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PubMed1994

Pattern and degree of methylation in ribosomal RNA genes of Cucurbita pepo L.

Methylation with respect to its degree and distribution throughout the 18S, 5.8S and 25S rRNA gene clusters (rDNA) and within single rDNA repeats in seedlings of the higher plant Cucurbita pepo L. (zucchini) was investigated. In this plant, which is characterized by several thousand repeats, at least 70% are completely or nearly completely methylated in CpGs and to a lower degree in CpNpGs. Detailed methylation analysis revealed that a fraction of about 3-4% of all repeats is hypomethylated near the transcription initiation site (TIS) which may indicate the fraction of active repeats in C. pepo. However, a different fraction (3-4% of all repeats) which is not methylated in all sites tested (including those at the TIS) is present in C. pepo and may thus represent active but differentially methylated rDNA. The results are discussed in context of recent models on methylation and gene activity.

DNA, Ribosomal↗
PubMed1994

Purification and partial amino-acid sequence of gibberellin 20-oxidase from Cucurbita maxima L. endosperm.

Gibberellin (GA) 20-oxidase was purified to apparent homogeneity from Cucurbita maxima endosperm by fractionated ammonium-sulphate precipitation, gel-filtration chromatography and anion-exchange and hydrophobic-interaction high-performance liquid chromatography (HPLC). Average purification after the last step was 55-fold with 3.9% of the activity recovered. The purest single fraction was enriched 101-fold with 0.2% overall recovery. Apparent relative molecular mass of the enzyme was 45 kDa, as determined by gel-filtration HPLC and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, indicating that GA 20-oxidase is probably a monomeric enzyme. The purified enzyme degraded on two-dimensional gel electrophoresis, giving two protein spots: a major one corresponding to a molecular mass of 30 kDa and a minor one at 45 kDa. The isoelectric point for both was 5.4. The amino-acid sequences of the amino-terminus of the purified enzyme and of two peptides from a tryptic digest were determined. The purified enzyme catalysed the sequential conversion of [14C]GA12 to [14C]GA15, [14C]GA24 and [14C]GA25, showing that carbon atom 20 was oxidised to the corresponding alcohol, aldehyde and carboxylic acid in three consecutive reactions. [14C]Gibberellin A53 was similarly converted to [14C]GA44, [14C]GA19, [14C]GA17 and small amounts of a fourth product, which was preliminarily identified as [14C]GA20, a C19-gibberellin. All GAs except [14C]GA20 were identified by combined gas chromatography-mass spectrometry. The cofactor requirements in the absence of dithiothreitol were essentially as in its presence (Lange et al., Planta 195, 98-107, 1994), except that ascorbate was essential for enzyme activity and the optimal concentration of catalase was lower.

Amino Acid Sequence↗
PubMed1991

Differentiation of Fusarium solani f. sp. cucurbitae races 1 and 2 by random amplification of polymorphic DNA.

We have used a PCR-based technique, involving the random amplification of polymorphic DNA (RAPD), to assess genome variability between 21 isolates from F. solani f. sp. cucurbitae races 1 and 2. Based on RAPD marker patterns the isolates fell into two distinct groups corresponding to mating populations MPI and MPV. Four isolates that could not be assigned to one or other mating population by traditional means were distinguished by RAPD patterns. Seven polymorphic RAPD products were used to probe Southern blots of MPI and MPV genomic DNA. Six of the seven probes hybridized to single-copy sequences and five of the seven probes showed specificity for one or other mating population. We suggest that not only is the technique a rapid and reliable tool for isolate-typing of fungi but it also provides a rapid method for obtaining species- or race-specific hybridization probes.

Base Sequence↗
PubMed1992

High efficiency transformation of Fusarium solani f. sp. cucurbitae race 2 (mating population V).

A cosmid vector, suitable for library construction and DNA transformation in filamentous fungi, has been constructed and a reliable and highly efficient PEG-mediated DNA transformation system for F. solani f. sp. cucurbitae, based on resistance to hygromycin B, has been developed for use with this vector. This transformation system yielded 10(4) transformants per micrograms of DNA when using 10(7) protoplasts. Factors important in achieving high efficiency included: the maintenance of an osmoticum in all transformation steps, PEG 4000 concentration, and the ratio of transforming vector DNA to protoplasts. Approximately 60% of transformants stably integrated vector DNA. Molecular analysis revealed multiple copies of the plasmid integrated into the genome at one or more sites. The frequency of transformation achieved will facilitate the isolation of genes from this fungus by complementation.

