Cytosolic triosephosphate isomerase is a single gene in rice.

A cDNA clone encoding rice (Oryza sativa L.) cytosolic triosephosphate isomerase (TPI), an important glycolytic enzyme, was isolated and characterized. The clone (pRTPI-6) contains an open reading frame of 759 base pairs, encoding a polypeptide chain of 253 amino acid residues (M(r) 27,060). The identity of this clone was defined by its high homology (85% nucleotide sequence and 89% amino acid sequence identical match) with a maize mRNA sequence encoding the cytosolic TPI and with TPIs from other species. Genomic DNA blot analysis using the cDNA as a probe showed that the cytosolic TPI gene is present as a single copy per haploid rice genome, as opposed to that found in maize, in which multiple TPI gene copies exist. A single TPI mRNA species of about 1100 nucleotides was detected by gel blot hybridization analysis of RNA isolated from root, culm, and leaf tissues, indicating that its expression is ubiquitous. Based on sequence comparison and molecular analysis, we propose that the chloroplast-located TPI may be encoded by divergent structural nuclear genes in rice.     

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     

Purification and differential expression of enolase from maize

Enolase was purified from maze ( Zea mays L. inbred B73)seeds to a 55 and 56 kDa protein doublet based upon sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Purification included ammonium sulfate-precipitation, gel filtration, Mono Q, and Phenyl Superose chromatography. Two-dimensional gels further resolved the 56 kDa protein into three isoselectric forms. Polyclonal antibodies raised against the purified proteins, were found to bind specifically to both the 55 and 56 kDa proteins during purification. Theses antibodies did not recognized a 56 kDa protein when the strain was complemented with maize enolase (pZM245). Maize enolase antibodies recognized a extracts indicated that the 55 kDa form of enolase was more abundant in roots. Enolase protein levels remained unchanged in maize roots after 24 h of anaerobiosis, even though the specific activity of enolase increased to twice its initial levels. A plastid form of enolase in maize could not be found as either enolase activity or protein (with immunoblots).     

Structure and expression of the lignin O-methyltransferase gene from Zea mays L.

The isolation and characterization of cDNA and homologous genomic clones encoding the lignin O-methyltransferase (OMT) from maize is reported. The cDNA clone has been isolated by differential screening of maize root cDNA library. Southern analysis indicates that a single gene codes for this protein. The genomic sequence contains a single 916 bp intron. The deduced protein sequence from DNA shares significant homology with the recently reported lignin-bispecific caffeic acid/5-hydroxyferulic OMTs from alfalfa and aspen. It also shares homology with OMTs from bovine pineal glands and a purple non-sulfur photosynthetic bacterium. The mRNA of this gene is present at different levels in distinct organs of the plant with the highest accumulation detected in the elongation zone of roots. Bacterial extracts from clones containing the maize OMT cDNA show an activity in methylation of caffeic acid to ferulic acid comparable to that existing in the plant extracts. These results indicate that the described gene encodes the caffeic acid 3-O-methyltransferase (COMT) involved in the lignin biosynthesis of maize.     

cDNA cloning and nucleotide sequence of rat muscle-specific enolase (beta beta enolase)

The nucleotide sequence of rat muscle-specific enolase cDNA was determined by sequencing three cDNA clones encoding this enolase isozyme. The nearly full-length cDNA consists of 13-bp 5'- and 84-bp 3'-noncoding regions and a poly(A) tail in addition to a 1302-bp coding region encoding a polypeptide composed of 434 amino acid residues. The deduced primary structure of this enolase isozyme is about 80% similar to those determined previously for rat neuron-specific and non-neuronal enolase isozymes. Southern blot analysis suggested strongly the existence of a single copy of the muscle-specific enolase gene per haploid genome. The mRNA for this enolase isozyme was detected in rat skeletal muscle on day 1 after birth and its level increased rapidly during 10-30 days after birth without any change in its size (1500 bases).     

