Characterization and expression of an mRNA encoding a wound-induced (Win) protein from ethylene-treated tomato leaf abscission zone tissue

A cDNA clone (TAB7) encoding a putative woundinduced (Win) proteinhas been isolated from a tomato (Lycopersicon esculentum Mill.cv. Ailsa Craig) leaf abscission zone cDNA library using a differentialscreening strategy. The clone has a high degree of homologyat the amino acid level to both the potato win1 and 2 genes,Hevea brasiliensis hevein and Nicotiana tabacum PR-4a and PR-4bproteins. The mRNA encoded by TAB7 is up-regulated within 12h of exposure to ethylene (10µl l^–1) and its expressionincreases steadily within the cells comprising the leaf abscissionzone and to a lesser extent in the adjacent non-zone tissue.This rise precedes the onset of cell separation. Southern analysisindicates that the mRNA is encoded by either a single gene ora small gene family. The role of the protein during abscissionis discussed. Key words: Lycopersicon esculentum, abscission zone, ethylene, tomato, wound-induced proteins     

Sequence Analysis of Cloned cDNA and Proteolytic Fragments for Nitrate Reductase from Spinacia oleracea L.

Proteolytic fragments were obtained by limited proteolysis of120 kDa nitrate reductase from Spinacia oleracea L. using trypsinand Staphylococcus aureus V8 protease. Determination of NH₂-terminalsequences in 9 to 14 Edman degradation steps allowed the exactlocalization of the fragments within the amino-acid sequenceof spinach nitrate reductase was deduced from the nucleotidesequence of cDNA clone pSPNR117 which was initially identifiedby hybridization to squash nitrate reductase cDNA clone [Crawford,¹N. M., Campbell, W. H. and Davis, R. W. (1986) Proc. Natl. Acad.Sci. USA 83: 8073] and anti spinach nitrate reductase polyclonalantibodies. This clone has a 2324 base insert, and the aminoacid sequence deduced from its open reading frame, which contains640 residues. The predicted sizes 42.5 and 30 kDa were in reasonableagreement with previous determination of the apparent molecularsizes of the FAD-cyt-chrome b557-binding, and FAD-binding fragments,respectively. Arginine residue was the cleavage site for trypsin and glutamicacid was for S. aureus V8 protease. The amino acid residueswithin the linker regions which connect the functional domains,could be cleaved with trypsin or S. aureus V8 protease may bewell conserved in the amino acid sequences deduced from thenitrate reductase cDNA sequences. A sequence identity of 61.2-80.1 % was found in the amino acidsequences deduced from the cDNA sequences as obtained by spinachand other higher plant nitrate reductases. However, the aminoacid sequences surrounding the proteolytic cleavage sites ofnitrate reductase had poor homology. (Received March 30, 1991; Accepted July 24, 1991)     

Evidence for No Proteolytic Processing during Transport of Isocitrate Lyase into Glyoxysomes in Castor Bean Endosperm

The amino-terminal sequence of isocitrate lyase purified fromcastor bean endosperm glyoxysomes was compared with that deducedfrom the nucleotide sequence of cDNA for the enzyme [Plant Mol.Biol. (1987) 8: 471]. The isolated active enzyme lacked sixamino acid residues in the amino terminus, although the enzymeimmunoselected from a tissue homogenate with trichloroaceticacid had the amino-terminal part. Thus the six amino acid residuesseem to be eliminated during enzyme purification and the enzymeis transported into glyoxysomes without proteolytic processing. (Received August 31, 1987; Accepted November 30, 1987)     

Molecular characterization of four chitinase cDNAs obtained fromCladosporium fulvum-infected tomato

