Gene, 111 (1992) 223-228 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00

223

GENE 06304

Short Communications

RAmy2A; a

novel 0c-amylase-encoding gene in rice

(Nucleotide sequence; introns; molecular evolution; RNA blot; polymerase chain reaction) Ning Huang, Stephen J. Reinl and Raymond L. Rodriguez Department of Genetics, University of California, Davis, CA 95616 (U.S.A.) Received by R. Wu: 17 September 1991 Revised/Accepted: I I November/12 November 1991 Received at publishers: 27 November 1991

SUMMARY

The structure and expression of the u-amylase-encoding gene, RAmy2A, are described. This only representative of the Amy2 subfamily in rice differs from other cereal ~-amylase-encoding genes in several respects. It contains the largest introns of all the cereal u-amylase-encoding genes ,examined to date. Moreover, the second of three introns in this gene contains a long inverted repeat sequence that can potentially form a large and stable stem-loop structure in the unspliced RNA transcript. Finally, RAmy2A is constitutively expressed at very low levels in germinated seeds, root, etiolated leaves, immature seeds and callus. This is in marked contrast to the Amy2 genes c¢ wheat and barley which are highly expressed in the aleurone layer of the germinated seeds.

INTRODUCTION

u-Amylases in rice, barley and wheat are encoded by multigene families. In wheat, the ~-amylase-encoding genes (~Amy) are classified into three subfamilies with eleven to 14 genes in Amyl, ten to eleven genes in Amy2 and three to four genes in Amy3 (Huttly et al., 1988). In barley, only two subfamilies, Amy1 (high pl, seven genes) and Amy2 (low pI, three to four genes) have been identified (Khl.~rsheed and Rogers, 1988; Knox et al., 1987; Rahmatullah et al., 1989; Whittier et al., 1987). Several rice z/lmy genes have been recently isolated and characterized (Huang et al.,

Correspondenceto: Dr. R.L. Rodriguez, Department of Genetics, University of California, Davis, CA 95616 (U.S.A.) Tel. (916)752-3263; Fax (916) 752-1185. Abbreviations: aa~ amino acid(s); ABA, abscisic acid; Amy (aAmy), ~,amylase gene subfamily: bp, base pair(s)', GA, gibberellic acid; kb, kilc~ base(s) or !~% bp; nt, nucleotide(s); PCR, polymerase chain reaction; RAiny, rice ~rAmygene(s).

1990b; O'Neill et al., 1990). Most of these genes fall into either the Amyl or Amy3 subfamilies (Huang et al., 1990a,b; Sutliff et al., 1991). One gene, however, does not fit into either subfamily based on restriction mapping data and hybridization studies. This gene, which is represented by genomic clone ~OSg9C, has been placed in the hybridization Group 4 (Huang et al., 1990b) and it is the only ~my gene that maps to chromosome 6 in rice (Ranjhan et al., 1991). In this report we describe the characterization of this unique rice ~my gene. On the basis of nt sequence similarity to other cereal u-amylases, this gene appears to belong to the Amy2 subfamily and for this reason it was designated, R~my2A. In spite of its sequence similarity, RAmy2A differs from all other cereal o~tmygenes in a number of ways. The significance of these differences in terms of gene expression and evolution will be discussed. EXPERIMENTAL AND DISCUSSION

(a) Southern-blot analysis of the rice Group-4 ~Amy gene Of the seven overlapping genomic clones making up the hybridization Group 4 (Huang et al., 1990b), three clones,

224

RAmy2Acontains introns of unusual size and structure The most striking of RAmy2A was the unusual size of its introns. Of the 16 cereal 7Amy genes examined in this study, nine had two introns and six had three introns (Table I). Moreover, intron location is fixed in all the cereal ~4my genes (Huang et al., 1990b). By aligning the RAmy2A sequence with two rice ~4my cDNAs, pOS 103 and pOS 137 (O'Neill et al., 1990), RAmy2A was found to contain three introns in the expected positions (Huang et al., 1990b). With the exception of RAmy2A, average intron size ranges from 102 bp in the first position, 227 bp in the second position and 96 bp in the third position. As can be seen in Table I (columns I1, I2 and I3), the sizes of the second and third introns of RAmy2A are unusually large. The second intron, which is 6.6 times larger than the average intron at that position, is the largest yet found in any plant x4my gene. The third intron of RAmy2A is 5.6 times larger than its counterpart in the other cereal ~Amy genes. The presence of large introns in RAmy2A explains the unusual Southern-blot results mentioned above (see section a). Another interesting feature of RAmy2A was the presence of a long inverted repeat sequence beginning at position 1613 and ending at position 1991 ofintron 2 (Fig. 2). This sequence has the potential to form a large and stable stemloop structure in the unspliced RNA transcript. The stem was 170 bp long on each side with 85~o complementarity and a calculated free energy of formation of -134 kcal. The loop of this structure was 39 bp. There was also a 9-bp perfect direct repeat, typical of a transposable element, at the base of the stem-loop structure. A similar transposonlike sequence has been found in the 5'-flanking region of a wheat ~Amy gene (Martienssen and Baulcombe, 1989). There is, however, no homology between these two transposon-like sequences. (c)

