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- Publisher
- Springer Journals
- Copyright
- Copyright © European Molecular Biology Organization 1990
- ISSN
- 0261-4189
- eISSN
- 1460-2075
- DOI
- 10.1002/j.1460-2075.1990.tb08114.x
- Publisher site
- See Article on Publisher Site
Abstract
The EMBO Journal vol.9 no. 2 pp. 323 - 332, 1 990 of two Dark-induced and organ-specific expression in Pisum sativum asparagine synthetase genes amino acid to be discovered in plants (Vauquelin was the first Fong-Ying Tsai and Gloria M.Coruzzi and the for its Robiquet, 1896), enzyme responsible synthesis Laboratory of Plant Molecular Biology, The Rockefeller University, remains poorly understood to date. Asparagine, synthesized 1230 York Ave., New York, NY 10021, USA from asparatate and glutamine, is the major nitrogen Communicated by J.H.Weil amino acid in faced with conditions of excess transport plants ammonia rather than nitrate. During normal plant growth, of cDNAs for Nucleotide sequence analysis asparagine under a of conditions of ammonia excess occur variety has uncovered two synthetase (AS) of Pisum sativum circumstances which include growth on fertilizers, during distinct AS mRNAs (AS1 and AS2) encoding polypeptides seed nitrogen-fixation, during germination and during human AS that are highly homologous to the enzyme. In formation in senescing plants (Lea and Fowden, 1975). residues of both AS1 and AS2 poly- The amino-terminal for to of certain species, asparagine can account up 86% domain peptides are identical to the glutamine-binding and transported nitrogen in the above contexts (Lea Miflin, indicating that the full-length of the human AS enzyme, in a are also 1980). Levels of asparagine transported plant encode AS AS1 and AS2 cDNAs glutamine-dependent affected by external factors such as light. Since asparagine of nuclear DNA shows that AS1 and enzymes. Analysis than it is a more has a higher N:C ratio glutamine, AS2 are each encoded in P.satvum. Gene- by single genes in in economical nitrogen transport compound plants grown specific Northern blot analysis reveals that dark of the dark, when carbon skeletons are limiting. Analysis treatment induces high-level accumulation of AS1 mRNA exudate in Pisum sativum reveals that levels of phloem in leaves, while light treatment represses this effect are in exudates from dark-treated asparagine higher phloem as much as 30-fold. the dark-induced Moreover, versus light-grown plants (Urquhart and Joy, 1981). to be a accumulation of AS1 mRNA was shown Consistent with these results is the finding that AS enzyme phytochrome-mediated response. Both AS1 and AS2 detected in extracts of leaves is also higher when activity pea in of mRNAs also accumulate to high levels cotyledons are in the dark (Joy et al., 1983). plants grown in root nodules. germiinating and nitrogen-fiing sedings Although asparagine plays a crucial role in plant nitrogen of correlate well with These patterns AS gene expression transport, the enzyme responsible for its biosynthesis is as a the physiological role of asparagine nitrogen poorly characterized and has not been purified to homo- transport amino acid during plant development. geneity. The inability to purify the AS enzyme from plants Key words: asparagine synthetase/gene expression/light is due, in part, to the fact that the plant AS enzyme is regulation/nitrogen assimilation/plants extremely unstable in partially purified extracts (Rognes, 1975; Huber and Streeter, 1984, 1985). In addition, plant extracts contain contaminating asparaginase activity (Streeter, 1977) and specific non-protein inhibitors of AS (Joy et al., Introduction which the difficulties in 1983) compound assaying for AS In Escherichia coli and yeast, the genes for enzymes along enzyme activity in vitro. The inability to purify the plant amino acid biosynthetic pathways have been well charac- has it to precisely characterize AS enzyme made impossible terized by combined genetic, biochemical and molecular the number of AS in their subcellular isozymes plants, approaches. Until recently studies concerning plant amino localizations and the gene(s) encoding AS. corresponding acid biosynthetic enzymes have been limited to biochemical To circumvent the problems encountered via biochemical approaches. Traditionally, biochemical investigations have studies on we have used a molecular plant AS, biological been directed at characterizing plant nitrogen metabolic to clone AS cDNAs from P.sativwn. These approach directly enzymes in terms of their reaction mechanism, number of AS cDNA clones now be used in a 'reverse- plant may isozymes, subcellular localizations and roles during plant biochemical' approach to characterize the encoded AS development (Miflin, 1980). For several enzymes such as as well as to examine the regulated expression of enzymes nitrate reductase (Crawford et al., 1986), nitrite reductase their genes in higher plants. Here, we report corresponding (Back et al., 1988) and glutamine synthetase (Cullimore the isolation and characterization of two classes of plant AS et et al., 1984; Tingey al., 1987), detailed biochemical but cDNAs and AS2) which encode homologous (ASI studies have provided the basis for the isolation and DNA were distinct AS polypeptides. Gene-specific probes characterization of their corresponding genes. However, for to monitor the levels of ASI and used steady-state AS2 many other important nitrogen metabolic enzymes in higher mRNAs in leaves of or and in light- dark-grown plants plants, difficulties encountered in biochemical purification various during plant development. These studies have organs have prevented the characterization of the enzymes and hence shown that the of the AS in are expression genes plants their cognate genes. of increased affected and conditions by light developmental of the AS One amino acid biosynthetic enzyme that has proven to nitrogen transport. The regulated expression genes is with data be particularly recalcitrant to biochemical analysis plant shows a correlation previous physiological strong While asparagine synthetase (AS; EC6.3.5.4). asparagine transport during plant development. concerning asparagine Oxford University Press F.