An improved method for generating subtracted cDNA libraries using phage lambda vectors.
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Bacteriophage lambda↗
Biomedical subjects
L Willmitzer
Publications and source records attributed to L Willmitzer.
Ac/Ds transposon mutagenesis in Arabidopsis thaliana: mutant spectrum and frequency of Ds insertion mutants.
Using a two-component Ac/Ds system consisting of a stabilized Ac element (Acc1) and a non-autonomous element (DsA), 650 families of plants carrying independent germinal DsA excisions/transpositions were isolated. Progenies of 559 of these Acc1/DsA families, together with 43 families of plants selected for excision/transposition of wild-type (wt) Ac, were subjected to a broad screening program for mutants exhibiting visible alterations. This resulted in the identification of 48 mutants showing a wide variety of mutant phenotypes, including embryo lethality (24 mutants), chlorophyll defects (5 mutants), defective seedlings (2 mutants), reduced fertility (5 mutants), reduced size (3 mutants), altered leaf morphology (2 mutants), dark green, unexpanded rosette leaves (3 mutants), and aberrant flower or shoot morphology (4 mutants). To whether these mutants were due to transposon insertions, a series of Southern blot experiments was performed on 28 families, comparing in each case several mutant plants with others showing the wild-type phenotype. A preliminary analysis revealed in 4 of the 28 families analyzed a common, novel DsA fragment in all mutant plants, which was present only in heterozygous plants with wt phenotype, as expected for DsA insertion mutations. These four mutants included two showing embryo lethality, one with dark green, unexpanded rosette leaves and stunted inflorescences, and one with curly growth of stems, leaves and siliques. Further evidence for DsA insertion mutations was obtained for one embryo lethal mutant and for the stunted mutant, while in case of the second embryo lethal mutant, the DsA insertion could be separated from the mutant locus by genetic recombination.
Arabidopsis↗
Cloning and electrophysiological analysis of KST1, an inward rectifying K+ channel expressed in potato guard cells.
Potassium uptake by guard cells represents part of the osmotic motor which drives stomatal opening. Patch-clamp measurements have identified inward rectifying K+ channels capable of mediating K+ uptake in guard cells and various other plant cell types. Here we report the molecular cloning and characterization of a voltage-dependent K+ channel (KST1) from potato (Solanum tuberosum L.) guard cells. In situ hybridization shows expression of kst1 in guard cells. Two-electrode voltage-clamp and patch-clamp studies of the gene product after cRNA injection into Xenopus oocytes identified KST1 as a slowly activating, voltage-dependent, inward rectifying K+ channel. The single channel current voltage curve was linear in the range -160 to +20 mV, with a deduced single channel conductance of 7 pS in symmetrical 100 mM K+. This channel type, modulated by pH changes within the physiological range, required ATP for activation. In line with the properties of a K(+)-selective channel, KST1 was permeable to K+, Rb+ and NH4+ and excluded Na+ and Li+. Cs+ at submillimolar concentrations blocked the channel in a voltage-dependent manner. Related studies on potato guard cell protoplasts confirmed the biophysical characteristics of the kst1 gene product (KST1) in the heterologous expression system. Therefore, KST1 represents a major K+ uptake channel in potato guard cells.
Amino Acid Sequence↗
Cloning and expression analysis of sucrose-phosphate synthase from sugar beet (Beta vulgaris L.).
A cDNA clone encoding a sucrose-phosphate synthase from sugar beet (BvSPS 1) has been isolated by screening a tap root-specific cDNA library using a heterologous SPS cDNA from spinach. The 3635 bp sugar beet cDNA codes for a 1045 amino acid polypeptide with a predicted molecular mass of 118 kDa. The deduced amino acid sequence of sugar beet SPS shows homologies with SPS from maize (71% identity) and spinach (77% identity). Genomic Southern blot analysis suggests that BvSPS 1 is a low-copy-number gene. RNA blot analysis of sink and source leaves, root and tap root tissue shows that SPS 1 is expressed in an organ-specific manner, being predominantly active in tap root. Incubation of detached leaves of sugar beet in light in glucose-containing media leads to an accumulation of the SPS transcript, while sucrose feeding reduces the steady-state level of the mRNA.
Amino Acid Sequence↗
Molecular cloning and characterization of novel isoforms of potato ADP-glucose pyrophosphorylase.
