cDNA and promoter sequences for MCM3 homologues from maize, and protein localization in cycling cells

A partial cDNA from maize, ROA, encoding a protein homologous to the MCM3 family of essential factors for the initiation of DNA replication, has been isolated previously. In the present work, a longer version of the original ROA cDNA, encoding a full-length protein, was isolated and termed ZmROA1. In addition, three other closely related cDNAs, ZmROA2, ZmROA3 and ZmROA4, were also isolated. ZmROA2 end ZmROA3 appear to encode full-length proteins, whereas ZmROA4 a partial polypeptide. Two clusters of basic amino acids comprising putative nuclear localization signals were identified in the N-terminal domain of these proteins, together with a potential leucine zipper. Immunofluorescence studies on cycling meristematic root-tip cells revealed that these proteins are localized in the nucleus throughout interphase with a pattern overlapping that of chromatin. However, as chromatin condenses at prophase, ZmROA proteins become increasingly distinct from chromatin and appear totally dissociated from the segregating chromosomes during mitosis. This behaviour is consistent with a role in restricting DNA replication to only one round in each cell cycle. A 4.7 kb genomic sequence was also isolated, comprising part of the ZmROA2 gene, with approximately 3.2 kb of promoter sequence, and 600 bp of 5' sequence of the ZmROA2 cDNA, including the first four exons and three introns. Several putative regulatory elements were identified in the promoter sequence. This is the first report on promoter and cDNA sequences encoding full-length MCM3 homologues from higher plants and the distribution of such proteins during the plant cell cycle.Key words: Cell cycle, DNA replication licensing factor, MCM proteins, plant.      

Expression of nutritionally well-balanced protein, AmA1, inSaccharomyces cerevisiae

Food yeast.Saccharomyces cerevisiae, is a safe organism with a long history of use for the production of biomass rich in high quality proteins and vitamins. AmA1, a seed storage albumin fromAmaranthus hypochondriacus, has a well-balanced amino acid composition and high levels of essential amino acids and offers the possibility of further improving food and animal feed additives. In order to find an effective means of expressingAmA1 in yeast, the gene was cloned into an episomal shuttle vector. Four different promoters were tested: the glyceraldehyde-3-phosphate dehydrogenase promoter, galactose dehydrogenase 10 promoter, alcohol dehydrogenase II promoter, and a hybrid ADH2-GPD promoter. The recombinantAmA1 genes were then introduced into the yeastSaccharomyces cerevisiae 2805. Northern and Western blot analyses of the yeast under appropriate conditions revealed thatAmA1 was expressed by all four promoters at varying levels. An enzyme-linked immunosorbent assay demonstrated that the amount of AmA1 protein in the recombinant yeast was 1.3–4.3% of the total soluble proteins. The highest expression level was obtained from the hybrid ADH2-GPD promoter.     

Sequence of a cDNA encoding nitrite reductase from the tree Betula pendula and identification of conserved protein regions

Summary The sequence of an mRNA encoding nitrite reductase (NiR, EC 1.7.7.1.) from the tree Betula pendula was determined. A cDNA library constructed from leaf poly(A)⁺ mRNA was screened with an oligonucleotide probe deduced from NiR sequences from spinach and maize. A 2.5 kb cDNA was isolated that hybridized to an mRNA, the steady-state level of which increased markedly upon induction with nitrate. The nucleotide sequence of the cDNA contains a reading frame encoding a protein of 583 amino acids that reveals 79% identity with NiR from spinach. The transit peptide of the NiR precursor from birch was determined to be 22 amino acids in size by sequence comparison with NiR from spinach and maize and is the shortest transit peptide reported so far. A graphical evaluation of identities found in the NiR sequence alignment revealed nine well conserved sections each exceeding ten amino acids in size. Sequence comparisons with related redox proteins identified essential residues involved in cofactor binding. A putative binding site for ferredoxin was found in the N-terminal half of the protein.These sequence data appear in the EMBL/GenBank/DDBJ nucleotide sequence data bases under the accession number X60093     