-Y.Tsai and G.M.Coruzzi Results in vitro anchored by polymerase chain reaction (A-PCR) Isolation and characterization of two distinct AS an using oligonucleotide primer complementary to the 5' end cDNAs from pea of XcAS201 as described in Materials and methods. The A cDNA clone encoding human AS (pH131) (Andrulis restriction of the map full length cDNA of AS2 (cAS2) was et al., 1987) was used to isolate cDNAs deduced from the encoding plant AS cDNA overlapping partial clones XcAS201 from a pea nodule cDNA library. From 50 positive clones and pcAS801. identified out of 2 x 105 clones screened, eight clones The nucleotide sequences of the full-length AS 1 and AS2 (XcAS301 -XcAS308) were randomly selected for further cDNAs are shown in 2. Figure pcASl is 2200 nt long and analysis. Restriction mapping and nucleotide with the first sequence starting in-frame methionine encodes a protein analysis of these clones revealed that all eight contained of 586 amino acids with a predicted mol. wt. of 66.3 kd. cDNA inserts which correspond to overlapping portions of The 3' non-coding region of ASI cDNA is 333 nt long and a single mRNA species A cDNA contains a tail (AS1) (Figure IA). con- poly(A) (Figure 2A). cAS2 is 2002 nt long taining the 5' end of the AS 1 mRNA was and encodes a of 583 (XcAS907) protein amino acids with a predicted mol. synthesized in vitro using an oligonucleotide com- wt of 65.6 kd. The 3' non-coding primer region of cAS2 is 141 nt plementary to the 5' end of XcAS301 (see Materials and long and contains a poly(A) tail (Figure 2B). methods). The restriction maps of the three Nucleotide sequence homologies among overlapping pea cDNAs of AS1, cDNA AS2 and clones which include the entire AS 1 human AS cDNA (pH131) (Andrulis coding region et al., 1987) are shown in were Figure 1A. The ASI compared using the 'fasta' computer program composite full-length (Pear- son cDNA, pcAS1 (2.2 kb), was constructed and Lipman, 1988). The two AS by sequential pea cDNAs (ASI and ligation of are the restriction fragments (fragments a, b and AS2) highly homologous to each other at the c) nucleotide from the three level within their overlapping cDNA clones XcAS907, XcAS301 coding regions (81 %) and completely and in in XcAS305 respectively as indicated lA. divergent the 3' non-coding The Figure regions. overall A second of nucleotide type AS coding sequence (AS2) was detected homology between either AS cDNA of pea and in peas when a DNA from the AS cDNA of human fragment the coding region of is -50-55% within the coding an ASl cDNA was used to screen a regions. Neither pea pea genomic AS cDNA shares significant homology library. cDNA clones the mRNA were to the encoding AS2 asparagine synthetase (asnA) gene of E. coli subsequently (Nakamura isolated from a root cDNA an et al., 1981) pea library using AS2 genomic (not shown). fragment as a DNA The cDNA The deduced amino acid probe. longest AS2 clone, sequences for the pea AS 1, pea XcAS201, which contains a 1.5 kb cDNA AS2 and human AS polypetides are insert, was compared in Figure 3. selected for further A cDNA The polypeptides encoded AS analysis (Figure iB). by 1 and AS2 cDNAs share containing an the 5' end of the AS2 mRNA was overall homology of 86% at the amino (pcAS801) synthesized acid level. A A. AS 1 cDNAs pcAS EL S H Bg Bm po loi XcAS907 (E) XcAS301 XcAS305 B. AS2 cDNAs cAS2 FL -Hfl Bm Bs Bm HBsE i pcAS801 XcAS201 200bp Fig. Restriction maps of ASI and AS2 cDNA clones. E, EcoRI; S, SstI, Bm, BamHI; Bg, Bglll; H, HinclI; Bs, BstNI. E- respresents an EcoRI site in which was XcAS301 in the of and destroyed process cloning selection. bars the Open represent coding regions of each cDNA. (A) pcASl is a ASI (2.2 kb) composite full-length cDNA constructed from restriction fragments b and of cDNA (a, c) overlapping clones XcAS907, XcAS301 and XcAS305. cDNA (B) Overlapping clones and XcAS201 for AS2 mRNA pcAS801 were used to derive the restriction map of full-length cAS2. 324 synthetase genes in peas Asparagine 1 CTA CGT GTT GCT TCT TCC ACA CTC TTT GCT CCT AGT TTT TCG TGT CTT GTT TTC TTT ATC CTC TTC TCA TTC TCT TTG GTT CTT 84 1 M C G I L A V L G C S D D S 0 A K R V R I L E L S 25 85 CAA ATC ATA ATG TGT GGC ATA CTT GCT GTA CTT GGT TGC TCT GAT GAT TCA CAA GCT AAA CGA GTT CGC ATA CTC GAG CTT TCT 168 26 R R L K H R G P D W S G L H 0 H G D N Y L A H 0 R L A I 53 169 CGC AGA TTG AAG CAC CGT GGG CCA GAC TGG AGT GGG CTC CAC CAA CAT GGT GAT AAC TAT TTG GCT CAT CAA AGG TTA GCC ATT 252 54 V D P A S G D Q P L F N E D K S I I V T V N G E I Y N H 81 253 GTT GAT CCT GCT TCT GGT GAT CAA CCT CTC TTC AAT GAA GAC AAA TCA ATT ATT GTC ACG GTG AAT GGA GAA ATC TAC AAT CAT 336 82 E E L R K Q L P N H K F F T Q C D C D V I A H L Y E E H 109 337 GAA GAG CTC AGA AAA CAA TTG CCC AAT CAC AAG TTT TTT ACA CAA TGT GAC TGT GAT GTT ATT GCA CAC CTG TAC GAG GAA CAT 420 110 G E N F V D N L D G I F S F V L L D T R D N S F I V A R 137 421 GGA GAA AAT TTT GTG GAT ATG TTA GAC GGT ATA TTT TCG TTT GTT CTG CTG GAT ACT CGT GAC AAC AGT TTC ATA GTT GCG AGG 504 138 D A I G V T S L Y I G W G L D G S V W I A S E L K G L N 165 505 GAT GCT ATA GGT GTT ACT TCC TTG TAC ATT GGT TGG GGA CTA GAT GGT TCT GTT TGG ATT GCA TCA GAA TTG AAA GGA CTG AAT 588 166 D E C E H F E V F P P G H L Y S S K E R E F R R W Y N P 193 589 GAT GAA TGT GAA CAT TTC GAA GTT TTT CCG CCC GGT CAC TTA TAC TCG AGC AAA GAA AGA GAG TTT CGT CGA TGG TAT AAT CCT 672 194 P W F N E A I I P S T P Y 0 P L V L R N A F E K A V I K 221 673 CCA TGG TTC AAT GAG GCT ATT ATT CCG TCA ACA CCT TAT GAT CCT CTA GTT TTG AGG AAC GCG TTT GAG AAG GCT GTG ATA AAG 756 222 R L M T D V P F G V L L S G G L D S S L V A S V T A R Y 249 757 AGG TTG ATG ACC GAT GTG CCT TTC GGG GTT TTA CTA TCG GGA GGT TTG GAT TCA TCG TTG GTC GCG TCT GTC ACT GCT AGA TAC 840 250 L A G T K A A K 0 W G A K L P S F C V G L K G A P D L K 277 841 CTT GCT GGT ACA AAA GCT GCT AAG CAG TGG GGA GCA AAA TTG CCC TCT TTC TGT GTA GGC CTT AAG GGC GCA CCT GAC CTA AAG 924 278 A G K E V A D F L G T V H H E F E F T I 0 D G I D A I E 305 925 GCT GGA AAG GAG GTA GCA GAT TTC TTA GGA ACT GTC CAT CAT GAA TTT GAG TTT ACT ATC CAG GAC GGT ATA GAT GCA ATT GAA 1008 306 D V I Y H T E T Y D V T T I R A A T P M F L M S R K I K 333 1009 GAT GTC ATC TAT CAC ACA GAA ACA TAT GAT GTT ACT ACG ATA AGG GCT GCA ACA CCT ATG TTT CTG ATG TCT CGT AAG ATC AAA 1092 334 S S G V K W V I S G E G S D E I F G G Y L Y F H K A P N 361 1093 TCA TCC GGA GTC AAA TGG GTG ATT TCT GGA GAA GGA TCT GAT GAG ATC TTT GGA GGG TAT TTG TAT TTC CAT AAG GCG CCA AAC 1176 362 R E E F H 0 E T C