AOSg6A, 2OSg9C and AOSg7E, are located within a span of 26 kb of the rice genome (Fig. 1). Attempts to locate the coding region on these clones by Southern-blot analysis indicated that this u,4my gene was about 3.5 kb, or about 75% larger than other cereal ~Amy genes (data not shown). Since 3.5 kb is not large enough to contain two closely linked genes such as RAmy3D and RAmy3E (Huang et al., 1990a), we concluded that this particular ~lmy gene was novel and thus worthy of further investigation. (b) Salient features of the nt sequence of RAmy2A To elucidate the structure of RAmy2A and to understand its evolutionary relationship to other cereal x4my genes, appropriate restriction fragments from genomic clone ,~OSg9C were subcloned into sequencing vectors (Fig. 1). A total of 4128 bp of DNA, which included a 580-bp 5' region and an 88-bp 3' region, were sequenced from both orientations (Fig. 2). The nt sequence information was used to deduce the aa sequence (443 aa) of the protein encoded by this gene and to determine the size and locations of its introns. On the basis of its sequence similarity to Amy2 genes in wheat and barley (see section d), this gene was named RAmy2A. Examination of the 5'-flanking region revealed a consensus TATA box located about 140 bp upstream from the translational start codon. The 5'-flanking region also contained two conserved sequences known as 'amylase boxes' (Huang et al., 1990b) located at nt 280 and 330 in Fig. 2. These boxes have been implicated by others as playing an important role in the transcription of cereal 0~4my genes (J. Yamaguchi, M. Lanahan and T.D. Ho, personal comm.). Finally, two ATG codons, located eight codons apart, were identified at the beginning of the coding sequence for this gene. Comparison of the aa sequence to that of other cereal ~-amylases suggests that the first of these codons is the translational start site.

RAmy2A

T S

R

'

'

I

'

4

R ~SBTX= T S"T I

'-i

"

8

1"2

r

V

/

/ 4kb 9CS4

SI/•

TR

"llil

I

R

"

i

16 ~,OSg6A

20

~" X

5.2kb ~

RTS I

III

24

24.07 kb

XOSg9C ~

X~

I

XOSgTE "'

9CHR ~ 9CRHF

Fig. 1. A compositemap of the RAmy2Alocusin rice. Sevenz,lmygenomiccloneswereisolatedfrom a rice M202genomiclibrary(Huanget al., 1990b) and subjectedto restrictionenzymemapping. The threeclones formingthe maximumcontiguoussequencesspanning this regionof the rice genomeare shown beneath the restrictionmap, The blackenedboxesrepresentthe four exons and the open boxesrepresentthe three introns. The arrow abovethe R.4my2Agoneindicatesthe directionof transcription.Two overlappingfragments(the4-kb Sail fragmentand the 5,2-kbEcoR|-Hindlllfragment)from clone).OSBgCweresubclonedinto FBluescriptfor nt sequencing.The arrowsbelowthe subclonesshowthe directionand the coverage,,~fthe nt sequence analysis. B, BamHi:H, HindllI: R, EcoRh S, Salh T. Sstl'~X, Xbal.

