Molecular cloning and analysis of a cDNA coding for the bifunctional dihydrofolate reductase-thymidylate synthase of Daucus carota

Molecular cloning of dihydrofolate reductase-thymidylate synthase (DHFR-TS) of Daucus carota was achieved by immunoscreening of a cDNA library obtaining a 2 kbp clone which contains an open reading frame of 1528 bp. Comparison of the deduced amino acid sequence with those from other sources revealed the presence of motifs typical of DHFR and TS thus confirming the bifunctional nature of the carrot protein. As in other organisms, a higher degree of conservation was observed in the TS domain. Analysis of the dhfr-ts gene content in carrot revealed the presence of several copies per diploid genome.     

A novel bifunctional aspartate kinase-homoserine dehydrogenase from the hyperthermophilic bacterium,Thermotoga maritima

ABSTRACT

The orientation of the three domains in the bifunctional aspartate kinase-homoserine dehydrogenase (AK-HseDH) homologue found in Thermotoga maritima totally differs from those observed in previously known AK-HseDHs; the domains line up in the order HseDH, AK, and regulatory domain. In the present study, the enzyme produced in Escherichia coli was characterized. The enzyme exhibited substantial activities of both AK and HseDH. L-Threonine inhibits AK activity in a cooperative manner, similar to that of Arabidopsis thaliana AK-HseDH. However, the concentration required to inhibit the activity was much lower (K_0.5 = 37 μM) than that needed to inhibit the A. thaliana enzyme (K_0.5 = 500 μM). In contrast to A. thaliana AK-HseDH, Hse oxidation of the T. maritima enzyme was almost impervious to inhibition by L-threonine. Amino acid sequence comparison indicates that the distinctive sequence of the regulatory domain in T. maritima AK-HseDH is likely responsible for the unique sensitivity to L-threonine.

Abbreviations: AK: aspartate kinase; HseDH: homoserine dehydrogenase; AK–HseDH: bifunctional aspartate kinase–homoserine dehydrogenase; AsaDH: aspartate–β–semialdehyde dehydrogenase; ACT: aspartate kinases (A), chorismate mutases (C), and prephenate dehydrogenases (TyrA, T).     

Molecular characterisation and expression of a wound-inducible cDNA encoding a novel cinnamyl-alcohol dehydrogenase enzyme in lucerne (Medicago sativa L.)

A lucerne (alfalfa, Medicago sativa) stem cDNA library was screened with a cinnamyl-alcohol dehydrogenase (CAD) cDNA probe from tobacco (Nicotiana tabacum cv. Samsun). Two distinctly different cDNA clones (54% identical) were isolated and identified as putative CAD-encoding cDNAs by comparison of their nucleotide sequences with those of CAD-encoding DNA sequences from other plant species. One of the cDNAs, MsaCad2, was found to be 99.4% identical at the nucleotide level to the previously isolated lucerne cad cDNA which encodes a CAD isoform involved in lignin biosynthesis. The other cDNA, MsaCad1, has not been reported previously in lucerne, and encodes a protein related to the ELI3 class of elicitor-inducible defence-related plant proteins. The MsaCad1- and MsaCad2-encoded proteins were expressed in Escherichia coli and CAD1 was shown to be active with a range of cinnamyl, benzyl and aliphatic aldehyde substrates, while CAD2 was specific for the cinnamyl aldehydes only. Each of the respective genes is present as one or two copies. The MsaCad1 gene is expressed most actively in stem and floral tissue, whereas MsaCad2 is most actively expressed in stem, hypocotyl and root tissue. In stem tissue, expression of both genes occurs predominantly in internodes 4 and 5 (from the apex). MsaCad2, in contrast to MsaCad1, is not significantly expressed in the top three internodes of the stem. Both MsaCad1 and MsaCad2 are wound-inducible, and the wound-responsiveness of each gene is modulated by salicylic acid.     

Identification of a cDNA encoding 6-phosphogluconate dehydrogenase from a human heart cDNA library

A full-length cDNA clone for human 6-phosphogluconate dehydrogenase (PGD) was isolated from a human adult heart cDNA library. The clone encoded an open reading frame of 483 amino acids. When the amino acid sequences of human PGD and sheep PGD were aligned, 94.2% identity between these two proteins was found. Its calculated molecular weight is 53,149 daltons. The predicted isoelectric point is 6.85. When the secondary structure of human PGD was examined by the PROSIS software, 36% α-helix and 9% β-sheet were found.     