R K I K A L H R Y D C L R A N K S T Y 389 1177 AGG GAA GAG TTT CAC CAA GAA ACA TGC CGC AAG ATC AAA GCT CTT CAT AGA TAT GAT TGT TTG AGA GCC AAT AAA TCA ACA TAT 1260 390 A W G L E A R V P F L D K D F I K V A M D I D P E F K M 417 1261 GCA TGG GGT CTA GAA GCT AGA GTA CCA TTT TTG GAC AAG GAC TTT ATC AAG GTT GCA ATG GAC ATT GAT CCT GAG TTT AAA ATG 1344 418 I K H D E G R I E K W I L R K A F D D E E N P Y L P K H 445 1345 ATA AAA CAT GAT GAA GGA AGA ATT GAG AAA TGG ATT CTA AGA AAG GCC TTT GAT GAT GAA GAG AAT CCA TAT CTG CCT AAG CAC 1428 446 I L Y R 0 K E 0 F S D G V G Y G W I D G I K D H A A K H 473 1429 ATT TTA TAT AGG CAG AAG GAA CAA TTC AGT GAT GGA GTT GGA TAT GGC TGG ATA GAT GGC ATC AAG GAC CAT GCT GCA AAA CAT 1512 474 V T D R M M F N A S H I F P F N T P N T K E A Y Y Y R M 501 1513 GTC ACT GAC AGA ATG ATG TTC AAT GCT TCT CAC ATC TTT CCT TTC AAC ACT CCA AAT ACC AAA GAA GCA TAT TAC TAT AGA ATG 1596 502 I F E R F F P 0 N S A R L T V P G G P S V A C S T E K A 529 1597 ATC TTT GAA AGG TTT TTC CCT CAG AAC TCG GCA AGG CTT ACA GTT CCT GGA GGA CCT AGT GTT GCA TGC AGC ACA GAG AAA GCT 1680 530 I E W D A S W S N N L D P S G R A A L G V H V S A Y E H 557 1681 ATT GAA TGG GAT GCT TCA TGG TCA AAC AAC CTG GAT CCT TCT GGT AGA GCA GCA CTT GGA GTG CAT GTT TCA GCT TAT GAA CAC 1764 558 Q I N P V T K G V E P E K I I P K I G V S P L G V A I 0 585 1765 CAA ATC AAC CCA GTT ACA AAA GGT GTA GAG CCA GAG AAG ATT ATA CCA AAG ATA GGA GTT TCT CCT CTT GGA GTT GCC ATT CAA 1848 586 T * 587 1849 ACC TAG TAT GAG ACA TAG CAA GTA TTA CTT GCT TAA AAA ACC AAG ATA TTA TTA TAC TAT TAG TAT TCA ATA AAA AGA ATA ACA 1932 1933 TAA AGG GAA AAT TTG CCT GTT ATG TAT TTT ATC CAG GTA CAG GTA CAT TTG TAT GTA TAA GCC TTT CTA CTT AGC TGT ATT TAT 2016 2017 GTG TTT TGA TGT TGT GTA ATC CAC ATC TTG TCT TTG CTT TTA ATT GAT GTG GTG ATT TGA ACA CTT TCA GAT TGT AAT TTG GCT 2100 2101 TTT TAA GAA GAG TTG TGT ATT ATG TTA AAT TTG AGT GCA AGT TTC ACT ATT TGA ATA CTA CTT ATA AAT ATA TGT CTT TAC ATT 2184 2185 AAA AAA AAA AAA AAA A 2200 1 TT CCA AAG CCA TTA TTA GTA TTA CAA CTA CAT ACA TAT TTT CTT CTT AGT TTA TTC CAA ATT CTG TCT TTG ATT TCA TTA TCG 83 1 M C G I L A V L G C S D P S R A K R V R V L 22 84 TAT AAA ACA TAA ACA ACA ATG TGT GGT ATA CTT GCT GTT CTT GGT TGT TCT GAT CCT TCT CGA GCC AAG AGA GTT CGT GTG TTG 167 23 E L S R R L K H R G P E W S G L H 0 H G D C Y L A 0 0 R 50 168 GAA CTT TCA CGC AGA TTG AAG CAC CGA GGC CCT GAA TGG AGT GGG CTC CAC CAA CAT GGT GAT TGT TAT TTG GCA CAA CAA CGG 251 51 L A I V D P A S G D Q P L F N E D N P S I V T V N G E I 78 252 TTA GCC ATA GTT GAT CCT GCT TCT GGT GAT CAA CCT CTC TTC AAT GAA GAC AAT CCG TCA ATT GTC ACG GTA AAC GGA GAG ATT 335 79 Y N H E D L R K 0 L S N H T F R T G S D C D V I A H L Y 106 336 TAC AAT CAT GAA GAT CTC AGG AAA CAG TTG TCT AAT CAC ACG TTT AGG ACC GGA AGT GAT TGT GAT GTT ATT GCG CAT TTG TAC 419 107 E E Y G E D F V D N L D G I F S F V P L D T R D N S Y I 134 420 GAG GAA TAT GGA GAA GAC TTT GTG GAT ATG TTG GAT GGT ATA TTT TCG TTT GTT CCA TTG GAT ACT CGT GAC AAC AGT TAT ATT 503 325 F.-Y.Tsai and G.M.Coruzzi 135 V A R D A I G V T S L Y I G W G L D G S V W I S S M K 162 504 GCT AGA GAT GCG ATT GGT GTA ACT TCT TAC ATT GTG CTA GGT TGG GGA TTA GAT GGT TCG GTT TGG ATT TCG TCG ATG 587 GAA AAA 163 G L N D D C E H F E C F P P G H L Y S S K D S G F R R 190 588 GGT TTG AAC GAT GAT TGT GAA CAT TTC GAG TGT TTT CCA CCT GGT CAT TTG TAT TCG AGC GAT AGT GGC TTT AGA AGA TGG 671 AAA 191 Y N P S W Y S E A I P S A P Y D P L A L R H A F E K A V 218 672 TAT AAT CCT TCT TGG TAC TCT GAG GCT ATT CCG TCG GCT CCT TAT GAT CCT CTT GCT TTG AGG CAC GCC TTC GAG AAG GCG GTG 755 219 V K R L T D V P F G V L L M S G G L D S S L V A S I T S 246 756 AGG TTG ATG ACA GAT GTA CCT TTC GTT CTA GTA AAA GGT CTA TCC GGA GGT TTG GAC TCG TCA TTG GTT GCA TCC ATC ACT TCT 839 247 R Y L A T T K A A E Q W G S K L H S F C V G L E G S P D 274 CGC TAC CTA GCA ACC ACG GCG GCT TGG GGA TCA 840 AAA GAA CAA AAA CTA CAT TCA TTC TGC GTT GGA CTC GAG GGC TCA CCT GAT 923 275 L K A G K E V A D Y L G T V H H E F T F T V Q D G I D A 302 924 CTT AAG GCT GGA AAA GAA GTT GCA GAT TAT CTC GGA ACC GTT CAT CAT GAG TTT ACC TTT ACT GTT CAG GAT GGT ATA GAT GCA 1007 303 I E D V I Y H V E T Y D V T S I R A S T P F L M M S R K 330 1008 ATT GAG GAT GTT ATA TAC CAT GTT ACA TAT GAT GTT ACT TCA ATT AGA GCA AGC ACG CCT ATG TTT CTC ATG TCG GAA AGG AAG 1091 331 I K S L G V K W V I S G G S D E I F G G Y L E Y F H K A 358 1092 ATT TCA CTT GGT GTC TGG GTG ATC TCC GGT GGA TCC GAT GAG ATC GGC AAA AAA GAA TTT GGA TAT CTG TAC TTT CAC AAG GCA 1175 359 P N K F H T C R K I K A L H Q Y D E E E E C Q R A N K S 386 1176 CCG AAC GAG TTT CAC ACT TGC CGC ATC GCA AAG GAA GAA GAA AAG AAA CTG CAC CAA TAT GAT TGC CAG AGA GCT TCG 1259 AAT AAA T Y A W G L E A R V P F L D K A 387 F I N V A N I P N M D E 414 1260 ACT TAT GCT TGG GGT TTA GCT AGA GTT CCG TTT CTG GAC GCG TTT ATC GTT GCG ATG AAT ATT GAT CCT GAA AAG AAT GAG AAT 1343 415 K M I K R D E G R I E K Y I L K A F N P Y L P R D D E E 442 1344 AAA ATG ATA AAA CGA GAT GAA GGA CGA ATT GAG AAG TAT ATT TTG AGG AAG GCA TTT GAT GAC GAG AAT CCT TAT CTG CCA GAA 1427 443 K H I L Y R Q K E Q F S D G V G Y S W I D G L K A H A A 470 1428 CAC ATT TTG TAT AGG CAG TTC AGT GAT GGA GTT GGT TAT AGC TGG ATT GAT GGT CTT GCT CAT GCT GCA AAG AAA GAA CAA AAA 1511 471 K H V T D K M L N A G N I F P H N T P N T K A Y Y Y M E 498 GAT ATG GGT ATC 1512 AAA CAT GTG ACC AAA ATG CTT AAT GCT AAT TTC CCG CAC AAC ACA CCA AAC ACA GCA TAC TAC TAC 1595 AAG GAA 499 M I F E R F F P Q N S A L T V P G G P T R R V A C S T A 526 1596 AGA ATG ATC TTT GAG CGG TTC TTC CCT CAG TCG GCA AGA CTA ACT GTT CCC GGA GGA CCA ACG GTT GCA TGT AGC ACA AAC GCA 1679 527 K A V E D A A W S N N L D P S G R A A L G V H D S A Y W 554 1680 GCT GTT GAG TGG GAT GCT GCT TGG TCA CTC GAT CCT TCT GGT AGA GCA GCA CTC GGA GTT CAT GAT TCA AAA AAC AAC GCT TAT 1763 555 N H N K V N K T V E F E K I I P L A A P V L A I Q E E E 582 1764 CAT GTC AAC ACT GTA GAG TTT GAG ATT ATA CCA CTG GCC GCT CCT GTC GAG GAA AAC AAC AAA AAA AAG GAA CTT GCC ATC CAG 1847 583 G * 584 GGC TAG CAG CTA TGG GGA ATG ACT GTG CTA TGA AGA TTG ACT 1848 TTT CAA GAA GAA TAA TAA AAA TAA CAT ATA TGA AGA ATT TGC 1931 CTT CTG TTT TTT ATC CGG GGC ACA ATG CTA TAT ATA GAT AM GCT TTA AAT 1932 AAT GAA AAT AAA AAA 2002 AAA AA 2. Nucleotide of cDNAs ASI and AS2. Nucleotide of cDNAs and cAS2 are shown in Fig. sequences encoding pea sequences pcASl (A) (B) the mRNA sense. The