Structure and expression of a sugarcane gene encoding a housekeeping phosphoenolpyruvate carboxylase

A gene (SCPEPCD1) encoding phosphoenolpyruvate carboxylase (PEPC) was isolated from the C-4 monocot sugarcane (Saccharum hybrid var. H32-8560). SCPEPCD1 is ca. 6800 bp long, with 10 exons. The entire gene sequence from –1561 to 262 bp downstream of the putative poly(A) addition signal is reported. A low-level, essentially constitutive pattern of expression, amino acid sequence similarities to other housekeeping PEPC enzymes, and the absence of DNA sequence elements conserved in the upstream region of maize and sorghum C-4-specific PEPC genes indicate that SCPEPCD1 encodes a housekeeping PEPC. Despite this, a motif proposed to act as a phosphorylation site in light-mediated activation of photosynthetic PEPC enzymes [10] is present in the SCPEPCD1 protein; evidence is presented for the presence of this site in other housekeeping PEPC proteins.     

The phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum: molecular cloning, nucleotide sequence, and expression

Summary The ppc gene of Corynebacterium glutamicum encoding phosphoenolpyruvate (PEP) carboxylase was isolated by complementation of a ppc mutant of Escherichia coli using a cosmid gene bank of chromosomal c. glutamicum DNA. By subsequent subcloning into the plasmid pUC8 and deletion analysis, the ppc gene could be located on a 3.3 kb SalI fragment. This fragment was able to complement the E. coli ppc mutant and conferred PEP carboxylase activity to the mutant. The complete nucleotide sequence of the ppc gene including 5 and 3 flanking regions has been determined and the primary structure of PEP carboxylase was deduced. The sequence predicts a 919 residue protein product (molecular weight of 103154) which shows 34% similarity with the respective E. coli enzyme. Present address: Institut für Biotechnologie 1 der Kernforschungsanlage, Postfach 1913, D-5170 Jülich, Federal Republic of Germany     

Differential expression of individual genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase in Lemna gibba

The gene family encoding the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase in the monocot Lemna gibba contains approximately twelve members. We have isolated six of these genes from a genomic library, and sequenced five of the coding regions. The transit peptide nucleotide sequences are conserved, but less highly than the mature polypeptide coding sequence. The mature polypeptide amino acid sequences are identical to each other and to the sequence deduced from a cDNA clone derived from a seventh gene. Each of the five fully characterized genomic sequences contains a single intron in precisely the same position as the second intron of several dicots. The intron sequences differ in length and are less conserved than the coding sequences.The 3-untranslated regions of the different genes have been sequenced and used to prepare gene-specific probes. These probes have been used to study the expression levels of individual rbcS sequences. Expression of six of the seven genes can be detected in total RNA isolated from plants grown in continuous light. The levels of RNA encoded by each expressed gene are regulated by the action of phytochrome, but there is variability in the amount of expression of each RNA.     

High-level salivary gland expression in transgenic mice

A 7.1 kb mini-gene construct containing cloned DNA from the murine parotid secretory protein (PSP) gene with 6.2 kb of the promoter, has previously been shown to direct specific mRNA expression to the salivary glands in transgenic mice. However, the level of transgene expression in the parotid gland was only a few percent of the endogenous level. This indicated that elements necessary for high-level expression are still to be found. In this study, we have searched for such regulatory elements in additional flanking regions by using a 25 kb clonedPsp ^b fragment containing the complete structural gene, 11.4 kb of 5-flanking sequence, and 2.5 kb 3-flanking sequence as a transgene. To distinguish the expression of the transgene from that of the endogenous gene, we took advantage of an allelic difference, using an oligonucleotide that recognized the mRNA fromPsp ^b and the transgene but not that from the other allele,Psp ^a . The expression of the transgene was examined in animals homozygous forPsp ^a . Three independent integrations all exhibited a level of parotid-gland-specific expression that corresponded to that of the endogenous gene. Thus, sequences responsible for this high-level PSP mRNA expression are situated within the genomic DNA of the transgene.     