ADP-glucose pyrophosphorylase (AGPase) is one of the major enzymes involved in starch biosynthesis in higher plants. We report here the molecular cloning of two cDNAs encoding so far uncharacterized isoforms (AGP S2 and AGP S3) of the potato enzyme. Sequence analysis shows that the two polypeptides are more homologous to previously identified large subunit polypeptides from potato and other plant species than to small subunit isoforms. This observation suggest that AGP S2 and AGP S3 represent novel large subunit polypeptides. agpS2 is expressed in several tissues of the potato plant, including leaves and tubers. Expression was stronger in sink leaves than in source leaves, indicating developmental regulation. In leaves, agpS2 expression was induced 2- to 3-fold by exogenous sucrose; therefore, agpS2 represents a new sucrose-responsive gene of starch metabolism. Expression of agpS3 was restricted to tubers: no agpS3 expression could be seen in leaves of different developmental stages, or when leaves were incubated in sucrose. Therefore, agpS3 represents the only AGPase gene so far characterized from potato, which is not expressed in leaves. Conversely, all four AGPase isoforms known from potato are expressed in tubers.
Amino Acid Sequence↗
Inhibition of flower formation by antisense repression of mitochondrial citrate synthase in transgenic potato plants leads to a specific disintegration of the ovary tissues of flowers.
The tricarboxylic acid (TCA) cycle constitutes a major component of the mitochondrial metabolism of eucaryotes, including higher plants. To analyze the importance of this pathway, we down-regulated mitochondrial citrate synthase (mCS; EC 4.1.3.7), the first enzyme of the TCA cycle, in transgenic potato plants using an antisense RNA approach. Several transformants were identified with reduced citrate synthase activity (down to approximately 6% of wild-type activity). These plants were indistinguishable from wild-type plants in the greenhouse during vegetative growth. A major change, however, was seen upon initiation of the generative phase (flower formation). In the case of transgenic plants with a strong reduction in citrate synthase activity (< 30% of wild-type levels), flower buds formed > 2 weeks later as compared with wild-type plants. Furthermore, flower buds from these plants did not develop into mature flowers but rather were aborted at an early stage of development. Microscopic analysis showed that in these cases ovaries disintegrated during flower development. We conclude that the TCA cycle is of major importance during the transition from the vegetative to the generative phase.
Citrate (si)-Synthase↗
Isolation and expression analysis of cDNA clones encoding a small and a large subunit of ADP-glucose pyrophosphorylase from sugar beet.
The cDNA cloning of a small and a large subunit of ADP-glucose pyrophosphorylase (AGPase) from sugar beet is reported. The deduced amino acid sequences are highly homologous to previously identified AGPase polypeptides from other plant species. Both subunits are encoded by low copy genes. When RNA gel blot experiments were performed, strongest expression was detected in sink and source leaves of greenhouse-grown sugar beet plants. A lower expression was found in other tissues tested, i.e. in the hypocotyl, the tap root and roots. In these tissues, slightly higher transcript levels were found for the small subunit gene than for the large subunit gene.
Allosteric Regulation↗
Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.).
Sink strength of growing potato tubers is believed to be limited by sucrose metabolism and/or starch synthesis. Sucrose synthase (Susy) is most likely responsible for the entire sucrose cleavage in sink tubers, rather than invertases. To investigate the unique role of sucrose synthase with respect to sucrose metabolism and sink strength in growing potato tubers, transgenic potato plants were created expressing Susy antisense RNA corresponding to the T-type sucrose synthase isoform. Although the constitutive 35S CaMV promoter was used to drive the expression of the antisense RNA the inhibition of Susy activity was tuber-specific, indicating that independent Susy isoforms are responsible for Susy activity in different potato organs. The inhibition of Susy leads to no change in sucrose content, a strong accumulation of reducing sugars and an inhibition of starch accumulation in developing potato tubers. The increase in hexoses is paralleled by a 40-fold increase in invertase activities but no considerable changes in hexokinase activities. The reduction in starch accumulation is not due to an inhibition of the major starch biosynthetic enzymes. The changes in carbohydrate accumulation are accompanied by a decrease in total tuber dry weight and a reduction of soluble tuber proteins. The reduced protein accumulation is mainly due to a decrease in the major storage proteins patatin, the 22 kDa proteins and the proteinase inhibitors. The lowered accumulation of storage proteins is not a consequence of the availability of the free amino acid pool in potato tubers. Altogether these data are in agreement with the assumption that sucrose synthase is the major determinant of potato tuber sink strength. Contradictory to the hypothesis that the sink strength of growing potato tubers is inversely correlated with the tuber number per plant, no increase in tuber number per plant was found in Susy antisense plants.
Base Sequence↗
A cDNA clone for an ATP-sulfurylase from Arabidopsis thaliana.
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Arabidopsis↗
Cloning and expression analysis of the cytosolic NADP(+)-dependent isocitrate dehydrogenase from potato. Implications for nitrogen metabolism.