deduced amino acid is denoted above the in nucleotide the standard one-letter code. Amino acids are sequence sequence numbered with the first in-frame methionine as 1. The translation termination in codons each clone are as *. starting designated of the AS and human AS of either ASI or AS2 cDNAs. The results shown comparison pea polypeptides sequences reveals an overall of 47% which extends in 4 reveals that in each a homology along Figure digestion only single the entire AS There are several of genomic DNA restriction to each polypeptide. regions high fragment hybridizes probe. local shared between the AS and In the DNA which homology (>80%) pea addition, genomic fragments hybridize human AS acid residues to either ASI or AS2 cDNA are distinct. Similar polypeptides (amino 116-128; probes 218 352 and 486-500 results were obtained with DNA 3' -243; 340-348; -360; 392-401; fragments containing in the ASI In the first four amino of or cAS2 These pea protein). particular, non-coding sequences pcASl (not shown). acids of the human AS which results indicate that contain a for ASI and protein (Met-Cys-Gly-Ile), peas single gene have been shown to be the a glutamine binding site (Heeke distinct single gene for AS2. and are conserved in both the Schuster, 1989), perfectly ASI and AS2 A of between pea proteins. region divergence 'Dark-induced' accumulation of AS 1 mRNA in leaves the AS and human AS occurs at amino acid pea proteins Previous biochemical studies have shown that AS enzyme residues 165-234 of the human AS protein. This stretch activity increases when plants are grown in the dark (Joy of amino acids is not found in either ASI or AS2 To whether this in pea poly- et aL., 1983). address increase AS enzyme and be the result of modification peptide may gene (deletion activity reflects an increase in AS gene expression in the or evolution of versus animal insertion) during plant AS. dark, gene-specific probes derived from 3' non-coding regions of ASI and AS2 cDNAs were used in Northern blot AS 1 and AS2 are encoded by single genes in pea experiments to detect AS mRNAs in leaves of plants grown Southern blot was used to examine the number of under different 5 and ASI mRNA analysis light regimes (Figures 6). ASI and in P.sativum. genes encoding AS2 Pea genomic (2.2 kb) accumulates to high levels in leaves of mature dark- DNA with four restriction was digested enzymes fractionated adapted green plants (Figure 5A, lanes 2 and 3). However, on a 0.7% and Southern blots were when these are to agarose gel probed with plants transferred continuous white light, 32P-labeled cDNA the the levels of fragments containing coding steady-state ASi mRNA decrease dramatically 326 Asparagine synthetase genes in peas 10 50 AS2 MCGILAVLGCSDPSRAKRVRVLELSRRLKHRGPE --- -WSGLHQHGDCYLAQQRLAIVDPA AS1 MCGILAVLGCSDDSQAKRVRILELSRRLKHRGPD --- -WSGLHQHGDNYLAHQRLAIVDPA AShuman MCGIWALFG - SDDCLS - -VQCLS -AMKIAHRGPDAFRFENVNGYTNCCFGFHRLAVVDPL 10 50 AS2 SGDQPLFNEDNPSI -VTVNGEIYNHEDLRKQLSNHTFRTGSDCDVIAHLYEEYG - EDFVD AS1 SGDQPLFNEDKSII -VTVNGEIYNHEELRKQLPNHKFFTQCDCDVIAHLYEEHG - ENFVD AShuman FGMQPIRVKKYPYLWLCYNGEIYNHKKMQQHF- EFEYQTKVDGEII HLYDKGGIEQTIC MLDGIFSFVPLDTRDNSYIVARDAIGVTSLYIGWGLDGSVWISSEMKGLNDDCEHFECFP AS2 ASI MLDGIFSFVLLDTRDNSFIVARDAIGVTSLYIGWGLDGSVWIASELKGLNDECEHFEVFP AShuman MLDGVFAFVLLDTANKKVFLGRDTYGVRPLFKAMTEDGFLAVCSEAKGLVTLKHSATPFL AS2 - ------------- PGHLYSSKDSGFRRWYNPSWYSEA- IPSAPYDPLALRHAFE ------------------- PGHLYSSKEREFRRWYNPPWFNEAIIPSTPYDPLVLRNAFE AS1 AShuman ..... EVLDLKPNGKVASVEMVKYHHCRDVPLHALYDNVEKLFPGFEIETVKNNLRILFN AS2 KAVVKRLKTDVPFGVLLSGGLDSSLVASITSRYLATTKAAEQWGSKLHSFCVGLEGSPDL ASI KAVIKRLMTDVPFGVLLSGGLDSSLVASVTARYLAGTKAAKQWGAKLPSFCVGLKGAPDL AShuman NAVKKRLMTDRRIGCLLSGGLDSSLVA --- -ATLLKQLKEAQV-QYPLQTFAIGMEDSPDL AS2 KAGKEVADYLGTVHHEF-rFTVQDGIDAIEDVIYHVETYDVTSIRASTPMFLMSRKIKSLG KAGKEVADFLGTVHHEFEFTIQDGIDAIEDVIYHTETYDVTTIRAATPMFLMSRKIKSSG ASI LAARKVADHIGSEHYEVLFNSEEGIQALDEVIFSLETYDITTVRASVGMYLISKYIRKNT AShuman 300 350 AS2 VKWVI -SGEGSDEIFGGYLYFHKAPNKEEFHEETCRKIKALHQYDCQRANKSTYAWGLEA AS1 VKWVI SGEGSDEIFGGYLYFHKAPNREEFHQETCRKIKALHRYDCLRANKSTYAWGLEA AShuman DSVVIFSGEGSDELTQGYIYFHKAPSPEKAEEESERLLRELYLFDVLRADRTTAAHGLEL AS2 RVPFLDKAFINVAMNIDPENKMIKRDEGRIEKYILRKAFDDEENPYLPKHILYRQKEQFS RVPFLDKDFIKVAMDIDPEFKMIKHDEGRIEKWILRKAFDDEENPYLPKHILYRQKEQFS ASI RVPFLDHRFFSYYLSLPPEMRIPK- -NG-IEKHLLRETF-- EDSNLIPKEILWRPKEAFS AShuman AS2 DG ---VGYSWIDGLKAHAAKHVTDKMMLNAGNIFPHNTPNTKEAYYYRMIFERFFPQNSA ASI DG ---VGYGWIDGIKDHAAKHVTDRMHFNASHIFPFNTPNTKEAYYYRMIFERFFPQNSA DGITSVKNSWFKILQEYVEHQVDDAMHANAAQKFPFNTPKTKEGYYYRQVFERHYPGRAD AShuman AS2 RLTVPGGPTVACSTAKAVEWDAAWSNNLDPSGRAALGVHDSAYENH-NKVNKTVEFEKII AS1 RLTVPGGPSVACSTEKAIEWDASWSNNLDPSGRAALGVHVSAYEHQINPVTKGVEPEKII AShuman WLSHYWMPKWINATDPSARTLTHYKSAVKA AS2 P-LEAAPVELAIQG PKIGVSPLGVAIQT AS1 3. Amino acid sequence homology of plant AS and human AS polypeptides. The deduced amino acids sequences encoded by pea pcASl, pea Fig. cAS2 and human AS cDNA (pH131) (Andrulis et al., 1987) are compared. Double dots denote identities between pea ASI and pea AS2 or pea ASI and human AS sequences as shown. Dashes in the amino acid sequences represent deletions used to maximize homology of AS proteins. Amino acid 'fasta' (Pearson and Lipman, 1988). is according to computer program alignment to almost undetectable levels (Figure 5A, lanes 4 and 5). respectively). The steady-state levels of ASI mRNA in leaves In both the dark-induced and light-repressed of dark-adapted plants (Figure 5A, lane 3) are 30-fold higher mature plants, accumulation of AS 1 mRNA can be detected 6 h after than the AS1 mRNA levels present in leaves of light-grown changing the light conditions (Figure 5A, lanes 2 and 4 plants (Figure 5A, lane 5). As a control, mRNA for cytosolic 327 F.-Y.Tsai and G.M.Coruzzi ... ....