Cloning and Characterization of a cDNA Encoding Aspartate ... · isolated clone was an in-frame fusion of the cDNA with the vector. A convenient enzyme gel activity stain was available

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.).

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 immuno- complexed with aspartate aminotransferase-P1-specific antibodies. l h e complete aspartate aminotransferase-P1 cDNA 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). Com- plementation of the mutation was achieved using this construct.

AAT (EC 2.6.1.1) catalyzes the transamination reaction:

Aspartate + 2-Oxoglutarate Oxaloacetate + Glutamate

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 chlo- roplasts, 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 Uni- harvest 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

This work was supported by a postgraduate fellowship awarded to C.S.W. by the Horticulture and Food Research Institute of New Zealand.

* Corresponding author; fax 64-6-354-6731.

nodule development (Reynolds and Famden, 1979). Isoform- specific monoclonal antibodies have been raised against L. angustifolius AAT-Pl and AAT-P2 proteins (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.

MATERIALS AND METHODS

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 200- PL aliquots at -2OOC prior to use.

Construction and Screening of a Root-Tip cDNA Library

Total RNA was isolated from 3-d-old developing root tips using the acid phenol/guanidinium isothiocyanate method

Abbreviations: AAT, aspartate aminotransferase; IPTG, isopro- pylthio-P-D-galactoside; MAb 285E5, monoclonal antibody produced by the hybridoma 285E5; PAG, polyacrylamide gel.

41 7

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41 8 Winefield et al. Plant Physiol. Vol. 104, 1994

described by Chomczynski and 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 Giga- pack I1 Gold packaging extracts (Stratagene). Screening of the cDNA library and subsequent immunocomplexing experiments 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. Subse- quent 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 (Stra tagene) .

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 Bio- chemical). Universal fonvard and reverse primers (New Eng- land Biolabs) were used in a11 sequencing reactions except in two cases where primers (GGTGCTTACCGAACTGAGGA and GGATTGAATGCTGAGCAAGT) were generated (Ap- plied Biosystems model 38 1A oligonucleotide synthesizer) 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 E. coli XLI Cells

E. coli cells were made competent with CaCL and transformed according to Sambrook et al. (1989). Transformed cells were grown to a cell density of approximately 10' cells mL-'. IPTG was added to a final concentration of 0.5 m ~ , and the culture was grown for a further 3 h at 3OoC to induce the synthesis of the phagemid-encoded protein. Cells were harvested and resuspended (0.2 M potassium phosphate, 34 m~ L-aspartate, and 50 pg mL-' pyridoxal-5-phosphate, pH 7.6), and cell lysates were prepared by sonication. Crude cell extracts were fractionated on 7.5% (w/v) nondenaturing PAGs and subsequently stained for AAT activity with Fast violet B (Decker and Rau, 1963).

In immunoprecipitation experiments crude E. coli and plant extracts (10 pL) were incubated with 10 pL of the MAb 2B5E5 (100 pg mL-') for 1 h at 37OC prior to fractionation on a nondenaturing PAG and staining for activity using Fast violet B (Decker and Rau, 1963).

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). Sup- plements (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 cDNA

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 ex- amined 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 AbO0 of the culture samples was determined.

Northern Analysis of S. cerevisiae Strain BRSCSC Complemented with the AAT-Pl cDNA

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 from lupin root nodules was isolated according to the method of Chomczynski and Sacchi (1987). RNA from S. cerevisiae cells or from lupin root nodules was separated by formalde- hyde-formamide agarose gel electrophoresis (Sambrook et al., 1989) and subsequently transferred to a Hybond N+ nylon membrane (Amersham) by capillary transfer in 20x SSC. RNA was fixed to the membrane by immersion in 0.05 M NaOH according to the manufacturer's protocol. The membrane was prehybridized/hybridized with a solution of 1 m~ EDTA, 0.5 M Na2HP04 (pH 7.2), and 7% SDS (Church and Gilbert, 1984). The northem blot was hybridized with an AAT-Pl cDNA (EcoRI-HindIII fragment) probe. The probe was radioactively labeled using random priming (Sambrook et al., 1989) and [ L U - ~ ~ P I ~ A T P (Amersham). Following hybrid- uation, the membrane was subjected to a high-stringency wash (0.1X SSC at 65OC for 30 min) prior to autoradiography.



