Molecular characterization of Arabidopsis thaliana cDNAs encoding three purine biosynthetic enzymes

Glycinamide ribonucleotide (GAR) synthetase, GAR transformylase and aminoimidazole ribonucleotide (AIR) synthetase are the second, third and fifth enzymes in the 10-step de novo purine biosynthetic pathway. From a cDNA library of Arabidopsis thaliana, cDNAs encoding the above three enzymes were cloned by functional complementation of corresponding Escherichia coli mutants. Each of the cDNAs encode peptides comprising the complete enzymatic domain of either GAR synthetase, GAR transformylase or AIR synthetase. Comparisons of the three Arabidopsis purine biosynthetic enzymes with corresponding enzymes/polypeptide-fragments from procaryotic and eucaryotic sources indicate a high degree of conserved homology at the amino acid level, in particular with procaryotic enzymes. Assays from extracts of E. coli expressing the complementing clones verified the specific enzymatic activity of Arabidopsis GAR synthetase and GAR transformylase. Sequence analysis, as well as Northern blot analysis indicate that Arabidopsis has single and monofunctional enzymes. In this respect the organization of these three plant purine biosynthesis genes is fundamentally different from the multifunctional purine biosynthesis enzymes characteristic of other eucaryotes and instead resembles the one gene, one enzyme relationship found in procaryotes.     

Isolation of several cDNAs encoding yeast peroxisomal enzymes

Several candidate clones carrying partial cDNAs for yeast peroxisomal enzymes, such as catalase, carnitine acetyltransferase, isocitrate lyase, malate synthase and acyl-CoA oxidase, were efficiently isolated at a single plating from a phage lambda gt11 recombinant cDNA library prepared with poly(A)-rich RNA from an n-alkane-grown yeast, Candida tropicalis, with a mixture of antibodies against the respective purified enzymes. Among them, one candidate clone carrying partial cDNA for catalase was subcloned and subjected to nucleotide sequence analysis. We succeeded in determining that the amino acid sequence deduced from the nucleotide analysis included the sequences derived from the two peptide fragments obtained from the purified enzyme.     

Isolation of two cDNAs encoding E2 ubiquitin-conjugating enzymes from Pavlova lutheri (Haptophyceae)

Two novel cDNAs, Plubc1 and Plubc2, encoding ubiquitin-conjugatingenzyme E2, were isolated from a Pavlova lutheri cDNA library. They areeach encoded by single copy genes in thealgae genome. Sequence comparison withplant, yeast and algal E2 sequences showedthat PlUBC1 and PlUBC2 are members of newE2 subfamilies. Time-course expressionanalysis of the two cDNAs revealed thatPlubc1 is transitionallyover-expressed at the end of theexponential phase of growth of the culture,while Plubc2 is constitutivelyexpressed at the same level throughout thecell growth. The phylogenetic study and thedifferent expression patterns suggest thatthese two enzymes could exhibit differentphysiological functions in P. lutheri.The partial sequence of the 18S rRNA geneand the full-length cDNA sequence of Plubc1 and Plubc2 reported in thispaper will appear in the Genbank databaseunder the accession numbers AY135218,AY135219 and AY135220 respectively.     

Cloning of two genes encoding potassium transporters in Neurospora crassa and expression of the corresponding cDNAs in Saccharomyces cerevisiae

Two Neurospora crassa genes, trk-1 and hak-1, encode K+ transporters that show sequence similarities to the TRK transporters described in Saccharomyces cerevisiae and Schizosaccharomyces pombe, and to the HAK transporters described in Schwanniomyces occidentalis and barley. The N. crassa TRK1 and HAK1 transporters expressed by the corresponding cDNAs in a trk1 delta trk2 delta mutant of S. cerevisiae exhibited a high affinity for Rb+ and K+. Northern blot analysis and comparison of the kinetic characteristics of the two transporters in the trk1 delta trk2 delta mutant with the kinetic characteristics of K+ uptake in N. crassa cells allowed TRK1 to be identified as the dominant K+ transporter and HAK1 as a transporter that is only expressed when the cells are K+ starved. The HAK1 transporter showed a high concentrative capacity and is identified as the K(+)-H+ symporter described in N. crassa, whereas TRK1 might be a K+ uniporter. Although the co-existence of K+ transporters of the TRK and HAK types in the same species had not been reported formerly, we discuss whether this co-existence may be the normal situation in soil fungi.     