Base Sequence↗
PubMed1976

Autoradiographic and ultrastructural study of Cucurbita pepo root cells during their growth and differentiation.

Cortex cells of the root meristem of Cucurbita pepo (0.0-0.5 mm from the cap junction), in the 3-4, 5-6 and 7-8 mm segments above the root tip, and the cells of the first three layers of lateral part of root cap were the object of the present study. The volume of cortex cells increases more than 20 times in the 7-8 mm segment as compared with meristematic cells, and the volume of cytoplasm about sevenfold. The largest increment of the cytoplasmic volume occurs between 0.5-6.0 mm. In consecutive root segments the sustained increase of the volume of nuclei takes place. By applying autoradiography the following processess have been investigated: DNA synthesis (3H thymidine uptake), template activity of DNA (3H actinomycin D(3H AMD)-binding), RNA synthesis (3H uridine incorporation), and protein synthesis (3H leucine). In the root cap cells and in segments where meristematic activity is over, DNA is replicated by endomitosis. On the basis of nuclear labelling it appears that nuclei in the 3-4 mm segment reach 4C ploidy state, but in the 7-8 mm segment half of the nuclei reach the 8C ploidy state. Most of the root cap cells are 4C, the remaining cells are 8C. Considering the uptake of 3H thymidine into nucleoli one may suppose that in the root cap cells nucleolar DNA is underreplicated, and to a lesser degree in 5-6 and 7-8 mm segments, while in 3-4 mm segment DNA is overreplicated as compared to meristem cells. Measurements of nucleolar volume, 3H uridine uptake, 3H AMD binding and quantity of granular component, indicate that the most noticeable nucleolar activity takes place in meristematic zone and in root parts showing the highest increase of cytoplasmic volume (3-4 and 5-6 mm segments). 3H leucine is still incorporated intensely into 7-8 mm segment, in which the concentration of ribosomes is low, however they are present in the form of polysomes. Comparison of 3H thymidine uptake into nuclear DNA with 3H AMD binding and 3H uridine incorporation into nuclei indicates that endomitotic DNA replication results in an increase of DNA template activity in root cap cells as well as in 3-4 and 5-6 mm segments; in the 7-8 mm segment binding of 3H AMD slightly decreases, while 3H uridine incorporation is considerably reduced. Divergence between the ploidy state, 3H AMD binding and 3H uridine incorporation can be due to the increment of the condensed chromatin area in differentiated cells. Plastids and mitochondria reach full maturity in 3-4 mm segment. The increasing volume density of ER and diminishing volume density of Golgi structures is accompanied by differentiation of cortex cells.

Cell Differentiation↗
PubMed1996

Variability of fatty acid content in pumpkin seeds (Cucurbita pepo L.).

Pumpkin (Cucurbita pepo L.) seed oil is a common salad oil which is produced in Slovenia, Hungary and the southern parts of Austria. It is dark green and has a high content of free fatty acids. The seed itself can be eaten. Due to its colour and the foam formation, the oil cannot be used for cooking. The content of vitamin E, especially gamma-tocopherol, is very high. The oil content of the pumpkin seed is about 50%. The variability in the oil content is very high resulting from a broad genetic diversity. Thus a breeding programme for increasing the oil productivity is very promising. The four dominant fatty acids are palmitic, stearic, oleic and linoleic acids. These four fatty acids make up 98 +/- 0.13% of the total amount of fatty acids, others being found at levels well below 0.5%.

Austria↗
PubMed1996

The sterols of Cucurbita moschata ("calabacita") seed oil.

From the sterol fraction of seed oil from commercial Cucurbita moschata Dutch ("calabacita") delta 5 and delta 7 sterols having saturated and unsaturated side chain were isolated by chromatographic procedures and characterized by spectroscopic (1H and 13C-nuclear magnetic resonance, mass spectrometry) methods. The main components were identified as 24S-ethyl 5 alpha-cholesta-7,22E-dien-3 beta-ol (alpha-spinasterol); 24S-ethyl 5 alpha-cholesta-7,22E,25-trien-3 beta-ol (25-dehydrochondrillasterol); 24S-ethyl 5 alpha-cholesta-7,25-dien-3 beta-ol; 24R-ethyl-cholesta-7-en-3 beta-ol (delta 7-stigmastenol) and 24-ethyl-cholesta-7, 24(28)-dien-3 beta-ol (delta 7,24(28)-stigmastadienol).

