifolius. In addition, we show expression and correct assembly of L. angustifolius AAT-P1 subunits in Escherichia coli and complementation of a Saccharomyces cerevisiae AAT mutant with the plant AAT-Pl cDNA.
A root tip cDNA library, constructed in the X Zap II expression vector, was immunoscreened with a monoclonal antibody raised against aspartate aminotransferase-P, from Lupinus angustifolius 1. vai Uniharvest. One 1452-base pair clone was isolated. l h e encoded protein sequence had high homology to both plant and animal aspartate aminotransferase sequences. l h e clone was converted to the phagemid form and expressed in Escherichia coli. The expressed protein was enzymically active and could be immunocomplexed with aspartate aminotransferase-P1-specific antibodies. l h e complete aspartate aminotransferase-P1cDNA was cloned into the yeast expression vector pEMBL-yex4 and transformed into Saccharomyces cerevisiae strain BRSCSC, which possesses a mutated aspartate aminotransferase gene (the asp5 mutation). Complementation of the mutation was achieved using this construct.
AAT (EC 2.6.1.1) catalyzes the transamination reaction: Aspartate
+ 2-Oxoglutarate
Oxaloacetate + Glutamate
MATERIALS AND METHODS
and plays a key role in carbon and nitrogen metabolism in plants. The enzyme has been shown to be involved in the shuttling of reducing equivalents from the cytoplasm to chloroplasts, mitochondria, glyoxysomes, and peroxisomes via the malate aspartate shuttle (Wightman and Forest, 1978; Givan, 1980). AAT is thought to be involved in the transamination of oxaloacetate formed by PEP carboxylase in the mesophyll cells of Cq plants with low malic enzyme levels. The aspartate produced is then transferred to the mitochondria of the bundle sheath cells and retransaminated to oxaloacetate (Hatch and Mau, 1973). Up to five separate isoforms of AAT have been reported in plants, differing both kinetically and in levels of expression (Wightman and Forest, 1978). In the nitrogen-fixing root nodules of legumes, two isoforms of AAT have been described that were found to differ in their kinetic properties and expression characteristics throughout nodule development (Ryan et al., 1972; Reynolds et al., 1981; Griffith and Vance, 1989). AAT-P2 from Lupinus angustifolius L. var Uniharvest has been shown to be expressed in an inducible manner concomitantly with the onset of biological nitrogen fixation (Reynolds and Famden, 1979). AAT-Pl from L. angustifolius is expressed in a constitutive manner throughout
Plant Material
Lupinus angustifolius L. var Uniharvest seeds were surface sterilized and germinated on sterile agar plates for 3 d at 25OC, at which time the root tips (approximately 3 cm in length) were harvested, frozen in liquid nitrogen, and stored at -8OOC. Plants were grown and infected with Rhizobium lupini NZP2257 and the nodules were harvested as described by Reynolds and Famden (1979). Nodule Crude Extracts
Crude extracts of 21-d-old nodule tissue were prepared in the following manner: 1 g of fresh or 1 g of harvested and -80°C-frozen tissue was crushed in 2 mL of extraction buffer (50 m~ Tris-HC1, 0.4 M SUC,50 wg mL-' pyridoxal-5-phosphate). The extract was centrifuged at 8000g for 15 min at 4OC. The supernatant was then removed and stored in 200PL aliquots at -2OOC prior to use. Construction and Screening of a Root-Tip c D N A Library
Total RNA was isolated from 3-d-old developing root tips using the acid phenol/guanidinium isothiocyanate method
This work was supported by a postgraduate fellowship awarded to C.S.W. by the Horticulture and Food Research Institute of New Zealand.
Abbreviations: AAT, aspartate aminotransferase; IPTG, isopropylthio-P-D-galactoside; MAb 285E5, monoclonal antibody produced by the hybridoma 285E5; PAG, polyacrylamide gel.