Structural characterization,expression analysis and evolution of the red/far-red sensing photoreceptor gene,phytochrome C (<Emphasis Type="Italic">PHYC</Emphasis>), localized on the ‘B’ genome of hexaploid wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Phytochromes are a family of red/far-red light perceiving photoreceptors.The monocot phytochrome family is represented by three members, PHYA, PHYB and PHYC. We have isolated and characterized the first PHY gene member (TaPHYC) from common wheat, Triticum aestivum var. CPAN1676. It codes for a species of the photoreceptor, phyC, which is known to be light-stable in all plants analyzed so far. A sequence of 7.2 kb has been determined, which includes 3.42 kb of coding region.This is the second full-length PHYC gene sequenced from a monocot (first was from rice). TaPHYC gene shares structural similarities with the rice PHYC containing four exons and three introns in the coding region. The 5 UTR is 1.0-kb-long and harbors an upstream open reading frame (URF) encoding 28 aa. Southern blot analysis of TaPHYC indicates that it represents single locus in the wheat genome, although the possibility of additional loci cannot be completely ruled out. Chromosomal localization using nullisomic–tetrasomic lines of Triticum aestivum var. Chinese Spring places TaPHYC on chromosome 4B. PHYC represents a constitutively expressed gene in all the organs tested and under light/dark conditions. However, PHYC was found to be developmentally regulated showing maximal expression in 3-day-old dark-grown seedlings, which declined thereafter. In silico analysis has also been done to compare TaPHYC gene with the partial sequences known from other wheat species and cultivars. The presence of a topoisomerase gene immediately downstream of the PHYC gene, both in rice and wheat genomes, presents yet another example of synteny in cereals and its possible significance has been discussed.     

Organization and expression of the two homologous genes encoding the NADP-malate dehydrogenase in Sorghum vulgare leaves

Summary We previously described the isolation and the nucleotide sequence of a nuclear gene from sorghum (NMDHI; 4.6 kb) encoding the NADP-malate dehydrogenase. Further analysis led us to identify a second homologous gene (NMDH II; 4.8 kb) located within the same 12.3 kb genomic clone (LM17); these two genes are tandemly organized, in direct orientation. This second gene was entirely sequenced and comparison with the first gene showed that the positions on the 14 exons and 13 introns are conserved in both genes. The analysis of the genomic organization and copy number in the Sorghum vulgare genome revealed that there are no additional homologues and there is only one copy each of NMDH I and NMDH II. The isolation of two different cDNA clones in a previous work suggested that both genes were probably expressed. Analysis of specific mRNA accumulation during the greening process using synthetic oligonucleotide probes showed that the NMDH I gene is induced in the presence of light while the NMDH II gene seems to be constitutively expressed at low level.Abbreviations Cab chlorophyll a/b binding protein - CTAB N-cetyl-N,N,N-trimethyl-ammonium bromide - NADP-MDH NADP-dependent dehydrogenase - RbcS ribulose-1,5-bisphosphate carboxylase small subunit - SSC 0.15 M NaCl, 0.015 M sodium citrate pH 7.6     

Isolation of Xenopus HGF gene promoter and its functional analysis in embryos and animal caps

Previously, we isolated Xenopus HGF (hepatocyte growth factor) cDNA and showed in Xenopus embryos that expression of this gene starts at the late gastrula stage mainly in the ventral mesoderm, and furthermore that the expression is induced in animal cap by activin A and bFGF (basic fibroblast growth factor). Here we have cloned the Xenopus HGF gene, covering a 14 kb 5-upstream region and a 0.2 kb 5-coding region. Within about 0.5 kb of the 5-flanking region, the Xenopus HGF gene contained a TATA-like element AATGAAA, one putative NF-1 binding site, two NF-IL-6 binding motif sequences, one putative TGF--dependent inhibitory element (TIE) and one AP-1 binding site. A recombinant circular plasmid consisting of a 1.7 kb HGF promoter region and the bacterial chloramphenicol acetyltransferase (CAT) gene was first expressed at the late gastrula stage in the ventral mesoderm, as was the endogenous HGF gene. The expression of the fusion gene was induced in animal cap cells by activin A and bFGF although induction by the latter was not so strong. Using a series of 5-deletion constructs introduced into animal caps, silencer elements, which seem to be essential for the gene's regionally correct expression, and the element responsible for induction by activin were found. The results show that the HGF gene promoter isolated here contains elements which may endow the gene with the regulative function for its temporally and spatially regulated expression, although the element necessary for induction by bFGF seems to be missing.     

