Rice ubiquitin promoters: deletion analysis and potential usefulness in plant transformation systems

Strong constitutive promoters form a cornerstone for basic and applied research using transgenic plants. GUS (beta-glucuronidase) expression levels from constructs containing RUBQ1 or RUB2 rice ubiquitin promoters were 8- to 35-fold higher in transgenic rice [Oryza sativa (L.)] plants, respectively, when compared to the 35S promoter. Deletion analysis of the 5'-upstream region of RUBQ2 revealed a putative enhancer region that produced a 2.4-fold increase in transient GUS expression. Southern blot analysis showed that three to seven copies of the GUS gene were stably inserted into R0 and R1 plants and inherited in a monogenic fashion.     

Self cloning in Micromonospora olivasterospora of fms genes for fortimicin A (astromicin) biosynthesis.

Summary We have cloned the seven genes that are responsible for biosynthesis of the antibiotic fortimicin A (FTM A) using a recently developed self-cloning system that employs the plasmid vector pMO116 for Micromonospora olivasterospora. Five chimeric plasmids that restored FTM A production in M. olivasterospora mutants blocked at different biosynthetic steps were isolated by shotgun cloning. Secondary transformation using other non-producing mutants showed that two additional FTM A biosynthetic genes were included on these plasmids, and that at least four of the genes were clustered. Interestingly AN38-1, a non-producing mutant that had a defect in dehydroxylation of a precursor of FTM A, was complemented by the DNA fragment containing a neomycin resistance gene that had been cloned from a neomycin-producing strain (Micromonospora sp. FTM A non-producing strain) in the course of constructing the plasmid vector pM0116. These results clearly show that this novel gene cloning system in Micromonospora is of practical use.     

Enzymatic reduction of (+)-dihydroflavonols to flavan-3,4-cis-diols with flower extracts from Matthiola incana and its role in anthocyanin biosynthesis

A cell-free extract from flowers of Matthiola incana catalyzes a NADPH-dependent stereospecific reduction of (+)-dihydrokaempferol to 3,4-cis-leucopelargonidin (5,7,4-trihydroxyflavan-3,4-cis-diol). The pH-optimum of this reaction is around 6. The rate of reaction with NADH was about 50% of that found with NADPH. (+)-Dihydroquercetin and (+)-dihydromyricetin were also reduced by the enzyme preparation to the corresponding flavan-3,4-cis-diols. Correlation between the genotype of M. incana and the presence of dihydroflavonol 4-reductase is strong evidence that this enzyme is involved in anthocyanin biosynthesis.     

Evidence for multiple carboxymethylcellulase genes in Pseudomonas fluorescens subsp. cellulosa

Summary A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA was constructed in bacteriophage 47.1 and recombinants expressing carboxymethylcellulase (CMCase) activity isolated. A 7.3 kb partial EcoRI fragment, a 9.4 kb EcoRI fragment and a 5.8 kb HindIII fragment were subcloned from three different phages into pUC18 to yield recombinant plasmids pJHH1, pJHH3 and pGJH2 respectively. Cells of Escherichia coli harbouring these plasmids expressed CMCase activity. The positions of the CMCase genes in the three plasmids were determined by subcloning and transposon mutagenesis. pJHH1 contained two distinct DNA regions encoding CMCases, which were controlled by the same promoter. All four cloned enzymes cleaved p-nitrophenyl--D-glucopyranoside, although at a very low rate, but none exhibited exoglucanase activity. In common with other extracellular enzymes cloned in E. coli, all the CMCases were exported to the periplasmic space in the enteric bacterium. The carboxymethylcellulase genes encoded by pJHH1 and pJHH3, were subject to glucose repression in E. coli.Abbreviations SSC 0.15 M NaCl, 0.015 M sodium citrate - Sm^r resistance to streptomycin - Km^r resistance to kanamycin - Ap^r resistance to ampicillin - Tc^r resistance to tetracycline - Cm^r resistance to chloramphenicol - CMCase carboxymethylcellulase     

A membrane-bound nitrate reductase encoded by the narGHJI operon is responsible for anaerobic respiration in Halomonas maura

