Plant Molecular Biology 14: 1031-1040, 1990. © 1990 Kluwer Academic Publishers. Printed in Belgium. 1031

The b-32 protein from maize endosperm: characterization of genomic sequences encoding two alternative central domains

Hans Hartings, 1 Nadia Lazzaroni, 1 Paolo Ajmone M a r s a n , 1 Anna Aragay, 2 Richard Thompson, 3 Francesco Salamini, 3 Natale Di F o n z o , 1 Jaume P a l a u 4 and Mario Motto 1. Istituto Sperimentale per la Cerealicoltura, Sezione di Bergamo, Via Stezzano 24, 24100 Bergano, Italy

(*author for correspondence); 2 Centre d'lnvestigacio i Desenvolupament, CSIC, Carrer Girona Salgado 18, E-08034 Barcelona, Spain; 3 Max-Planck-Institut far Ziichtungsforschung, D-5000 KOln, Federal Republic of Germany; 4 Present address: Dept. Enginyeria quimica i Bioquimica, Univ. Barcelona, Imperial Tarraco 1, E-43005 Tarragona, Spain

Received 29 December 1989; accepted in revised form 26 February 1990

Key words: b-32 protein, genomic cloning, protein domain, zein regulation

Abstract

As derived from a cDNA clone, the structure of the b-32 protein of Zea mays, a putative regulatory factor of zein expression, has a central acidic region separated by two domains covered by secondary structure motifs. In this work, three b-32 genomic clones were selected from two genomic libraries obtained from the maize inbred lines W64A and A69Y. The nucleotide sequences of the complete coding region of each b-32 gene, as well as long stretches of their 5' and 3' flanking regions, were determined. Introns are not present in the b-32 genomic sequences. Minor variations among the three genes and an earlier reported b-32 cDNA indicates that they constitute a gene family showing a characteristic polymorphism. Such a polymorphism is highly evident in large segments of the upstream regulatory sequences. Interestingly, when compared with cDNA (W64A) or with gene b-32.120 (W64A), the genes b-32.129 (W64A) and b-32.152 (A69Y) show three jumps of the reading frame in the central part of the coding region, resulting in a completely different sequence of the b-32 protein central domain. In all cases, variations in the N- and C-terminal domains account only for microheterogeneity.

Introduction

The protein b-32 of maize endosperm has been described as a putative regulatory factor for the synthesis of zeins, the major group of storage proteins [5, 14]. Several reported findings [14] point in this direction: a) there is both a temporal and quantitative coordination between the syn- thesis of b-32 protein and the accumulation of storage proteins in the endosperm tissue; and b) the 02 and 06 mutants induce a concomitant

decrease in the production of zein polypeptides and of protein b-32. This is in agreement with the observation that the endosperm tissue of these mutants contains a very low level of mRNA cod- ing for both polypeptide species [5, 6].

Recently, the cDNA coding for the b-32 protein has been cloned and the complete amino acid sequence of the protein derived [6]. From struc- tural analysis of the b-32 protein one can predict the existence of one acidic central domain and two terminal domains full of secondary structure

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motifs, that would fold up giving rise to a globular shape [6].

It has been postulated [ 15] that the b-32 pro- tein could be involved in a multiple step regulatory process of the maize endosperm tissue during development. In such a process, zein genes would occur at the end of a cascade regulatory pathway. In order to get an insight into this possibility, we have cloned and sequenced three b-32 genes from the inbred lines W64A and A69Y. Our results indicate that the b-32 genes form a gene family showing polymorphism. Sequence heterogeneity affecting the whole central domain of the protein, as well as variations at the 5' flanking region between genes of the two inbred lines, afford evidence for a complex role of b-32 genes and b-32 products within a regulated regulatory pathway.

formed as described by Yamamoto etal. [16]. Southern analysis was performed essentially as described by Maniatis etal. [10], using [~-3Zp]dCTP nick-translated DNA as described in the Nick Translation Manual by Amersham International.

Construction of a A69 Y genomic library

Lambda EMBL4 Bam HI arms were prepared as described by Frischhauf et aL [7]. DNA of the inbred line A69Y was partially digested with Barn HI and restriction fragments ranging in size from 12 to 20 kb were purified on a 10-40~ sucrose gradient [10], ligated into the lambda arms and plated on Escherichia coli K803.