Developmental regulatory elements in the 5′ flanking DNA of the Drosophila choline acetyltransferase gene

Summary Choline acetyltransferase (ChAT, EC 2.3.1.6) catalyzes the production of the neurotransmitter acetylcholine, and is an essential factor for neurons to be cholinergic. We have analyzed regulation of the Drosophila ChAT gene during development by examining the -galactosidase expression pattern in transformed lines carrying different lengths of 5 flanking DNA fused to a lacZ reporter gene. The largest fragment tested, 7.4 kb, resulted in the most extensive expression pattern in embryonic and larval nervous system and likely reflects all the cis-regulatory elements necessary for ChAT expression. We also found that 5 flanking DNA located between 3.3 kb and 1.2 kb is essential for the reporter gene expression in most of the segmentally arranged embryonic sensory neurons as well as other distinct cells in the CNS. The existence of negative regulatory elements was suggested by the observation that differentiating photoreceptor cells in eye imaginal discs showed the reporter gene expression in several 1.2 kb and 3.3 kb transformants but not in 7.4 kb transformants. Furthermore, we have fused the 5 flanking DNA fragments to a wild type ChAT cDNA and used these constructs to transform Drosophila with a Cha mutant background. Surprisingly, even though different amounts of 5 flanking DNA resulted in different spatial expression patterns, all of the positively expressing cDNA transformed lines were rescued from lethality. Our results suggest that developmental expression of the ChAT gene is regulated both positively and negatively by the combined action of several elements located in the 7.4 kb upstream region, and that the more distal 5 flanking DNA is not necessary for embryonic survival and development to adult flies. Correspondence to: P.M. Salvaterra     

Regulatory factors involved in gene expression (subunits of ribulose-1,5-bisphosphate carboxylase) in mustard (Sinapis alba L.) cotyledons

Phytochrome-controlled appearance of ribulose-1,5-bisphosphate carboxylase (RuBP-Case) and its subunits (large subunit LSU, small subunit SSU) was studied in the cotyledons of the mustard (Sinapis alba L.) seedling. The main results were as follows: (i) Control of RuBPCase appearance by phytochrome is a modulation of a process which is turned on by an endogenous factor between 30 and 33 h after sowing (25° C). Only 12 h later the process begins to respond to phytochrome. (ii) The rise in the level of RuBP-Case is the consequence of a strictly coordinated synthesis de novo of the subunits. (iii) While the levels of translatable mRNA for SSU are compatible with the rate of SSU synthesis the relatively high LSU mRNA levels are not reflected in the rates of in-vivo LSU or RuBPCase syntheses. (iv) Gene expression is also abolished in the case of nuclear-encoded SSU if intraplastidic translation and concomitant plastidogenesis is inhibited by chloramphenicol, pointing to a plastidic factor as an indispensable prerequisite for expression of the SSU gene(s). (v) Regarding the control mechanism for SSU gene expression, three factors seem to be involved: an endogenous factor which turns on gene expression, phytochrome which modulates gene expression, and the plastidic factor which is an indispensable prerequisite for the appearance of translatable SSU mRNA.Abbreviations CAP chloramphenicol - cFR continuous farred light - LSU large subunit of RuBPCase - NADP-GPD NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) - Pfr far-red-absorbing form of phytochrome - pSSU precursor of SSU - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - SSU small subunit of RuBPCase     

Inhibition of the circadian rhythm of CO2 metabolism in Bryophyllum leaves by cycloheximide and dinitrophenol