The halophilic bacterium Halomonas maura is capable of anaerobic respiration on nitrates. By insertional mutagenesis with the minitransposon Tn-5 we obtained the mutant Tc62, which was incapable of anaerobic respiration on nitrates. An analysis of the regions adjacent to the transposon allowed us to characterize the membrane-bound anaerobic-respiratory nitrate reductase narGHJI gene cluster in H. maura. We identified consensus sequences for fumarate and nitrate reductase regulator (FNR)-like protein-binding sites in the promoter regions of the nar genes and consensus sequences corresponding to the NarL binding sites upstream of the nar genes. RT-PCR analysis showed that the narGHJI operon was expressed in response to anaerobic conditions when nitrate was available as electron acceptor. This membrane-bound nitrate reductase is the only enzyme responsible for anaerobic respiration on nitrate in H. maura. In this article we discuss the possible relationship between this enzyme and a dissimilatory nitrate-reduction-to-ammonia process (DNRA) in H. maura and its role in the colonization of the rhizosphere.     

Generation and characterization of transgenic poplar plants overexpressing a cotton laccase gene

Laccases are copper-containing glycoproteins, which are widespread in higher plants as multigene families. To gain more insight in the function of laccases in plants, especially potential role in lignification, we produced transgenic poplar plants overexpressing a cotton laccase cDNA (GaLAC1) under the control of the cauliflower mosaic virus 35S promoter. As compared with untransformed control plants, transgenic plants exhibited a 2.1- to 13.2-fold increased laccase activity, whereas plant growth rate and morphological characters remained similar to control plants. A 2.1–19.6% increase in total lignin content of the stem was found in transgenic plants. Moreover, transgenic plants showed a dramatically accelerated oxidation rate of phenolics, without obvious change in total phenolic content. Our data suggested that GaLAC1 may participate in lignin synthesis and phenolic metabolism in plants. The present work provided a new genetic evidence for the involvement of plant laccases in lignification.     

Short communication: Construction of a cloning vector for Paenibacillus polymyxa and P. azotofixans

A cloning vector that could replicate in Paenibacillus polymyxa, P. azotofixans and Bacillus subtilis was constructed using two Staphylococcus aureus plasmids. The recombinant plasmid confers chloramphenicol and erythromycin resistance and contains unique restriction sites for PvuII and BclI. The stability of pRJ45 was analysed.     

A new DNA-cloning vector forHaemophilus influenzae Rd.

A new, high-efficiency, DNA-cloning vector pJ1-8 was derived in two steps from the chimeric plasmid pD7 consisting of RSF 0885(amp ^r) andHaemophilus influenzae chromosomal DNA. pJl-8 has only oneEcoRI site and a molecular weight of only 2.5 × 10⁶. No detectableamp ^r transformation was obtained with pJl-8 DNA. However,amp ^r transformation increases markedly ifHaemophilus influenzae chromosomal DNA segments are spliced into it, providing a very facile assay for detecting inserts.     

Inhibitors of aldose reductase and advanced glycation end-products formation from the leaves of Stelechocarpus cauliflorus R.E. Fr.

Two dihydroflavonol glycosides, engeletin and astilbin, were isolated from an EtOAc extract of the leaves of Stelechocarpus cauliflorus R.E. Fr. (Annonaceae). The inhibitory activity of engeletin against a recombinant human aldose reductase (IC₅₀ value=1.16 μM) was twice that of quercetin as a positive control (2.48 μM), and 23 times greater than that of astilbin (26.7 μM). Engeletin inhibited the enzyme uncompetitively. Astilbin was about as potent as the positive control, quercetin, in its inhibition of advanced glycation end-products formation. These flavonoids displayed therapeutic potential in the prevention and treatment of diabetic complications.     

A killer-rescue system for self-limiting gene drive of anti-pathogen constructs

A number of genetic mechanisms have been suggested for driving anti-pathogen genes into natural populations. Each of these mechanisms requires complex genetic engineering, and most are theoretically expected to permanently spread throughout the target species' geographical range. In the near term, risk issues and technical limits of molecular methods could delay the development and use of these mechanisms. We propose a gene-drive mechanism that can be self-limiting over time and space, and is simpler to build. This mechanism involves one gene that codes for toxicity (killer) and a second that confers immunity to the toxic effects (rescue). We use population-genetic models to explore cases with one or two independent insertions of the killer gene and one insertion of the rescue gene. We vary the dominance and penetrance of gene action, as well as the magnitude of fitness costs. Even with the fitness costs of 10 per cent for each gene, the proportion of mosquitoes expected to transmit the pathogen decreases below 5 per cent for over 40 generations after one 2:1 release (engineered:wild) or after four 1:2 releases. Both the killer and rescue genes will be lost from the population over time, if the rescue construct has any associated fitness cost. Molecular approaches for constructing strains are discussed.     