Materials and methods

Enzymes and chemicals

Restriction enzymes were obtained from Bethesda Research Laboratories; sequenase en- zyme was purchased from United States Biochemical; the nick translation kit was ob- tained from Amersham International. All en- zymes were used as indicated by the manu- facturer.

Deoxynucleotides and dideoxynucleotides were obtained from PL Biochemicals; sequence primer and reverse sequence primer were pur- chased from Amersham International; [ct- 35S]dATP 1000 mCi and [~-32p]dCTP 3000 mCi were from Amersham International.

The phagemids pGEM3Zf( + ) and pGEM3Zf( - ), helper phages R408 and M 13K07 as well as bacterial strains JM109 and NM522 were obtained from Promega Biolabs.

DNA preparation and Southern blot

Genomic DNA was prepared as described in Marotta et al. [ 11 ]. Purification of bacteriophage lambda and extraction of phage DNA was per-

Selection of clones containing the b-32 protein

A genomic library of the inbred line W64A (a kind gift from Dr A. Viotti), cloned, after partial di- gestion with Sau 3A, in the lambda vector EMBL4, was screened using the full-length cDNA clone b-32.66 [6] as a probe. From 30 positive clones, 3 clones were further purified and partially identified using a number of restriction endonucleases. Two of these clones (b-32.120 and b-32.129) contained two Hind III-Hind III fragments that hybridized with the cDNA probe. From both clones a 2 kb Hind III-Hind III frag- ment (hybridizing to the 5' end of the b-32.66 clone) and a 650 bp Hind III-BglII fragment (hybridizing to the 3' end of the b-32.66 clone) were subcloned into the phagemid pGEMZf( + ) and restriction fragments were prepared for se- quence analysis.

Upon screening the A69Y genomic library, 3 clones with different insert lengths were further purified. Restriction analysis indicated that the three clones contained the same central Barn HI- Barn HI fragment flanked by a variable number of Bam HI-Barn HI fragments differing in size. From one of these clones (b-32.152) two Hind III-HindIII fragments (sized 1.6 and 0.6 kb), both hybridizing with the cDNA clone

b-32.66, were subcloned into the phagemid pGEM3Zf( + ).

DNA sequence analys~

Dideoxy chain termination sequencing was per- formed using the phagemids pGEM3Zf( + ) and p G E M 3 Z f ( - ) in combination with the Se- quenase (modified T7 D N A polymerase) enzyme. About 8 0 ~ of all sequence data was obtained sequencing both strands, while the complete se- quence was obtained by sequencing at least two identical clones from each subcloning. Electro- phoresis was performed using 5~o acrylamide wedge-shaped gels of 40 cm length.

Fragments to be sequenced were subcloned in one of the p G E M 3 Z f vectors. Single colonies were grown in 2 ml of LB containing 50 #g/ml of ampicillin for 16 h at 37 °C and the presence of the insert was tested extracting plasmid DNA on 1.5 ml of the culture as described by Maniatis etaL [10]. 20 #1 of the culture of the positive clones were added to 1 ml of LB containing 50 #g/ml of ampicillin and grown at 37 °C until the OD6o o reached 0.2; then 10 #1 of helper phage R408 or M13K07 were added (m.o.i. = 20). After the cells were vigorously shaken for 8 h at 37 ° C, they were removed by centrifugation at 13000 rpm for 10 min in an Eppendorfcentrifuge. 100 #1 of supernatant was added together with 50 #1 of NM522 (overnight culture) to 3 ml of LB and grown for 16 h at 37 °C with vigorous shaking. Cells were removed by centrifugation at 13 000 rpm for 10 min in an Eppendorfcentrifuge. Phagemid particles were precipitated on ice for 30min by adding 0.25 volume of a 2.5 M NaCl/20~o polyethylene glycol (PEG6000) solu- tion to the supernatant. The phagemid particles were then collected by centrifugation at 13 000 rpm for 10 min in an Eppendorf centrifuge and resuspended in 500/zl ofTE. D N A was puri- fied extracting three times with an equal volume of a mixture of phenol/chloroform/isoamyl alco- hol (25 : 24 : 1) and precipitated on ice for 30 min by adding 0.1 volume of 3 M Na acetate pH 5.6 and 600 ~tl of isopropanol. Then the DNA was

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collected by centrifugation at 13000rpm for 15 min in an Eppendorf centrifuge, washed with 600/~1 of isopropanol, dried and resuspended in 25 #1 of TE. The method yielded 3-5 #g of very clean single strand DNA, 600-700 ng of which were used directly for the sequence reactions.