Pea dehydrins: identification,characterisation and expression

An antiserum raised against dehydrin from maize (Zea mays) recognised several polypeptides in extracts of pea (Pisum sativum) cotyledons. A cDNA expression library was prepared from mRNA of developing cotyledons, screened with the antiserum and positive clones were purified and characterised. The nucleotide sequence of one such clone, pPsB12, contained an open reading frame which would encode a polypeptide with regions of significant amino acid sequence similarity to dehydrins from other plant species.The deduced amino acid sequence of the pea dehydrin encoded by B12 is 197 amino acids in length, has a high glycine content (25.9%), lacks tryptophan and is highly hydrophilic. The polypeptide has an estimated molecular mass of 20.4 kDa and pI=6.4. An in vitro synthesised product from the clone comigrates with one of the in vivo proteins recognised by the antiserum.A comparison of the pea dehydrin sequence with sequences from other species revealed conserved amino acid regions: an N-terminal DEYGNP and a lysine-rich block (KIKEKLPG), both of which are present in two copies. Unexpectedly, pea dehydrin lacks a stretch of serine residues which is conserved in other dehydrins.B12 mRNA and dehydrin proteins accumulated in dehydration-stressed seedlings, associated with elevated levels of endogenous abscisic acid (ABA). Applied ABA induced expression of dehydrins in unstressed seedlings. Dehydrin expression was rapidly reversed when seedlings were removed from the stress or from treatment with ABA and placed in water.During pea cotyledon development, dehydrin mRNA and proteins accumulated in mid to late embryogenesis. Dehydrin proteins were some of the most actively synthesised at about the time of maximum fresh weight and represent about 2% of protein in mature cotyledons.     

Characterization of a maize G-box binding factor that is induced by hypoxia

G-box cis-acting DNA sequence elements are present in the promoter region of a number of signal-inducible plant genes. In many cases this motif is essential for gene expression. Maize nuclear extracts contain a protein complex that binds specifically to the G-box sequence. Previously, a protein called GF14 was described that is physically associated with the G-box binding complex, but is not a DNA-binding factor in and of itself. This paper reports the isolation of a cDNA encoding a maize G-box binding factor (GBF). The deduced amino acid sequence indicates that maize GBF1 is a basic region-leucine zipper protein. GBF1 binds to the G-box element with specificity similar to that of the binding activity in nuclear extracts. Furthermore, maize GBF1 and the factor detected in nuclear extract are identical in their molecular weight and are immunologically related. GBF1 mRNA accumulates rapidly in hypoxically induced maize cells prior to the increase in Adh1 mRNA levels. Taken together with results that indicate that GBF1 binds to the hypoxia-responsive promoter of maize Adh1, these observations suggest that GBF1 may be one of the factors involved in the activation of Adh1.     

Direct genetic selection of a maize cDNA for dihydrodipicolinate synthase in an Escherichia coli dapA- auxotroph

Summary Dihydrodipicolinate synthase (DHPS; EC 4.2.1.52) is the first committed enzyme in the lysine branch of the aspartate-derived amino acid biosynthesis pathway and is common to bacteria and plants. Due to feedback inhibition by lysine, DHPS serves in a regulatory role for this pathway in plant metabolism. To elucidate the molecular genetic characteristics of DHPS, we isolated a putative full-length cDNA clone for maize DHPS by direct genetic selection in an Escherichia coli dapA ^–auxotroph. The maize DHPS activity expressed in the complemented E. coli auxotroph showed the lysine inhibition characteristics of purified maize DHPS, indicating that the cDNA encoded sequences for both the catalytic function and regulatory properties of the enzyme. The N-terminal amino acid sequence of purified maize DHPS was determined by direct sequencing and showed homology to a sequence within the cDNA, indicating that the clone contained the entire coding region for a mature polypeptide of 326 amino acids plus a 54 amino acid transit peptide sequence. The molecular weight of 35854, predicted from the deduced amino acid sequence, was similar to the 38 000 M_r determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for the purified enzyme from maize. DHPS mRNAs complementary to the cDNA were detected in RNA isolated from developing maize endosperm and embryo tissues. Southern blots indicated the presence of more than one genomic sequence homologous to DHPS per haploid maize genome.     

Molecular cloning and expression of the hot pepper ERabp1 gene encoding auxin-binding protein

The 22 kDa auxin-binding proteins in higher plants have received considerable attention as candidates for an auxin receptor. A cDNA clone Ca-ERabp1 of hot pepper (Capsicum annum) was isolated using the oligonucleotides as PCR primers. The cDNA codes for a polypeptide related to the major 22 kDa auxin-binding protein from maize and Arabidopsis ERabp1. The deduced amino acid sequence contains an endoplasmic reticulum retention signal, the KDEL sequence located at the C-terminal end, and has two possible auxin-binding sites, HRHSCE and YDDWSVPHTA conserved sequences. Northern hybridization analysis revealed that the Ca-ERabp1 gene is differentially expressed in total RNA isolated from different organs of a pepper plant, showing the highest level of expression in fruits but barely detectable in leaves and roots.     