Alternative gene for biotin sulfoxide reduction inEscherichia coli K-12

A gene at 42 min on theEscherichia coli chromosome, identified as the locus of pseudoreversion of knockout mutations in the biotin sulfoxide reductase gene,bisC, has 64% base sequence identity withbisC. This makes it a member of a multigene family of molybdopterin enzymes that includes genes for anaerobic reduction of trimethylamine oxide (torA) and dimethylsulfoxide (dmsA). Disruption of this gene eliminates the background activity of biotin sulfoxide reduction observed inbisC mutants. Sequence comparison of the new gene (bisZ) withbisC indicates that certaints mutants ofbisC arise by gene conversion between the two loci.     

Construction and evaluation of pMycoFos, a fosmid shuttle vector for Mycobacterium spp. with inducible gene expression and copy number control

Molecular tools for Gram-positive bacteria such as Mycobacterium are less well-developed than those for Gram-negatives such as Escherichiacoli. This has slowed the molecular-genetic characterisation of Mycobacterium spp, which is unfortunate, since this genus has high medical, environmental and industrial significance. Here, we developed a new Mycobacterium shuttle vector (pMycoFos, 12.5 kb, Km^R) which combines desirable features of several previous vectors (controllable copy number in E. coli, inducible gene expression in Mycobacterium) and provides a new multiple cloning site compatible with large inserts of high-GC content DNA. Copy number control in E. coli was confirmed by the increased Km^R of cultures after arabinose induction and the greater DNA yield of vector from arabinose-induced cultures. Measurement of beta-galactosidase activity in pMycoFos clones carrying the lacZ gene showed that in Mycobacterium smegmatis mc²-155, expression was inducible by acetamide, but in E. coli EPI300, the expression level was primarily determined by the vector copy number. Examination of protein profiles on SDS-PAGE gels confirmed the beta-galactosidase assay results. Construction of a fosmid library with the new vector confirmed that it could carry large DNA inserts. The new vector enabled the stable cloning and expression of an ethene monooxygenase gene cluster, which had eluded previous attempts at heterologous expression.     

Effects of constitutive expression of nitrate reductase in transgenic Nicotiana plumbaginifolia L. in response to varying nitrogen supply

Transformed Nicotiana plumbaginifolia plants with constitutive expression of nitrate reductase (NR) activity were grown at different levels of nitrogen nutrition. The gradients in foliar NO ₃ ^– content and maximum extractable NR activity observed with leaf order on the shoot, from base to apex, were much decreased as a result of N-deficiency in both the transformed plants and wild type controls grown under identical conditions. Constitutive expression of NR did not influence the foliar protein and chlorophyll contents under any circumstances. A reciprocal relationship between the observed maximal extractable NR activity of the leaves and their NO ₃ ^– content was observed in plants grown in nitrogen replete conditions at low irradiance (170 mol photons·m^–2 ·s^–1). This relationship disappeared at higher irradiance (450 mol photons·m^–2·S^–1) because the maximal extractable NR activity in the leaves of the wild type plants in these conditions increased to a level that was similar to, or greater than that found in constitutive NR-expressors. Much more NO ₃ ^– accumulated in the leaves of plants grown at 450 mol photons·m^–2·s^–1 than in those grown at 170 mol photons·m^–2·s^–1 in N-replete conditions. The foliar NO ₃ ^– level and maximal NR activity decreased with the imposition of N-deficiency in all plant types such that after prolonged exposure to nitrogen depletion very little NO ₃ ^– was found in the leaves and NR activity had decreased to almost zero. The activity of NR decreased under conditions of nitrogen deficiency. This regulation is multifactoral since there is no regulation of NR gene expression by NO ₃ ^– in the constitutive NR-expressors. We conclude that the NR protein is specifically targetted for destruction under nitrogen deficiency. Consequently, constitutive expression of NR activity does not benefit the plant in terms of increased biomass production in conditions of limiting nitrogen.Abbreviations Chl chlorophyll - N nitrogen - NR NADH-nitrate reductase - WT wild type     

