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D Grierson

Publications and source records attributed to D Grierson.

At least 55 records · Page 3Linked to original sources
PubMed1991

Identification of a tomato gene for the ethylene-forming enzyme by expression in yeast.

The ethylene-forming enzyme (EFE), which catalyzes the last step in the biosynthesis of the plant hormone ethylene, has never been purified and no molecular probes are available. Recently, a putative cDNA clone for tomato EFE (pTOM13) has been identified by inhibiting ethylene synthesis with an antisense gene expressed in transgenic plants. A direct test of its function has been made by expression of a pTOM13 gene in Saccharomyces cerevisiae. After cloning artefacts were discovered in the 5' region of the cDNA, a corrected cDNA (pRC13) was created by the fusion of the 5' end of a genomic clone to the 3' end of the cDNA and expressed in S. cerevisiae. Cultures of transformed yeast converted 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene, whereas control cells did not. This EFE activity displays similar characteristics to EFE found in plant tissue: it converts the trans isomer of the ACC analogue 1-amino-2-ethylcyclopropane-1-carboxylic acid to 1-butene in preference to the cis isomer, and it is strongly inhibited by cobaltous ions and 1,10-phenanthroline. Furthermore, information gained from the activity of effectors on yeast EFE activity supports the hypothesis that EFE is one of a group of hydroxylase enzymes.

Amino Acid Oxidoreductases↗
PubMed1990

Expression of a truncated tomato polygalacturonase gene inhibits expression of the endogenous gene in transgenic plants.

Tomato plants were transformed with a chimaeric polygalacturonase (PG) gene, designed to produce a truncated PG transcript constitutively. In these plants expression of the endogenous PG gene was inhibited during ripening, resulting in a substantial reduction in PG mRNA and enzyme accumulation. This inhibition was comparable to that achieved previously using antisense genes. The expression of the truncated gene in ripe fruit was substantially lower than its expression in green fruit. Thus expression of both the endogenous and truncated genes is reduced in ripe fruit in which both are active. The implication of this observation is discussed in relation to the possible mechanism whereby sense constructs inhibit gene expression.

Blotting, Northern↗
PubMed1988

Identification and sequence determination of a cDNA clone for tomato pectin esterase.

Cell wall softening during tomato fruit ripening is brought about through the action of a number of pectolytic enzymes. We have reported previously the cloning and characterisation of the cDNA for the major cell wall softening (degrading) enzyme, polygalacturonase [Grierson, D., Tucker, G. A., Keen, J., Ray, J., Bird, C. R. and Schuch, W. (1986) Nucleic Acids Res. 14, 8595-8603]. We have now isolated a cDNA clone for tomato pectin esterase, an enzyme also implicated in cell wall softening. Here we report the structure of this cDNA and compare it with the structure of pectin esterase derived from amino acid sequence experiments [Markovic, O. and Jornvall, H. (1986) Eur. J. Biochem. 158, 455-462]. We have used the pectin esterase cDNA clone to analyse pectin esterase gene expression during development and ripening of normal and mutant fruit.

Amino Acid Sequence↗
PubMed1987

Structure and expression of an ethylene-related mRNA from tomato.

Messenger RNAs homologous to a cDNA clone (pTOM 13) derived from a ripe-tomato-specific cDNA library are expressed during tomato fruit ripening and after the wounding of leaf and green fruit material. Both responses involve the synthesis of the hormone ethylene. Accumulation of the pTOM 13--homologous RNA during ripening is rapid and sustained, and reaches its maximum level in orange fruit. Following mechanical wounding of tomato leaves a pTOM 13--homologous RNA shows rapid induction within 30 minutes, which occurs before maximal ethylene evolution (2-3 h). This RNA also accumulates following the wounding of green tomato fruit. Northern blot analysis of poly(A)+ RNA indicates that the length of the mRNA is about 1400 nucleotides. Nucleotide sequence analysis showed the cDNA insert to contain the complete coding region of the pTOM 13 protein (33.5 kD) and an unusual 5' structure of ten dT-nucleotides. Hybridisation of the pTOM 13 cDNA insert to Southern blots of tomato DNA indicates the presence of only a small number of homologous sequences in the tomato genome.

Amino Acid Sequence↗
PubMed1986

Sequencing and identification of a cDNA clone for tomato polygalacturonase.

The 2a isoenzyme of tomato polygalacturonase was purified from ripe fruit and characterised. The N-terminal amino acid sequence of the protein was determined in order to identify polygalacturonase cDNA clones. The nucleotide sequence of a ripening-related cDNA (pTOM 6) was determined and found to encode the N-terminal sequence of mature polygalacturonase 2a. The complete open reading frame encodes a polypeptide of molecular weight 50,051, including a putative pre-sequence of 71 amino acids.

Amino Acid Sequence↗
PubMed1985

Detection of a subgenomic mRNA for gene V, the putative reverse transcriptase gene of cauliflower mosaic virus.

Polypeptides synthesized in vitro in rabbit reticulocyte lysates, directed by poly(A)+ RNA isolated from turnip leaves infected with cauliflower mosaic virus (CaMV), were analysed by polyacrylamide gel electrophoresis. Following translation of virus-specific RNA purified by hybrid-selection using CaMV DNA immobilized on DBM papers, the polypeptides observed included the viral gene VI inclusion body protein P62, and a larger product, P75, together with several smaller polypeptides. By translating RNA hybrid-selected with restriction fragments encompassing the CaMV genome, a mRNA for P75 has been mapped to gene V. These results, together with sucrose gradient ultracentrifugation studies, suggest that a CaMV gene V mRNA is a sub-genomic transcript of approximately 2.5Kb and 22S. The expression strategy of the CaMV genome is discussed in the light of our findings.