Lupin Aspartate Aminotransferase-P, cDNA 419

RESULTS

Isolation of a cDNA Clone Encoding AAT-P, from aRoot-Tip cDNA Expression Library

A cDNA library, constructed using mRNA from 3-d-old L.angustifolius developing root tips, was immunoscreened usingthe MAb 2B5E5 (Jones et al., 1994). This MAb is monospecificfor the AAT-P] isoform and possibly recognizes a confor-mational epitope (Jones et al., 1994). One positive clone wasidentified and rescreened using MAb 2B5E5 prior to furtheranalysis.

Expression of cDNA-Encoded Protein in F. coli XLI andDemonstration of AAT Activity

The immunoscreen of the cDNA library indicated that theisolated clone was an in-frame fusion of the cDNA with thevector. A convenient enzyme gel activity stain was available(Decker and Rau, 1963), which allowed us to analyze theexpression of the cDNA clone (converted to phagemid form)in E. coli. The results of this experiment are shown in Figure1. Fractionation of the crude extract from lupin root noduleson native PAGs (Fig. 1, lane 1) shows the presence of AAT-PI and AAT-P2 protein. Preincubation of a crude noduleextract with MAb 2B5E5 resulted in the MAb 2B5E5/AAT-PI antibody antigen complex remaining close to the bottomof the loading wells (Fig. 1, lane 2). Because MAb 2B5E5 hasbeen shown not to inhibit enzyme activity (Jones et al., 1994),the band remained visible. As a negative control, crudeextracts of nontransformed E. coli cells were fractionated inthe same manner as were the crude plant extracts. The E. coliAAT enzyme is seen in Figure 1, lanes 3 and 4. Preincubationof the £. coli crude extract with MAb 2B5E5 did not affectthe £. coli enzyme (Fig. 1, lane 4). An additional enzymeactivity band was detectable in crude extracts of £. colitransformed with the cDNA clone (Fig. 1, lane 5). Preincu-bation of these extracts with MAb 2B5E5 resulted in theremoval 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 en-coding AAT. Both nodule and E. coli crude extracts were fraction-ated on a 7.5% native PAG and stained for activity using Fast violetB (see "Materials and Methods"). Immunocomplexing of AAT pres-ent in the crude nodule and E. coli extracts (10 jil_) was achievedby incubating with 10 ML of MAb 2B5E5 (100 Mg ml"1) for 1 h at37°C prior to electrophoresis. Lanes 1, 3, and 5, Crude extracts ofnodule, E. coli XLI, and E. coli XLI transformed with the AAT-P,cDNA clone, respectively. Lanes 2, 4, and 6, The same extractspreincubated with MAb 2B5E5.

Figure 2. Growth curves of the 5. cerevisiae strain BRSCS6 trans-formed with the yeast expression vector pEMBL-yex4, with andwithout the AAT-P, cDNA insert. Cultures were grown on definedmedium 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 ofaspartate; •, pEMBL-yex4 with the AAT-P, insert grown in theabsence of aspartate; O, pEMBL-yex4 without an insert grown inthe presence of aspartate; •, pEMBL-yex4 without an insert grownin the absence of aspartate.

tion of a high mol wt antibody/antigen complex (Fig. 1,lane 6).

Complementation of the asp5 Mutation of 5. cerevisiaeStrain BRSCS6 with the Complete AAT-P, cDNA

S. cerevisiae BRSCS6 cells transformed with the AAT-PicDNA construct in the pEMBL-yex4 expression vector grewboth in the presence and absence of 100 jig mL"1 aspartate(Fig. 2). Cells transformed with pEMBL-yex4, without aninsert, exhibited significant growth only in the presence ofadded aspartate (Fig. 2). A small amount of growth wasrecorded for this transformant in the absence of aspartate,which is consistent with the observation that the asp5 muta-tion is leaky (Jones and Fink, 1982).