Chromatography, High Pressure Liquid↗
PubMed1995

Growth of Cucurbita maxima L. plants in the presence of the cycloartenol synthase inhibitor U18666A.

Squash, like other Cucurbitaceae, have unique sterol profiles that offer an excellent opportunity to examine the relationship between sterol biosynthesis and plant growth. To determine the effect of sterol biosynthesis inhibition on squash growth, Cucurbita maxima seedlings with and without cotyledons were subjected to increasing concentrations of the cycloarternol synthase (EC 5.4.99.8) inhibitor 3 beta-(2-diethylaminoethoxy)androstenone (U18666A). Inhibition of shoot growth was concentration-dependent (from 0, 2, 5, 10, and 20 microM); plants with intact cotyledons grew to 26.4, 23.7, 21.6, 20.0, and 15.6 cm, respectively, at the above inhibitor concentrations, compared to 25.5, 19.4, 17.0, 12.0, and 11 cm for plants with severed cotyledons. In plants with severed cotyledons, 10 and 20 microM U18666A caused rapid necrosis of the first two, newly emerged, primary leaves, and halted new leaf formation. Secondary root formation was initially affected at all inhibitor concentrations regardless of whether cotyledons were present or not. Vegetative tissue showed a decrease in the accumulation of the major squash sterol, 7,22-stigmastadienol, accompanied by increased accumulation of minor sterol components. Sterol profiles in cotyledons were unaltered. The data show that sterols are crucial for maintaining plant growth and viability, but do not address the cotyledonary effect on growth with respect to sterol biosynthesis.

Androstenes↗
PubMed1999

Analysis of phloem protein patterns from different organs of Cucurbita maxima Duch. by matrix-assisted laser desorption/ionization time of flight mass spectroscopy combined with sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Sieve tubes mediate the long-distance transport of nutrients and signals between source and sink organs of plants. To detect mobile phloem proteins that are differentially distributed in source and sink organs of Cucurbita maxima, we used both one-dimensional gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Both techniques revealed that phloem protein patterns depend on the sampling site: whilst several proteins were consistently observed in all phloem samples studied others appeared to occur in a organ-specific manner. For a characterization and identification of distinct phloem polypeptides, two approaches were chosen. First, protein bands resolved by SDS-PAGE were eluted from the polyacrylamide gel and the masses of the proteins were then determined by MALDI-TOF MS. Second, proteins resolved by SDS-PAGE were subjected to proteolytic degradation and the resulting peptides were analyzed by MALDI-TOF MS: the masses of the proteolytic peptides were used for a database search. By the latter approach, three mobile phloem compounds were identified as the phloem-specific protein PP2 (D.E. Bostwick et al., 1992, The Plant Cell 4, 1539-1548) a chymotrypsin and an aspartic proteinase inhibitor. None of the other polypeptides studied corresponded to any of the protein sequences present in the database. Furthermore, MALDI-TOF MS analyses indicated that some of the mobile phloem proteins occur in a covalently modified form and that the extent of the modification depends upon the plant organ.

Amino Acid Sequence↗
PubMed1977

Evidence about the catecholoxidase activity of the enzyme ascorbate oxidase extracted from Cucurbita pepo medullosa.

Pure ascorbate oxidase (L-ascorbate:oxygen oxidoreductase, EC 1.10.3.3) isolated from Cucurbita pepo medullosa, which is known to be specific for ascorbic acid, shows a secondary catecholoxidase activity at approx. pH 6.7. This activity was tested against natural and synthetic compounds possessing a catechol-like structure. Among natural compounds (+)-catechin furnishes the same complex oxidation mixture obtained with other oxidases. Among synthetic compounds, 3,5-di-t-butylcatechol and 4-t-butylcatechol give the corresponding o-quinones. The significance of this secondary activity in the darkening process of fruits and vegetables which contain ascorbate oxidase is also discussed.

Ascorbate Oxidase↗
PubMed1978

Phosphoenolpyruvate carboxykinase and gluconeogenesis in cotyledons of Cucurbita pepo.