Characterization of a cDNA encoding a proline-rich 14 kDa protein in developing cortical cells of the roots of bean (Phaseolus vulgaris) seedlings

A cDNA clone, corresponding to mRNAs preferentially expressed in the roots of bean (Phaseolus vulgaris L.) seedlings, was isolated. This clone contains a 381 bp open reading frame encoding a polypeptide of 13.5 kDa, designated PVR5 (Phaseolus vulgaris root 5). The amino acid sequence of this clone is rich in proline (13.5%) and leucine (12.7%) and shares significant amino acid sequence homology with root-specific and proline-rich proteins from monocots (maize and rice), and proline-rich proteins from dicots (carrot, oilseed rape, and Madagascar periwinkle). The precise biological roles of these polypeptides are unknown. PVR5 mRNA accumulation is developmentally regulated within the root, with high levels at the root apex and declining levels at distances further from the root tip. In situ hybridization shows that PVR5 mRNA specifically accumulates in the cortical ground meristem in which maximal cell division occurs. Southern blot analysis suggests that genomic DNA corresponding to PVR5 cDNA is encoded by a single gene or a small gene family.     

Isolation and characterization ofo-diphenol-O-methyltransferase cDNA clone in hot pepper (Capsicum annuum L.)

A cDNA clone,CaOMTl encoding ano-diphenol-O-methyltransferase (OMT), which is involved in capsaicin biosynthesis, was isolated by screening of a cDNA library prepared from the mRNA of pepper (Capsicum annuum L.) pericarp. Nucleotide sequence analysis ofCaOMTl revealed that it had an open reading frame of 1080 bp which encodes a polypeptide with a predicted molecular weight of 39,430 D, corresponding well with the size of the known OMT’s of tobacco, poplar, aspen, alfalfa, and cabbage. It also had five conserved boxes which appear in all known OMT’s. The nucleotide sequence ofCaOMTl had 89–74% identity with the OMT cDNA’s of tobacco, aspen, alfalfa, and poplar, but a relatively lower identity of 59% with the OMT cDNA of maize. Amino acid sequence analysis also revealed that CaOMT1 has high identity with the known OMT’s which have a substrate ofo-diphenolic compounds, especially 5-hydroxyferulic acid and caffeic acid. It supportsCaOMTl which encodes an OMT. Southern blot analysis suggested thatCaOMTl might exist in the form of multiple copies in the pepper genome.CaOMTl is expressed preferentially in pepper fruit and its expression levels increased during pepper fruit development, but decreased during fruit ripening, suggesting that theCaOMTl gene is fruit development-related.CaOMTl is the first reported cDNA clone for enzymes related to the phenlypropanoid pathway in pepper.     

Abundance of an mRNA encoding a high mobility group DNA-binding protein is regulated by light and an endogenous rhythm

A cDNA clone encoding an HMG1 protein from Pharbitis nil was characterized with regard to its sequence, genomic organization and regulation in response to photoperiodic treatments that control floral induction. The HMG1 cDNA contains an open reading frame of 432 nucleotides encoding a 144 amino acid protein of approximately 16 kDa. The predicted polypeptide has the characteristic conserved motifs of the HMG1 and HMG2 class of proteins including an N-terminal basic region, one of two HMG-box domains, and a polyacidic carboxy terminus. Within the HMG-box region, Pharbitis HMG1 deduced amino acid sequence shares 47%, 67% and 69% identity with its animal, maize, and soybean counterparts, respectively. Southern blot hybridization analysis suggests that HMG1 is a member of a multigene family. Analysis of mRNA abundance indicates that the HMG1 gene is expressed to higher levels in dark-grown tissue, such as roots, and at lower levels in light-grown tissue, such as cotyledons and stems. Following the transition to darkness, the levels of HMG1 mRNA in cotyledons were initially stable, however, after a lag time of 8 h or more, HMG1 mRNA increased in abundance to a peak level at 20 h. A second peak in mRNA levels was observed about 24 h later, indicating that the expression of the HMG1 gene is regulated by an endogenous circadian rhythm. Abundance of the HMG1 mRNA during a dark period was dramatically affected by brief light exposure (night break), a treatment which inhibits floral induction. These data indicate that the expression of HMG1 is regulated by both an endogenous rhythm and the light/dark cycle and are consistent with a role for HMG1 in maintaining patterns of circadian-regulated gene expression activated upon the transition from light to darkness.     