Results

Identification of b-32 genomic clones

Upon screening of a maize W64A genomic library with the c D N A clone b-32.66, corresponding to a full-length b-32 sequence, 30 positive clones were selected out of which 3 were further con- sidered. Two recombinant phages, coded as b-32.120 and b-32.129, contained a Hind lII- Hind III and a Hind III-BgllI fragments, hy- bridizing with both ends of the b-32.66 cDNA. These fragments were analyzed in detail, using a number of different restriction enzymes.

Fig. 1. Southern gel blotting analysis of inbred lines W64A (Lane 1) and A69Y (Lane 2). 8 #g of genomic DNA was digested with Barn HI, electrophoresed on a 0.8% agarose gel, transferred to nitrocellulose and hybridized to nick-

translated b-32.66 DNA.

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120: AGCTTG•TTAGTCTTGT•CAAGATTACAAGGGACTGACT•G•CC•AA•TATTGGGGAT•GGAAGAACCAT•CTTTT•TGGGAAGATATGTGGAATAAGGGGCAC•C GGCTTCACTCAAT -1169

129: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . -1167

120: AT~TGAGTTATT~TTCTTTG~TTGTAACAG~AAA~TTAC~GT~AAAGAAGCTAT~CAAAAGGAA~AA~TTATTGAAAATTTTCAG~TT~TCTGTCGGTG~AAG~TATGAA~C -1049

129: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -1047

120: l~TGA~TTAGA~GCAAc~TGGGGG~AAATCATGGTAG~CAACA~AAATGATG~TTGGAAA~T~ATTTGGGGAG~TGATAATTT~TCTAcAAAGAAAACTTATAGA~ATTT~TGGGT~AGG - 929

129: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 927

120: CT~AGGTT~ACTAGAT~TT~AGATCG~Tl~TGGAAAAATAAGTGTCAAC~AAAA~ATAAGGTTTTTTTGGCTTTGG~TAAAGAA~AGA~T~AATA~AAGAGATATGCTGAGGAG~C - 809

129: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 807

120: ATGAAT•TAGAGT•TTA•A•TTGTGAGAATTG•AT•TGG•AAAAGGAGGAAAC••TTTAT•AT•TTTT•CT•AAGTGTAATTAT•GCGAAGG•TTGCTGGAATTCTATTGGTATGACGCC - 689

129: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 687

152: AGCTTCGGAAGAGGTTC - 690

120: ••CAAGAATTA•TMTC•TGTGGA•G•TG•AGTAAAT•TAATG•AG•AG•T•AATGTTT•CTTCT•TATGGAAATTGT•ATT•T•ATGACCTGGAGCATTTGGAAATGTCGTAATG•CTG - 569

129: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 567

152: GT~AT~TTGCT~AAGATGAC~AGAAC~GAAGCCTC~G~G~AGAGAG~l-F~AT~AGGAAGGCTT~TACAATGAGGA~GACTGGTATGAGGATTTCCATCATGGAAA~GC~C~T~ - 570

120: GCTTTTT~AGGATAAGGA~AACAGTG~AG~ATTGTAAAAATGAGTTTG~AAAGGAATTATAT~TGGT~A~GCTTAGAG~TAAAGGACGATT~GATT~GACAATAC~l~TGGC~CA - 449

