bluuebunny/crossref_metadata_2025_split huggingface dataset:
Site‐specific integration of DNA into wild‐type and mutant lox sites placed in the plant genome
Authors: Albert Henrik , Dale Emily C. , Lee Elsa , Ow David W. 
Published: March 2003

SummaryThe bacteriophage P1 Cre—lox site‐specific recombination system has been used to integrate DNA specifically at lox sites previously placed in the tobacco genome. As integrated molecules flanked by wild‐type lox sites can readily excise in the presence of Cre recombinase, screening for mutant lox sites that can resist excisional recombination was performed. In gene integration experiments, wild‐type and mutant lox sites were used in conjunction with two strategies for abolishing post‐integration Cre activity: (i) promoter displacement of a cre‐expression construct present in the target genome; and (ii) transient expression of cre. When the promoter displacement strategy was used, integrant plants were recovered after transformation with constructs containing mutant lox sequences, but not with constructs containing wild‐type lox sites. When cre was transiently expressed, integrant plants were obtained after transformation with either mutant or wild‐type lox sites. DNA rearrangements at the target locus were less frequent when mutant lox sites were used. DNA integration at the genomic lox site was usually without additional insertions in the genome. Thus, the Cre—lox site‐specific recombination system is useful for the single‐copy integration of DNA into a chromosomal lox site.
Strength and tissue specificity of chimeric promoters derived from the octopine and mannopine synthase genes
Authors: Ni Min , Cui Decai , Einstein Jane , Narasimhulu Soma , Vergara Claudia E. , Gelvin Stanton B. 
Published: March 2003

SummaryTo investigate promoter strength and the tissue‐specific patterns of expression, chimeric promoters incorporating subdomains of mannopine synthase (mas2′) and octopine synthase (ocs) promoter and activator regions were affixed to a β‐glucuronidase reporter gene and the constructions introduced into tobacco plants. Addition of a trimer of the ocs upstream activating sequence (UAS) to a mas promoter/activator region resulted in highly elevated levels of GUS activity in all tissues examined. In leaf tissue, this chimeric promoter is approximately 156‐fold and 26‐fold stronger than are the CaMV 35S and the ‘enhanced’ double CaMV 35S promoters, respectively. Expression of GUS activity directed by the mas and ocs promoters/activators is limited to specific cell types. Addition of the ocs or mas UAS to the mas or ocs promoter/activator regions modulated these expression patterns. The addition of a trimer of the ocs UAS to the mas promoter/activator region resulted in a transcriptional control element that directed GUS expression in most cell types. In addition to the strong expression in transgenic tobacco plants, this novel promoter directed higher levels of GUS expression than did the CaMV 35S promoter in transiently transformed tobacco leaf discs and suspension culture cells, as well as in cassava and cowpea explants. It is proposed that the strong promoter containing a trimer of the ocs UAS affixed to the mas promoter/activator will be useful for the very high level constitutive expression of linked genes in a wide variety of plant species.
In vitro assembly of cellulose/xyloglucan networks: ultrastructural and molecular aspects
Authors: Whitney Sarah E.C. , Brigham Jennifer E. , Darke Arthur H. , Reid J.S. Grant , Gidley Michael J. 
Published: March 2003