A novel method for the cloning of chromosomal mutations in a single step: Isolation of two mutant alleles of envZ, an osmoregulatory gene from Escherichia coli

Summary We have developed a simple, rapid and powerful method for the cloning of chromosomal mutations from total cellular DNA in a single step using a plasmid carrying the clined wild-type locus of interest and a convenient selectable marker such as antibiotic resistance. This method relies upon the ability of the cloned wild-type gene to form a heteroduplex with the mutant chromosomal locus. The plasmid from primary transformants can be screened rapidly by size; more than 50% of plasmids of the correct size contained the mutant locus. When this method was used to clone two chromosomal mutations in the envZ gene of Escherichia coli, a locus which encodes a membrane-bound sensory protein involved in the osmoregulation of outer membrane porin biosynthesis, more than 50% of the retransformants from the plasmids selected by size were found to exhibit the mutant phenotype. Preliminary characterization of these mutant alleles is discussed. This novel and powerful method should be generally applicable in any system where the cloned locus is available.This work was presented at the 86th Annual Meeting of the American Society for Microbiology, March 1986, Washingnton, D.C.     

New proteins orthologous to cerato-platanin in various <Emphasis Type="Italic">Ceratocystis</Emphasis> species and the purification and characterization of cerato-populin from <Emphasis Type="Italic">Ceratocystis populicola</Emphasis>

Natural variants of cerato-platanin (CP), a pathogen associated molecular pattern (PAMP) protein produced by Ceratocystis platani (the causal agent of the plane canker stain), have been found to be produced by other four species of the genus Ceratocystis, including five clones of Ceratocystis fimbriata isolated from different hosts. All these fungal strains were known to be pathogenic to plants with considerable importance in agriculture, forestry, and as ornamental plants. The putative premature proteins were deduced on the basis of the nucleotide sequence of genes orthologous to the cp gene of C. platani; the deduced premature proteins of Ceratocystis populicola and Ceratocystis variospora reduced the total identity of all the others from 87.3% to 60.3%. Cerato-populin (Pop1), the CP-orthologous protein produced by C. populicola, was purified and characterized. Pop1 was a well-structured α/β protein with a different percentage of the α-helix than CP, and it self-assembled in vitro in ordered aggregates. Moreover, Pop1 behaved as PAMP, since it stimulated poplar leaf tissues to activate defence responses able to reduce consistently the C. populicola growth. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Anaerobic induction of the maize<Emphasis Type="Italic"> GapC4</Emphasis> promoter in poplar leaves requires light and high CO<Subscript>2</Subscript>

The maize (Zea mays L.) glyceraldehyde-3-phosphate dehydrogenase gene 4 (GapC4) promoter confers anaerobic gene expression in tobacco (Nicotiana tabacum L.), potato (Solanum tuberosum L.) and Arabidopsis thaliana (L.) Heynh. Here we have investigated its expression in hybrid poplar (Populus tremula × P. alba). Our results show that the promoter is not expressed in leaves and stems under normoxic conditions while anaerobiosis induces reporter gene expression in leaves up to a level observed for the STLS-1 promoter from potato that is shown to confer leaf-specific gene expression in transgenic poplar. Anaerobic induction is cell autonomous and requires a CO₂ atmosphere and light. As in tobacco, the GapC4 promoter in poplar is wound inducible. The induction by CO₂ and light may reflect a natural situation because flooding, a natural cause of anaerobiosis, is often accompanied by high CO₂ concentrations in the floodwater. Our results show that the GapC4 promoter is suitable as an anaerobic reporter and as an inducible gene expression system in poplar.Abbreviations CaMV cauliflower mosaic virus - GapC4 glyceraldehyde-3-phosphate dehydrogenase gene 4 - GUS -glucuronidase - 4-MU methylumbelliferone - STLS-1 stem- and leaf-specific promoter 1