- 447 129 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

152: TAG~TrTTATTAAGAGGTTAGAAATTAGTGGGATGACATAACTTTTAGGG~TAATTTGAGAGCTACAAAAC~GGAGGGATTGGAGGGGCTA~TCCCT~C~A~C~TA~A - 450

120: GCATTGGCAGTAC~TCTCGTCAA 11 I I ~C~TTTTGTAATTTTGTAATTTATAATTTACTTTC~TGTATG~TTcTAA~T~TGT~GTTTTAAAATTTTAATAAAATTTCAGTAGGGGC~G - 329

- 327 129: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

152: GCCTCTrTAATCC~TTTCGGl-FTTGTGGCTCC~AAACTAG~cCTTAGTGGGAGATGATAATGGATAGT~AGTGGAAGATGATGTGGATGGCTTGC~TcA~TGG~C~ - 330

120: ~CT~TGT~TATAAAAAAAAGTTGA~GTGAATAGATTT~ATTAAGAGGTTGGATGTTAGTGGGATGA~ATGA~TATTAGTAGGA~GAGATGATGTG~G~AGT~T~TA - 209

- 207 129: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

152: AGTTAGTAATTGTGTTTAGCG...A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . - 210

120: TGGATGAGTTTTTGCTTTCATCGAAA GGI-FGGAAGGTTAGTATGATGACATGGCTAATATAGATACATAGAT ATAGACTACCAACATGGCTGCATGCCCCCAAGCTCTCCCACTA - 94

- 94 129: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

152: . . . . . . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ATAG . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - 93

120: TATATAT~T~TGGTAGCA~AT~AT~CAATT~A~AATG~TTA~AAAAACA~ATCCAT~ATTl-rAGCTGTTGATAcAAAAGAG~TAGAAGGGAGATG~GC~GAGACAAAT~AGAGTTGAGT + 27

129: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + 27

152: . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . + 27

120: GATC TTATGGCGCAAA~AAA~AAA~TA~TACCAAAGTTcA~TGAAAT~TTCC~GTGGAGGA~GTGAA~TAC~l-rA~AGCGCCTTCAT~GCGTCGGT~cGGAAAGA~GTGAT~ + 144

+ 144 129: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

152: . . . . TTA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + 147

120: AAAcACTGCAcCGACCATAAAGGGAT•TT•CAGcccGTGCTGcCA•CGGAGAAGAAGGTACcc•AGCTGTGGTTcTA•ACGGAGCTCAAAACTAGGA•CAG•T•cATCACGCTCG•CAl•A + 264

+ 264 129: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

+ 267 152: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1035

120: CGCATGGACAACCTGTACCTCGTGGGC'I-[CAGGACCCCGGGCGGGGTGTGGTGGGAGTT GGCAAGGCCGGCC~ACACCCACCTCCTCGGCGACAACCCCAGGTGGCTCGGCTTCGGCGGC + 383

129: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + 3 8 4

152: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + 387

120: AGGTACCAGGACCTCATCGGCAACAAGGGTCTGGAGACCGTCACCATGG6CCGCGC GAGATGACCAGGGCCGTCAACGACCTGGCGAAGAAGAAGAAGATGGCGACACTGGAGC.~GGAG + 502

129: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + 504

1 5 2 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + 5 0 7

120: GAGGTGCAGATGCAGATGCAGATGCCGGAGGC G•TGAA•TGG•GGCGGCGGCGGCGG•GGCTGACCCACAGGCCGACACGAAGAGCAAGCTGGTGAAGCTGGTGGTCATGGTGTGCGAG + 621

129: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : . . . . . . . . . . . . . . . . . . . . . . . . . + 624

152: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . T . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + 624

120: G••CTGC•GTTCAACACCTT•TCCC•CACGGTGGAC•C•G••TTCAACAGcCA•CAC•••GT•ACCTT•ACC•T6ACGCAGGG•AA•CAG•T•CA•AA•TGG•ACAGGATCTcC'AAGGC• + 741

129: . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + 744

152: . . . . . . . . . . . . . . . . . . G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . + 744

120: GCCTrCGAGTGGGCCGACCACC~CACCGCTGTC.~ATC~CCGACATGCAGAAGCTTGGCATCAAGGATAAGAACGAAGCAGCGAGGAT~GTTGCGCTCGTT~TC~CTACTGC~T + 861