Northern analysis of total RNA isolated from the BRSCS6cells transformed with the pEMBL-yex4 expression vector,with and without the AAT-Pi cDNA insert, and RNA fromthe wild-type S. cerevisiae (strain XE-101-1A) cells and lupinroot nodules, showed that only RNA from lupin root nodulesand yeast cells containing the AAT-Pi cDNA construct pos-sessed a transcript homologous to the AAT cDNA probe (Fig.3). The transcript present in the yeast transformed with theAAT-Pi cDNA construct (Fig. 3, lane 3) was approximately70 bp larger than that found in the plant tissue (Fig. 3, lane4). This is consistent with the transcription start sites on theexpression vector being between 50 and 80 bp upstream ofthe cDNA cloning site (Cesareni and Murray, 1987). Toobtain a balanced signal for the northern analysis, we dem-onstrated that 1 Mg of RNA isolated from the BRSCS6 cellscontaining the vector and cDNA insert gave the same signalas 15 Mg of the lupin root nodule RNA (Fig. 3). www.plantphysiol.orgon February 9, 2020 - Published by Downloaded from

Copyright © 1994 American Society of Plant Biologists. All rights reserved.


420 Winefield et al. Plant Physiol. Vol. 104, 1994

1 2 3 4

fusion transcriptAAT-Pi transcript

Figure 3. Northern analysis of total RNA isolated from transformedS. cerev/s/ae strain BRSCS6 cells, wild-type (XE-101-1 A) S. cerevisiaecells, and lupin root nodules. The probe used corresponded tonucleotides 1 to 704 of the cDN A clone. Lane 1, Fifteen microgramsof total RNA isolated from XE-101-1A cells. Lane 2, Fifteen micro-grams of total RNA isolated from BRSCS6 cells transformed withthe yeast expression vector pEMBL-yex4 (no insert). Lane 3, Onemicrogram of total RNA isolated from BRSCS6 cells transformedwith the AAT-P, cDNA/pEMBL-yex4 construct. Lane 4, Fifteenmicrograms of total RNA isolated from lupin root nodules.

Sequence Analysis of the AAT-P] Clone

The AAT-Pi cDNA clone was sequenced and found tocontain an open reading frame of 1296 bp (Fig. 4). A possibleinitiating ATG was found at nucleotides 37 to 39 (Fig. 4).The flanking sequences, with the exception of the cytosine atnucleotide 34, are homologous to the consensus sequencesfound to flank the initiating ATG in other plants (Cavenerand Ray, 1991). The open reading frame from the proposedinitiating Met to the termination codon at nucleotides 1297to 1299 encodes a 420-amino acid polypeptide with a calcu-lated Mr of 45,781 (Fig. 4).

Alignment of the translated open reading frame againsttranslated nucleic acid sequences from the GenBank database using the FASTA search program (Pearson and Lipman,1988) revealed that the L angustifolius AAT-Pi sequenceshared 82.3% identity with Panicum miliaceum cytosolicAAT2 (Taniguchi et al., 1992), 88.6% amino acid sequenceidentity with Medicago sativa cytosolic AAT1 (Udvardi andKahn, 1991), and 81.6% identity with D. carota cytosolic AAT(Turano et al., 1992) (Fig. 5). The L. angustifolius AAT-Pj hadonly 56.4% sequence identity with the plastid AAT-P2 fromL angustifolius (Reynolds et al., 1992), 52.4% and 52.1%identity with mitochondrial enzymes AA3 and AAT1, re-spectively, from P. miliaceum (Taniguchi et al., 1992), 50.4%identity with the chloroplast AAT from Glycine max (Wads-worth et al., 1993), and 57.5% homology with M. sativaplastid AAT2 (Gantt et al., 1992) (alignments not shown). Langustifolius AAT-Pi shared sequence identity of approxi-mately 50% with the other animal and bacterial AAT se-quences found in the GenBank data base.

DISCUSSION

A cDNA clone encoding the constitutively expressed andcytosol-located isoform of AAT in L. angustifolius (AAT-Pi)

was isolated from a root-tip cDNA library. MAbs againstAAT-Pi were previously shown, and confirmed here, not tocross-react with the plastid AAT-P2 (Jones et al., 1994; Fig.1). The immunocomplexing and expression experiments pre-sented here (Fig. 1) show that the expressed cDNA clone iscoding for AAT-P]. The two AAT enzyme activity bands inFigure 1, lane 5, correspond to the AAT-Pi enzyme fromlupin root nodules (Fig. 1, lane 1) and the native AAT enzymefrom £. co/i (Fig. 1, lane 3). This suggests that E. coli cantranscribe the plant AAT cDNA, translate the mRNA, andcorrectly assemble the plant enzyme, even in the presence ofthe E. coli native enzyme. A high degree of amino acidsequence identity (88.6, 82.3, and 81.6%) with three plantcytosol AAT sequences (Fig. 4) and the immunocomplexingof 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