1. The aim of this work was to investigate the role of phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating) EC 4.1.1.49) in the conversion of fat to sugar by the cotyledons of seedlings of Cucurbita pepo. 2. The enzyme was partially purified from the cotyledons of 5-day-old seedlings. The Michaelis constants for oxaloacetate and ATP were 56 and 119 micron, respectively. The decarboxylation reaction was optimum at pH 7.4. A range of intermediary metabolites did not affect the activity of the enzyme, but 3-mercaptopicolinic acid at micron concentrations was an effective inhibitor. 3. Centrifugation of extracts of 5-day-old cotyledons sedimented appreciable proportions of the ribuloseibisphosphate carboxylase, isocitrate lyase and fumarate hydratase present but very little of the phosphoenolpyruvate carboxykinase. 4. Measurements of phosphoenolpyruvate carboxykinase of cotyledons during germination showed that the maximum catalytic activity exceeded, and changed coincidently with, the rate of gluconeogenesis. 5. 3-Mercaptopicolinic acid inhibited gluconeogenesis from [1-14C]- and [2-14C]acetate supplied to excised cotyledons. The detailed distribution of 14C indicated inhibition of the conversion of oxaloacetate to phosphoenolpyruvate. 6. It is concluded that in marrow cotyledons phosphoenolpyruvate carboxykinase is in the soluble phase of the cytoplasm and catalyses a component reaction of gluconeogenesis.

Gluconeogenesis↗
PubMed1989

The refined 2.0 A X-ray crystal structure of the complex formed between bovine beta-trypsin and CMTI-I, a trypsin inhibitor from squash seeds (Cucurbita maxima). Topological similarity of the squash seed inhibitors with the carboxypeptidase A inhibitor from potatoes.

The stoichiometric complex formed between bovine beta-trypsin and the Cucurbita maxima trypsin inhibitor I (CMTI-I) was crystallized and its X-ray crystal structure determined using Patterson search techniques. Its structure has been crystallographically refined to a final R value of 0.152 (6.0-2.0 A). CMTI-I is of ellipsoidal shape; it lacks helices or beta-sheets, but consists of turns and connecting short polypeptide stretches. The disulfide pairing is CYS-3I-20I, Cys-10I-22I and Cys-16I-28I. According to the polypeptide fold and disulfide connectivity its structure resembles that of the carboxypeptidase A inhibitor from potatoes. Thirteen of the 29 inhibitor residues are in direct contact with trypsin; most of them are in the primary binding segment Val-2I (P4)-Glu-9I (P4') which contains the reactive site bond Arg-5I-Ile-6I and is in a conformation observed also for other serine proteinase inhibitors.

Animals↗
PubMed1990

A new protein inhibitor of trypsin and activated Hageman factor from pumpkin (Cucurbita maxima) seeds.

A protein inhibitor (CMTI-V; Mr 7106) of trypsin and activated Hageman factor (Factor XIIa), a serine protease involved in blood coagulation, has been isolated for the first time from pumpkin (Cucurbita maxima) seeds by means of trypsin-affinity chromatography and reverse phase high performance liquid chromatography (HPLC). The dissociation constants of the inhibitor complexes with trypsin and Factor XIIa have been determined to be 1.6 x 10(-8) and 4.1 x 10(-8) M, respectively. The primary structure of CMTI-V is reported. The protein has 68 amino acid residues and one disulfide bridge and shows a high level of sequence homology to the Potato I inhibitor family. Furthermore, its amino terminus consists of an N-acetylates Ser. The reactive site has been established to be the peptide bond between Lys44-Asp45. The modified inhibitor which has the reactive site peptide bond hydrolyzed inhibits trypsin but not the Hageman factor.

Amino Acid Sequence↗
PubMed1992

Natural abundance 15N NMR assignments delineate structural differences between intact and reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III.

15N NMR assignments were made to the backbone amide nitrogen atoms at natural isotopic abundance of intact and reactive-site (Arg5-Ile6) hydrolyzed Cucurbita maxima trypsin inhibitor III (CMTI-III and CMTI-III*, respectively) by means of 2D proton-detected heteronuclear single bond chemical shift correlation (HSBC) spectroscopy, utilizing the previously made sequence-specific 1H NMR assignments (Krishnamoorthi et al. (1992) Biochemistry 31, 898-904). Comparison of the 15N chemical shifts of the two forms of the inhibitor molecule revealed significant changes not only for residues located near the reactive-site region, but also for those distantly located. Residues Cys3, Arg5, Leu7, Met8, Cys10, Cys16, Glu19, His25, Tyr27, Cys28 and Gly29 showed significant chemical shift changes ranging from 0.3 to 6.1 ppm, thus indicating structural perturbations that were transmitted throughout the molecule. These findings confirm the earlier conclusions based on 1H NMR investigations.

Amino Acid Sequence↗
PubMed1975

Location of gluconeogenesis from phosphoenolpyruvate in cotyledons of Cucurbita pepo.