Salt-inducible betaine aldehyde dehydrogenase from sugar beet: cDNA cloning and expression

Members of the Chenopodiaceae, such as sugar beet and spinach, accumulate glycine betaine in response to salinity or drought stress. The last enzyme in the glycine betaine biosynthetic pathway is betaine aldehyde dehydrogenase (BADH). In sugar beet the activity of BADH was found to increase two- to four-fold in both leaves and roots as the NaCl level in the irrigation solution was raised from 0 to 500 mM. This increase in BADH activity was paralleled by an increase in level of translatable BADH mRNA. Several cDNAs encoding BADH were cloned from a gt10 libary representing poly(A)⁺ RNA from salinized leaves of sugar beet plants, by hybridization with a spinach BADH cDNA. Three nearly full-length cDNA clones were confirmed to encode BADH by their nucleotide and deduced amino acid sequence identity to spinach BADH; these clones showed minor nucleotide sequence differences consistent with their being of two different BADH alleles. The clones averaged 1.7 kb and contained an open reading frame predicting a polypeptide of 500 amino acids with 83% identity to spinach BADH. RNA gel blot analysis of total RNA showed that salinization to 500 mM NaCl increased BADH mRNA levels four-fold in leaves and three-fold in the taproot. DNA gel blot analyses indicated the presence of at least two copies of BADH in the haploid sugar beet genome.     

Proteolytic and partial sequencing studies of the bifunctional dihydrofolate reductase-thymidylate synthase from Daucus carota

The bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) of Daucus carota has been further characterized as regards molecular weight, amino acid composition, protease digestion and microsequencing of proteolytic peptides. Data reported in this paper demonstrate that the carrot protein has a calculated M _r of 124000 thus indicating that, contrarily to what has previously been suggested, it occurs as a dimer of identical subunits. Results of partial amino acid microsequencing show the presence of sequences highly homologous with those of the active sites of both DHFR and TS from other organisms confirming, at the structural level, the bifunctional nature of the carrot protein. As in the case of Leishmania tropica DHFR-TS, incubation of the carrot protein with V8 protease led to a rapid loss of TS activity while retaining that of DHFR. However the pattern of proteolysis did not allow to establish whether the sequence of domains is DHFR-TS as in Leishmania, or vice versa. Low homology of other amino acid sequences, as judged by computer analysis, and absence of common epitopes indicate an apparent divergence between carrot and leishmanian proteins.     

Isolation and characterization of a soybean cDNA clone encoding the plastid form of aspartate aminotransferase

Five aspartate aminotransferase (EC 2.6.1.1; AAT) isozymes were identified in soybean seedling extracts and designated AAT1 to AAT5 based on their rate of migration on non-denaturing electrophoretic gels. AAT1 was detected only in extracts of cotyledons from dark-grown seedlings. AAT3 and AAT4 were detected in crude extracts of leaves and in cotyledons of seedlings grown in the light. AAT2 and AAT5 were detected in all tissues examined. A soybean leaf cDNA clone, pSAT17, was identified by hybridization to a carrot AAT cDNA clone at low stringency. pSAT17 had an open reading frame which could encode a 50 581 Da protein. Alignment of the deduced amino acid sequence from the pSAT17 open reading frame with mature AAT protein sequences from rat disclosed a 60 amino acid N-terminal extension in the pSAT17 protein. This extension had characteristics of a plastid transit peptide.A plasmid, pEXAT17, was constructed which encoded the mature protein lacking the putative chloroplast transit polypeptide. Transformed Escherichia coli expressed a functional soybean AAT isozyme, which comigrated with the soybean AAT5 isozyme during agarose gel electrophoresis. Differential sucrose gradient sedimentation of soybean extracts indicated that AAT5 specifically cofractionated with chloroplasts. Antibodies raised against the pEXAT17-encoded AAT protein specifically reacted with the AAT5 isozyme of soybean and not with any of the other isozymes, indicating that the soybean cDNA clone, pSAT17, encodes the chloroplast isozyme, AAT5.     

Identification and characterisation of cDNA clones encoding cinnamyl alcohol dehydrogenase from tobacco

Cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) is an enzyme involved in lignin biosynthesis. We have previously isolated pure CAD enzyme as two closely related polypeptides of 44 and 42.5 kDa from tobacco stems. In this paper, we report partial amino acid sequences of these two polypeptides. Based on the peptide sequences mixed oligonucleotides were used to screen a tobacco stem cDNA library and CAD cDNA clones encoding the two polypeptides were identified. DNA sequence comparisons indicate very high sequence identity between these clones both in the coding and in the 5 and 3 untranslated sequences. The close similarity between the two CAD genes leads us to suggest that they do not represent different isoforms but are the same gene from each of the two parental lines of Nicotiana tabacum cv. Samsun. Sequence comparisons with alcohol dehydrogenase 1 (ADH1) from yeast shows sequence similarities of ca. 30%, while comparisons with maize, barley and potato ADH1 sequences show similarities of not more than 23%.Abbreviations CAD cinnamyl alcohol dehydrogenase (EC 1.1.1.195) - ADH alcohol dehydrogenase (EC 1.1.1.1)     

Characterization of the non-specific lipid transfer protein EP2 from carrot (Daucus carota L.)