129: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + 864

152: . . . . . . . . . . . . . . T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + 858

120: GCCGCTGCCGCTACTGCTGCCAGTGCTGACAACGACGATGACGAGGCCTGA. TCA T CGACACATCATGATCTGCTGCTGCACTTAA TATG'I-i'CGTATACAAATAAATACACC + 972

129: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + 979

152: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~ . . C . . . . . . . . . . . . . . . A.GCAA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TI'AC . . . . . . . . . . . . . . . . . . . . . . . . . . + 978

120: CCGCGTACG GTGTT~CTTATATAAATTTTA~A~TACATA.`~TAAAATAAAATAAT~]~] ]AAAAAAT~TTAAGTTTAG~AACTAAA~TTTAGACAAGA~T~CCATA +1091

129: . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . +1098

152: .G . . . . . . . C . . . . . . . . . . . . . GGTCTAAAATGTAGCCAGTAAATTTTAAACTAC••T••AT•-[C1-rGAGGCTCTATAAACAATAGATTTAAAGTGTTATGTAAAGTTTAGACAACCTTAT +1098

120: CGAAGTTTTGAATCCGAGAAGAGAATAATGAC~'AATATAAA~CAGGATCGAGTTGCCAT~CT~TT~CAATT~TA~GTAG~MTTG~C~GC~TC~CCTCG~ +1211

129: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . +1218

152: ATAAATTTTAGACTACATAAATA +1121

120: AGCTTGTCCATGCACATAGAAACGCATGTAGGCCATATAAGTAGTC GTTGAAT/L~GCAAATACTGCATCTGTTCCAAAATAAAATCTGTi"GTACCiII]IATACTAGA +1319

129: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ATATAAGTAGTC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . +1338

120: TTCATACAATAGTrAATGTATGTGTTATATGT~TAGGTTCATAATCATC~ATATAAATATGGATATAAAAATCTAGAGATAAAGTGACAATTAT~-rTAAGACGGATGGAG~TAC +1439

1 2 9 : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . + 1 4 5 8

120: ATACCTA +1446

129: . . . . . . . +1465

Fig. 2. Alignment of three b-32 DNA sequences relative to clone b-32.120. CATAGA and TATA boxes as well as start (ATG), stop (TGA) and polyadenilation signals are underlined. The three one base insertions found in clones b-32.129 and b-32.152 are

marked with an asterisk.

1036

Upon screening of a A69Y genomic library with the cDNA clone b-32.66 a number of positive clones carried a central Bam HI restriction frag- ment, hybridizing with the cDNA probe. Two Hind III fragments of clone b-32.152 hybridizing with the cDNA probe were subcloned into the phagemid pGEM3Zf( + ).

Southern blot analysis showed that at least three copies of the gene are present in the W64A genome on three Bam HI restriction fragments of 5.5, 7.0 and 7.5 kb, and in A69Y on two Bam HI restriction fragments of 7.5 and 11 kb(Figure 1). The 11 kb A69Y Barn HI fragment corresponds in size to a similar fragment in clone b-32.152. Correspondence of other fragments, genomic and cloned, was not conclusive.

Nucleotide sequence of the b-32.120 gene

Suitable restriction fragments were subcloned into the phagemids p G E M 3 Z f ( + ) and p G E M 3 Z f ( - ) and sequenced according to the dideoxy chain termination method of Sanger et al. [ 13 ], using the Sequenase enzyme. The complete nucleotide sequence of the b-32.120 gene as well as its 5' and 3' flanking regions is presented in Fig. 2.

A comparison with the sequence of the cDNA clone b-32.66 [6] revealed the absence of introns in the genomic clone. The translatable sequences of the cDNA clone and of the genomic clone can be perfectly aligned with the exception of 1 nu- cleotide at position + 805 (Fig. 2). This nucleo- tide substitution implies an amino acid change. In addition, a complete homology is also observable for the 5' untranslated region of the transcript as far as it is present in the cDNA clone. However, a number of differences can be observed com- paring the 3' untranslated region of both clones.