* Corresponding author; fax 64-6-354-6731. 41 7
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described by Chomczynskiand Sacchi (1987). Poly(A)+RNA in this fraction was enriched by one pass over an oligo(dT)spun column. An expressing cDNA library was constructed in the X Zap I1 (Stratagene) vector system using 5 pg of poly (A)+ RNA according to the manufacturer's protocols. The constructed X cDNA clones were packaged using Gigapack I1 Gold packaging extracts (Stratagene). Screening of the cDNA library and subsequent immunocomplexingexperiments were carried out using an MAb (2B5E5) that was raised against AAT-Pl protein purified from L. angustifolius root nodules. The production and characterization of the MAb 2B5E5 will be described elsewhere (Jones et al., 1994). Clones (6.5 x 105)were screened in an Escherichia coli XLI blue (Stratagene) background with the MAb 2B5E5. Subsequent rescreens of putative positives were conducted using the same MAb. Positive cDNA clones were excised with the helper phage R408 (Stratagene) and recircularized to form phagemid subclones in the pBluescript SK- vector (Stratagene). D N A Sequencing
cDNA inserts from positive clones were recloned into pUCll9. Double-stranded sequencing was performed using the dideoxy chain termination method of Sanger et al. (1977), with the use of the Sequenase enzyme (United States Biochemical). Universal fonvard and reverse primers (New England Biolabs) were used in a11 sequencing reactions except in two cases where primers (GGTGCTTACCGAACTGAGGA and GGATTGAATGCTGAGCAAGT) were generated (Applied Biosystems model 38 1A oligonucleotidesynthesizer) to intemal cDNA sequences corresponding to nucleotides 190 to 209 and 1150 to 1169, respectively. Protein Sequence Alignments
FASTA (Pearson and Lipman, 1988) searches were conducted using the GenBank sequence data base, and sequence comparisons were conducted using the CLUSTAL multiple sequence alignment program (Higgins and Sharp, 1988) and assembled in the HOMED data base (Stockwell, 1988). Expression of Putative AAT-PI in Transformed
Plant Physiol. Vol. 104, 1994
Crowth of Saccharomyces cerevisiae Strains
The AAT (asp5) mutant of Saccharomyces cerevisiae strain BRSCS6 (a, aspl, asp5, ade2, leu2, trp4) was available for use in the complementation experiments. Construction and characterization of this strain will be described elsewhere (B.D. Reddington, E. Vincze, J.M.J. Dickson, P.H.S. Reynolds, R.T.M. Poulter, K.J.F. Famden, unpublished data). Yeast strains for transformation (BRSCS6) and wild-type AAT analysis (XE-101-1A a, aspl, trp4) were grown on YPD (1% Difco yeast extract, 2% dextrose, 2% Bactopeptone) media. Yeast strains were checked for auxotrophic markers by growth on a medium containing yeast nitrogen base minus amino acids (lx, Difco), 2% Glc (sole carbon source). Supplements (adenine, Trp, and aspartate, each at 100 pg mL-') were added separately or in combination as required. Transformation of S. cerevisiae Strain BRSCSC with the AAT-P1 c D N A
S. cerevisiae strain BRSCS6 was transformed with the expression vector pEMBL-yex4 (Cesareni and Murray, 1987) containing the AAT-P' complete cDNA and pEMBL-yex4 without any insert as a control. Spheroplasting and the PEG transformation method of Burgers and Percival (1987) were used to transform this strain. Transformants were checked for auxotrophic markers by growth on defined media as previously described. Complementation of the BRSCS6 asp5 mutation was examined by growing the transformants at 3OoC with constant shaking in 250 mL of defined media containing the required supplements. Two cultures were grown for each transformant. Aspartate (100 pg mL-') was added to only one of the cultures. A11 cultures had 3% Gal added to fully activate the Gal-inducible promoter present on pEMBL-yex4 (Cesareni and Murray, 1987). Samples (1 mL) were taken at regular intervals and AbO0of the culture samples was determined. Northern Analysis of S. cerevisiae Strain BRSCSC Complemented with the AAT-Pl c D N A