A full-length cDNA (icdh-1) encoding a cytosolic NADP(+)-dependent isocitrate dehydrogenase (ICDH-1) from potato (Solanum tuberosum L.) has been isolated. Analysis of the deduced protein sequence revealed considerable homologies with the corresponding proteins from other eukaryotes such as tobacco, alfalfa, soybean, cattle, pig, and yeast. The gene was transcribed in all tissues tested, with the highest amount of icdh-1 transcript being found in green tissues, in flowers, and in roots. In leaves, enzyme activities were dependent on the age, with fully mature leaves showing the highest level of RNA expression and enzyme activity. This observation may indicate that NADP(+)-dependent ICDH is not only involved in amino acid biosynthesis via the glutamine synthetase/glutamine oxoglutarate aminotransferase cycle but also in cycling, redistribution, and export of amino acids. The latter assumption has been strengthened by our finding of a preferential expression of NADP(+)-dependent ICDH in leaf veins. Under in vivo conditions, the expression pattern paralleled the enzyme activity, indicating coarse control on the RNA level. Experiments carried out with detached leaves revealed an influence of light, nitrate, and sucrose on icdh-1 transcript levels and in some cases also on NADP(+)-dependent ICDH activity. In darkness, nitrate or sucrose induced icdh-1 mRNA expression. Leaves kept under starvation conditions exhibited a decrease of their protein content, whereas icdh-1 expression and ICDH activity increased significantly.
Amino Acid Sequence↗
Mitochondrial citrate synthase from potato: predominant expression in mature leaves and young flower buds.
A cDNA clone encoding mitochondrial citrate synthase (EC 4.1.3.7), the first enzyme of the tricarboxylic-acid cycle, was isolated from potato (Solanum tuberosum L.) and expression of the enzyme analyzed. The deduced amino-acid sequence of the potato mitochondrial citrate synthase showed high similarity to known citrate synthases from fungi, mammals and Arabidopsis thaliana. The expression pattern of this clone was determined by Northern blot analysis. Expression was detected in all tissues analyzed. The highest level of expression was found in green flower buds. In photosynthetic tissues, stronger mRNA expression was detected in mature than in immature leaves. This rise in expression with leaf age was accompanied by an increase in citrate-synthase activity. Within flowers, expression was severalfold stronger in anthers than in ovaries, indicating a role of mitochondrial citrate synthase during anther or pollen development. A comparatively low level of transcript was detected in underground heterotrophic tissues, such as stolons, tubers and roots. When tubers were stored at low temperature (4 degrees C), mitochondrial citrate-synthase gene expression increased slightly. From the data obtained, we conclude that expression of the mitochondrial citrate-synthase gene is regulated by developmental and environmental factors. The relatively high expression in leaves is in line with the assumption that mitochondria play an important role in photosynthetically active tissues.
Amino Acid Sequence↗
A novel DNA binding protein with homology to Myb oncoproteins containing only one repeat can function as a transcriptional activator.
A cDNA clone encoding a novel Myb-related protein, designated MybSt1, was isolated from a potato cDNA expression library by South Western screening using the CaMV 35S promoter domain A as a probe. Sequence comparison shows a small region with some homology to the highly conserved DNA binding domain of the c-myb proto-oncogene consisting of three imperfect repeats. The Myb motif of the MybSt1 protein is distinct from the plant Myb DNA binding domain described so far. In contrast to the known plant Myb proteins, with two repeats required for the DNA binding activity, the clone mybSt1 contains only one such repeat. Nevertheless, the Myb-related protein MybSt1 is able to bind to DNA in a sequence-specific manner. In addition to the Myb-like region, the protein MybSt1 contains an acidic segment in its central region as well as a proline-rich region near the C-terminus. Applying the random binding site selection technique, high-affinity DNA binding sites for MybSt1 were identified, sharing the core motif GGATA. In transient expression assays using plant protoplasts, clear evidence was obtained for this myb clone functioning as a transcriptional activator.
Amino Acid Sequence↗
Cloning and characterization of a cathepsin D inhibitor gene from Solanum tuberosum L.
A DNA clone encoding a cathepsin D inhibitor CathInh was isolated from a potato genomic library using a CathInh cDNA as hybridization probe. The amino acid sequence of the coding region is nearly identical with a CathInh cDNA and CathInh proteins previously isolated from a tuber-specific cDNA library and from tubers, respectively. Analysis of GUS activity resulting from expression of chimeric CathInh promoter-GUS genes in transgenic potato plants revealed expression exclusively confined to potato tubers. No GUS activity could be detected in any other organ of the transgenic plants either constitutively or after wounding or treatment with abscisic and jasmonic acid (JA). Interestingly, part of the promoter region of the CathInh gene, essential for GUS activity in tubers, shows striking similarity to promoter regions of tuber-specific class I patatin genes.
Amino Acid Sequence↗
Isolation and characterization of two cDNA clones encoding ATP-sulfurylases from potato by complementation of a yeast mutant.