225 ATT~Tc~T~TTAcAG~TGTTTAcTGTAGcATcA~TAGGcT~T~TG~TT~TTAGTcTc~TAcATTcGTcTcAcG~TTAGTcc~c~TATGAATGTcTTTTATT~AcT12o cTAcGTTT~TAT~AT~TTAGTATcc~TTcsATGTGATAc~AGTTA~J~cTc~GTTTTAGT~¢~cT~cGGTTcAT~AGGATAcAAG~TATcAscA~GTGTTGATT

~o

~GTAGGTTCAT~AGG~G~AGT~TAGCAC~TAA~GAGTTGGGTAT~T~TATA~AGCAGTTGT~C~CAA~G~CTCT~CAT~A~T~cAT~G~GG~TCTAAAAAAG~T

360

TCATC~TCCTCCATTTT~GARAAGGAAAG~TT~TC~TTCTGCCCTGCCGCAGCTGCGTCC~ACCCTACACGCGTAGCCATCTTCTGC~TCATCAAAACACcTGT480 • 5'-GCTATAGAGCTAG~TGGCTGCTGCC-3' ~TCATCTTCTG~TT~T~,ATTCAGAGTT~GAGCA~GTGTCCCTGCTTCTGTGCAGA~A~GATC~GCTATAGAGCTAGCCTGGCTGCTGC~TGGC~CCGGACGACGCCT 600 M A T G R R L 7

CTCCATGATCCTcCTCCTCCTcCTTCTC~CTTGGCTTcC~c~C~TTCTCTTC~GGTACG~G~AGCCACG~CTG~cTGAAGTT~GACC~TTTCAGT~AGTTTcTGAA

720 27

S M I L L L L L L G I A S G D K I L F Q 3'-TT~CCCTCAGCACCTCC~CGTTCC-5'

C~GCTGTTGCGTTCTCTT~GGGGTTC~CTGG~GTCGTGG~G~GAGCGGAGGGTGGTACAACCT~T~TGGGGAAGGTCGACGA~TCGTCGCCGCCGGCGT~CGCACGTCTG840 G F N W E S W R O S G G W Y N L L M G K V D D I V A A G V T H V N 6 0 GCTGCCGCCGCCGTCG~CTCCGTCTCCACGCAAGGTCGGTTTCTC~GCTGCTCCTCCTTGTCTGA~A~A~ACT~TGAA~T~TGG~TTGGCGAAATTTGG~TCCTGC~GCGTG

960

L P P P S H S V S T Q

71

GAGT~G~CGCT~CT~TACc~GATTGCG'~G~TGGAT~TGATTGTCACTTTGGCCTTT~TTCATTTGGGTTGr~.TGA~TGTTTCACTGATTTAGTTGAGG~TTTT 1080 TGGCTAGAT~TATCA~CT~TGCGT~GGGTC~TC~TCTTGC~CCCCATTCT~T~TTATACA~TCG~AATA~AAAGGT~GCTGTCAG~AGTTAGAAAAAAA~TT~TGA

1200

TATAT~TTAGTGT~T~TGAGTACTAGT~TT~G~T~T~CAGGA~AAA-~%TG~TTTTTTTT~A~TTT~GAAGT~cT~TATG~A~TTTATTAT~TGTTTTTATTTTCT~ 1320 ~AAC~TP~TA~T~CTGTACTT~CTGTGGGT~CG~A~ACATATC~TTTCT~AACATTTAAACTGGATGTGC~A~AT~TGTGTAAATTT~GTATACTTTATCCGTTCCACTT

1440

CGGGTATTCA~AATA~ATA~CT~GGGTCTATTTGGCAT~CTCTAGCT~AGAA~T~AGCTTCAGGTC~CCTTT~CG~A~TGGA~TCAACCA~AC~TTTTAGCTCCATA

1560

GAA~TTG~G~GATTTTA~A~GTTG~GTT~TTAGGACGTGACATACAC~GAGCGTA~CTATGCACACAC~C~CTC~GTGTACA~CCGTGTACAACA~CTAAAAATTATCACA 1680 AAA~ATTTTAGAAAAATTCATA~T~A~TT~TAGTATTA~T~TA~GTA~AAGTCG~ATCTTCARAT~ATT~TACATA~G~T~CA~AAAA~TA~AATT~GACAAATTTG~8~ C~CCTTA~cT~GATTTTTTTTTGTT~GG~TAAA~T~TG~TTTGA~GTT~GATTTT~A~TGT~TAc~TTGAAAGTA~TGTAT~TTTTTTcTA~TTTT192~