- 5. blot of Fig. Northern mRNA levels in ASI leaves of dark- analysis versus adapted from the 3' light-grown peas. gene-specific probe of was used to detect non-coding region pcASl mRNA ASI Fig. 4. (2.2 Southern blot kb) analysis of AS genes in P.sativum. in Pea nuclear RNA from leaves of dark or treated (D) As a (L) DNA was light pea digested with the plants. following restriction enzymes: S, SstI of control, GS mRNAs were also cytosolic (1.4 detected on the kb) (lanes 1 and 5); E, EcoRI (lanes 2 and 6); B, BamHI (lanes 3 and 7); blots with cDNA GS299 Northern et probe al., H, HindHI (Tingey 1988). (A) (lanes 4 and 8), resolved by gel Total electrophoresis, transferred RNA from leaves of (20 in ytg) continuous white peas to grown nitrocellulose and probed with radioactive probes derived for from the 14 transferred to light (lane the dark for 6 h days 1), or 3 coding region of (lane 2) either pcASI (A) or cAS2 (B). and back to (lane continuous white days 3) for 6 h or (lane light 4) Total (lane 5). RNA from day (B) leaves of (20 24 or 31 Lg) 10, 17, old Controls were in day plants. continuous white GS (1.4 grown for kb) monitored on the 10, same blot revealed light no dramatic 24 or 31 17, lanes ('L', Dark-treated days 1, 3, 5, 7). were changes in mRNA plants levels in response to the light treatments. incontinous white for 21 or grown 28 and light 7, 14, then days AS2 mRNA levels were also transferred to detected on the dark for an replicate blots with additional 3 days ('D', lanes, 2, 4, 6, 8) before a DNA probe from the 3' non-coding of region cAS2. harvesting. These experiments revealed that AS2 mRNA (2.2 kb) is present at much lower levels than AS 1 mRNA in leaves of dark- grown plants and is undetectable in leaves of grown plants (not shown). Northern blots were also performed on RNA isolated from plants at various developmental stages which were grown in continuous white light (Figure 5B, lanes 5 1, 3, and 7) and then transferred to the dark (Figure 5B, lanes 2, 4, 6 and 8). The results of these experiments reveal that the dark- induced accumulation of ASI mRNA occurs in plants of all developmental stages but is most dramatic in mature plants. The dark-induced increase of AS1 mRNA varies from 5-fold in 10 day old plants (Figure SB, compare lanes 1 and 2) to > 20-fold in 31 day old plants (Figure SB, compare lanes and 8). As a control, Northern blots reprobed with a DNA probe encoding a cytosolic form of glutamine synthetase (GS) (Tingey et al., 1988) reveal that the mRNA for cytosolic GS (1.4 kb) is relatively unaffected by the different light treatments (Figure SB, lower band). Phytochrome mediates the 'dark-induced' accumulation 6. of AS1 mRNA blot Northern for Fig. mRNA levels in analysis ASI etiolated pea leaves ASl treated with mRNA different accumulates to Detection of high levels in light regimes. ASI mRNA leaves of etiolated was as described in 5. mRNA for GS seedlings Figure chloroplast was (Figure 6A, lane (GS2) 1) and decreases to almost detected with cDNA GS185 et probe Total (Tingey al., 1988). undetectable levels (A) when plants were transferred to RNA from leaves of (20 in /g) the dark for 7 peas grown days (lane continuous white light (Figure and 6A, lane 2). transferred to the The accumu- 1) for 72 h Total light (lane RNA 2). (B) lation of AS 1 from mRNA (20 leaves of during dark in the dark treatment is Ag) for 9 in direct peas grown (lane and days 1) treated with a contrast to of red the subsequently then back to light-induced pulse the dark accumulation of mRNA light put for the for 3 h treated with a (lane 2), of red followed a form pulse light chloroplast of GS2 by (1.5 kb) (Tingey et pulse al., 1988) in of far-red then back into the dark for 3 h light put or (lane these 3) samples (Figure 6A, lower bands). transferred to the continuous white for 8 h light (lane 4). 328 in Asparagine synthetase genes peas In order to determine whether the plant photoreceptor N x ~~~I phytochrome is involved in mediating the dark-induced expression of AS 1 gene in 1 mRNA peas, AS was examined in etiolated plants treated with light regimes known to activate or inactivate phytochrome (Figure While 1 6B). AS mRNA accumulates to high levels in etiolated plants (Figure 6B, lane 1) the levels of ASI mRNA decrease dramatically in plants treated with a red-light pulse (Figure 6B, lane 2). The repression of ASI expression by red light is partially reversed by a subsequent pulse of far-red light (Figure 6B, lane 3). The effects of red and far-red light pulses on accumulation of AS 1 mRNA were h detected within 3 after light treat- ment. These results show that the dark-induced accumulation of ASl mRNA is at least in mediated, part, through the chromophore phytochrome. AS 1 and AS2 mRNAs accumulate in developmental contexts of increased nitrogen mobilization To determine whether the level of AS mRNA increases in contexts where large amounts of asparagine are synthesized for nitrogen transport, the steady-state levels of ASl and AS2 mRNAs were monitored in nitrogen-fixing root nodules of peas and in cotyledons of germinating pea seedlings (Figure 7). Asparagine serves as a major nitrogen transport amino acid during germination and (Dilworth Dure, 1978; Capdevila and Dure, 1977; Kern and Chrispeels, 1978). The results of the gene-specific Northern blots for AS reveals that both AS1 and AS2 mRNAs accumulate to high levels Northern blot of ASI and AS2 mRNA levels in Fig. analysis (A) of and root in cotyledons germlinating seedlings (B) nitrogen-fixing cotyledons of germinating pea seedlings (Figure 7A). nodules. DNA from 3' the Gene-specific fragments non-coding region While ASI mRNA can be detected after 10 days of of or cAS2 was used to detect the AS I mRNA or pcASlI (2.2 kb) germination (Figure 7A, lane 5, upper panel), AS2 mRNA AS2 mRNA on Northern blots. mRNA (2.2 As a for the kb) control, is detected earlier (4-6 days of germination) (Figure 7A, (3-subunit of mitochondrial ATPase was also detected (2.1 kb) (Boutry and Total RNA extracted from lanes 2 and 3, lower panel). There is a > Chua, 1985). (A) (20 of 20-fold increase ug) cotyledons 2-18 (lanes RNA pea seedlings germinated days 1-8). (B) Poly(A)+ of both ASl and AS2 mRNAs in cotyledons during a isolated from roots of uninfected (lane and (1 1) peas nitrogen- jig) germination time course lanes 2 and (Figure 7A, compare root nodules (lane fixing 2). 