S. cerevisiae cells were grown in defined liquid culture to mid-log phase (A600 = 0.8-1.2). Total RNA was isolated according to the method of Schmitt et al. (1990). Total RNA E. coli cells were made competent with CaCL and transfrom lupin root nodules was isolated accordingto the method formed according to Sambrook et al. (1989). Transformed of Chomczynski and Sacchi (1987). RNA from S. cerevisiae cells were grown to a cell density of approximately 10' cells mL-'. IPTG was added to a final concentration of 0.5 m ~ , cells or from lupin root nodules was separated by formaldehyde-formamide agarose gel electrophoresis (Sambrook et and the culture was grown for a further 3 h at 3OoC to induce al., 1989) and subsequently transferred to a Hybond N+ the synthesis of the phagemid-encoded protein. Cells were nylon membrane (Amersham) by capillary transfer in 20x harvested and resuspended (0.2 M potassium phosphate, 34 SSC. RNA was fixed to the membrane by immersion in 0.05 m~ L-aspartate, and 50 pg mL-' pyridoxal-5-phosphate, pH M NaOH according to the manufacturer's protocol. The mem7.6), and cell lysates were prepared by sonication. Crude cell brane was prehybridized/hybridizedwith a solution of 1 m~ extracts were fractionated on 7.5% (w/v) nondenaturing EDTA, 0.5 M Na2HP04(pH 7.2), and 7% SDS (Church and PAGs and subsequently stained for AAT activity with Fast Gilbert, 1984). The northem blot was hybridized with an violet B (Decker and Rau, 1963). AAT-Pl cDNA (EcoRI-HindIII fragment) probe. The probe In immunoprecipitation experiments crude E. coli and plant was radioactively labeled using random priming (Sambrook extracts (10 pL) were incubated with 10 pL of the MAb 2B5E5 et al., 1989) and [ L U - ~ ~ P I ~(Amersham). ATP Following hybrid(100 pg mL-') for 1 h at 37OC prior to fractionation on a uation, the membrane was subjected to a high-stringency nondenaturing PAG and staining for activity using Fast violet wash (0.1X SSC at 65OC for 30 min) prior to autoradiography. B (Decker and Rau, 1963). E. coli XLI Cells
Lupin Aspartate Aminotransferase-P, cDNA
419
RESULTS Isolation of a cDNA Clone Encoding AAT-P, from a Root-Tip cDNA Expression Library A cDNA library, constructed using mRNA from 3-d-old L. angustifolius developing root tips, was immunoscreened using the MAb 2B5E5 (Jones et al., 1994). This MAb is monospecific for the AAT-P] isoform and possibly recognizes a conformational epitope (Jones et al., 1994). One positive clone was identified and rescreened using MAb 2B5E5 prior to further analysis. Expression of cDNA-Encoded Protein in F. coli XLI and Demonstration of AAT Activity The immunoscreen of the cDNA library indicated that the isolated clone was an in-frame fusion of the cDNA with the vector. A convenient enzyme gel activity stain was available (Decker and Rau, 1963), which allowed us to analyze the expression of the cDNA clone (converted to phagemid form) in E. coli. The results of this experiment are shown in Figure 1. Fractionation of the crude extract from lupin root nodules on native PAGs (Fig. 1, lane 1) shows the presence of AATPI and AAT-P2 protein. Preincubation of a crude nodule extract with MAb 2B5E5 resulted in the MAb 2B5E5/AATPI antibody antigen complex remaining