1. The aim of this work was to discover the location of the enzymes that convert phosphoenolpyruvate to fructose 6-phosphate during gluconeogenesis in fatty seeds. Cotyledons of 5-day-old dark-grown seedlings of marrow (Cucurbita pepo) were used as experimental material. 2. Cotyledons were separated into palisade and mesophyll tissue. Extracts of the two tissues had comparable activities of gluconeogenic enzymes. 3. Extracts of cotyledons were fractionated by density gradient centrifugation to yeild mitochondria and glyoxysomes, and by gel filtration to yield proplastids. The isolated organelles retained their characteristic ultrastructure and appreciable amounts of marker enzymes. The proportions of the total activities of phosphoglyceromutase and fructose-1, 6-diphosphatase recovered in the mitochondrial and glyoxysomal preparations were insignificant. The same was true for the activities of phosphoglyceromutase and phosphopyruvate hydratase found in the proplastid preparations. 4. Extracts of a number of other gluconeigenic plant tissues were centrifuged at 2500 times g to yield particulate preparations. None of these preparations contained a significant proportion of the total activity of phosphoglyceromutase. 5. It is suggested that gluconeogenesis from phosphoenolpyruvate in plants occurs in the cytoplasm.

Cell Fractionation↗
PubMed1978

Identification of the regulatory steps in gluconeogenesis in cotyledons of Cucurbita pepo.

1. The aim of this work was to discover the steps at which the conversion of oxaloacetate to glucose 6-phosphate during gluconeogenesis is regulated in the cotyledons of 5-day-old seedlings of Cucurbita pepo. 2. We estimated the maximum catalytic activities of all the enzymes in the above sequence and also the amounts of their substrates present in vivo. The results show that the reactions catalysed by fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase are the only ones in the sequence that are substantially displaced from equilibrium in vivo. 3. We also determined the effects of 3-mercaptopicolinic acid, an inhibitor of gluconeogenesis, on the amounts of the gluconeogenic intermediates present in vivo. The results show that the enzyme system, fructose-1,6-bisphosphatase: phosphofructokinase, and the system phosphoenolpyruvate carboxykinase: phosphoenolpyruvate carboxylase make major contributions to the regulation of gluconeogenesis in the cotyledons. 4. Possible mechanisms for the above regulation are discussed.

Fructose-Bisphosphatase↗
PubMed1989

The Cucurbita maxima ribosomal DNA intergenic spacer has a complex structure.

The nucleotide (nt) sequence of the 5508-nt intergenic spacer (IGS), between the 25S- and the 18S-coding regions of Cucurbita maxima rDNA, was determined. The fragment sequenced is 6142 nt long and includes 472 nt of 25S- and 162 nt of 18S-coding regions. The IGS has a complex primary structure, composed of five repetitive families (A-E) and three unique domains. It is dominated by the presence of nine, tandemly-repeating units of approximately 250 nt (repeat D), each unit containing four copies of an internal subrepeat (repeat E). The repetitive units show sequence variability consisting of nt changes, insertions and deletions. Upstream of the nine D repeats and between two copies of the B repeat is a 575-nt region, highly G + C rich (83%) and heavily biased toward C (58%) in the sense strand. Within this region are six repetitive units, averaging 42 nt (repeat C) each, containing but a single A nt. Downstream from the terminus of the 25S-coding sequence, are two tandem copies of the 103-nt A repeat. The IGS of C. maxima is longer and more complex than that of other plant IGSs described to date. The 600 nt at the 5' portion of cucurbit IGS is more conserved in evolution than the remainder, as revealed by comparison of C. maxima and C. pepo IGS restriction maps and by nucleotide sequence comparison of C. maxima and Cucumis sativa IGSs.

Base Sequence↗
PubMed1991

Synthesis of a wild-type and three mutant Cucurbita maxima trypsin inhibitor-encoding genes by a single-strand approach.

A single-strand approach to gene assembly, based on a modification of an in vitro complementary oligodeoxyribonucleotide template-directed ligation of the desired sequence to a linearized vector [Chen et al., Nucleic Acids Res. 18 (1990) 871-878], is described. The gene coding for the wild-type Cucurbita maxima trypsin inhibitor of 29 amino acid residues [Bode et al., FEBS Lett. 242 (1989) 285-292], as well as three mutant forms of the gene, in which two of the three disulfide bonds have been replaced singly or as a pair, have been synthesized in a single synthesis run with minimal manual intervention. Subsequent to ligation to pUC9 and in vivo gapped duplex repair by Escherichia coli, their sequences have been verified.

Amino Acid Sequence↗