Several sequences related to the putative con- trol elements discussed by Breathnach and Chambon [3] and by Joshi [8] are found in the 5' flanking region of the b-32.120 gene. A TATA box typical for plant genes (TATATATAT) is found at position - 95. Upstream from the TATA box, at position - 143, a motif (CATAGA) resembling

both the CAAT or AGGA box consensus sequences [1, 12] was found. The transcription start point of the gene has been located at position - 4 2 and the observed sequence motif coincides

with that for the genes corresponding to maize storage proteins 28 kDa G-2 [1] and pML1 [2]. In addition, this sequence is in good agreement with the consensus sequence for the transcription start reported for plant genes [ 8 ]. As described for the b-32.66 cDNA clone, one conventional poly- adenylation signal is present in the 3' region of the gene [ 6 ].

Finally, notable is a sequence element (TCT- TATGGC/GCAAACAAACAAAAAAA/TAG- TAC) found in the coding region from + 30 to + 61 that displays a high degree of homology (92 ~o) with a motif relevant to the control of the replication rate of DNA [9]. Whether this se- quence constitutes a real control element within the maize genome is a matter deserving further research.

Nucleotide sequence of the b-32.129 and b-32.152 genes

The experimental procedure for sequencing was the same as that in the case of clone b-32.120.

The b-32.129 gene shows overall homology with b-32.120 (Fig. 2), the most outstanding variations being three one-nucleotide insertions within the coding region at positions + 324, + 440 and + 535, as well as several short deletions and one insertion at different positions of the 3' flanking region. An interesting feature of the in- sertions in the coding region is the fact that they cause three jumps of the reading frame, thus changing the polypeptide sequence (of 72 residues) in the central region of the protein.

A comparison of the amino acid sequence of the central domain corresponding to both reading frames is presented in Fig. 3. As reported earlier [6], the sequence of the central domain derived from clones b-32.66 (cDNA) and b-32.120 (genomic) is highly hydrophilic and very rich in acidic residues. The sequence of the central domain of b-32 protein corresponding to the ge-

1037

120: Met Ala Glu Thr Asn Pro Glu Leu Ser Asp Leu Met Ala Gin Thr Asn Lys Lys I le Val Pro Lys Phe Thr Glu I le Phe Pro Val 29

129: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

152: . . . . . . . . . . . . . . . . . . . . . Ser . . . . . . . . . I le . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

120:

129:

152:

Glu Asp Val Ash Tyr Pro Tyr Ser Ala Phe l le Ala Ser Val Arg Lys Asp Val l le Lys His Cys Thr Asp His Lys Gly l le Phe Gin 59

59

60

120: Pro Val Leu Pro Pro Glu Lys Lys Val Pro Glu Leu Trp Phe Tyr Thr Glu Leu Lys Thr Arg Thr Ser $er l le Thr Leu Ala l le Arg 89

129: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . . . . . . . . . . . . . . . 89

152: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

120: Met Asp Asn leu Tyr Leu Val Gly Phe Arg Thr Pro Gly Gly Val Trp Trp Glu Leu Ala Arg Pro Ala Thr Pro Thr Ser Ser Ala Thr 119

129: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phe Gly Lys . . . Gly Asp . . . His Leu Leu Gly I18

152: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phe Gly Lys . . . Gly Asp . . . His Leu Leu Gly 119

120: Thr Pro Gly Gly Ser Ala Ser Ala Ala Gly Thr Arg Thr Ser Set Ala Thr Arg Val Trp Arg Pro Set Pro Trp Ala Ala Arg Asp Asp 149

129: Asp ASh Pro Arg Trp Leu Gly Phe Gly . . . Arg Tyr Gin Asp Leu I le Gly Ash Lys Gly Leu Glu Thr Val Thr Met Gly . . . Ala Glu 148

152: Asp Asn Pro Arg Trp Leu Gly Phe Gly . . . Arg Tyr Gin Asp Leu l le Gly Ash Lys Gly Leu Glu Thr Val Thr Met Gly . . . Ala Glu 149

120: Gin Gly Arg Gin Arg Pro Gly Glu Glu Glu Glu Asp Gly Asp Thr Gly Gly Gly Gly Gly Ala Asp Ala Asp Ala Asp Ala Gly 177