close to the bottom of the loading wells (Fig. 1, lane 2). Because MAb 2B5E5 has been shown not to inhibit enzyme activity (Jones et al., 1994), the band remained visible. As a negative control, crude extracts of nontransformed E. coli cells were fractionated in the same manner as were the crude plant extracts. The E. coli AAT enzyme is seen in Figure 1, lanes 3 and 4. Preincubation of the £. coli crude extract with MAb 2B5E5 did not affect the £. coli enzyme (Fig. 1, lane 4). An additional enzyme activity band was detectable in crude extracts of £. coli transformed with the cDNA clone (Fig. 1, lane 5). Preincubation of these extracts with MAb 2B5E5 resulted in the removal of the extra activity-stained band due to the forma-
AAT-Pi-< AAT-P2-; E. coli AAT-i
Figure 1. Native PAGE analysis of an expressed cDNA clone encoding AAT. Both nodule and E. coli crude extracts were fractionated on a 7.5% native PAG and stained for activity using Fast violet B (see "Materials and Methods"). Immunocomplexing of AAT present in the crude nodule and E. coli extracts (10 jil_) was achieved by incubating with 10 ML of MAb 2B5E5 (100 Mg ml"1) for 1 h at 37°C prior to electrophoresis. Lanes 1, 3, and 5, Crude extracts of nodule, E. coli XLI, and E. coli XLI transformed with the AAT-P, cDNA clone, respectively. Lanes 2, 4, and 6, The same extracts preincubated with MAb 2B5E5.
Figure 2. Growth curves of the 5. cerevisiae strain BRSCS6 transformed with the yeast expression vector pEMBL-yex4, with and without the AAT-P, cDNA insert. Cultures were grown on defined medium in the presence and absence of aspartate (100 /tg ml"1) and growth was monitored at 600 nm. Cells transformed with: D, pEMBL-yex4 containing the AAT-P, insert grown in the presence of aspartate; •, pEMBL-yex4 with the AAT-P, insert grown in the absence of aspartate; O, pEMBL-yex4 without an insert grown in the presence of aspartate; •, pEMBL-yex4 without an insert grown in the absence of aspartate.
tion of a high mol wt antibody/antigen complex (Fig. 1, lane 6). Complementation of the asp5 Mutation of 5. cerevisiae Strain BRSCS6 with the Complete AAT-P, cDNA S. cerevisiae BRSCS6 cells transformed with the AAT-Pi cDNA construct in the pEMBL-yex4 expression vector grew both in the presence and absence of 100 jig mL"1 aspartate (Fig. 2). Cells transformed with pEMBL-yex4, without an insert, exhibited significant growth only in the presence of added aspartate (Fig. 2). A small amount of growth was recorded for this transformant in the absence of aspartate, which is consistent with the observation that the asp5 mutation is leaky (Jones and Fink, 1982). Northern analysis of total RNA isolated from the BRSCS6 cells transformed with the pEMBL-yex4 expression vector, with and without the AAT-Pi cDNA insert, and RNA from the wild-type S. cerevisiae (strain XE-101-1A) cells and lupin root nodules, showed that only RNA from lupin root nodules and yeast cells containing the AAT-Pi cDNA construct possessed a transcript homologous to the AAT cDNA probe (Fig. 3). The transcript present in the yeast transformed with the AAT-Pi cDNA construct (Fig. 3, lane 3) was approximately 70 bp larger than that found in the plant tissue (Fig. 3, lane 4). This is consistent with the transcription start sites on the expression vector being between 50 and 80 bp upstream of the cDNA cloning site (Cesareni and Murray, 1987). To obtain a balanced signal for the northern analysis, we demonstrated that 1 Mg of RNA isolated from the BRSCS6 cells containing the vector and cDNA insert gave the same signal as 15 Mg of the lupin root nodule RNA (Fig. 3).
Winefield et al.