The extracellular protein EP2 was previously identified as non-specific lipid transfer protein based on its cDNA-derived amino acid sequence. Here, the purification of the EP2 protein from the medium of somatic embryo cultures is described. After two cycles of ion-exchange and gel permeation chromatography, a single silver-stained protein band with an apparent molecular mass of 10 kDa was observed on SDS-PAGE. This protein band was recognized by the antiserum raised against a EP2--galactosidase fusion-protein. Employing a fluorescent phospholipid analog, it was shown that the purified EP2 protein is capable of binding phospholipids and is able to enhance their transfer between artificial membranes. Employing a gel permeation assay, it could be demonstrated that the EP2 protein is also capable of binding palmitic and oleic acid as well as oleyl-CoA. Because in plants these fatty acids are used as precursor molecules for cutin, these results are in support of the proposed role of the EP2 protein to transport cutin monomers from their site of synthesis through the cell wall of epidermal cells to sites of cutin polymerization.     

Isolation of a cDNA encoding a protease from Perinereis aibuhitensis Grube

The cDNA encoding a protease of Perinereis aibuhitensis Grube （PPA） was cloned. The deduced amino acid sequence analysis showed that the protein had 49% identity to the C-terminal amino acid 169-246 of serine protease of Heterodera glycines. Northern blotting analysis indicated that the cDNA could hybridize with mRNA of approximately 260 bases isolated from the marine earthworm. The cDNA was amplified by polymerase chain reaction and cloned into pMAL-p2 to construct expression vector pMAL-PPA. pMAL-PPA was introduced into Escherichia coli BL21（DE3） and overexpression of PPA fused with maltose binding protein was achieved by isopropyl-β-D-thiogalactopyranoside induction. The fusion protein was purified by affinity chromatography on an amylose resin column and ion-exchange chromatography on a diethylaminoethyl-Sepharose 4B column. Rabbits were immunized with the purified protein and antiserum was prepared. The antibody could react with a protein of approximately 9 kDa extracted from the marine earthworm as shown by Western blotting analysis. The activity analysis of the recombinant PPA suggested that it was probably a plasminogen activator.     

Isolation and characterization of a cDNA encoding for a lysozyme from the gut of the reduviid bug Triatoma infestans

We have isolated and characterised a Triatoma infestans cDNA encoding a lysozyme. A 174-bp fragment was amplified by PCR using degenerate oligodeoxyribonucleotide primers derived from the known amino acid sequences of lysozyme from other insects. This PCR fragment was used to screen a cDNA gut library of T. infestans. A clone containing the 3'-end of the lysozyme cDNA (219 bp) was isolated and sequenced. RACE was used to amplify the 5'-end of the lysozyme cDNA. After sequencing the complete lysozyme cDNA, the deduced 417 amino acid sequence showed high identity (40-50%) with other chicken-type lysozymes. The amino acid residues responsible for the catalytic activity and the binding of the substrate were essentially conserved. The expression pattern of the lysozyme gene in bugs at different molting and feeding states showed that this gene was upregulated in the digestive tract directly after the molt and after feeding. Additionally, this lysozyme gene was expressed differently in the different regions of the digestive tract, strongly in the cardia and stomach, the anterior regions of the midgut, and only traces of lysozyme mRNA could be detected in the small intestine, the posterior region of the midgut.     

Chitinase profiles in mature carrot (Daucus carota) roots and purification and characterization of a novel isoform

The profile of chaitinases (EC 3.2.1.14) in mature carrot ( Daucus carota L. cv. Eagle) roots was studied. Multiple chitinase bands (8–10) were observed in native and sodium dodecylsulfate-denaturing polyacrylamide gels. The molecular masses of these chitinases were estimated to be from 20 000 to 40 000. One major chitinase was purified and found to be an acidic protein with pI at 4.3 and a molecular mass of 39 500. The optimum pH for enzymatic activity was around 5 and the optimum temperature was 25°C. The enzyme was stable at pHs below 8 and temperatures below 60°C. The protein did not have a chitin-binding domain, but showed some similarity to the amino acid composition of tobacco class I chitinase. The N-terminal amino acid sequence did not resemble any of the described classes of chitimases. This chitinase did not possess lysozyme activity and showed antifungal activity when tested against Trichoderma sp.     

Proliferation-dependent pattern of expression of a dihydrofolate reductase-thymidylate synthase gene from Daucus carota

The pattern of expression of a carrot dhfr-ts gene was evaluated in different plant organs, in somatic embryos, and in hypocotyl explants induced to dedifferentiate in vitro by the addition of the synthetic auxin 2,4 dichorophenoxyacetic acid. The promoter of this gene was also placed upstream of a uidA (GUS) reporter gene and, using biolistic and protoplasts transient expression assays, was shown to drive a particularly high level of expression in actively growing suspension cells. The results from these expression analyses combined with the presence of putative cell cycle-related cis-acting elements in the dhfr-ts promoter, strongly point to a cell division-dependent expression of this gene.