129: Met Thr ... Ala Val Asn Asp Leu Ala Lys Us Lys Lys Met Ala'... Leu Glu Glu Glu Glu Val Gln Met Gin Met Gin Met Pro Glu 178

152: Met Thr ... Ala Val Asn Asp Leu Ala Lys Lys Lys Lys Met Ala ... Leu Glu Glu Glu Glu Val Gin Met Gin Met lln Met Pro Glu 179

120: Gly Ala Glu Leu Ala Ala Ala Ala Ala Ala Ala Asp Pro Gin Ala Asp Thr Lys Ser Lys Leu Val m.ys Leu Val Val Met Val Cys Glu 207

129: Ala . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208

208 152: Ala . . . Asp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

120: Gly Leu Arg Phe Ash Thr Leu Set Arg Thr Val Asp Ala Gly Phe Asn Set Gin His Gly Val Thr Leu Thr Val Thr Gin Gly Lys Gin 237

129: . . . . . . . . . . . . . . . . . . Val . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238

152: . . . . . . . . . . . . . . . . . . Val . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238

120: Val Gin Lys Trp Asp Arg l le Ser Lys Ala Ala Phe Glu Trp Ala Asp His Pro Thr Ala Val l le Pro Asp Met Gln Lys Leu Gly l le 267

129: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268

152: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268

120: Lys Asp Lys Asn Glu Ala Ala Arg I le Va] Ala Leu Val Lys Asn Gin Thr Thr Ala Ala Ala Ala Ala Ala Thr Ala Ala Set AIa Asp 297

129: . . . . . . . . . . . . . . . . . . . . . . . . Leu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298

152: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296

120: Ash Asp Asp Asp G1u A1a 303

129: . . . . . . . . . . . . . . . . . . 304

152: . . . . . . . . . . . . . . . . . . 302

Fig. 3. Protein alignment obtained from the coding regions of the three b-32 clones relative to clone b-32.120. N-terminal and C-terminal domains are conserved, whereas central domains are varied.

1038

-1282

120

FIR AT-rich PIR

I+L -142 -95 -42 CAT TATA $ AT6

IZ~

129 ' ' I

T T C

-707 -309

I I '' I C C C divergent s e q u e l a e i i i

152 +324 +441 +537

I::=

Fig. 4. Structure of clones b-32.120, b-32.129 and b-32.152. The position of the CAT and TATA boxes, of the putative start of transcription (S) and the respective distances relative to the translation start (ATG) are indicated. Clones b-32.120 and b-32.129 furthermore establish a far inverted repeat (FIR), a proximal inverted repeat (PIR), and a 82 bp long poly(dA/dT) rich region, indicated by diagonally striped boxes, all absent in clone b-32.152. The three 1-base insertions in clones b-32.129 and b-32.152 are indicated below the respective coding regions together with their positions relative to the translation start of clone b-32.120.

The altered central domain in the b-32.129 and b-32.152 coding regions is indicated by a filled box.

nomic clones b-32.129 and b-32.152 is also hy- drophilic, but substantially less acidic and more basic in comparison with the central domain de- rived from the other two genes. Such variations in sequence must correlate with variations in secon- dary structure motifs. In this sense, predictions using a standard procedure [4] show for clones b-32.66 [6] and b-32.120 the presence of a short a-helix flanking the C-terminal part of the central domain. However, according to secondary struc- ture predictions for clones b-32.129 and b-32.152, the a-helix would extend about twenty residues more going into the N-terminal direction of the polypeptide chain. This would confer a higher rigidity to the central part of the b-32 polypeptide molecule for b-32.129 and b-32.152 clones, in comparison with that for clones b-32.66 and b-32.120.

The b-32.152 gene from the inbred line A69Y shows several duplications and a long inverted repeat upstream from the CATAGA motif sug- gesting, in comparison with the two W64A genes, an extensive polymorphic divergence of upstream regulatory sequences (Fig. 4). However, the coding region of the b-32.152 gene resembles that of the b-32.129 gene in the fact that it shows three one-nucleotide insertions at the same relative positions (Fig. 2 and 3).