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fusion transcript AAT-Pi transcript
Figure 3. Northern analysis of total RNA isolated from transformed S. cerev/s/ae strain BRSCS6 cells, wild-type (XE-101-1 A) S. cerevisiae cells, and lupin root nodules. The probe used corresponded to nucleotides 1 to 704 of the cDN A clone. Lane 1, Fifteen micrograms of total RNA isolated from XE-101-1A cells. Lane 2, Fifteen micrograms of total RNA isolated from BRSCS6 cells transformed with the yeast expression vector pEMBL-yex4 (no insert). Lane 3, One microgram of total RNA isolated from BRSCS6 cells transformed with the AAT-P, cDNA/pEMBL-yex4 construct. Lane 4, Fifteen micrograms of total RNA isolated from lupin root nodules.
Sequence Analysis of the AAT-P] Clone The AAT-Pi cDNA clone was sequenced and found to contain an open reading frame of 1296 bp (Fig. 4). A possible initiating ATG was found at nucleotides 37 to 39 (Fig. 4). The flanking sequences, with the exception of the cytosine at nucleotide 34, are homologous to the consensus sequences found to flank the initiating ATG in other plants (Cavener and Ray, 1991). The open reading frame from the proposed initiating Met to the termination codon at nucleotides 1297 to 1299 encodes a 420-amino acid polypeptide with a calculated Mr of 45,781 (Fig. 4). Alignment of the translated open reading frame against translated nucleic acid sequences from the GenBank data base using the FASTA search program (Pearson and Lipman, 1988) revealed that the L angustifolius AAT-Pi sequence shared 82.3% identity with Panicum miliaceum cytosolic AAT2 (Taniguchi et al., 1992), 88.6% amino acid sequence identity with Medicago sativa cytosolic AAT1 (Udvardi and Kahn, 1991), and 81.6% identity with D. carota cytosolic AAT (Turano et al., 1992) (Fig. 5). The L. angustifolius AAT-Pj had only 56.4% sequence identity with the plastid AAT-P2 from L angustifolius (Reynolds et al., 1992), 52.4% and 52.1% identity with mitochondrial enzymes AA3 and AAT1, respectively, from P. miliaceum (Taniguchi et al., 1992), 50.4% identity with the chloroplast AAT from Glycine max (Wadsworth et al., 1993), and 57.5% homology with M. sativa plastid AAT2 (Gantt et al., 1992) (alignments not shown). L angustifolius AAT-Pi shared sequence identity of approximately 50% with the other animal and bacterial AAT sequences found in the GenBank data base. DISCUSSION
A cDNA clone encoding the constitutively expressed and cytosol-located isoform of AAT in L. angustifolius (AAT-Pi)
Plant Physiol. Vol. 104, 1994
was isolated from a root-tip cDNA library. MAbs against AAT-Pi were previously shown, and confirmed here, not to cross-react with the plastid AAT-P2 (Jones et al., 1994; Fig. 1). The immunocomplexing and expression experiments presented here (Fig. 1) show that the expressed cDNA clone is coding for AAT-P]. The two AAT enzyme activity bands in Figure 1, lane 5, correspond to the AAT-Pi enzyme from lupin root nodules (Fig. 1, lane 1) and the native AAT enzyme from £. co/i (Fig. 1, lane 3). This suggests that E. coli can transcribe the plant AAT cDNA, translate the mRNA, and correctly assemble the plant enzyme, even in the presence of the E. coli native enzyme. A high degree of amino acid sequence identity (88.6, 82.3, and 81.6%) with three plant cytosol AAT sequences (Fig. 4) and the immunocomplexing of the expressed cDNA protein (Fig. 1, lane 6), confirms that