Maize phosphoenolpyruvate carboxylase. Cloning and characterization of mRNAs encoding isozymic forms

The isozymic forms of maize phosphoenolpyruvate carboxylase (P-enolpyruvate carboxylase) involved in photosynthetic CO2 fixation were shown by protein gel blot analysis to consist of 100-kDa subunits. The nonautotrophic isoform found in roots is comprised of 96-kDa subunits and is about 50-100-fold less prevalent. Further analysis of P-enolpyruvate carboxylase isoforms made use of cloned cDNA probes. Two cDNA clones were isolated from a library constructed from maize leaf poly(A) RNA. The largest clone was complementary to about 25% of P-enolpyruvate carboxylase mRNA, which is 3.4 kilobases in length. The quantity of P-enolpyruvate carboxylase mRNA in green, mature leaf tissue was estimated to be 0.20% of poly(A) RNA, whereas P-enolpyruvate carboxylase mRNA in roots was about 100-fold less prevalent. We used thermal denaturation of a P-enolpyruvate carboxylase cDNA probe hybridized to RNA gel blots to estimate the degree of sequence difference between mRNAs encoding different P-enolpyruvate carboxylase isoforms. There appear to be at least two prevalent P-enolpyruvate carboxylase mRNAs in green leaves which are significantly different in sequence, as are P-enolpyruvate carboxylase mRNAs in roots and shoots. The hybridization pattern of maize genomic DNA Southern blots indicates that P-enolpyruvate carboxylase is encoded by a small gene family.     

玉米苹果酸脱氢酶基因的分离与结构分析

以一个玉米（ＺｅａｍａｙｓＬ．）杂种一代超亲表达的ｃＤＮＡ片段为探针,从玉米幼苗期ｃＤＮＡ文库中筛选到一个全长１２８７ｂｐ的ｃＤＮＡ克隆。序列分析表明,该ｃＤＮＡ编码细胞质苹果酸脱氢酶,推导的氨基酸序列与龙须海棠（Ｍｅｓｅｍｂｒｙａｎｔｈｅｍｕｍ　ｃｒｙｓｔａｌｌｉｕｍ　Ｌ．）及拟南芥（Ａｒａｂｉｄｏｐｓｉｓ　ｔｈａｌｉａｎａ（Ｌ．）Ｈｅｙｎｈ．）同一编码基因的氨基酸序列同源性分别为９０％和８４％。这是禾谷类作物中首次克隆的编码细胞质苹果酸脱氢酶的完整基因。     

Isolation and Characterization of a cDNA Encoding Maize Cyto solic Malate Dehydrogenase

A cDNA fragment derived from a gene over-expressing in hybrid maize ( Zea mays L. ) was isolated with RT-PCR and used as probe to screen cDNA library of hybrid maize seedlings. A positive cDNA clone ZH02 corresponding to the full-length mRNA sequence was obtained, which was shown to have an open reading frame encoding 332 a.a. DNA and proteinase database search revealed that the deduced amino acid sequence of ZH02 has high similarity with the cMDH of Mesembryaathemum crystallium L. and Arabidopsis thaliana (L.) Heynh. up to 90% and 84%, respectively. This is the first report of the full-length gene coding for the cereal cMDH.     

Isolation and sequencing of cDNA clones encoding ethylene-induced putative peroxidases from cucumber cotyledons

A cDNA library from ethephon-treated cucumber cotyledons (Cucumis sativus L. cv. Poinsett 76) was constructed. Two cDNA clones encoding putative peroxidases were isolated by means of a synthetic probe based on a partial amino acid sequence of a 33 kDa cationic peroxidase that had been previously shown to be induced by ethylene. DNA sequencing indicates that the two clones were derived from two closely related RNA species that are related to published plant peroxidase sequences. Southern analysis indicates that there are 1–5 copies in a haploid genome of a gene homologous to the cDNA clones. The deduced amino acid sequences are homologous with a tobacco (55% sequence identity), a horseradish (53%), a turnip (45%), and a potato (41%) peroxidase. The cloned sequences do not encode the 33 kDa peroxidase from which the original synthetic probe was been derived, but rather other putative peroxidases. An increase in the level of mRNA is evident by 3 hours after ethephon or ethylene treatment and plateaus by 15 hours.