Expression of an engineered acidic-subunit 11S globulin of amaranth carrying the antihypertensive peptides VY,in transgenic tomato fruits

The acidic-subunit of amarantin, main seed storage protein of Amaranthus hypochondriacus, carrying four antihypertensive biopeptides Val-Tyr was expressed in the fruit of transgenic tomato plants. Immunoblot analyses indicate that the expressed recombinant protein was stably accumulated at levels up to 12.71 % with respect to total protein content of transgenic fruits. There was a remarkable change in total protein content (5–22 % increase) of transgenic tomato fruits compared to non-transformed samples. Specific increases of the essential amino acids valine (31–40 %), tyrosine (29–34 %), isoleucine (21–31 %), leucine (28–31 %) and phenylalanine (28–29 %) were also detected in some transgenic lines versus wild type lines. Protein hydrolysates from transgenic tomato fruits showed in vitro inhibition of the angiotensin converting enzyme, with IC₅₀ values that ranged from 0.376 to 3.241 μg ml^?1; this represents an increase of up to 13-fold in the inhibitory activity compared with the protein hydrolysates of non-transformed fruits. These results suggest the possible application of transgenic tomato fruit for massive production of this engineered version of amarantin, which could be especially useful in the prevention and control of hypertension.     

Molecular Cloning of a Phosphoenolpyruvate Carboxylase cDNA from Tropical Epiphytic CAM Orchid

A full-length cDNA encoding phosphoenolpyruvate carboxylase (PEPC) was isolated from tropical epiphytic CAM orchid Mokara Yellow. The cDNA designated as Mpepc1 is 3 450 bp in length with an open reading frame of 2 862 bp encoding 954 amino acids. The deduced amino acid sequence of Mpepc1 shows 83 % identity with pepc2 of sorghum, 82 % with pepc1 and pepc2 of maize and 81 % with pepc of Arabidopsis thaliana. RT-PCR analysis showed that Mpepc1 was expressed in mature leaves, immature leaves, and aerial roots of M. Yellow. No expression was detected in the flower.     

Isolation and characterisation of a maize cDNA that complements a 1-acyl sn-glycerol-3-phosphate acyltransferase mutant of Escherichia coli and encodes a protein which has similarities to other acyltransferases

We selected cDNA plasmid clones that corrected the temperature-sensitive phenotype of Escherichia coli strain JC201, which is deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity. A plasmid-based maize endosperm cDNA library was used for complementation and a plasmid that enabled the cells to grow at 44°C on ampicillin was isolated. Addition of this plasmid (pMAT1) to JC201 restored 1-acyl-sn-glycerol-3-phosphate acyltransferase activity to the cells. Total phospholipid labelling showed that the substrate for the enzyme, lysophosphatidic acid, accumulated in JC201 and was further metabolised to phosphatidylethanolamine in complemented cells. Membranes isolated from such cells were able to convert lysophosphatidic acid to phosphatidic acid in acyltransferase assays. The cDNA insert of pMAT1 contains one long open reading frame of 374 amino acids which encodes a protein of relative molecular weight 42 543. The sequence of this protein is most similar to SLC1, which is thought to be able to acylate glycerol at the sn-2 position during synthesis of inositol-containing lipids. Homologies between the SLC1 protein, the 1-acyl-sn-glycerol-3-phosphate acyltransferase of E. coli (PlsC) and the maize ORF were found with blocks of conserved amino acids, whose spacing was conserved between the three proteins, identifiable.     

Overexpression of serine acetyltransferase in maize leaves increases seed‐specific methionine‐rich zeins

Maize kernels do not contain enough of the essential sulphur‐amino acid methionine (Met) to serve as a complete diet for animals, even though maize has the genetic capacity to store Met in kernels. Prior studies indicated that the availability of the sulphur (S)‐amino acids may limit their incorporation into seed storage proteins. Serine acetyltransferase (SAT) is a key control point for S‐assimilation leading to Cys and Met biosynthesis, and SAT overexpression is known to enhance S‐assimilation without negative impact on plant growth. Therefore, we overexpressed Arabidopsis thaliana AtSAT1 in maize under control of the leaf bundle sheath cell‐specific rbcS1 promoter to determine the impact on seed storage protein expression. The transgenic events exhibited up to 12‐fold higher SAT activity without negative impact on growth. S‐assimilation was increased in the leaves of SAT overexpressing plants, followed by higher levels of storage protein mRNA and storage proteins, particularly the 10‐kDa δ‐zein, during endosperm development. This zein is known to impact the level of Met stored in kernels. The elite event with the highest expression of AtSAT1 showed 1.40‐fold increase in kernel Met. When fed to chickens, transgenic AtSAT1 kernels significantly increased growth rate compared with the parent maize line. The result demonstrates the efficacy of increasing maize nutritional value by SAT overexpression without apparent yield loss. Maternal overexpression of SAT in vegetative tissues was necessary for high‐Met zein accumulation. Moreover, SAT overcomes the shortage of S‐amino acids that limits the expression and accumulation of high‐Met zeins during kernel development.     