Sulfur plays an important role in plants, being used for the biosynthesis of amino acids, sulfolipids and secondary metabolites. After uptake sulfate is activated and subsequently reduced to sulfide or serves as donor for sulfurylation reactions. The first step in the activation of sulfate in all cases studied so far is catalyzed by the enzyme ATP-sulfurylase (E.C. 2.7.7.4.) which catalyzes the formation of adenosine-5'-phosphosulfate (APS). Two cDNA clones from potato encoding ATP-sulfurylases were identified following transformation of a Saccharomyces cerevisiae mutant deficient in ATP-sulfurylase activity with a cDNA library from potato source leaf poly(A)+ RNA cloned in a yeast expression vector. Several transformants were able to grow on a medium with sulfate as the only sulfur source, this ability being strictly linked to the presence of two classes of cDNAs. The clones StMet3-1 and StMet3-2 were further analyzed. DNA analysis revealed an open reading frame encoding a protein with a molecular mass of 48 kDa in the case of StMet3-1 and 52 kDa for StMet3-2. The deduced polypeptides are 88% identical at the amino acid level. The clone StMet3-2 has a 48 amino acid N-terminal extension which shows common features of a chloroplast transit peptide. Sequence comparison of the ATP-sulfurylase Met3 from Saccharomyces cerevisiae with the cDNA StMet3-1 (StMet3-2) reveals 31% (30%) identity at the amino acid level. Protein extracts from the yeast mutant transformed with the clone StMet3-1 displayed ATP-sulfurylase activity. RNA blot analysis demonstrated the expression of both genes in potato leaves, root and stem, but not in tubers.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Signals involved in the wound-induced expression of the proteinase inhibitor II gene of potato.
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Abscisic Acid↗
Functional analysis of a leucine aminopeptidase from Solanum tuberosum L.
A protein encoded by a potato cDNA homologous to a leucine aminopeptidase (LAP) from bovine lens (Hildmann et al. 1992) was expressed in Escherichia coli cells and biochemically characterized by hydrolysis of leucine p-nitroanilide. Activity was highest under alkaline conditions with an optimum at about pH 10. Maximal activities were measured at 65 degrees C. Apart from leucine p-nitroanilide the enzyme could also efficiently hydrolyze the p-nitroanilides of arginine and methionine. Complete inhibition of the enzyme was achieved by incubating bacterial extracts with bestatin and EDTA, which classifies the enzyme as a metalloprotease belonging to the same group as the homohexameric LAPs from mammals. Protein blots showed low constitutive expression of the LAP in all organs of potato plants: buds, flowers, tubers, roots and leaves. An increase in steady-state protein that was paralleled by an increase in total LAP activity was observed in leaf extracts after supplying jasmonic acid via the petioles. Plants containing the cDNA in antisense orientation behind the constitutive Cauliflower Mosaic Virus 35S promoter showed nearly complete reduction of the corresponding mRNA in leaves. However, in these plants LAP activities were only decreased by about 20% as compared to non-transgenic potato plants, while after feeding with jasmonic acid the activity of transgenic plants was reduced to about 5% of that of non-transgenic plants also induced by jasmonic acid. There was no phenotypic difference between wild-type and LAP antisense plants.
Amino Acid Sequence↗
Analysis of the expression of potato uridinediphosphate-glucose pyrophosphorylase and its inhibition by antisense RNA.
The expression of the enzyme UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) from potato (Solanum tuberosum L.) was analysed with respect to sink-source interactions and potato tuber storage. The highest level of expression was found in developing tubers, the strongest sink tissue. Storage of mature tubers at low temperatures led to an increase of the steady-state level of UGPase mRNA, implicating a role of this enzyme in the process of "cold-sweetening". Transgenic plants were created expressing UGPase antisensee RNA under the control of the 35S promoter of the Cauliflower Mosaic Virus with the polyadenylation signal of the octopine-synthase gene. Regenerated plants were tested for reduction of UGPase at the RNA, protein and activity levels. Plants with a 95%-96% reduction of UGPase activity in growing tubers showed no change in growth and development. Also, carbohydrate metabolism in tubers of these plants was not substantially affected, indicating that only 4% of the wild-type UGPase activity is sufficient for the enzyme to function in plant growth and development.
Base Sequence↗
Characterization of a gene that is expressed in leaves at higher levels upon tuberisation in potato and upon flowering in tobacco.
A partial cDNA clone has been isolated which represents a gene that is induced in potato (Solanum tuberosum L.) leaves upon tuber initiation. The gene is also present in tobacco (Nicotiana tobacum L.) where it is induced upon flowering, and in tomato (Lycopericon esculentum, L.) where it is induced when the plant starts to form fruit. This provides further evidence for the similarity of the floral-induction and tuber-induction pathways, and raises the possibility that this gene may be involved in a common step in both of these pathways. Alternatively, it could be involved in a secondary process that is induced during tuberisation, flowering and fruit development.
Base Sequence↗