SummaryFeatures of the interaction between cellulose and xyloglucan have been studied using the cellulose‐producing bacterium Acetobacter aceti ssp. xylinum (ATCC 53524) and tamarind seed xyloglucan. Direct microscopic evidence is provided for the generation of cross‐bridges between cellulose ribbons produced in the presence of xyloglucan but not carboxymethyl‐cellulose. Cross‐bridge lengths are very similar to those observed for de‐pectinated onion cell walls. Similar cross‐bridge lengths are observed following mixing of isolated A. xylinum cellulose and xyloglucan, showing that network formation can be an abiotic process. The level of incorporation of xyloglucan in an actively growing system (ca. 38% of cellulose) is an order of magnitude higher than that observed in mixtures of isolated polymers and is comparable with cell wall levels.NMR spectroscopy suggests that 80–85% of incorporated xyloglucan is segmentally rigid with the backbone adopting an extended ‘cellulosic’ conformation and probably aligned with cellulose chains. The remaining xyloglucan is more mobile and is assigned to cross‐bridges with, on average, a twisted backbone conformation. No evidence for specific involvement of side‐chain residues in binding is found, and the observation of cross‐bridges with a non‐fucosylated xyloglucan shows that fucose residues are not essential for network formation. Xyloglucan causes cellulose ribbons to become more amorphous and to have a decreased 1α/1β crystallite ratio without any significant alteration in ribbon diameter.Based on the findings that levels of xyloglucan incorporation, the presence and lengths of cross‐bridges, and the modification of cellulosic molecular organization are all similar to those found in plant cell walls, we suggest that A. aceti ssp. xylinum is a more useful model for primary plant cell walls and their assembly than has previously been appreciated.
Cloning and functional analysis of a cDNA encoding a novel 139 kDa starch synthase from potato (Solanum tuberosum L.)
Authors: Abel Gernot J.W. , Springer Franziska , Willmitzer Lothar , Kossmann Jens 
Published: March 2003

SummaryThree isoforms of starch synthase were shown to be present in soluble potato tuber extracts by activity staining after native gel electrophoresis. An antibody directed against a domain conserved in starch synthases was used to clone a cDNA for one of these isoforms by screening a tuber‐specific expression library. A partial cDNA of 2.6 kbp was obtained and used to isolate a full‐length cDNA of 4167 bp. The deduced amino acid sequence identifies the protein as a novel type of starch synthase from potato with a molecular mass of 139.2 kDa for the immature enzyme including its transit peptide. This novel isoform was designated SS III. An analysis of the expression pattern of the gene indicates that SS III is equally expressed in tubers of different developmental stages as well as in sink and source leaves. In several independent transgenic potato lines, where the expression of SS III was repressed using the antisense approach, the activity of a specific starch synthase isoform was reduced to non‐detectable levels as determined through activity staining after native gel electrophoresis. The reduction of this isoform of starch synthase leads to the synthesis of a structurally modified starch in the transgenic plants: there is a drastic change in granule morphology and an increased level of covalently linked phosphate.
The structures of integration sites in transgenic rice
Authors: Takano Makoto , Egawa Hitomi , Ikeda Joh‐E , Wakasa Kyo 
Published: March 2003

SummaryExtensive genomic sequencing and sequence motif analysis have been conducted over the integration sites of two transgenic rice plants, #478 and #559, carrying the luciferase gene and/or hygromycin phosphotransferase gene. The transgenes reside in a region with inverted structure and a large duplication of rice genome over 2 kb. Integration was found at the AT‐rich region and/or at the repetitive sequence region, including a SAR‐like structure, retrotransposon and telomere repeats. The presence of a patch of sequence homology between plasmid and target DNA, and a small region of duplication involving the target DNA around the recombination site, implicated illegitimate recombination in the process of gene integration. Massive rearrangement of genomic DNA including deletion or translocation was also observed at the integration site and the flanking region of the transgene. The recognition sites of DNA topoisomerases I or II were observed in the rearranged sequences. Since only three junctions of transgenic rice were implicated in the illegitimate recombination and extensive rearrangement of the rice genome, rice protoplasts may be active in this process.
Cinnamoyl CoA reductase, the first committed enzyme of the lignin branch biosynthetic pathway: cloning, expression and phylogenetic relationships
Authors: Lacombe Eric , Hawkins Simon , Van Doorsselaere Jan , Piquemal Joël , Goffner Deborah , Poeydomenge Odile , Boudet Alain‐M. , Grima‐Pettenati Jacqueline 
Published: March 2003