Isolation of plant-growth-promoting Bacillus strains from soybean root nodules

Endophytic bacteria reside within plant tissues and have often been found to promote plant growth. Fourteen strains of putative endophytic bacteria, not including endosymbiotic Bradyrhizobium strains, were isolated from surface-sterilized soybean (Glycine max. (L.) Merr.) root nodules. These isolates were designated as non-Bradyrhizobium endophytic bacteria (NEB). Three isolates (NEB4, NEB5, and NEB17) were found to increase soybean weight when plants were co-inoculated with one of the isolates and Bradyrhizobium japonicum under nitrogen-free conditions, compared with plants inoculated with B. japonicum alone. In the absence of B. japonicum, these isolates neither nodulated soybean, nor did they affect soybean growth. All three isolates were Gram-positive spore-forming rods. While Biolog tests indicated that the three isolates belonged to the genus Bacillus, it was not possible to determine the species. Phylogenetic analysis of 16S rRNA gene hypervariant region sequences demonstrated that both NEB4 and NEB5 are Bacillus subtilis strains, and that NEB17 is a Bacillus thuringiensis strain.     

Cloning and expression of cDNAs encoding plant V-ATPase subunits in the corresponding yeast null mutants

Complementation of yeast null mutants is widely used for cloning of homologous genes from heterologous sources. We have used this method to clone the relevant V-ATPase genes from lemon fruit and Arabidopsis thaliana cDNA libraries. The pH levels are very different in the vacuoles of the lemon fruit and the A. thaliana, yet both are the result of the activity of the same enzyme complex, namely the V-ATPase. In order to investigate the mechanism that enables the enzyme to maintain such differences in pH values, we have compared the subunit composition of the V-ATPase complex from both sources. Towards this end, we have constructed a cDNA library from lemon fruit and cloned it into a similar shuttle vector to the one of the A. thaliana cDNA library, which is commercially available. In this work, we report the cloning and expression of VMA10 from both sources, two isoforms of the lemon proteolipid (VMA3) and the lemon homologue of yeast VPH1/STV1 subunit, LEMAC.     

Isolation of two cDNAs encoding functional human cytoplasmic cysteinyl-tRNA synthetase

Cysteinyl-tRNA synthetase catalyzes the addition of cysteine to its cognate tRNA. The available eukaryotic sequences for this enzyme contain several insertions that are absent from bacterial sequences. To gain insights into the differences between the bacterial and eukaryotic forms, we previously studied the E. coli cysteinyl-tRNA synthetase. In this study, we sought to clone and express the full-length gene for the human cytoplasmic cysteinyl-tRNA synthetase. Although a gene encoding the human enzyme has been described, the predicted protein sequence, consisting of 638 amino acids, lacks homology with other eukaryotic enzymes in the carboxyl-terminus. This suggested that a further investigation was necessary to obtain the definitive sequence for the human enzyme. Here we report the isolation of a full-length cDNA that encodes a protein of 748 amino acids. The predicted protein sequence shows considerable similarity to other eukaryotic cysteinyl-tRNA synthetases in the carboxyl-terminus. We also found that approximately 20% of the mRNA encoding the cytoplasmic cysteinyl-tRNA synthetase contained an insertion of 8 bases in the 3' coding region of the mRNA. This insertion arises from an alternative splicing between the last two exons of the gene. The alternative splicing alters the reading frame and results in the replacement of the carboxy-terminal 44 amino acids with a novel sequence of 22 amino acids. Expression of the full-length and alternative forms of the enzyme in E. coli generated functional proteins that were active in aminoacylation of human cytoplasmic tRNA(Cys) with cysteine.     