TTGGTGA~TTTT~AGTTGGTGTG~CGTGTGTACACGTGTGGGACTGTGTGCATAG~TATGTTGCCG~CATACACACATGTAGTGAGTATGCAT~GGCATGC~TATTTATATC 2040

TTGGTCTCTACTTTATCTTACATCATTA~TGT~.AA~GTG~TTTCATATcTC~T~C~TTTACcATTATT~TATTTTCGTATCTTTCTCTACR~CACTATGGCT~TTCTATC

2160

TTTTTTTATTC~%T~TGTATGTCc~T~C~cT~TTTTGTTATATATTCA~T~TATCAT~T~G~GcC~`A~CTA~"~u~.CTTAGCcATGTAGTATCCTTTTGGTCTTGGTT2280 TGGTTATGcACCARAACG~GCGATTTTA~GGTTCGTGTA~A~TTGcATC~C~ATCC~TTTAcTCGTTATTC~TGTGGC~TGT~GTG~G~GTA~TGCCTGGGCG~TGTA 2400 G Y M P G R L ¥ 7 9 ~CTTG~TGCGTCTAGGTACGGcACGTCGATG~GTTG~GTCGTT~TCAGCGCG~C~CGG~%GGG~TTCAGGC~TCGCTGACGT~T~T~AA~ACCGCTGCGC~ACTA D L D A S R Y G T S M E L K S L I S A L H G K G I Q A I A D V V I N H R C A D Y I I 9

2520

C~G~CAGC~GCGGC~CTA~TGCAT~TTT~GG~GGCACACCT~CGGC~C~C~CTGGGGCCCCCA~T~TCTGCCGCGATGATACCCAGTTCTCC~CGG~CAGGC~CCT K D S R G I Y C I F E G G T P D G R L D W G P H M I C R D D T Q F S D G T G N L I 5 9

2640

C~ACCGGC~C~CTTCGC~T~C~CATT~~TGGT~GTC~G~G~T~CCGA~GGCT~TCTGGCTC~GTCTGAC~GGTTGG~TCGATGCGTG276~ D T G A D F A A A P . D I D H L N G V V Q R E L T ' D W L L W L K S D E V G F D A W I 9 9

GCGGCTC~CTTC~RAGGGGGTACTCGCcG~G~GGCC~GGTGTAcATT~GGG~C~CG~T~G~TGGC~T~CGGA~T~G~A¢TC~TGG~GTACGGCGGAGACGG28~ R L D F A R G Y S P E V A K V Y

E G T T P V G L A V A E L W D S M A Y G G D G 2 3 9

G~CCG~GTAC~T~G~C~A~cCGG~GGCG~GGTG~CTGGGTG~CAGGGTG~T~CGGC~C~CGGG~T~TGTTC~CTT~C~C~%c~G~T~TG~.CAL3~ K

P

E

Y

N

~

D

A

H

R

Q

A

L

V

D

W

V

D

R

V

G

G

T

A

S

A

G

M

V

F

D

F

T

T

K

G

MNT279

GGCGGTG~GGGG~GCTGTG~CGGCTGATC~C~G~GGGG~AGGCA~G~GGT~TCGGG~GTG~C~CA~AGGCTGT~CCTTCGTCGAC/~CCACGA~CTGGCTC~C~A3~20 A V E G E L W R L I D Q Q G K A

G V I G W W P A K A V T F V D N H D T G S T Q 3 1 9

A~GATGTGGCCATTCCCCTCC~C~GGTCATG~G~CTACGCcTACATCCTCACC~TCCC~CATGCATCGT~GTACTCCTACTACTAC~CCTCTGTTTTT~.TA~T32~0 ~ M W ~ F P S D K V M O G Y A Y L T H P G N P C I 345 GACA~GTCGATTTTTTAT~CATGTTT~TCATTGTCTTATTT-.~u~T~TGT~TTAT~TTTAT~TGTTATG~TTGTTTTATCACTC~u~GTACTTT~GTATGATTTATATG 3360 TTATA~TTTACATA~z~`%TT~T~ATTA~TGGTT~TTGACTCTCC.`'AAT~.CTATT~TTGATTATT~TCA~T~G~GACTAT~;~'TATTTTTTTT~G~CACT ~G3480 GT~TAGcTATATTTGTGAc¢¢TA~GA~TcAGTAGT~`AcATATTTAT~TTATTTGT~TTTTT~T~G"~GT~c~u~GTTT~TATTG~TA~T~Tc~TTT~T36~ GCTATTTATTAGCCGT~TA¢TTTTT~CCCTTATTTA~AGTTGTCTACTAAACTTG~T~TGCA~TGTGTACATAT~TAT~cTCATCA~TCTAAAATTGCAc~T~TG

3720

TGCCTGTATATATGTACATGTA~TCTAC~CCATTTCTT~ATTGGGGGTTA~A~A~A~TCGCGG~CTGGTGG~GTGA~¢~CGGAACGGCGTGACGGCGACGAGCTCGCTCA3840 V y D H F F D W G L K E Q I A A L V A V R Q R N G V T A T S S L K 3 7 6