The circadian rhythm of CO₂ output in darkened leaves of Bryophyllum fedtschenkoi R. Hamet and Perrier can be inhibited by cycloheximide (10^-6 mol) and 2,4-dinitrophenol (10^-5 mol) applied via the transpiration stream. After having been suppressed by 10^-6 M cycloheximide, the rhythm can be reinitiated with a 12-h exposure to light. Experiments using ¹⁴CO₂ show that cycloheximide abolishes the rhythm by inhibiting the dark fixation of CO₂. Cycloheximide inhibits malate accumulation and acidification of the leaves, but does not affect the amount of the CO₂-fixing enzyme phosphoenol-pyruvate carboxylase (PEP-C, EC 4.1.1.31) which can be extracted from the leaves during the 45 h of the experiment. Cycloheximide has no direct effect on the activity of the enzyme as measured in the assay. PEP-C from desalted leaf extracts was inhibited by L-malate (K_i=0.4 mmol). The most likely explanation for the inhibitory effect of cycloheximide and dinitrophenol is that they cause changes in tonoplast properties which result in a redistribution of malate from the vacuole to the cytoplasm. An increase in malate concentration in the cytoplasm will lead to inhibition of PEP-carboxylase, and hence the suppression of the rhythm of CO₂ output.Abbreviations CAM crassulacean acid metabolism - PEP-C phosphoenol-pyruvate carboxylase - MDH malate dehydrogenase - CHM cycloheximide - DNP 2,4-dinitrophenol - LD light-dark-cycle - DD continuous darkness     

The sequence of a pea vicilin gene and its expression in transgenic tobacco plants

A 5.5 kb Eco RI fragment containing a vicilin gene was selected from a Pisum sativum genomic library, and the protein-coding region and adjacent 5 and 3 regions were sequenced. A DNA construction comprising this 5.5 kb fragment together with a gene for neomycin phosphotransferase II was stably introduced into tobacco using an Agrobacterium tumefaciens binary vector, and the fidelity of expression of the pea vicilin gene in its new host was studied. The seeds of eight transgenic tobacco plants showed a sixteen-fold range in the level of accumulated pea vicilin. The level of accumulation of vicilin protein and mRNA correlated with the number of integrated copies of the vicilin gene. Pea vicilin was confined to the seeds of transgenic tobacco. Using immunogold labelling, vicilin was detected in protein bodies of eight out of ten embryos (axes plus cotyledons) and, at a much lower level, in two out of eleven endosperms. Pea vicilin was synthesized early in tobacco seed development; some molecules were cleaved as is the case in pea seeds, yielding a major parental component of M _r50000 together with a range of smaller polypeptides.     

Structure and expression of the three MHC-linked HSP70 genes

A duplicated locus encoding the major heat shock-induced protein HSP70 is located in the major histocompatibility complex (MHC) class III region 92 kilobases (kb) telomeric to the C2 gene. Nucleotide sequence analysis of the two intronless genes, HSP70-1 and HSP70-2, has shown that they encode an identical protein product of 641 amino acids. A third intronless gene, HSP70-Hom, has also been identified 4 kb telomeric to the HSP70-1 gene. This encodes a more basic protein of 641 amino acids which has 90% sequence similarity with HSP70-1. In order to investigate the expression of the three (MHC)-linked HSP70 genes individually by northern blot analysis, we have isolated locus-specific probes from the 3 untranslated regions of the genes. The HSP70-1 and HSP70-2 genes have been shown to be expressed at high levels as a 2.4 kb mRNA in cells heat-shocked at 42°C. HSP70-1 is also expressed constitutively at very low levels. The HSP70-Hom gene, which has no heat shock consensus sequence in its 5 flanking sequence, is expressed as a 3 kb mRNA at low levels both constitutively and following heat shock.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M34267-9. Address correspondence and offprint requests to: R. D. Campbell.     

Systemic induction of a potato pin2 promoter by wounding,methyl iasmonate,and abscisic acid in transgenic rice plants