7). The same Northern blot with DNA for reprobed probe vitro a cytosolic form of glutamine synthetase et and In this we have (GS) (Tingey al., (Lea Miflin, 1980). paper, directly 1988) reveals that mRNA for cytosolic GS accumulates cloned AS cDNAs a DNA plant using heterologous probe earlier (2-4 days) than either AS mRNA human AS et Two (not shown). encoding (Andrulis al., classes 1987). of AS cDNAs AS mRNA levels were also examined in and that encode but nitrogen-fixing (ASlI AS2) homologous distinct AS were root nodules of pea where asparagine serves as a major obtained from cDNA libraries. proteins pea for The between the I compound nitrogen transport from nodules to the rest AS and A52 cDNAs are homologies pea of 81 and 86% at nucleotide and amino the plant (Scott et al., 1976; Reynolds et al., 1982). RNA acid levels respectively. from cDNAs for and A52 of nitrogen-fixing root nodules and roots of uninfected ASlI are shown to Full-length pea plants were in Northern blot with encode whose sizes and amino probed experiments gene- acid are proteins sequences AS in excellent with that deduced for the human specific probes (Figure 7B). These experiments reveal AS agreement that both ASl and mRNAs accumulate to The AS 1 and A52 AS2 very high cDNAs and human AS protein. pea levels in nodules cDNA share an overall nucleotide of nitrogen-fixing (Figure 7B, lane 2) 50-55% homology compared to uninfected roots lane The their entire that are (Figure 7B, 1). along coding sequence. Regions highly induction of AS1 nmRNA in nodules to roots is conserved between the AS and human AS compared pea polypeptides >80 20-fold while that of AS2 mRNAs is 5-fold. The lower % at amino acid most include only ( level) likely important sites for For fold induction of AS2 mRNA may reflect the basal the first four amino higher enzyme activity. example, levels in acids in the human AS present uninfected roots lane As which (Figure 7B, 1). protein (Met-Cys-Gly-Ile), a the have been shown to be the control, Northern blot was reprobed with a DNA site and probe glutamine-binding for the 13-subunit of for et the mitochondrial ATPase and Heeke (Boutry important enzyme activity (Andrulis al., 1987; which is at levels in roots of and are conserved in both the Chua, 1985) expressed equal Schuster, 1989), perfectly pea uninfected and nodules ASI and A52 The of plants nitrogen-fixing (Figure 7B, proteins. high degree sequence lower panel). between the AS and human AS homology pea proteins the conclusion that the full and ASI A2 supports length cDNAs encode AS of glutamine-dependent peas. Discussion The of two but distinct AS significance homologous While is an in asparagine important nitrogen transport amino polyRpNtides plantris intriging. TheaASirl and A2r acid in the involved in cDNAs of higher plants, enzyme its synthesis pea may encode two distinct subunits of a single is poorly characterized to date due to in AS enzyme instability holoenzyme (heterologous holoenzyme); or each subunit 329 F.-Y.Tsai and G.M.Coruzzi as may assemble into a separate AS holoenzyme (homologous whether it represents a post-transcriptional response or of unknown holoenzyme of either AS1 or AS2 subunits). These two has been shown for another dark-induced gene In direct contrast to the are not mutually exclusive. Partially purified function (Okubara et al., 1988). possibilities of AS1 mRNA in the plant AS enzyme preparations have been shown to utilize dark-induced accumulation leaves, form of glutamine synthetase glutamine as a preferred substrate; however, ammonia can mRNA for the chloroplast in the in a also be used as a substrate in the same preparations albeit (GS2) accumulates light phytochrome-mediated Parallel molecular studies with higher Km values (Scott et al., 1976; Huber and response (Tingey et al., 1988). of binding for dark-induced accumulation of AS1 Streeter, 1984, 1985). The existence glutamine on the mechanisms of the AS I and AS2 proteins accumulation of GS2 mRNA will sites at the amino terminus pea mRNA and light-induced and AS2 genes encode glutamine- encoding nitrogen metabolic implies that both ASI uncover how two genes forms of AS. It is possible, however, that these a common pathway are regulated by light dependent enzymes along are able to utilize ammonia as a substrate in via phytochrome in opposite fashions. AS enzymes revealed levels of under conditions of ammonia excess. It is interesting Previous biochemical studies have high vivo contexts where large to note that glutamine versus ammonia-dependent forms of AS activity in two developmental for AS are encoded by separated genes in E. coli (Felton et al., amounts of asparagine are synthesized nitrogen transport: and Dure, 1980; Humbert and Simoni, 1980) and yeast (Jones, 1978; in cotyledons of germinating seedlings (Capdevila 1978; Kern and Chrispeels, 1978) Ramos and Wiame, 1980). Therefore, we cannot exclude 1977; Dilworth and Dure, root nodules et al., 1976; the possibility that plants might contain another distinct AS and in nitrogen-fixing (Scott Previous Previous studies also showed that gene for ammonia-dependent form of AS. Reynolds et al., 1982). AS activity can D treatment abolished the induction of AS biochemical studies have also shown that actinomycin fractions of in of germinating cotton seedlings, be detected in both soluble and proplastid activity cotyledons of soybean (Boland et al., 1982). that AS in cotyledons is regulated at nitrogen-fixing nodules indicating expression encoded by AS 1 and AS2 cDNAs of pea are level (Capdevila and Dure, 1977; The proteins the transcriptional with those AS since neither of them contains a Dilworth and Dure, 1978). Consistent findings, most likely cytosolic of both ASI and AS2 It is possible that peas contain another distinct we have shown that the accumulation transit peptide. levels in of for plastid AS. mRNAs are induced to high cotyledons gene studies of AS Northern blot analysis has revealed that the steady-state germinating pea seedlings. Comparative mRNAs in this context show that the steady- levels of AS I and AS2 mRNAs parallel asparagine synthesis mRNAs and GS mRNA for GS accumulate earlier in various developmental contexts. For example, previous state levels of cytosolic asparagine is the major both AS mRNAs in of physiological studies have shown that than those of cotyledons germinating amino acid in plants grown in the dark These results that glutamine nitrogen transport seedlings (not shown). suggest and that AS activity can be act as a metabolic signal to induce (Urquhart and Joy, 1981), synthesized by GS may expression in this developmental context. The dark treatment et al., 1983). Here, AS gene enhanced by (Joy mRNAs of AS 1 and AS2 also accumulate to very high levels have demonstrated that in peas the increase of experiments in root nodules of peas in a parallel fashion with cytosolic in the dark is due, at least in part, to an