GCA CGA GCT CTT TCA AAT CAT CTT CTT AAT CAT CAA ATG GCT TCC GTT AAT N TCC GTT TCC GTT TCT CCA ACC GCT TCT TCT GAT TCC GTT TTC GCT CAT V S P T A S S D S V F A H S V S CTT CCT GCT CCT GAA GAT CCT ATT CTC GGG GTG ACT GTG GCT TAT AAC P G V T V A Y N E D L GAT CCA AGC CCA GTT AAG CTC AAT TTA GGT GTT GGT GCT TAC CGA ACT P K L N L G V G A Y R T D P S GTT CTG AAT GTA GTG AGG CGT GTT GAA CAG CAA GAA GGA AAA CCT P R N V V E E G K V L 0 Q CGC AAC AAG GAG TAT CTT CCA ATT GTT GGG GTC GTG AAT GAA GCG A K E P I V G V V N E R N Y L GAT TTC AAC AAA AGT GCT AGG CTC ATT TTT GGT GCT GAC AGC CCT K L I F D S D F N S A R G A P GTG ACC ACT GTT CAA TGC TTG TCT GGT ACT GGT ATT CAA GAG AAC G N V T T V L S G T I Q E Q C GAA TTT CTA GCT AGA CAC TAT CAC CAG AGG ACT TTA AGA GTT GGG R V G E F L A R H Y H R T L Q TAT ATT CCT CAA ACA TGG GGT AAT CAC CCA AAG ATT TTC ACC TTA H P L Y I P T W G N K I F T Q GGG TTA TCT GTC ACA TAT CGC TAT TAT GCT CCG GCA ACA AGA GGG Y G L S V T Y R Y A P A G CTA GAA GAT CTT GGT TCT GCC CCA GAC TTT GAA TCC L L E D L G S S ATT GTT TTG CTA CAT GCA TGC GCA CAT AAC CCT ACT GGT GTT GAT CCA ACC H K I V L L A C A H P T G V D P T ACT GAG CAA TGG GAG CAG ATT AGG CTG TTG AGA TCA AAA GCT TTG TTA L R T E Q W E I R L S K A L L 0 TTT GCT AGT GGA AGT CTA GAT ATA CCT TTC TTT GAC AGT GCT TAT CAA D S F D I Y Q A S GCT GAT GGG GGT GAA TTG CTG CTT GAT GCA CAG GCT GTT TTG TTT D A L F A D G G E L L Q A GCC CAG AGT TAT AAC ATG GGT CTA TAT GGA GAA CGT GTT GGC GCC N M A A Q CTA AGC ATT GTC TCC AAG TCA GCT GAT GTT GCA AGC AGG GTT GAG AGC CAG L S I V S V K S A D A S V s o GTG AAA CTT GTG GTT AGG CCA ATG TAC ATT CAT GGT GCA AAC CCT V K L V P M Y I H G A TCC ATT GTG GCT ATT CTC AGG GAC TTG TAC AAT GAG TGG CAT S I V I L R L Y N E W H ATT GAG CTG AAG GCA ATG GCT GAT CGC AAA ATG CGC CAA CAA CTT D L K A M K H 0 L Q TTT GAT GCT TTG CAA TCC AGA GGC ACA CCT GGT GAT TGG AGT . CAC ATT ATC I I F D A L Q W D S H AAG CAG . 3A ATG TTT ACT TTC ACA GGA TTG AAT GCT GAG CAA GTT TCC K Q 3 M F T F T G L N Q *G GAG TAC CAT ATA TAC TTG ACA TCT GAT GGG AGG TTC TTA F L T K E Y H I Y L T S D G R ATG GCT GGT CTG AGT TCC AAA ACA GTT CCT CAT GCT GAT GCA ATA CAT K T V P H A D A I H GCA GCT GTA ACC AGA GTT GTC TAA AAC ATG TTA ATT ACA GTT TTC TAT A A V T R V TTG CCC CTT GTT GGA GGA CAT CCA TTA TTT TCG TTC AAT AAT TAC GGA CAT CAT AAC ATT ATA TCT CAT CAT TAT GAC ATT TTT GTT CAG TTA TCT TAA TAT SGC ATC ATA AAA AAA AAA AAA AAA
CAA Q CTT L AAA K GAG E CTT L GCC A GCT A TCA S ATA I GCT A CTT
51 5 102 22 153 39 204 56 255 73 306 90 357 107 408 124
459 HI 510 15B 561 175 612 192 663 209 714 226 765 213 816 260 867 277 918
294 969 311
1020 328 1071 345 1122 362 1173 379 1224 396 1275 413 1326 420 1377 1428 1452
Figure 4. Nucleotide and deduced amino acid sequence of the AAT-Pi cDNA clone. Double-stranded sequencing was carried out on pUC119 subclones as described in "Materials and Methods." The sequences that are underlined with a single line represent the sequences surrounding a possible translation initiation start (nucleotides 31-42) and putative poly(A) addition signals (nucleotides 1364-1369 and 1424-1429). The double-underlined region represents the pyridoxal-5-phosphate binding motif (Wightman and Forest, 1978). The sequence was determined for both strands except for nucleotide 637. Sequencing in the forward direction resulted in a cytosine being scored without ambiguity. Sequencing in the reverse direction resulted in a band (stop) in all four lanes.