Cloning and characterization of two maize cDNAs encoding Cinnamoyl-CoA Reductase (CCR) and differential expression of the corresponding genes

Cinnamoyl-CoA Reductase (CCR, EC 1.2.1.44) catalyses the first step of the lignin pathway. Two full-length cDNAs identified by sequence analysis as CCR-encoding cDNAs were isolated from a maize root cDNA library. These two cDNAs designated ZmCCR1 and ZmCCR2 exhibit 73% sequence conservation at the nucleotide level for their coding regions and are relatively divergent at their 5- and 3-untranslated regions. They both contain a common signature which is thought to be involved in the catalytic site of CCR. Northern blot analysis indicated that ZmCCR2 was expressed at very low levels in roots whereas ZmCCR1 was widely expressed in different organs. The high level of ZmCCR1 gene expression along the stalk suggests that the corresponding enzyme is probably involved in constitutive lignification.     

Isolation and characterization of the membrane envelope enclosing the bacteroids in soybean root nodules

The membrane envelope enclosing the bacteroids in soybean root nodules is shown by ultrastructural and biochemical studies to be derived from, and to retain the characteristics of, the host cell plasma membrane. During the early stages of the infection process, which occurs through an invagination, Rhizobium becomes surrounded by the host cell wall and plasma membrane, forming the infection thread. The cell wall of the infection thread is degraded by cellulolytic enzyme(s), leaving behind the enclosed plasma membrane, the membrane envelope. Cellulase activity in young nodules increases two- to threefold as compared to uninfected roots, and this activity is localized in the cell wall matrix of the infection threads. Membrane envelopes were isolated by first preparing bacteroids enclosed in the envelopes on a discontinuous sucrose gradient followed by passage through a hypodermic needle, which released the bacteroids from the membranes. This membrane then sedimented at the interface of 34--45% sucrose (mean density of 1.14 g/cm3). Membranes were characterized by phosphotungstic acid (PTA)-chromic acid staining. ATPase activity, and localization, sensitivity to nonionic detergent Nonidet P-40 (NP-40) and sodium dodecyl sulfate (SDS) gel electrophoresis. These analyses revealed a close similarity between plasma membrane and the membrane envelope. Incorporation of radioactive amino acids into the membrane envelope proteins was sensitive to cycloheximide, suggesting that the biosynthesis of these proteins is primarily under host-cell control. No immunoreactive material to leghemoglobin antibodies was found inside or associated with the isolated bacteroids enclosed in the membrane envelope, and its location is confined to the host cell cytoplasmic matrix.     

Appearance of purine-catabolizing enzymes in fix and fix root nodules on soybean and effect of oxygen on the expression of the enzymes in callus tissue

The appearance of enzymes involved in the formation of ureides, allantoin, and allantoic acid, from inosine 5′-monophosphate was analyzed in developing root nodules of soybean (Glycine max). Concomitant with development of effective nodules, a substantial increase in specific activities of the enzymes 5′-nucleotidase (35-fold), purine nucleosidase (10-fold), xanthine dehydrogenase (25-fold), and uricase (200-fold), over root levels was observed. The specific activity of allantoinase remained constant during nodule development. With ineffective nodules the activities were generally lower than in effective nodules; however, the activities of 5′-nucleotidase and allantoinase were 2-fold higher in ineffective nodules unable to synthesize leghemoglobin than in effective nodules. Since the expression of uricase has been shown to be regulated by oxygen (K Larsen, BU Jochimsen 1986 EMBO J 5: 15-19), the expression of the remaining enzymes in the purine catabolic pathway were tested in response to variations in O₂ concentration in sterile soybean callus tissue. Purine nucleosidase responded to this treatment, exhibiting a 4-fold increase in activity around 2% O₂. 5′-Nucleotidase, xanthine dehydrogenase, and allantoinase remained unaffected by variations in the O₂ concentration. Hence, the expression of two enzymes involved in ureide formation, purine nucleosidase and uricase, has been demonstrated to be influenced by O₂ concentration.     