Complexity of the b-32 gene family

As shown by the results of Southern analysis, at least three Bam HI restriction fragments hy- bridize with the cDNA clone b-32.66, thus indi- cating the presence of more than one b-32 gene. The small differences encountered among the cDNA and the genomic sequences are an indica- tion of the existence of a number of closely related b-32 genes.

Discussion

The cloning and sequencing of three b-32 genes from two different inbred lines, as well as the Southern analysis, demonstrate that the b-32 genes form a polymorphic gene family that, in the case of W64A, is constituted by at least three genes.

As a general feature, the coding region of the two isolated W64A genes and the previously re- ported cDNA are extremely similar and most of the observed nucleotide variations account for an amino acid replacement (in some cases, conserva- tive) or for an insertion/deletion of 1-2 residues within the amino acid sequence. In principle, these variations should not substantially alter the

tertiary structure of the b-32 protein. However, the genes b-32.129 (W64A) and b-32.152 (A69Y) differ from the b-32.120 gene (W64A) and b-32.66 cDNA clone (W64A) in three 1-nucleotide in- sertions, occuring in the central part of the coding region. The reading frame changes three times within 219 nucleotides and, as a result, the trans- lation into the central domain of the b-32 protein changes markedly from one to the other set of clones. This contrasts with the preservation in all cases of the N-terminal and C-terminal domains.

All b-32 genes described in this paper appear to possess characteristics typical of functional genes. The two b-32 genes isolated from the inbred line W64A are very similar with regard to the flanking sequences and they possess the same motifs that apparently are relevant for gene ex- pression. In addition to the typical elements that participate in the activation of the transcriptional machinery (CATAGA and TATA boxes), a long first ATG codon. Two additional motifs are present, corresponding to a stretch of DNA rich in A + T is present at about 410 bp upstream of the proximal inverted repeat (PIR in Fig. 4) and to a far inverted repeat (FIR in Fig. 4), flanking the A + T-rich element.

A comparison between gene b-32.129 (W64A) and gene b-32.152 (A69Y) is of special interest. From the coding sequence they could be con- sidered as polymorphic genes from two different inbred lines showing a high level of homology. However, the 5' flanking region upstream from the CATAGA motif shows great divergence, since the b-32.152 gene possesses several dupli- cations and long inverted repeats, when com- pared with the b-32.129 gene. At present, we have no further sequence of the 5' upstream region of the b-32.152 gene. However, the present data indicate that an important polymorphic variation exists between the two genes and may suggest a different regulatory mechanism for each case.

Based on genetic evidence, the b-32 protein was credited with a positive regulatory role in zein deposition [14, 5]. The results presented still do not exclude the possibility that the b-32 family consists of genes that may play a role as inter- mediate elements of an unknown regulatory

1039

chain. The fact that the mutants 02 and 06 control the expression of both b-32 and zein genes, makes it interesting to investigate a possible mechanism of zein gene regulation in which the b-32 genes and their protein products might be involved.

It would be of great interest to determine if both b-32 protein messages are present and active (giving rise to two differentiated albumin com- ponents) during maize endosperm development. Potential on-and-off regulation of each message at different times during development could result in a clue to the role of this protein in the expres- sion of storage protein genes and the control that the Opaque-2 locus exerts on them. We have carried out preliminary experiments (chemical cleavage of Tyr and Met residues) on b-32 protein from mature endosperm (40 days after polli- nation). Peptides were recovered, electrophores- ed, blotted on nitrocellulose paper and detected by an antibody reaction. Our results indicate that the b-32 component present in mature endo- sperm cells derives most, if not all, from clone b-32.120. Further research should be done in order to demonstrate the expression of clone b-32.129 as well as a possible timing in the appearance and translation of the two postulated mRNA species.

Acknowledgements

This work was supported by EEC contract BAP- 0214-I(A) within the framework of the Biotech- nology Action Programme and by the Ministero dell'Agficoltura e delle Foreste, Italy, special grant 'Tecnologie Avanzate in Agricoltura'. J.P. acknowledges a NATO fellowship for a sabbati- cal stay at the Istituto Sperimentale per la Cereali- coltura, Sezione di Bergamo.

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