CAA TCC GTT TCCQ S V SCTT CTT CCT GCTL L

AAA GATK DGAG GAAE ECTT GTGL V

GCC GATA DGCT ATTA ITCA TTAS L

ATA TATI Y

GCT GGGA GCTT GAC

CCA AGCP S

GGA AAAG K

AAT GAAN ETTC AACF N

CAA GAGQ E

AGA GTTR V

ATT CCTI P

TTA TCTL S

TTT GAA

ATT GTT TTG CTAI V L L

CAA TGGQ WTTT GAC

D

ACT GAGT ECCT TTC

GAT GCAD AGCC CAGA QCTA AGCL SGTG AAAV KTCC ATTS I

ATT GAG

CAG GCTQ A

AGT TAT

GTTVCCTP

CCAP

CCTP

GCGAAAAK

AACNGGGG

CAAQ

GTCV

CATH

GAGE

AGTS

GTT

TCT CCA ACC GCT TCTS P T A S

GAA GAT CCT ATT CTCE

GTTD

AATN

AATN

AAGK

AGGR

ACTT

CTAL

GGTG

CGCR

GATL L E D

GCA TGC GCA CATA C A H

CAG0

GCT

AAG CTCK L

GTT CTGV LCGC AACR N

AGT GCTS A

GTG ACCV T

GAA TTTE F

ACA TGGT W

ACA TATT Y

CTA GAA

ATT AGGI R

TAT CAAY Q

TTG TTTL F

AAC ATG GGT

TTAL

GTAV

GAGE

CTCL

GTTV

GCTA

AATN

TATY

CTTL

AACK

CTGL

TTTF

GCTA

CTA

TCT GAT TCC GTT TTC GCTS D S V F A

GGG GTG ACT GTG GCTG V T V A

GGT GTT GGT GCT TACG V G A Y

GTG AGG CGT GTT GAAV R

TAT CTTY L

ATT TTTI F

CAA TGCQ C

AGA CACR H

CAC CCAH P

TAT GCTY A

GGT TCTG S

CCT ACTP T

TTG AGAL R

GCT AGTA S

GAT GGGD G

TAT GGA

CCA ATTP I

GGT GCTG A

TTG TCTL S

TAT CACY H

AAG ATTK I

CCG GCAP A

GCC CCA

EGTTV

GACD

GGTG

CAGQ

TTCF

ACA

TATY

CGAR

CAGQ

GGGG

AGCS

ACTT

AGGR

ACCT

AGA

GGT GTT GAT CCAG V D P

TTGL

GATD

TCA AAAS K

GGA AGT

GCTA

CTA

GGT GAAG E

GAA CGT

TTGL

GTT

CTG

GGCN M

AAT 51N 5

CAT 102H 22

AAC 153N 39

ACT 204T 56

CAA 2550 73

GTC 306V 90

CCT 357P 107

GGT 408G 124

ACT 459T HI

TTA 510L 15B

GGG 561G 175

TCC 612S 192

ACC 663T 209

TTA 714L 226

ATA 765I 213

CTT 816L 260

GCC 867A 277

ATT GTCI V

CTT GTGL VGTG GCT

TCCS

GTT

AAGK

AGG

VATTI

CTG AAG GCA ATGL K A M

TCC

TCA GCTS A

CCA ATGP M

CTC AGGL R

GCT GAT

GATD

TACY

GAC

GTTV

CGC

GCA AGC AGG GTT GAGA S

AAC CCTV

AGA GGC ACA CCT GGTTTT GAT GCT TTG CAAF D A L Q

3A ATG TTT ACT TTC ACA GGA TTG3 M F T F T G L*G GAG

AAG CAG .K QTTC TTA TACF L T K E Y

ATG GCT GGT CTG AGT TCC

ATT CATI H

AAT GAGN E

CGC CAAQ

AGT CACS H

GAG CAAQ

CAT ATA TAC TTG ACA TCT GAT GGG AGGH I Y L T S D G R

D

TTGL

AAAK

GATD

AATN

TACY

ATGH

TGG .W

GCT

GCA GCT GTA ACC AGA GTTA A V T R VTTG CCC CTT GTT GGA GGACAT AAC ATT ATA TCT CATSGC ATC ATA AAA AAA AAA

AAA ACA GTT CCT CATK T V P H

GTC TAA AAC ATG TTA

GCTA

ATT

GAT GCAD A

ACA GTT

CAT CCACAT TATAAA AAA

TTAGAC

TTT TCG TTC AAT AAT TACATT TTT GTT CAG TTA TCT

AGC CAGs o

GGT GCAG A

TGG CATW H

CAA CTT0 L

ATT ATCI I

GTT TCC

ATA CATI H

TTC TAT

GGA CATTAA TAT

9182949693111020328107134511223621173379122439612754131326420137714281452

Figure 4. Nucleotide and deduced amino acid sequence of theAAT-Pi cDNA clone. Double-stranded sequencing was carried outon pUC119 subclones as described in "Materials and Methods."The sequences that are underlined with a single line represent thesequences surrounding a possible translation initiation start (nucle-otides 31-42) and putative poly(A) addition signals (nucleotides1364-1369 and 1424-1429). The double-underlined region repre-sents the pyridoxal-5-phosphate binding motif (Wightman and For-est, 1978). The sequence was determined for both strands exceptfor nucleotide 637. Sequencing in the forward direction resulted ina cytosine being scored without ambiguity. Sequencing in thereverse direction resulted in a band (stop) in all four lanes.