Isolation and characterization of two cDNAs from atlantic cod encoding two distinct psychrophilic elastases

The cDNAs encoding two different Atlantic cod elastases have been isolated and sequenced. The predicted amino acid sequences revealed two preproelastases, consisting of a signal peptide, an activation peptide and a mature enzyme of 242 and 239 amino acids. Amino acid sequence identity between the two cod elastases was 60.1% and identity with mammalian elastases ranged from 50–64%. The two cod elastases contain all the major structural features common to serine proteases, such as the catalytic triad His57, Asp102 and Ser195. Both cod elastases have a high content of methionine, consistent with previous findings in psychrophilic fish enzymes.     

Isolation and oxidative properties of mitochondria and bacteroids from soybean root nodules

Summary A method for the separation and purification of bacteroids and mitochondria from nodules of soybean roots is described. Cross contamination between these two oxidative fractions was easily assessible by using NADH oxidase and -hydroxybutyrate dehydrogenase respectively as specific mitochondrial and bacteroid markers. Bacteroid respiration was characterized by substantial endogenous respiration which could be reduced by keeping plants in the dark prior to isolation, and stimulated by uncoupler or organic acids. Nodule mitochondria readily oxidized external NADH and a range of tricarboxylic acid cycle intermediates, with good respiratory control. A major difference between nodule and root mitochondria was the former's high sensitivity to the inhibitors rotenone and cyanide. This indicates a reduced capacity for non-phosphorylating electron transport in nodule mitochondria, which may be related to the large energy demand during ammonia assimilation in nodule cells.     

Subcellular localization of enzymes involved in the assimilation of Ammonia by soybean root nodules

Ammonia, the primary product of nitrogen fixation is rapidly incorporated into a number of amino acids such as glutamate and aspartate. A novel enzyme system glutamine: 2-oxoglutarate aminotransferase oxidoreductase, which probably has an important role in ammonia assimilation has been detected, in the present studies, in the rhizobial fraction of soybean root nodules and in Rhizobium japonicum grown in culture. The role of this latter enzyme and other enzymes such as glutamate dehydrogenase, aspartate aminotransferase and alanine aminotransferase in ammonia assimilation by soybean nodules is discussed.     

Distinct properties of two glutamine synthetase isoforms in soybean root nodules

Glutamine synthetase (GS) in nodule cytosol plays a major role in the assimilation of the ammonium produced by biological nitrogen fixation. To characterize the GS protein family in Glycine max root nodules, the catalytical properties of 2 GS1 isoenzymes (GS1β1 and GS1γ1) were compared in this study. Although, GmGS1β1 and GmGS1γ1 have very similar kinetic characteristics, they also exhibit distinct enzymatic properties in terms of thermal stability and the transferase to synthetase activity (GSt/GSs) ratios. The results demonstrated that GmGS1γ1, which displayed lower thermal stability and GSt/GSs ratios than GmGS1β1, might be considered as superior isoform to participate in the efficient assimilation of ammonia only when it is needed. Also, it is proposed that the difference of enzymatic properties between isoforms contribute to their differential roles in ammonia assimilation under variable internal and external environments.     

Isolation and functional analysis of two Cistus creticus cDNAs encoding geranylgeranyl diphosphate synthase

Cistus creticus ssp. creticus is an indigenous shrub of the Mediterranean area. The glandular trichomes covering its leaf surfaces secrete a resin called “ladanum”, which among others contains a number of specific labdane-type diterpenes that exhibit antibacterial and antifungal action as well as in vitro and in vivo cytotoxic and cytostatic activity against human cancer cell lines. In view of the properties and possible future exploitation of these metabolites, it was deemed necessary to study the geranylgeranyl diphosphate synthase enzyme (GGDPS, EC 2.5.1.30), a short chain prenyltransferase responsible for the synthesis of the precursor molecule of all diterpenes. In this work, we present the cloning, functional characterisation and expression profile at the gene and protein levels of two differentially expressed C. creticus full-length cDNAs, CcGGDPS1 and CcGGDPS2. Heterologous yeast cell expression system showed that these cDNAs exhibited GGDPS enzyme activity. Gene and protein expression analyses suggest that this enzyme is developmentally and tissue-regulated showing maximum expression in trichomes and smallest leaves (0.5–1.0 cm). This work is the first attempt to study the terpenoid biosynthesis at the molecular level in C. creticus ssp. creticus.