Complementary DNA clones encoding acidic and basic isoforms of tomato chitinases were isolated fromCladosporium fulvum-infected leaves. The clones were sequenced and found to encode the 30 kDa basic intracellular and the 26 and 27 kDa acidic extracellular tomato chitinases previously purified (M.H.A.J. Joostenet al., in preparation). A fourth truncated cDNA which appears to encode an extracellular chitinase with 82% amino acid similarity to the 30 kDa intracellular chitinase was also isolated. Characterization of the clones revealed that the 30 kDa basic intracellular protein is a class I chitinase and that the 26 and 27 kDa acidic extracellular proteins which have 85% peptide sequence similarity are class II chitinases. The characterized cDNA clones represent four from a family of at least six tomato chitinases. Southern blot analysis indicated that, with the exception of the 30 kDa basic intracellular chitinase, the tomato chitinases are encoded by one or two genes. Northern blot analysis showed that the mRNA encoding the 26 kDa acidic extracellular chitinase is induced more rapidly during an incompatibleC. fulvum-tomato interaction than during a compatible interaction. This difference in timing of mRNA induction was not observed for the 30 kDa basic intracellular chitinase.     

Structure and expression of a cDNA encoding zeaxanthin epoxidase, isolated from a wilt-related tomato (Lycopersicon esculentum Mill.) library

Zeaxanthin epoxldase (ZE) catalyses two early steps in the abscisicacid (ABA) biosynthetic pathway. The sequence of a cDNA cloneencoding ZE from Nicotiana plumbaginifolia was reported In 1996and represented the first DNA sequence data on an ABA biosyntheticenzyme. The N. plumbaginifolia cDNA has been used to providea heterologous probe to isolate a ZE cDNA from tomato (Lycopersiconesculentum Mill.). DNA and amino acid sequence differences areconsidered in relation to putative functional domans withinthe enzyme. The results of northern analysis in tomato are discussedin relation to the effects of water stress on ZE mRNA levels. Key words: ABA biosynthesis, zeaxanthin epoxidase, tomato     

Rice Embryo Globulins: Amino-Terminal Amino Acid Sequences, cDNA Cloning and Expression

A globulin fraction prepared from rice embryos contained polypeptidesor polypeptide groups of 49 kDa (designated REG1), 46 kDa (designatedREG2), about 35 kDa, 32 kDa and 25 kDa. The amino-terminal sequencesof REG1 and the major polypeptide in the 35-kDa group were identical,suggesting that the REG1 polypeptide undergoes partial proteolyticprocessing that removes a carboxy-terminal region. A cDNA clone,designated pcREG2, encoding REG2 was isolated, and its nucleotidesequence was determined. The deduced amino acid sequence ofREG2 was found to be 68% identical to that of the maize GLB2globulin. Reg2 mRNA was present at high levels during embryodevelopment for up to 14 days after flowering (DAF). Lower levelswere found 20 DAF when the maturation of embryos was almostcompleted, and at the dry mature stage. Reg2 mRNA almost disappearedupon imbibition of isolated dry mature embryos but it was re-inducedat a low level by further treatment with ABA. The expressionof Reg2 was not induced by ABA in suspension-cultured cells,unlike that of Osem, one of the late embryogenesis abundantprotein (LEA) genes. (Received November 6, 1995; Accepted April 22, 1996)     

A cDNA Encoding the 57 kDa Subunit of a Cytokinin-Binding Protein Complex from Tobacco: the Subunit Has High Homology to S-Adenosyl-L-Homocysteine Hydrolase

130 kDa cytokinin-binding protein complex (CBP130) is a majorcytokinin binding entity in tobacco leaves (Nicotiana sylvestris)and contains two protein species of 57 and 36 kDa [CBP57 andCBP36, Mitsui and Sugiura (1993) Plant Cell Physiol. 34: 543].We have determined partial amino acid sequences of CBP57 andisolated cDNAs encoding the protein from a tobacco cDNA libraryusing an oligonucleotide probe. Sequence analysis revealed significanthomology between CBP57 and S-adenosyl-L-homocysteine hydrolasefrom other organisms, which catalyzes the reversible hydrolysisof S-adenosyl-L-homocysteine, a methyltransferase inhibitor.CBP57 contains an additional sequence of 41 amino acids whichis not present in animal and slime mold Sadenosyl-L-homocysteinehydrolases. This additional sequence is also found in the parsleyand Rhodobacter enzymes, suggesting that it is unique to photosyntheticorganisms. CBP57 is encoded by more than one nuclear genes intobacco. Northern and western blot analyses revealed that thelevel of expression of the genes is high in roots and low inleaves. They are also expressed in cultured tobacco cells. Wediscuss the possibility that at least some of the physiologicaleffects of cytokinin are mediated through the control of methylation/demethylationby regulating the intracellular concentration of S-adenosyl-L-homocysteinevia the hydrolase. (Received June 24, 1993; Accepted August 14, 1993)     