SummaryCinnamoyl CoA:NADP oxidoreductase (CCR, EC 1.2.1.44) catalyzes the conversion of cinnamoyl CoA esters to their corresponding cinnamaldehydes, i.e. the first specific step in the synthesis of the lignin monomers. The cloning of a cDNA encoding CCR in Eucalyptus gunnii (EUCCR) is reported here. The identity of the EUCCR cDNA was demonstrated by comparison with peptide sequence data from purified CCR and functional expression of the recombinant enzyme in Escherichia coli. Sequence analysis revealed remarkable homologies with dihydroflavonol‐4‐reductase (DFR), the first enzyme of the anthocyanin biosynthetic pathway. Moreover, significant similarities were found with mammalian 3β‐hydroxysteroid dehydrogenase and bacterial UDP‐galactose‐4‐epimerase, suggesting that CCR shared a common ancestor with these enzymes and can therefore be considered as a new member of the mammalian 3β‐hydroxysteroid dehydrogenase/ plant dihydroflavonol reductase superfamily. In Eucalyptus gunnii, CCR is encoded by one gene containing four introns whose positions are similar to those of introns I, II, III and V in DFR genes from dicots. In agreement with the involvement of CCR in lignification, the CCR transcript was shown to be expressed in lignified organs, i.e. root and stem tissues, and was localized mainly in young differentiating xylem. On the other hand, its abundance in Eucalyptus leaves suggests that monolignols may be precursors of end products other than lignins. This first characterization of a gene corresponding to CCR opens new possibilities to genetically engineer plants with lower lignin content. This is particularly important for woody plants such as Eucalyptus which are used for pulp making.
Co‐ordinated induction of mRNAs for extracellular invertase and a glucose transporter in Chenopodium rubrum by cytokinins
Authors: Ehneß Rainer , Roitsch Thomas 
Published: March 2003

SummaryExtracellular or cell wall invertase is regarded as crucial to supply sink tissues with carbohydrates via an apoplastic pathway. A cell wall invertase from Chenopodium rubrum was purified to homogeneity and the corresponding cDNA encoding CIN1 was identified via peptide sequences. The CIN1 mRNA was found to be highly induced by physiological concentrations of both adenine‐ and phenylurea‐derived cytokinins in suspension culture cells. This was paralleled both by a higher steady‐state protein level and a higher enzyme activity of the extracellular invertase. The cytokinin‐inducible accumulation of CIN1 mRNA in tissues of C. rubrum plants supports the physiological significance of this regulatory mechanism. In contrast to the extracellular sucrose cleaving enzyme, the mRNA levels of the two putative intracellular invertases CIN2 and CIN3 and of sucrose synthase were not elevated. In addition, it has been found that the accumulation of mRNA for one out of three hexose transporters present in the suspension culture cells is induced co‐ordinately with the mRNA for extracellular invertase by cytokinins. It has been shown that this regulatory mechanism results in higher uptake rates both for sucrose, via the hexose monomers, and for glucose. The increased level of both extracellular invertase and hexose transporters and the resulting higher carbohydrate supply are discussed with respect to the control of carbohydrate partitioning by plant hormones and the molecular basis for known physiological cytokinin responses such as the stimulation of cell division.
Possible involvement of differential splicing in regulation of the activity of Arabidopsis ANP1 that is related to mitogen‐activated protein kinase kinase kinases (MAPKKKs)
Authors: Nishihama Ryuichi , Banno Hiroharu , Kawahara Emi , Irie Kenji , Machida Yasunori 
Published: March 2003