Lupin Aspartate Aminotransferase-P, cDNA
42 1
Lupinus Panicum Medicago Daucus Lupinus Panicm Medicago Daucus Lupinus Panicum Medicago DaUCUS
Lupinus Panicum Medica90
Daucus Lupinus Panicum Medicago Daucus
deduced amino acid sequence against other plant cytosolic AAT sequences. Sequences were obtained through a FASTA search and aligned using t h e CLUSTAL multiple-alignment program and assembled using the HOMED sequence editor as described in "Materiais and Methods." Residues identical to the Lupinus sequence are indicated by a dot. The levels of sequence identity found between AAT-P, (Lupinus) and AAT2 (Panicum), AATl (Medicago),and the AAT from (Daucus) are 82.3, 88.6, and 81.6%, respectively. Figure 5. Multiple alignment of the AAT-P,
the isolated cDNA clone encodes cytosol-located isoenzyme AAT-PI. With the sequence identity between AAT isoenzymes in a particular organism being as low as 50% at the amino acid level (Christen et al., 1985), a convenient method of cloning diverged AAT isoenzymes in plants would be useful. A strain of S. cerevisiae possessing a mutated AAT (asp5) gene was available. This strain of S. cerevisiae was unable to grow in the absence of aspartate (Jones and Fink, 1982). We show here that the cloned AAT from lupin root tips can complement the asp5 mutation and allows growth in the absence of added aspartate (Fig. 2). Therefore, this strain of S. cerevisiae can be used to isolate other functional AAT cDNA clones. The small amount of growth observed for BRSCS6 cells that were not transformed with the vector containing the AAT cDNA insert, grown in the absence of added aspartate, is consistent with the observation that the asp5 mutation is leaky (Jones and Fink, 1982). The lack of substrate specificity of transaminases as a whole has been well described (Wightman and Forest, 1978; Givan, 1980; Christen et al., 1985). The growth observed here can be explained by other aminotransferases catalyzing limited oxaloacetate transamination to provide the strain with aspartate for some growth (Jones and Fink, 1982). An AAT transcript of the expected size was produced by BRSCS6 transformants containing the vector and AAT insert (Fig. 3). The increased size of the transcript compared with the native AAT-PI transcript is due to the four possible transcription start sites of the expression vector 50 to 80 bp upstream of the AAT insert site (Cesareni and Murray, 1987). To obtain a comparable signal in the northern analysis (Fig. 3), 1 jtg of RNA from the BRSCS6 transformants containing the vector and cDNA insert and 15 Pg from lupin root nodules were used. This difference in transcript abundance can possibly be accounted for by the high copy number and high level of expression obtained by the expression vector pEMBLyex4 (Cesareni and Murray, 1987).