Lupin Aspartate Aminotransferase-P, cDNA 42 1

Lupinus Panicum Medicago Daucus

Lup inus P a n i c m Medicago Daucus

Lup inus Panicum Medicago DaUCUS

Lupinus Panicum Medica90 Daucus

Lup inus Panicum Medicago D a u c u s

Figure 5. Multiple alignment of the AAT-P, 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.

the isolated cDNA clone encodes cytosol-located isoenzyme

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 comple- ment 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 (Wight- man 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 pEMBL- yex4 (Cesareni and Murray, 1987).

AAT-PI. 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 pro- posed 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 exam- ining 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 (AA- TATG), 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 SDS- PAGE 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



422 Winefield e t al. Plant Physiol. Vol. 104, 1994

84.1 % 52.2 90 identity

41.6 % Identity 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 the same animal were only 46% identical. This scheme of relatedness of similar, intra- cellularly located AAT sequences was extended to include those of human, rat, mouse, and horse (Cronin et al., 1990; Doonan, 1990). Sequence comparison here of the 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 and the cytosolic isoforms of M. sativa, D. carota, and P. miliaceum is approximately 84% (88.6, 81.6, and 82.3%, respectively; Figs. 5 and 6). Between L. angustifolius AAT-PI and the nodule proplastid- and chloroplast-associated forms of AAT from L. angustifolius (AAT- P2), M. sativa (AATZ), and G. max, respectively, the level of identity drops to approximately 54% (52.4, 57.5, and 50.4%, respectively; Fig. 6). Furthermore, when the nodule plastid- associated isoforms from L. angustifolius and M. sativa and the chloroplast form from G. max were compared with the 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 in 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.

LITERATURE ClTED

Burgers PMJ, Percival KJ (1987) Transformation of yeast sphero- plasts without cell fusion. Anal Biochem 163 391-397

Cavener DR, Ray SC (1991) Eukaryotic start and stop translation sites. Nucleic Acids Res 19 3185-3192

Cesareni G, Murray JAH (1987) Plasmid vectors carrying the rep- lication origin of filamentous single-stranded phages. In JK Setlow, ed, Genetic Engineering, Vol9. Plenum Publishing, New York, pp

Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162 156-159

Christen P, Graf-Hausner U, Bossa F, Doonan S (1985) Comparison of covalent structures of the isoenzymes of aspartate aminotransferase. In P Christen, DE Metzler, eds, Transaminases, Vol 2. Wiley, New York, pp 173-185

Church GM, Gilbert W (1984) Genomic sequencing. Proc Natl Acad Sci USA 81: 1991-1995

Cronin VB, Doyle JM, Doonan S (1990) Amino acid sequences of aspartate aminotransferases: the cytosolic isoenzymes from yeast and from human liver. Biochem SOC Trans 18 256

Decker LE, Rau EM (1963) Multiple forms of glutamate oxaloacetate transaminase in tissues. Proc SOC Exp Biol Med 112 144-149

Doonan S (1990) Aspartate aminotransferases and malate dehydro- genases: pattems of evolution. Biochem SOC Trans 1 8 167-169