increase AS activity mRNA. The accumulation of AS1 and AS2 mRNA in in levels of AS 1 mRNA. This dark-induced GS the steady-state nitrogen-fixing nodules may be the result of factors produced accumulation of AS1 mRNA occurs in leaves of both the of nodulation or/and by metabolic factor(s) etiolated seedlings and in mature dark-adapted green plants. by process in nodules. ASI mRNA such as ammonia or glutamine production the magnitude of dark-induced Moreover, DNA reveals that the Southern blot analysis of genomic accumulation increases significantly during plant develop- in of at least two genes, both dark-induced and gene family for AS peas is composed ment. Kinetic experiments reveal that 1 encode homologous but distinct gene in mRNA levels can be detected AS and AS2, which light-repressed changes ASI studies show that AS 1 and AS2 genes h (Figure 6B) and mature products. Expression within 3 in etiolated seedlings some similarities in expression patterns (e.g. induced after changing the light/dark conditions. share plants (not shown) of mRNA in and nodules); Thus the dark-induced accumulation of AS 1 mRNA is accumulation cotyledons they have distinct organ-specific patterns of significant for plants grown in a short dark however, physiologically AS1 mRNA accumulates to higher levels in at night). expression. period (e.g. mRNA accumulates of the ASI mRNA leaves to AS2, while AS2 The dark-induced accumulation compared 1 that are negatively to higher levels in roots than AS 1. In this respect, the AS classifies the AS gene with other genes where members as phytochrome (Otto et al., 1984; family resembles the GS gene family regulated by light such gene by distinct and 1988; Kay et al., 1989), proto- of a gene family may be differentially regulated Lissemore Quail, of individual genes in reductase et al., 1985) and an factors which modulate expression chlorophyllide (Mosinger et 1989). unidentified mRNA found in Lemna (Okubara et al., 1988). specific contexts during development (Coruzzi al., at the ASl As shown for (Lissemore and Quail, 1988; Kay Our continuing studies are aimed characterizing phytochrome reductase as well as defining the et 1989) and protochlorophyllide genes and AS2 genes and gene products al., of ASI mRNA DNA elements for differential (Mosinger et al., 1985), the repression cis-acting responsible of the AS 1 and AS2 genes during plant accumulation in the light is a phytochrome-mediated regulation whether the A comparative analysis of the AS and GS gene response. However, it remains to be determined development. of ASI reflects families will elucidate the molecular mechanisms responsible dark-induced (or light-repressed) expression or of coding as has been shown for for the co-ordinate induction repression genes a transcriptional response and Quail, 1988; Kay et al., 1989) for enzymes along a common nitrogen metabolic pathway phytochrome (Lissemore and reductase (Mosinger et al., 1985) in higher plants. protochlorophyllide 330 Asparagine synthetase genes in peas at 55°C for and (94'C for 45 s followed by annealing 1 min elongation and methods Materials at 72°C for 2 min). The amplified cDNAs were precipitated by ethanol, digested with EcoRI and BamHI, then separated on an A agarose gel. Growth of plant material predominant DNA fragment of - 600 bp was recovered from the obtained from Brother Seed agarose Seeds of P.sativum (var. 'Sparkle') Rogers Co. and into the EcoRI and BamHI sites of were imbibed and germinated in a Conviron environmental gel ligated pTZ19U (GenescribeTM). (Twin Falls, ID) The ligated DNA was introduced into E.coli XL1 blue. Clones of illumination of 1000 micro- containing chamber with a day length 16 h, AS2 sequences were isolated and sequenced by the dideoxy method [I = 1 a day/night cycle (Big- einsteins/m2/s einstein (E) mol of photons], at gin et al., 1983). of For etiolated plants, peas were grown for 7-9 days in black 21/18°C. contained in a dark environmental chamber. For germination lucite boxes DNA and RNA analyses seeds were imbibed in water and germinated in vermiculite. Nodules studies, Nuclear DNA from P.sativum was analyzed by Southern blot were isolated from 21 day old pea plants inoculated with Rhizobium analysis according to the method described in Tingey et al. (1987). Briefly, leguminosum strain 128C53 (Nitragin Co., Milwaukee, WI) as described pea nuclear DNA was digested with SstI, EcoRI, BamHI or HindIll, resolved previously (Tingey et al., 1987). by gel electrophoresis, transferred to nitroceilulose and probed with a 1373 bp For phytochrome induction experiments, 9 day old etiolated pea seedlings SstI-BamHI 32P-labeled fragment of pcASl (Figure IA, fragment 'b') or were irradiated with a 4 min pulse of red light (red fluorescent lamps, General a 872 bp BamHI-EcoRI 32P-labeled fragment of cAS2 (Figure IA, 3' end Electric F20T12R) at a fluence of 40 or were given a 4 min pulse jiE/m2/s of cAS2). The genomic Southern blot was performed at high stringency of red light followed by 12 min of far-red light (Westlake, FRF700) at (hybridized at 70°C and washed at 70°C in 0.1 x SSC and 0.1% for 3 h. For white SDS) the same fluence and were then returned to the dark to continuous white light such that cAS 1 and cAS2 cannot cross-hybridize to each other. Northern light treatment, etiolated seedlings were exposed blot analyses of RNA obtained from leaves, roots, nodules or for 8 h. cotyledons of P.sativum were performed according method described to the by Tingey total RNA or poly(A)+ RNA was denatured with Isolation of plant AS cDNAs et al. (1987). Briefly, glyoxal, resolved by gel electrophoresis, tranferred to nitrocellulose and ASI cDNA clones were selected from a Xgtl 1 cDNA library previously probed with a 423 bp BamHI-EcoRI 32P-labeled fragment of constructed from P.sativum (var. 