Characterisation of the S-adenosylmethionine decarboxylase (SAMDC) gene of potato

S-adenosylmethionine decarboxylase (SAMDC) is involved in the biosynthesis of the polyamines, spermidine and spermine. Recently, we reported the isolation of a putative cDNA clone of the SAMDC clone of potato (Plant Mol Biol 20; 641–651). In order to confirm that the potato genes does encode SAMDC, a complementation experiment with a yeast strain that possesses a null mutation in the SAMDC gene was performed. The yeast strain contains a deletion-insertion mutation in the SAMDC gene and has an absolute requirement for the addition of exogenous spermidine for growth. When the full-length potato cDNA was expressed in the mutant yeast strain there was no longer a requirement for exogenous spermidine. Immunoblotting experiments suggest that the potato SAMDC gene product has an apparent molecular mass of 39 kDa. Expression of the SAMDC gene was high in the young and actively dividing tissues and low in the mature and non-dividing tissues of both vegetative and reproductive organs. Additionally, isolation and characterisation of the corresponding genomic clone is reported. The gene has one intron in its 5-untranslated sequence but otherwise the transcribed portion is identical to the cDNA clone.     

Characterization of a cDNA encoding ricin E,a hybrid ricin-Ricinus communis agglutinin gene from the castor plant Ricinus communis

Two classes of ricin cDNA clones have been identified and sequenced. The cDNA clone pBL-1 closely matches in nucleotide sequence the ricin genomic clone pAKG previously described by Halling et al., 1985 (Nucl. Acids Res. 13:8019). A second group of cDNA clones, represented by pBL-3, encode a hybrid protein (ricin E), having a ricin-like A chain and N-terminal half of the B chain and an RCA (Ricinus communis agglutinin)-like C-terminal half of the B chain.     

Isolation of a full-length cDNA encoding calreticulin from a PCR library of in vitro zygotes of maize

A full-size cDNA clone (1614 bp) encoding calreticulin was isolated from a PCR-based cDNA library of maize in vitro zygotes. Calreticulin is a major Ca²⁺ storage protein located mainly in the lumen of the endoplasmic reticulum but also in the nucleus and/or cytoplasm of some cells. A differential screening between cDNA libraries originating from 104 in vitro zygotes (18 h after in vitro fertilization) and 128 unfertilized egg cells was performed to isolated newly expressed genes or genes expressed more abundantly after fertilization. The expression of the isolated cDNA clone is enhanced after fertilization and strongly correlated to cell division. Sequence comparison to a shorter maize calreticulin cDNA isolated from a conventional cDNA library proves the ability and reproducibility of the recently described method for PCR based cDNA library construction from a few plant cells [12]. It is further shown that calreticulins in maize are probably transcribed from a small gene family differentially expressed in abundance in diverse tissues. The deduced amino acid sequence encodes an acidic protein (pI 4.17) of 48 kDa sharing 77–92% and 50–54% homology to other plant and animal calreticulins, respectively. The described calreticulin gene represents to our knowledge the first cDNA clone isolated from a RT/PCR cDNA library originating from only a few plant cells and is the first gene isolated from zygotes of higher plants.     