Gantt JS, Larson RJ, Farnham MW, Pathirana SM, Miller SS, Vance CP (1992) Aspartate aminotransferase in effective and ineffective alfalfa nodules. Plant Physiol98 868-878

Givan CV (1980) Aminotransferases in higher plants. In PK Stumpf, EE Conn, eds, The Biochemistry of Plants, Vol5. Academic Press, New York, pp 329-357

Griffith SM, Vance CP (1989) Aspartate aminotransferases in alfalfa root nodules. I. Purification and partial characterization. Plant Physiol90 1622-1629

Hatch MD, Mau SL (1973) Activity, location and role of aspartate aminotransferase and alanine aminotransferase isoenzymes in leaves with C4 photosynthesis. Arch Biochem Biophys 156

Higgins DG, Sharp PM (1988) CLUSTAL: A package for performing multiple sequence alignments on a microcomputer. Gene 73

Jones EW, Fink GR (1982) Regulation of amino acid and nucleotide biosynthesis in yeast. In JN Strathem, EW Jones, JR Broach, eds, The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 181-300

Jones WT, Jones SD, Harvey D, Rodber KA, Ryan GB, Reynolds PHS (1994) Production and characterization of monoclonal antibodies against aspartate aminotransferase-P1 from lupin root nodules. Plant Physiol104 91-97

Jones WT, Reynolds PHS, Jones SD, Liddane CP, Rodber KA (1990) Production and characterization of monoclonal antibodies against aspartate aminotransferase-Pz from lupin root nodules. Plant Physiol94 1358-1364

Joshi CP (1987) Putative polyadenylation signals in nuclear genes of higher plants: a compilation and analysis. Nucleic Acids Res 15

Pearson WR, Lipman DJ (1988) Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 8 5 2444-2448

Reynolds PHS, Boland MJ, Farnden KJF (1981) Enzymes of nitrogen metabolism in legume nodules: partial purification and properties of the aspartate aminotransferases from lupine nodules. Arch Biochem Biophys 209 524-533

Reynolds PHS, Farnden KJF (1979) The involvement of aspartate aminotransferases in ammonium assimilation in lupin nodules. Phytochemistry 18: 1625-1630

Reynolds PHS, Smith LA, Dickson JMJJ, Jones WT, Jones SD, Rodber KA, Carne A, Liddane CP (1992) Molecular cloning of a cDNA encoding aspartate aminotransferase-P2 from lupin root nodules. Plant Mo1 Biol 19 465-472

Ryan E, Bodley F, Fottrell PF (1972) Purification and characterization of aspartate aminotransferases from soybean root nodules and Rhizobium japonicum. Phytochemistry 11: 957-963

135-154

195-206

237-244

9627-9640



Lupin Aspartate Aminotransferase-P, cDNA 423

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, Ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 7 4

Schmitt ME, Brown TA, Trumpower BL (1990) A rapid and simple method for preparation of RNA from Saccharomyces cereuisiae. Nucleic Acids Res 18: 3091-3092

Stockwell PA (1988) HOMED: a homologous sequence editor. Trends Biochem Sci 1 3 322-324

Taniguchi M, Sawaki H, Sasakawa H, Hase T, Sugiyama T (1992) Cloning and sequence analysis of cDNA encoding aspartate ami-

5463-5467

notransferase isoenzymes from Panicum miliaceum L., a C4 plant. Eur J Biochem 204 611-620

Turano FJ, Weisemann JM, Matthews BF (1992) Identification and expression of a cDNA clone encoding aspartate aminotransferase in carrot. Plant Physiol 100 374-381

Udvardi MK, Kahn ML (1991) Isolation and analysis of a cDNA clone that encodes an alfalfa (Medicago satiua) aspartate aminotransferase. Mo1 Gen Genet 231: 97-105

Wadsworth GJ, Marmaras SM, Matthews BF (1993) Isolation and characterization of a soybean cDNA clone encoding the plastid form of aspartate aminotransferase. Plant Mo1 Biol21: 993-1009

Wightman F, Forest JC (1978) Properties of plant aminotransferases. Phytochemistry 17: 1455-1471



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Cloning and Characterization of a cDNA Encoding Aspartate ... · isolated clone was an in-frame fusion of the cDNA with the vector. A convenient enzyme gel activity stain was available