One-step purification and structural characterization of a recombinant His-tag 11S globulin expressed in transgenic tobacco

Amarantin, an 11S globulin, is one of the most important storage proteins of amaranth seeds, with relevant nutritional-functional and nutraceutical characteristics. Its cDNA was cloned in-frame with a sequence encoding a polyhistidine tag and expressed under the direction of a 35S promoter in transgenic tobacco seeds. The presence of a (His)(6) tag on the polypeptide permitted a high-yield single-step purification using immobilized metal-ion affinity chromatography and rapid characterization. Purified His-tag amarantin accounted for up to 5% of total soluble seed protein. Biochemical characterization indicated that purified His-tag amarantin migrated with the expected molecular weight (53 kDa) and was correctly processed into an acidic polypeptide (32 kDa) with isoelectric point (pI) of 5.58 and a basic polypeptide (21 kDa) with pI of 9.24, linked by a disulfide bridge. Moreover, His-tag amarantin was assembled into both homo- and hetero-hexameric 11S structures. These results show that the His tag did not change the biochemical and physicochemical properties of amarantin. The strategy presented here for rapid and high-yield expression and purification procedure should facilitate structure-function studies for this nutritional protein.     

Isolation of two novel myb-like genes from Arabidopsis and studies on the DNA-binding properties of their products

Two novel myb-like genes (atmyb6 and atmyb7) were isolated from an Arabidopsis thaliana cDNA library. The entire proteins or the Myb domains encoded by the genes were expressed as fusion proteins in Escherichia coli. The DNA-binding domain of the murine c-Myb was also expressed in the same way for use in comparative studies. The fusion proteins were examined for their DNA-binding activity using the animal c-Myb DNA-binding site (MBS) and the binding site of the maize P gene product (PBS). The Myb domain of Atmyb6 bound to PBS more efficiently than to MBS. Complete Atmyb6 and Atmyb7 proteins preferentially bound to PBS but not MBS. This suggests that the in vitro binding consensus sequences for both Atmyb6 and Atmyb7 are similar to PBS. The binding of the Myb domain of Atmyb6 to both PBS and MBS raises the possibility that the protein recognizes multiple sequences in vivo. The third α-helix and three adjacent amino acids in the third repeat (R3) of c-Myb were replaced with the analogous sequence of Atmyb6 to create a chimeric Myb protein. This chimeric protein bound to PBS with a low affinity but failed to bind to MBS. Thus the binding pattern of the chimeric Myb protein is similar to that of the Atmyb6. This result suggests that the last 20 amino acids in the R3 repeat of Atmyb6 play a major role in DNA-binding.     

SUC1 and SUC2: two sucrose transporters from Arabidopsis thaliana; expression and characterization in baker's yeast and identification of the histidine-tagged protein

An important, most likely essential step for the long distance transport of sucrose in higher plants is the energy-dependent, uncoupler-sensitive loading into phloem cells via a sucrose-H⁺ symporter. This paper describes functional expression in Saccharomyces cerevisiae of two cDNAs encoding energy-dependent sucrose transporters from the plasma membrane of Arabidopsis thaliana, SUC1 and SUC2. Yeast cells transformed with vectors allowing expression of either SUC1 or SUC2 under the control of the promoter of the yeast plasma membrane ATPase gene (PMA1) transport sucrose, and to a lesser extent also maltose, across their plasma membranes in an energy-dependent manner. The K_M-values for sucrose transport are 0.50 mM and 0.77 mM, respectively, and transport by both proteins is strongly inhibited by uncouplers such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) and dinitrophenol (DNP), or SH-group inhibitors. The V_MAX but not the K_M-values of sucrose transport depend on the energy status of transgenic yeast cells. The two proteins exhibit different patterns of pH dependence with SUC1 being much more active at neutral and slightly acidic pH values than SUC2. The proteins share 78% identical amino acids, their apparent molecular weights are 54.9 kDa and 54.5 kDA, respectively, and both proteins contain 12 putative transmembrane helices. A modified SUC1-His6 cDNA encoding a histidine tag at the SUC1 C-terminus was also expressed in S. cerevisiae. The tagged protein is fully active and is shown to migrate at an apparent molecular weight of 45 kDa on 10% SDS—polyacrylamide gels.     

Fruit ripening-related expression of a gene encoding group 5 late embryogenesis abundant protein inCitrus

A cDNA and genomic clone (CuLEA5) encoding a group 5 late embryogenesis abundant protein (Lea5) was isolated from citrus fruit cDNA and genomic libraries. Sequence analysis indicated that the clone contains an open reading frame of 97 amino acids, and that the genomic structure is composed of two exons and one intron. A comparison of its amino sequence with other plant proteins showed that Lea5 proteins can be classified into two types - gymnosperm and angiosperm — based on a P-segment sequence designated by this study. Examination of its expression patterns indicated thatCuLEA5 has important roles during the development or ripening of seedless fruits and leaves inCitrus. The 5′-flanking region of the genomic DNA contains a number of putative hormonal- and stress-responsive elements. This is the first report that describes the expression ofLea5 during fruit ripening, as well as the sequence characteristics of its promoter region.