Isolation of a Tobacco cDNA Encoding Sar1 GTPase and Analysis of Its Dominant Mutations in Vesicular Traffic Using a Yeast Complementation System

The cDNA clone of NtSARl, a gene encoding the small GTPase Sar1pwhich is essential for vesicle formation from the endoplasmicreticulum (ER) membrane in yeast, has been isolated from Nicotianatabacum BY-2 cells. NtSAR1 as well as AtSAR1 cDNA isolated fromArabidopsis thaliana [d'Enfert et al. (1992) EMBO J. 11: 4205]could complement the lethality of the disruption of SARI inyeast cells in a temperature-sensitive fashion. They also suppressedyeast sec12 and sec16 temperature-sensitive mutations as yeastSARI does. Using this complementation system, we analyzed thephenotypes of several mutations in plant SAR1 cDNAs in yeastcells. The expression of NtSAR1 H74L and AtSAR1 N129I showeddominant negative effect in growth over the wild-type SARI,which was accompanied by the arrest of ER-to-Golgi transport.Such dominant mutations will be useful to analyze the role ofmembrane trafficking in plant cells, if their expression canbe regulated conditionally. (Received October 29, 1997; Accepted March 17, 1998)     

Cloning and sequence analysis of porcine myoglobin cDNA

Porcine myoglobin cDNA clones have been isolated from a cDNA library prepared from enriched heart-myoglobin mRNA. Sequence analysis revealed 59 nucleotides (nt) in the 5'-untranslated, 462 nt in the amino acid (aa)-coding, and 590 nt in the 3'-untranslated regions. The myoglobin cDNA showed a high G + C content (60%). When the nt sequence of the porcine myoglobin cDNA is compared with those of seal and human myoglobin cDNAs deduced from the corresponding genomic myoglobin genes [Blanchetot et al., Nature 301 (1983) 732-734; Weller et al., EMBO J. 3 (1984) 439-446; Akaboshi, Gene 33 (1985) 241-249], a high degree of homology is observed in the 5'-untranslated region and in parts of the 3'-untranslated region, as well as in the coding region.     

Isolation and sequencing of the 5′ end of the rat microtubule-associated protein (MAP1B)-encoding cDNA

We have isolated and sequenced the 5′ end of the cDNA encoding the rat microtubule-associated protein 1B (MAP1B). We found that this region is highly homologous to the corresponding regions of the human [Lien et al., 22 (1994) 273–280] and mouse [Noble et al., J. Cell Biol. 109 (1989) 3367–3376] MAPIB genes. The combination of the sequence that we are presenting with the previously published sequence [Zauner et al., Eur. J. Cell Biol. 57 (1992) 66–74], represents the complete rat MAP1B cDNA coding sequence.     

Structural studies on the tri- and tetrasaccharides isolated from porcine intestinal heparin and characterization of heparinase/heparitinases using them as substrates

We prepared a series of oligosaccharides from porcine intestinalheparin after extensive digestion with a mixture of Flavobacteriumheparinase as well as heparitinases I and II. Previously, wereported the structures of the two glycoserines derived fromthe carbohydrate-protein linkage region [Sugahara et al., J.Biol. Chem., 267, 1528–1533 (1992)] and three tetrasaccharidesderived from the antithrombin III-binding site [Yamada et al.,J. Biol. Chem., 268, 4780–4787 (1993)]. In this study,we determined the structures of 10 other tetrasaccharides anda trisaccharide by enzymatic digestion, fast atom bombardmentmass spectrometry and 500-MHz ¹H NMR spectroscopy. These tetrasaccharidesshare the common disulphated structure,     

Cinnamyl Alcohol Dehydrogenase from Aralia cordata: Cloning of the cDNA and Expression of the Gene in Lignified Tissues

A full-length cDNA clone encoding a subunit of cinnamyl alcoholdehydrogenase (CAD) was isolated from a perennial dicot, Araliacordata. The identity of the clone was demonstrated by two criteria:(i) the amino acid sequences of peptides derived from the purifiedCAD protein of A. cordata were highly homologous to regionsof the amino acid sequence deduced from the nucleotide sequenceof the cDNA; and (ii) a fusion protein expressed from