To address the question whether common signal(s) and transduction pathways are used to mediate a systemic wound response in monocot and dicot plants, a fusion of the potato proteinase inhibitor II gene (pin2) promoter and the bacterial -glucuronidase gene (Gus)-coding region was introduced into rice. In transgenic rice plants, the expression of the pin2-Gus fusion gene displays a systemic wound response, although the expression level is relatively low. Incorporation of the first intron from the rice actin 1 gene (Act1) into the 5-untranslated region of the pin2-Gus construct results in high-level, systemically wound-inducible expression of the modified construct in transgenic rice plants. Histochemical analysis shows that this high-level, wound-inducible expression is associated with the vascular tissue in both leaves and roots. Furthermore, the expression of the pin2-Act1 intron-Gus fusion gene in transgenic rice plants can be systemically induced by both methyl jasmonate (MJ) and the phytohormone abscisic acid (ABA). These results suggest that the signal(s) mediating the observed systemic wound response and certain steps of the transduction pathways are conserved between dicot and monocot plants. Transient expression assays show that the pin2-Act1 intron-Gus construct is also actively expressed in transformed cells and tissues of several other monocot plants. Thus, the wound-inducible pin2 promoter in combination with the rice Act1 intron 1 might be used as an efficient regulator for foreign gene expression in transgenic monocot plants.     

Effects of promoter,intron and enhancer elements on transient gene expression in sugar-cane and carrot protoplasts

Various chimaeric promoter regions coupled to the uidA -glucuronidase gene were evaluated for transient expression strength following electroporation into sugar-cane (monocot) and carrot (dicot) protoplasts. Multiple enhancer elements increased expression in sugar-cane, by up to 400-fold for the artificial Emu promoter relative to the CaMV 35S promoter. The relative expression strengths of promoters varied substantially between the species. Sugar-cane also differed in some respects from previously tested species in the family Poaceae. For example, in sugar-cane the nopaline synthase and CaMV 35S promoters were of equivalent strength, and insertion of Adh1 intron 1 into the 5 transcribed region decreased expression strength.     

Different mechanisms generating sequence variability are revealed in distinct regions of the hydroxyproline-rich glycoprotein gene from maize and related specie*

Summary The sequences of the genes coding for a hydroxyproline-rich glycoprotein from two varieties of maize (Zea mays, Ac1503 and W22), a teosinte (Zea diploperennis) and sorghum (Sorghum vulgare) have been obtained and compared. Distinct patterns of variability have been observed along their sequences. The 500 by region immediately upstream of the TATA box is highly conserved in theZea species and contains stretches of sequences also found in the sorghum gene. Further upstream, significant rearrangements are observed, even between the two maize varieties. These observations allow definition of a 5 region, which is common to the four genes and is probably essential for their expression. The 3 end shows variability, mostly due to small duplications and single nucleotide substitutions. There is an intron present in this region showing a high degree of sequence conservation among the four genes analyzed. The coding region is the most divergent, but variability arises from duplications of fragments coding for similar protein blocks and from single nucleotide substitutions. These results indicate that a number of distinct mechanisms (probably point mutation, transposon insertion and excision, homologous recombination and unequal crossing-over) are active in the production of sequence variability in maize and related species. They are revealed in different parts of the gene, probably as the result of the different types of functional constraints acting on them, and of the specific nature of the sequence in each region.The sequences reported in this paper have been deposited in the EMBL/GenBank Database (Bolt, Beranek, and Newman Laboratories, Cambridge, Mass., and EMBL, Heidelberg), accession nos. M36635 (maize Ac1503), X63134 (maize W22), X64173 (teosinte) and X56010 (sorghum)     

Expression of the Arabidopsis AtAux2-11 auxin-responsive gene in transgenic plants