AGAT~TGCTGCACGACGCCGACGCCTA~TcGccGA~TC~cGGCA~GGT~T~TGRA~TCG~TCCCGCTAcGACGTCTCCAGCCTCATCCCGCCCGGCT~cACC~GCCGCCC I

M

L

H

D

A

D

A

Y

V

A

E

I

D

G

K

V

V

M

K

I

G

S

R

Y

D

V

S

S

L

I

P

P

G

F

H

L

3960 A

ACGGCAAcGGCTACGcCGTCTG~AGRJ~GCGcCGcCGccGCCGCCGATCATCG~ccTcTTc~GcG~TcGCTcT~TTGA~AGcGATATGATGcTTTTGTGccACcTcTTAT~T G N G Y A V W E K S A A A A A D H R T S S S A S L * TTACTTCTGCTGTATA~GAAC~GG~C~CTACCTTCARAGGTAC~

A

H 418

4080 443 4128

Fig. 2. Nucleotide s ~ u e n c e of a rice R A ~ Q A gone. ~ c s~uencJng procedure w a s ~ e same as pr~ous]y described(Huang ¢t al., 1990b). Sequence data w e ~ ~ y z ~ using the S ~ u ~ c e An~ysis Soflw~e Pack~e (Dev~eux et ~.o 1984). The coding ~ o n of R,4.~QA was determined by s~uence ~i~ment to two cDNAs, pOS 103 and pOS 137 (O'Neill et ~., 1990). ~ e aa sequ~ce in single-letter code was d~ueed ~ r the coding re~ons and s h o ~ beneath the nt s~uence. ~ e putative TATA box is indicat~ in bold type ~ d the amylase ~ x e s ~ e underlined. ~ e double underlines indica[e direct repeats which flank the base of the stem-loop structure in intmn 2; the I ~ p is indicat~ by a w a ~ underline. ~ e s~uence, lenFh and location of two oligos u 8 ~ in RNA-PCR ( s ~ Fig. 3) were indicat~ a ~ v e the nt s ~ u ~ c e of exon 1 ~ d exon 2. ~ i s s~uence has been submitted to G~Bank und~ accession No. M741~7.

226 TABLE I Comparisons of intron (I) size and exon (E) sequence identity of RAmy2Ato other ~tAmygenesa Genes

Exon nt identitiesc

lntron sizes (nOb I1

12

I3

El-E4

El

E4

81

1503

544

1.000

1.000

1,000

100

253

96

W-Amy2/54

80 87

233 148

92 90

0.820 0.819 0.783 0.820

0.642 0,667 0.630 0.642

0,779 0.785 0.728 0.760

B-Amv6-4 B-Amy46 B.gKAmyl41 RAmylA W-Amyl/13

80 103 94 108 89

---93 --

106 106 106 84 108

0.748 0.745 0.733 0.750 0.739

0.568 0.556 0.556 0.506 0.506

0.687 0.671 0.667 0.694 0.647

RAmy3A RAmy3B RAmy3C RAmy3D RAmy3E W-Amy3/33

127 76 128 124 143 85

409 ------

91 99 90 88 85 104

0.716 0.692 0.687 0.724 0.714 0.684

0.543 0.519 0.469 0.444 0.457 0.469

0.597 0.561 0.592 0.638 0.628 0.613

RAmy2A B-Amy32b B-CIoneE

BogKAmy155

" The sources of nt sequences are as follows:RAmy2A(this study), Amy32b(Whittier et al., 1987),CloneE (Rogers and Milliman, 1983),gKAmy135and gKAmyl41(Knox et al., 1987),Amy2~54and Amyl/13(generouslyprovided by Dr. D. Baulcombeand C.M. Lazarus), Amy6-4and Amy46(Khursheed and Rogers, 1988), RAmylA (Huang et al., 1990b),RAmy3A,RAmy3Band RAmy3C(Sutliffet al., 1991), RAmy3Dand RAmy3E(Huang et al., 1990a), Amy3/33(Baulcombe et al., 1987). B, barley; W, wheat; R, rice. b Columns I 1, I2 and I3 are intron size of 15 x4mygenes. Note that CloneE is a eDNA. Dashes represent the absence of 12 in most of Amyl and Amy3 genes. Columns 5 to 7 are fractions of nt sequence identity of the RAmy2Agene to the other 15 x4mygenes. Calculations are based on the multiple sequence alignments performedusingthe SequenceAnalysisSoftwarePackage (Devereuxet ah, 1984).To maximizesequenceidentity and to avoid the introduction of arbitrary homology,gaps were inserted between codons only and the length of gaps are multiples of three. In the El-E4 column, the whole coding regions were compared. In columns El and E4, only exon I (El) and exon 4 (E4) were compared.