Sequence analysis of the clone revealed one open reading frame of 1296 bp (Fig. 4). At nucleotides 37 to 39, a putative initiating ATG was found. Cavener and Ray (1991) have analyzed the flanking sequences surrounding the translation start sites from eukaryotic mRNA sequences present in the GenBank data base. Their analysis of the data revealed a wide diversity in the sequences flanking translation start sites both within and between the major eukaryotic groups. For the 233 dicotyledonoous plants analyzed, the consensus for flanking sequences was AAAAAAAAAA AUG GC (boldface letters represent nucleotides that are highly conserved, other letters represent nucleotides preferred but not significantly conserved). The flanking sequences surrounding the proposed ATG in the AAT-PI cDNA clone were TAATCATCAA ATG GC. The cytosine at position -3 occurs in only 6% of the cases investigated by Cavener and Ray (1991). In the 3' noncoding region of the cDNA there are two sites that show homology to the consensus eukaryotic polyadenylation signal, AAUAAA (Joshi, 1987). This signal is usually found 10 to 30 nucleotides upstream from the poly(A) site. In examining plant polyadenylation signals, Joshi (1987) found that the unaltered consensus sequence occurred in only 39% of the cases investigated. The most likely signal in the AAT-PI cDNA sequence is found at nucleotides 1424 to 1429 (AATATG), approximately 10 nucleotides from the poly(A) sequence (Fig. 4). A second sequence fits the consensus more closely (AATAAT) but is situated further upstream of the poly(A) site (Fig. 4).The M,calculated from the open reachng frame of the cDNA is similar to the M,determined for the purified protein. Purified protein isolated from lupin root nodules has a subunit M,of 47,000, as determined by SDSPAGE analysis (Reynolds et al., 1981), whereas the M,for the cDNA-encoded protein reported here is 45,781. Animal AAT isoforms can be divided into two separate groups based on sequence comparisons. Christen et al. (1985) compared the pig and chicken mitochondxial and cytosolic AAT isoforms and found that the mitochondrial isoforms
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52.2 90identity 84.1 % Identity
41.6 % Identity
Figure 6. Separation of plant AAT isoforms o n the basis of amino acid sequence identity. Percentage identity was determined by FASTA searches of the GenBank data base.
were 88% identical a t the amino acid level and the cytosolic isoforms were 83% identical. However, the mitochondrial and cytosolic isoforms within t h e same animal were only 46% identical. This scheme of relatedness of similar, intracellularly located AAT sequences was extended to include those of human, rat, mouse, a n d horse (Cronin e t al., 1990; Doonan, 1990). Sequence comparison here of t h e available plant AAT sequences from L. angustifolius, M. sativa; D. carota, G. ma%,and P. miliaceum reveals a similar pattem of relatedness. The level of sequence identity between the cytosolic AAT-PI of L. angustifolius a n d t h e cytosolic isoforms of M. sativa, D. carota, a n d P. miliaceum is approximately 84% (88.6, 81.6, a n d 82.3%, respectively; Figs. 5 and 6). Between L. angustifolius AAT-PI a n d t h e nodule proplastid- and chloroplast-associated forms of AAT from L. angustifolius (AATP2), M. sativa (AATZ), and G. max, respectively, the level of identity drops t o approximately 54% (52.4, 57.5, and 50.4%, respectively; Fig. 6). Furthermore, when t h e nodule plastidassociated isoforms from L. angustifolius and M. sativa and the chloroplast form from G. max were compared with t h e mitochondrial isoforms from P. miliaceum, the level of sequence homology was only 48% (Fig. 6). In addition, this level of sequence identity between the plastid-associated AAT isoforms and AATl and AAT3 from P. miliaceum (48%) was similar to that found between the cytosolic and mitochondrial isoforms (51-52%, Fig. 6). These data suggest that cytosolic, plastid, and mitochondrial AAT isoforms i n different plants are members of three separate groups of AAT sequences.
ACKNOWLEDGMENTS
C;W. would like to thank Laura Smith for her advice on the constiuction of the cDNA library. Received June 14, 1993; accepted September 20, 1993. Copyright Clearance Center: 0032-0889/94/104/0417/07. The GenBank accession number for the sequence reported in this article is M92094.
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