Five constructions containing deletions of the promoter from an auxin-inducible gene of Arabidopsis thaliana, AtAux2-11, were fused to the coding region of the reporter gene LacZ, which encodes -galactosidase, and a polyadenylation 3-untranslated nopaline synthase sequence from Agrobacterium. These chimeric genes were introduced into Arabidopsis by Agrobacterium tumefaciens-mediated transformation, and expression of the gene was examined by spectrophotometric and histochemical analyses. A 600 bp fragment from the AtAux2-11 promoter conferred histochemical patterns of staining similar to the longest 5 promoter tested, a 3.0 kb fragment. Localization of AtAux2-11/LacZ activity in the transgenic plants revealed spatial and temporal expression patterns that correlated with tissues and cells undergoing physiological processes modulated by auxin. LacZ activity was expressed in the elongating region of roots, etiolated hypocotyls, and anther filaments. Expression was detected in the vascular cylinder of the root and the vascular tissue, epidermis, and cortex of the hypocotyl, and filament. The AtAux2-11/LacZ gene was preferentially expressed in cells on the elongating side of hypocotyls undergoing gravitropic curvature. Expression of the chimeric gene in the hypocotyls of light-grown seedlings was less than that in etiolated seedling hypcotyls. The AtAux2-11/LacZ gene was active in the root cap, and expression in the root stele increased at sites of lateral root initiation. Staining was evident in cell types that develop lignified cell walls, e.g. trichomes, anther endothecial cells, and especially developing xylem. The chimeric gene was not expressed in primary meristems. While the magnitude of expression increased after application of exogenous auxin (2,4-D), the histochemical localization of AtAux2-11/LacZ remained unchanged.Transgenic plants with a 600 bp promoter construct (–0.6 kb AtAux2-11/LacZ) had higher levels of basal and auxin-inducible expression than plants with a 3.0 kb promoter construct. Transgenic plants with a –500 bp promoter had levels of expression similar to the –3.0 kb construct. The –0.6 kb AtAux2-11/LacZ gene responded maximally to a concentration of 5 × 10^–6 to 5 × 10^–5 M 2,4-D and was responsive to as little as 5 × 10^–8 M. The evidence presented here suggests that this gene may play a role in several auxin-mediated developmental and physiological processes.co-first authors     

Isolation of the promoter of a root cap expressed pectinmethylesterase gene from Pisum sativum L. (rcpme1) and its use in the study of gene activity

A genomic clone of a pea pectinmethylesterase encoding gene, rcpme1, was isolated; the promoter region was found to include regions of homology to phenylalanine ammonia lyase (PAL) and nodulin gene promoters. Agrobacterium rhizogenes mediated hairy roots were used for rcpme1 expression and functional analysis in pea. Patterns of rcpme1 expression in cultured hairy roots, measured using uidA encoding -glucuronidase (GUS) as a reporter gene, were distinct from patterns which occur in normal pea roots. No reporter gene expression occurred in transgenic Arabidopsis thaliana, whose roots do not produce border cells. Border cell number from transgenic hairy roots expressing rcpme1 anti-sense mRNA under the control of its 2.75 kb 5 flanking sequence was reduced by > 50%. Nodulation genes of Rhizobium leguminosarum were used as a marker to document that roots with reduced production of border cells and other root cap exudates have a corresponding reduction in levels of biologically active signal molecules. Direct measurements were used to confirm that most of the exudate harvested from young, unwounded roots of normal pea plants is derived from the root tip region where rcpme1 is expressed. The potential application of the rcpme1 gene as a molecular marker for root exudate production is discussed.     

Molecular cloning and expression analysis of the mevalonate kinase gene from Arabidopsis thaliana

Mevalonate kinase (MVK), the enzyme that catalyzes the phosphorylation of mevalonate to produce mevalonate 5-phosphate, is considered as a potential regulatory enzyme of the isoprenoid biosynthetic pathway. The Arabidopsis thaliana MVK gene corresponding to the MVK cDNA previously isolated has been cloned and characterized. RNAse protection analysis indicated that the expression of the MVK gene generates three mRNA populations with 5 ends mapping 203, 254 and 355 nt upstream of the MVK ATG start codon. Northern blot analysis showed that the MVK mRNA accumulates preferentially in roots and inflorescences. Histochemical analysis, with transgenic A. thaliana plants containing a translational fusion of a 1.8 kb fragment of the 5 region of the MVK gene to the -glucuronidase (GUS) reporter gene, indicated that the MVK 5-flanking region directs widespread expression of the GUS gene throughout development, although the highest levels of GUS activity are detected in roots (meristematic region) and flowers (sepals, petals, anthers, style and stigmatic papillae). The expression pattern of the MVK gene suggests that the role of the encoded MVK is the production of a general pool of mevalonate-5-phosphate for the synthesis of different classes of isoprenoids involved in both basic and specialized plant cell functions. Functional promoter deletion analysis in transfected A. thaliana protoplasts indicated that regulatory elements between positions –295 and –194 of the MVK 5-flanking region are crucial for high-level MVK gene expression.