'Sparkle') nodule mRNA (Tingey et al., pcASl (Figure fragment 'c') or a 224 bp HincII-EcoRI 32P-labeled fragment 1987) as follows. Nitrocellulose filters containing denatured phage DNA IA, of cAS2 (Figure 3' end of cAS2) which have sp. act. - 1-2 x 108 corresponding to 250 000 individual plagues were incubated for 4 h at 45°C lB, c.p.m./Iug. The intensities of gene-specific mRNA were determined by in pre-hybridization buffer (6 x SSC, 10 x Denhardt's solution, 0.1% SDS, densitometer. For Northern blot analysis in which poly(A)+ RNA was 1 mM EDTA, 100 denatured salmon sperm DNA). Filters were then Agg/ml used, the induction fold was substracted by the intensities of p3-subunit of incubated for 24 h at 45°C in hybridization buffer (6 x SSC, 5 x Denhardt's solution, 0.1% SDS, 1 EDTA, 50 denatured mitochondrial ATPase mRNA. Sizes of mRNAs were estimated by migration mM jLg/mi relative to denatured DNA markers. salmon sperm DNA) plus 0.2 of 32P-labeled cDNA insert (sp. act. Ag 2 x 108 (1.7 kb HindIII fragment of pH 131) (Andrulis et al., c.p.m.jAg) Filters in 1 x SSC, 0.1% SDS for 15 min at room 1987). were washed followed by 15 min at 45°C and exposed to X-ray film. cDNA temperature, clones to the 5' of AS 1 niRNA were synthesized using corresponding end We thank Dr Irene Andrulis for supplying the human AS cDNA clones, a 40 base oligonucleotide primer complementary to the 5' end of cAS301 and Dr Janice W.Edwards for advice on A-PCR reactions and see Figure 2A). This oligonucleotide was annealed with S helpful (809-848 nt, Ag discussions. This research was NIH Grant GM 32877 and pea nodule poly(A)+ RNA and cDNA synthesis was performed using a supported by DOE DEFGO-289ER-14034. F.-Y.T. is by the Lucille cDNA Synthesis System (Bethesda Research Labs, Gaithersburg, MD). grant supported Charitable Florida. Following second strand synthesis, EcoRI linkers were added and the cDNA P.Markey Trust, Miami, fragments were ligated into Lambda ZAPII vector (Stratagene, La Jolla, CA). A genomic clone for the AS2 gene was identified when a DNA References fragment from the coding region of an AS 1 cDNA was used to screen a pea genomic library (Lycett et al., 1985). AS2 cDNAs were subsequently Chen,J.and Ray,P.N. (1987) Mol. Cell. Biol., 7, 2435-2443. Andrulis,I.L., isolated from a pea root cDNA library constructed in Xgtl 1 (Tingey et al., Back,E., Brukhart,W., Moyer,M., Privalle,L. and Rothstein,S. (1988) Mol. All cDNA of 1 1987). inserts both Xgtl and Lambda ZAPII clones were Gen. 20-26. Genet., 212, initially subcloned into pTZ18U or pTZ19U (GenescribeTM, US Biggin,M.D., Gibson,T.J. and Hong,G.F. (1983) Proc. NatI. Acad. Sci. Biochemical Restriction fragments of each cDNA Corp., Cleveland, OH7. 3963-3965. USA, 80, were then subcloned into Ml3mpl8 or M13mpl9 and the nucleotide Boland,M.J., Hanks,J.F., Reynolds,P.H.S., Blevins,D.G., Tolbert,N.E. determined by the dideoxy method (Biggin et al., 1983). sequence and Schubert,K.R. (1982) Planta, 155., 45-51. The cDNA clones containing the 5' end of AS2 mRNA were amplified Boutry,M. and Chua,N.-H. (1985) EMBO J., 4, 2159-2165. from pea nodule poly(A)+ RNA by anchored polymerase chain reaction Capdevila,A.M. and Dure,L. III (1977) Plant Physiol,,59, 268-273. et 1989). First strand cDNA was synthesized (A-PCR) technique (Loh al., Edwards,J.W., Tingey,S.V., Tsai,F.-Y. and Walker,E.L. Coruzzi,G.M., in a reaction mix containing 50 mM Tris-HCI pH 8.3, 75 mM KCI, 3 mM (1989) In The Molecular Basis ofPlant Development. Alan R. Liss, New MgC12, 50 mM dithiothreitol, 0.5 mM dNTP, 5 ug nodule poly(A)+ 223-232. York, pp. 200 U M-MLV reverse trancriptase (Bethesda Research Labs, RNA, Crawford,N.M., Campbell,W.H. and Davis,R.W. (1986) Proc.Natl. Acad. MD) and 1 oligonucleotide FY13 (5'-GGCCGAATT- Sci. Gaithersburg, USA, 83, 8073-8076. Ag CATACAAATGACCAGGTGGAAAACAC) which includes an EcoRI site Cullimore,J.V., Gebhardt,C., Saarelainen,R., Miflin,B.J., Idler,K.B. and plus sequences complementary to the 5' end of cAS201 (617-641 nt, see J. Barker,R.F. (1984) Mol. Appl. Genet., 2, 589-599. Figure 2B) at 37°C for 1 h. The reaction was stopped by phenol-chloroform and III Dilworth,M.F. Dure,L., (1978) Plant Physiol., 61, 698-702. extraction and the supernatant was passed through Linker 6 Quick SpinTM and 221-228. Felton,J., Michaelis,S. Wright,A. (1980) J. Bacteriol., 142, Columns (Boehringer Mannheim Biochemicals, Indianapolis, IN) to remove and J. Biol. Chem., 264, 5503-5509. Heeke,G.V. Schuster,M. (1989) excess linkers. After ethanol precipitation, the tailing reaction was performed and Plant Physiol., 74, 605-610. Huber,T.A. Streeter,J.G. (1984) according to manufacturer's instructions in 50 of reaction mix containing and Plant Sci., 42, 9-17. 1sl Huber,T.A. Streeter,J.G. (1985) 20 of 1 x 15 of dGTP, TdT buffer and U terminal deoxynucleotidyl Humbert,R. and Simoni,R.D. (1980) J. Bacteriol., 142, 212-220. jsM transferase Research Labs) at 37°C for 30 min. After J. (TdT) (Bethesda Bacteriol., 134, 200-207. Jones,G.E. (1978) -chloroform extraction and ethanol precipitation, the tailed cDNAs and phenol Joy,K.W., Ireland,R.J. Lea,P.J. (1983) Plant Physiol,, 73, 165-168. were redissolved in 20 of water and used as templates in an A-PCR and Plant 11 Kern,R. Chrispeels,M.J. (1978) Physiol., 62, 815-891. reaction. The A-PCR reaction was performed with Taq polymerase (Perkin- Kay,S.A., Keith,B., Shinozaki,K., Chye,M.-L. and Chua,N.-H. (1989) Elmer Norwalk CT) in 100 1l of a buffer containing 5 of the tailed Plant 351-360. Cetus, Cell, 1, cDNAs, 0.1 mM of dNTP, 2.5 sg of AS2 specific primer (FY13) and 2.5 and Proc. R. 13-26. Lea,P.J. Fowden,L. (1975) Soc., 192, Ag mix a 1:9 ratio of of an anchored primer containing AnC primer and In Miflin,B.J. (ed.),7he Biochemistry of Lea,P.J. Miflin,B.J. (1980) and An (5'-CAGGTCGACTCTAGAGGATCCCCCCCCCCCCCCC) Plants, Volume 5: Amino Acids and Derivatives. Academic Press, New A of six 569-604. primer (5'-CAGGTCGACTCTAGAGGATCCC). program cycles York, pp. Lissemore,J.L. and Mol. of low-stringency hybridization and amplification (940C for 45 s followed Quail,P.H. (1988) Cell. Biol., 8, 4840-4850. at for 1 and at 720C for 2 was by annealing 37°C min elongation min) Loh,E.Y., Elliott,J.F., Cwirla,S., Lanier,L.L. and Davis,M.M. (1989) followed 24 of and amplification 217-220. by cycles high-stringency hybridization Science, 243, 331 F.-Y.Tsai and G.M.Coruzzi Lycett,G.W., Croy,R.R.D., Shirsat,A.H., Richards,D.M. and Boulter,D. (1985) Nucleic Acids Res., 13, 6733-6743. Miflin,B.J. (1980) The Biochemistry of Plants, Vol. 5: Amino Acids and Derivatives. Academic Press, New York. 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Urquhart,A.A. and Joy,K.W. (1981) Plant 750-754. Physiol., 68, Vauquelin,L.N. and Robiquet,P.J. (1806) Ann. Chim., 57, 88-93. Received on September 11, 1989; revised on November 21, 1989
Journal
The EMBO Journal – Springer Journals
Published: Feb 1, 1990