SummaryThree types of Arabidopsis cDNA (cANP1, cANP2 and cANP3) have been isolated that encode putative protein kinases, designated ANP1, ANP2 and ANP3. These kinases exhibit a high degree of hornology to NPK1, a tobacco protein that is a member of the family of mitogen‐activated protein kinase kinase kinases (MAPKKKs), which appears to function in the proliferation of tobacco cells. The predicted amino acid sequences of the kinase domains in the amino‐terminal halves of the ANPs were more than 80% identical to that of NPK1, while the kinase‐unrelated regions in the carboxy‐terminal halves exhibited relatively low homology. Two species of cANP1 were identified, ANP1L cDNA (cANPIL) and ANP1S cDNA (cANP1S), which were derived from a single ANP1 gene: the former had an intron‐like sequence in the coding region for the kinase‐unrelated region, while the latter did not include such an intron‐like sequence. cANP1L encoded a putative protein with both kinase and kinase‐unrelated domains, resembling NPK1, whereas cANPIS encoded only the amino‐terminal kinase domain because the intron‐like sequence was absent, with resulting elimination of most of the kinase‐unrelated region. Genetic analysis with mutant yeast cells showed that over‐expression of cANPIL or of cANPIS activated the mating pheromone‐responsive signal pathway which is mediated by a MAP kinase cascade. Moreover, the extent of such activation by cANPIS was greater than that by cANPIL. These results predict that differential splicing of the intron‐like sequence in the ANP1 transcript might be at least one of the molecular mechanisms involved in the generation of active ANP1 protein kinase.
Molecular characterization of a phloem‐specific gene encoding the filament protein, Phloem Protein 1 (PP1), from Cucurbita maxima
Authors: Clark Anna M. , Jacobsen Karin R. , Bostwick Dwight E. , Dannenhoffer Joanne M. , Skaggs Megan I. , Thompson Gary A. 
Published: March 2003

SummarySieve elements in the phloem of most angiosperms contain proteinaceous filaments and aggregates called P‐protein. In the genus Cucurbita, these filaments are composed of two major proteins: PP1, the phloem filament protein, and PP2, the phloem lectin. The gene encoding the phloem filament protein in pumpkin (Cucurbita maxima Duch.) has been isolated and characterized. Nucleotide sequence analysis of the reconstructed gene gPP1 revealed a continuous 2430 bp protein coding sequence, with no introns, encoding an 809 amino acid polypeptide. The deduced polypeptide had characteristics of PP1 and contained a 15 amino acid sequence determined by N‐terminal peptide sequence analysis of PP1. The sequence of PP1 was highly repetitive with four 200 amino acid sequence domains containing structural motifs in common with cysteine proteinase inhibitors. Expression of the PP1 gene was detected in roots, hypocotyls, cotyledons, stems, and leaves of pumpkin plants. PP1 and its mRNA accumulated in pumpkin hypocotyls during the period of rapid hypocotyl elongation after which mRNA levels declined, while protein levels remained elevated. PP1 was immunolocalized in slime plugs and P‐protein bodies in sieve elements of the phloem. Occasionally, PP1 was detected in companion cells. PP1 mRNA was localized by in situ hybridization in companion cells at early stages of vascular differentiation. The developmental accumulation and localization of PP1 and its mRNA paralleled the phloem lectin, further suggesting an interaction between these phloem‐specific proteins.
Very‐long‐chain fatty acid biosynthesis is controlled through the expression and specificity of the condensing enzyme
Authors: Millar Anthony A. , Kunst Ljerka 
Published: March 2003

SummaryThe Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene encodes a putative seed‐specific condensing enzyme. It is the first of four enzyme activities that comprise the microsomal fatty acid elongase (FAE) involved in the biosynthesis of very‐long‐chain fatty acids (VLCFAs). FAE1 has been expressed in yeast and in tissues of Arabidopsis and tobacco, where significant quantities of VLCFAs are not found. The introduction of FAE1 alone in these systems is sufficient for the production of VLCFAs, for wherever FAE1 was expressed, VLCFAs accumulated. These results indicate that FAE1 is the rate‐limiting enzyme for VLCFA biosynthesis in Arabidopsis seed, because introduction of extra copies of FAE1 resulted in higher levels of the VLCFAs. Furthermore, the condensing enzyme is the activity of the elongase that determines the acyl chain length of the VLCFAs produced. In contrast, it appears that the other three enzyme activities of the elongase are found ubiquitously throughout the plant, are not rate‐limiting and play no role in the control of VLCFA synthesis. The ability of yeast containing FAE1 to synthesize VLCFAs suggests that the expression and the acyl chain length specificity of the condensing enzyme, along with the apparent broad specificities of the other three FAE activities, may be a universal eukaryotic mechanism for regulating the amounts and acyl chain length of VLCFAs synthesized.
Plant male germ line transformation
Authors: Touraev A. , Stöger E. , Voronin V. , Heberle‐Bors E. 
Published: March 2003