(d) RAmy2A is similar to the Amy2 genes of barley and wheat To establish the relationship of RAmy2A to other cereal a~Amygenes, both nt and aa sequences were compared, The degree of nt sequence identity in the coding regions o f RAmy2A and 15 other cereal 0~4my genes is shown in Table I (column E l - E 4 ) . The RAmy2A sequence was found to be most similar to the Amy2 genes (78-82%) in barley (Amy37.b, CloneE, and gKAmy155) and wheat (Amy2/54), and moderately similar to the Amy1 genes (around 7 2 75%). RAmy2A was the least similar to the Amy3 genes (68-72%). We found the same relationship between genes when exons 1 and 4 were compared separately (Table I, columns El and E4). Concerted evolution has made exons 2 and 3 so similar that they could not be used in a comparison to distinguish between the various 0~4my genes. Further evidence that RAmy2A is an Amy2 gene comes from a close examination of the aa sequences around aa 280. Rodriguez et al. (1991) have identified a pattern of aa deletions in this region (none for Arny2, one for Amyl and thrc~ for Am~,~) that can be used to classify the cereal z4my

genes into their respective subfamilies. According to this scheme, RAmy2A belongs to the Amy2 subfamily. (e) Low level and constitutive expression of the RAmy2A gene An earlier stud~ using R N A blot analysis was unable to detect RAmy2A m R N A in either germinated rice seeds or seed-derived callus tissue (Simmons et al., 1991). This is in contrast to barley and wheat, where the expression of Amy2 genes in isolated aleurone layers or germinated seeds is very high (Huang et al., 1984; Khursbeed and Rogers, 1988; Lazarus et al., 1985). Here, we investigated RAmy2A expression using the more sensitive technique o f R N A - P C R (Huang et al., 1990a). Totai RNA, isolated from five different rice tissues, was reverse-transcribed into e D N A and amplified with primers that flank intron 1 (Fig. 2). If RAmy2A was transcribed and correctly spliced, a P C R product of 139 bp should be produced. Any genomic D N A and/or unsplieed R N A that might be present in the R N A P C R reaction would generate a product o f 220 bp. These two products can be easily distinguished by polyacrylamide

227 gel electrophoresis. Both the 139-bp and 220-bp products were observed in all five R N A - P C R reactions (Fig. 3). This result provided evidence for RAmy2A expression in these tissues. It is, however, difficult to compare the relative levels of RAmy2A expression using this approach. Since RAmy2A transcripts could not be detected by R N A blot analysis (Simmons et al., 1991), we concluded that expression of this gene in rice is very low. The amplification of the 220-bp band in all five reactions indicated the presence of unspliced R N A and/or trace amounts o f genomic D N A in the R N A samples. The faint band that is visible between the 139-bp and 220-bp bands is probably due to

~.

w w ,

mere i n ~ ,-.

-220 -139

Fig. 3. Expression of a RAmy2Agene as detected by RNA-PCR. Total RNA was isolated from six-day-oldgerminated rice seeds (lane 1), roots (lane 2), etiolated young leaves (lane 3), two-month-old callus 0ane 5) and panicles ten days after flowering(lane 4), followinga procedure described by Kurrer et aL (1991). Lane 6 is RAmy2Aplasmid DNA amplified as a control. Methods. Primers were synthesized on a Cyclone Plus DNA Synthesizer (MilliGen/Biosearch). The sequence, length and location of the two primers used for RNA-PCR are shown in Fig. 2. The 3' primer was previously used as a common primer for other rice ¢,lmygenes (Huang et al., 1990a). The 5' primer is specific for RAmy2A.The primers were designed to flank the first intron so that the predicted RNA-PCR products could be distinguished from products amplified from unspliced RNA and genomic DNA (see section e). RNA-PCR amplification followed the procedure described in Huang et al. (1990a) with some modification to combine cDNA synthesis and cDNA amplificationinto a single step. The RNA-PCR reaction was performed in a TwinBlock (Ericomp) thermal cycler. The amplification reaction of 50 #l consisted of 0.5/~g of total RNA/1.5 units of reversetranscriptase/10 units of RNasin (Promega, Madison, WI)/0.4 #M each of two primers/0.2/~g per #l bovine serum albumin/1 mM each dNTP/1 unit of Taqpolymerase (US Biochemicals) in Taqpolymerase buffer [50 mM Tris pH 8.8/5 mM MgCI2/5mM KCI/ 15 mM (NH4)2SO4I.The reaction was allowed to run at 42 °C for 15 min, 50°C for 10 min to synthesize cDNA by reverse transcriptase. The same reaction was then allowed to run for 30 cycles ofcDNA amplificationwith DNA denaturation at 94°C for I min, primer annealing at 60°C for ! min and primer extension at 72°C for 2 min. The reaction was stopped after a final primer extension for 5 rain at 72°C. The PCR products were analyzed on a 7.5% polyacrylamide gel run in I x TAE buffer (40 mM Tris" acetate/I mM EDTA). The PCR products were visualized by both ethidiam bromide staining (not shown) and by Southern-blot analysis in which the separated PCR products were electro-blotted onto nylon membrane and probed with RAmy2A,labelled with [~.32p] dCTP using the ofigo-labellingprocedure (Feinberg and Vogelstein, 1983).

heterodimer o f c D N A (139 bp) and genomic D N A (220 bp) formed during P C R cycles.

(f) Evolutionary relationship of Amy2genes in rice, barley and wheat In spite of their similarity at the D N A and protein levels, RAmy2A differs from the other Amy2 genes in barley and wheat in four important ways. First, unlike wheat (Huttiy et al., 1988) and barley (Khursheed and Rogers, 1988) which have multiple Amy2 genes, RAmy2A is the sole representative of this subfamily in rice. Why the,4my2 subfamily in rice has been restricted to only one gene is not known. Second, due to its large introns, RAmy2A is almost twice the size of any barley or wheat Amy2 genes published so far. Third, RAmy2A was found to be constitutively expressed at very low levels in all tissues examined. Although low-level expression of Amy2 genes can be detected in immature wheat seeds and barley leaves, high-level expression of these genes can be detected in the aleurone layer of germinated wheat (Huttly et al., 1988) and barley (Jacobsen et al., 1986) seeds. Fourth, unlike the barley and wheat Amy2 genes which are stimulated by G A and inhibited by ABA (Huang et al., 1984; Huttly et al., 1988; Khursheed and Rogers, 1988; Nolan and He, 1988), RAmy2A expression appears to be refractory to these two hormones (E.E. Karrer and R.L.R., unpublished resUlts). In the case of the wheat Amy2/46 gene (Huttiy et al., 1988), expression is so low that its response to G A and ABA is difficult to measure. We believe these differences reflect the balance of mutations and natural selection during the course of Amy2 gene evolution. The number ofgenes in the Amy2 subfamily and their level of expression may reflect the way these species have adapted to their respective environments. These differences also raise many important questions regarding the role of RAmy2A in rice. Future studies to determine if, when and where the product of RAmy2A is expressed will require isozyme-specific analysis. The ability to express rice 0t-amylase genes in a yeast expression/ secretion system (Kumagai et al., 1990) should facilitate the studies. With an abundant source of pure RAmy2A isozyme, it should be possible to raise isozyme-specific antibodies and locate sites of RAmy2A expression using immunohistochemical methods.

ACKNOWLEDGEMENTS We would fike to acknowledge M. Cadle for her expert technical assistance, J. Litts and J. Chandler for the excellent advice and very helpful discussion they provided during the course of this project. We thank Drs. D. Baulcombe and C.M. Lazarus for providing us with two wheat ~rny nt sequences: Amy1~13and Amy2~54used in the compari-

228

son shown in Table I. We also express our appreciation to Joanne Brice for her critical reading of this manuscript. This research was supported by USDA-CRGO grant 8837262-4019 and California Competitive Technology grant C88-157.