SummaryA new method to produce transgenic plants ‐ male germ line transformation (MAGELITR) is reported. Unicellular tobacco microspores were isolated from excised anthers, and DNA carrying two marker genes was transferred biolistically. The bombarded microspores were matured in vitro for 6 days, and the mature pollen was used for in vivo pollination. Seeds were recovered and putative transformants were selected on the basis of their antibiotic resistance. Five kanamycin‐resistant plants were chosen for further analysis, four contained the first construct, one the second. Parallel experiments with bicellular immature pollen did not produce any transgenic plants. A detailed DNA blot and expression analysis confirmed the transgenic nature of the five plants, and a genetic analysis showed that the transgenes are transmitted to subsequent generations. MAGELITR is a fast, regeneration‐independent method, not prone to chimerism and somaclonal variation which should be genotype‐independent and may be applicable in a wide range of species once in vitro maturation of pollen is established.
Genetic transformation of dinoflagellates (Amphidinium and Symbiodinium): expression of GUS in microalgae using heterologous promoter constructs
Authors: Te Michael R. , Lohuis  , Miller David J. 
Published: March 2003

SummaryGenetic transformation of two dinoflagellates (Amphidinium sp., Symbiodinium microadriaticum) was achieved using plasmid constructs containing the neomycin phosphotransferase gene (nptII) fused to the Agrobacterium nos promoter, or the hygromycin B phosphotransferase gene (hpt) fused to the bidirectional Agrobacterium p1′2′ promoter. Gene transfer into intact (walled) dinoflagellate cells was achieved by agitation in the presence of silicon carbide (SiCa) whiskers. Transformation rates of 5–24 transformants per 107 cells were obtained. Southern hybridization of transformants revealed stable integration of multiple copies of the constructs. Activity of integrated copies of the β‐glucoronidase (GUS) reporter gene coupled to the cauliflower mosaic virus 35S promoter or the p1′2′ promoter was confirmed both histochemically and fluorometrically. This is the first report of successful application of heterologous and widely used promoter and reporter genes in microalgae, and is the first demonstration of transformation of a dinoflagellate. There appear to be no substantial barriers to transformation of Amphidinium and Symbiodinium, which must now be considered as the first of the dinoflagellate genera accessible to genetic manipulation.
Plasma membrane depolarization‐activated calcium channels, stimulated by microtubule‐depolymerizing drugs in wild‐type Arabidopsis thaliana protoplasts, display constitutively large activities and a longer half‐life in ton 2 mutant cells affected in the organization of cortical microtubules
Authors: Thion Laurence , Mazars Christian , Nacry Philippe , Bouchez David , Moreau Marc , Ranjeva Raoul , Thuleau Patrice 
Published: March 2003

SummaryDepolarization‐activated plasma membrane calcium channels have been suggested to play prominent roles in signal perception and transduction processes during growth and development of higher plants. The existence of such channels has recently been established in higher plant cells. However, patch–clamp experiments have shown that their activity is very low and decreases very rapidly after the establishment of the whole‐cell configuration, due most probably to protein–protein interactions involving microtubules. The present study takes advantage of the existence of Arabidopsis thaliana mutants referred to as ton 2 mutants reported to be affected in their microtubule organization, to address the physiological relevance of such a hypothesis based on a pharmacological approach. Patch–clamp studies showed that depolarization‐activated calcium channel activities in ton 2 protoplasts were 10‐fold higher and their relative half‐life three‐times longer than in wild‐type protoplasts. In addition, oryzalin and colchicine, which disrupt the microtubule organization, stimulated and stabilized calcium channel activities in wild‐type but remained ineffective on ton 2 protoplasts. However, although the microtubules appeared important in the regulation of calcium channels in A. thaliana, immunocytological staining of tubulin demonstrated that there was no visible difference in the general organization of microtubule networks or in the amount of microtubules bound to the plasma membrane in ton 2 and wild‐type protoplasts. It is suggested that the down‐regulation of calcium channels implicating microtubules involves additional component(s) corresponding probably to gene product(s) defective in ton 2 mutant cells.