REFERENCES Baulcombe, D.C., Huttly, A.K., Martienssen, R.A., Barker, R.F. and Jarvis, M.G.: A novel wheat '-amylase gene ('.Amy3). Mol. Gen. Genct. 209 (1987) 33-40. Devcreux, J., Haebcrii, P. and Smithies, O.: A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12 (1984) 387-395. Feinbcrg, A.P. and Vogclstcin, B.: A technique for radiolabeling DNA restriction cndonuclease fragments to high specific activity. Anal. Biochem. 132 (1983) 6-13. Huang, J., Sweglc, M., Dandekar, A.M. and Muthukrishnan, S.: Expression and regulation of a-amylase gene family in barley alcurones. J. Mol. Appl. Genet. 2 (1984) 579-588. Huang, N., Koizumi, N., Reinl, S. and Rodriguez, R.U: Structural organization and differentialexpression of rice a-amylase genes. Nucleic Acids Res. 18 (1990a) 7007-7014. Huang, N., Sutliff, T.D., Litts, J.C. and Rodriguez, R.L.: Classification and characterization of rice a-amylase multigene family. Plant Mol. Biol. 14 (1990b) 655-668. Huttly, A.K., Marticnssen, R.A. and Baulcombc, D.C.: Sequence heterogeneity and differential expression of the '-Amy2 gene family in wheat. Mol. Gen. Genet. 214 (1988) 232-240. Jacobsen, J.V., Hanson, A.D, and Chandler, P.C.: Water stress enhances expression of an '-amylase gene in barley leaves. Plant Physiol 80 (1986) 350-359. Karrer, E.E., Litts, J.C, and Rodriguez, R.L.: Differential expression of a-amylase genes in germinating rice and barley seeds. Plant Mol. Bio. 16 0991) 797-805. Kharsheed, B. and Rogers, J.C.: Barley ~-amylasc genes. Quantitative comparison of steady.state mRNA levels from individual members of the two different families expressed in aleurone cells. J. Biol. Chem. 263 (1988) 18953-18960.

Knox, C,A.P., Sonthayanon, B., Chandra, G.R. and Muthukrishnan, S.: Structure and organization of two divergent ~,-amylase genes from barley. Plant Mol. Biol. 9 (1987) 3-17. Kumagai, M.H., Shah, M., Terashima, M., Vrkljan, Z., Whitaker, J.R. and Rodfiguez, R.L.: Expression and secretion of rice a-amylase by Saccharomyces cerevisiae. Gcne 94 (1990) 209-216. Lazarus, C.M., Baulcombe, D.C. and Martienssen, R.A.: a-Amylase genes of wheat are two multigenc families which are differentially expressed. Plant Mol. Biol. 5 (1985) 13-24. Marticnssen, R.A. and Baulcombe, D.C.: An unusual wheat insertion sequence (WI S 1) lies upstream of an alpha-amylase gene in hcxaploid wheat, and carries a 'minisatellite' array. Mol. Gen. Genet. 217 (1989) 401-410. Nolan, R.C. and He, T.H.D.: Hormonal regulation of gene expression in barley aleurnne layers. Planta 174 (1988) 551-560. O'Neill, S.D., Kumagai, M.H., Majumdar, A., Huang, N., Sutliff, T.D. and Rodriguez, R.L.: The ,,-amylase genes in Oryza sativa: characterization of eDNA clones and mRNA expression during seed germination. Mol. Gun. Genet. 221 (1990) 235-244. Rahmatullah, R.J., Huang, J., Clark, K.L., Reeck, G.R., Chandra, G.R. and Muthukrishnan, S.: Nuclcotidv and predicted amino acid sequences of two different genes for high-pl '-amylase from barley. Plant Mol. Biol. 12 (1989) 119-121. Ranjhan, S., Litts, J.C., Foolad, M.R. and Rodrigucz, R.L.: Chromosomal localization and genomicorganization of ~-amylas¢ genesin rice (Oryza sativa L.). Thcor. Appl. Genet. 82 (1991) 481-488, Rodriguvz, R.L., Huang, N., Sutliff, T.D., Ranjhan, S., Karrer, E. and Litts, J.: Organization, structure and expression of the rice ,,-amylase multigene family. Second International Rice Genetic Symposium, Manila, 1991, pp, 417-429. Rogers, LC. and Milliman, C.: Isolation and sequence analysis of barley •"-amylase eDNA clone. J. Biol. Chem. 258 (1983) 8169-8174. Simmons, C.R., Huang, N., Cao, Y. and Rodriguez, R.L.: Synthesis and secretion of "-amylase by rice callus: evidence for differential gone expression, Biotechnol. Bioeng. 38 (1991) 545-551. Sutliff,T.D. Huang, N., Litts, J.C. and Rodrigucz, R.L.: Characterization of an '-amylase multigenc cluster in rice. Plant Mol. Biol. 16 (1991) 579-591. Whittier, R.F., Dian, D.A. and Rogers, LC.: Nucleotide sequence analysis of a-amylase and thiol proteasc genes that are hormonally regulated in barley aleurone cells. Nucleic Acids Res. 15 (1987) 25152535.