Chromosome Mapping↗
PubMed1984

Ethylene-independent and ethylene-dependent biochemical changes in ripening tomatoes.

Fruits of Lycopersicon esculentum Mill cv Sonatine stored in 6% CO(2), 6% O(2), and 88% N(2) for 14 weeks at 12 degrees C, exhibited a temporal separation of certain biochemical events associated with ripening.The specific activity of two citric acid cycle enzymes, citrate synthase and malate dehydrogenase, fell substantially during the first 2 weeks of storage when changes in organic acid concentration also occurred. During this period, lycopene, polygalacturonase, and ethylene were undetectable.When fruit were removed from store, ethylene was evolved and polygalacturonase and invertase activity were rapidly initiated as was synthesis of lycopene.To determine whether the changes in organic acid metabolism were affected by ethylene, fruit was kept at 22 degrees C in either a normal atmosphere or a normal atmosphere supplemented with 27 microliters per liter of ethylene, and it was shown that in both atmospheres similar quantitative changes to those described above occurred in the citric acid cycle enzymes specific activities before any detectable increase in the specific activities of invertase and polygalacturonase. These latter changes, together with pigment changes, occurred between 2 and 3 days earlier in fruit exposed to ethylene, compared with those kept in a normal atmosphere.

Journal Article↗
PubMed1983

Ultrastructure of tomato fruit ripening and the role of polygalacturonase isoenzymes in cell wall degradation.

Ultrastructural changes in the pericarp of tomato (Lycopersicon esculentum Mill) fruit were followed during ripening. Ethylene production was monitored by gas chromatography and samples analyzed at successive stages of the ripening process.Changes in the cytoplasmic ultrastructure were not consistent with the suggestion that ripening is a ;senescence' phenomenon. A large degree of ultrastructural organization, especially of the mitochondria, chromoplasts, and rough endoplasmic reticulum, was retained by ripe fruit.Striking changes in the structure of the cell wall were noted, beginning with dissolution of the middle lamella and eventual disruption of the primary cell wall. These changes were correlated with appearance of polygalacturonase (EC 3.2.1.15) isoenzymes. Application of purified tomato polygalacturonase isoenzymes to mature green fruit tissue duplicated the changes in the cell wall noted during normal ripening. Possible roles of the polygalacturonase isoenzymes in cell wall disorganization are discussed.

Journal Article↗
PubMed1982

Degradation of isolated tomato cell walls by purified polygalacturonase in vitro.

Cell wall preparations from green pericarp of normal and mutant Neverripe (Nr) and ripening inhibitor (rin) tomato (Lycopersicon esculentum Mill.) fruit were all equally degraded in vitro by a cell wall-bound protein extract from ripe normal tomatoes.Similar cell wall-bound protein extracts from ripe Nr fruit were not as effective and those from ripe rin fruit gave no cell wall degradation at all in vitro. This was correlated with the absence of polygalacturonase in rin and low activity of Nr extracts.Purified polygalacturonase was capable of in vitro cell wall degradation and it seems that this enzyme can account for the cell wall degradation observed with the total cell wall-bound protein extracts from ripe fruit.

Journal Article↗
PubMed1981

The conversion of tomato-fruit polygalacturonase isoenzyme 2 into isoenzyme 1 in vitro.

Polygalacturonase is extractable from ripe tomatoes in two isoenzyme forms, polygalacturonase 1 and 2. These isoenzymes have previously been shown to have substantially different properties although their polypeptides appear similar. Green fruit contain a heat-stable, non-dialysable factor capable of the conversion of polygalacturonase 2 in vitro into another isoenzyme which, on the basis of heat stability, molecular weight and density in caesium chloride, is equivalent to polygalacturonase 1. The amount of this factor extractable from tomato tissue increases during ripening.

Chemical Phenomena↗
PubMed1980

Changes in polygalacturonase isoenzymes during the 'ripening' of normal and mutant tomato fruit.

1. Polygalacturonase activity is not detectable in mature green tomato fruits but appears as fruits begin to change colour and continues to increase during the ripening period. There is a sequential appearance of two isoenzymes, polygalacturonase 1 and 2, during ripening. These isoenzymes have been purified and their properties compared. Polygalacturonase 1 has a Mr of 100,000, is 50% inactivated at 78 degrees C and has a density of 1.343 g cm-3 in caesium chloride. Polygalacturonase 2 has a Mr of 42,000, is 50% inactivated at 57 degrees C and has a density of 1.300 g cm-3 in caesium chloride. 2. Fruits from isogenic lines homozygous for the 'Neverripe' (Nr) mutation do not ripen normally and contain reduced amounts of polygalacturonase. Only polygalacturonase 1 is produced in Nr fruit. Tomatoes from isogenic lines homozygous for the 'ripening inhibitor' (rin) mutation do not ripen normally and produce very little detectable polygalacturonase. 3. Although polygalacturonases 1 and 2 have different properties they both give rise to a single polypeptide on electrophoresis in polyacrylamide gels in the presence of sodium dodecyl-sulphate (Mr = 46,000). A comparison of the major fragments produced by limited proteolysis of polygalacturonase 1 and 2 with chymotrypsin suggests that the polypeptides from the two isoenzymes are similar. The same conclusion was reached from a comparison of polygalacturonase 1 and 2 by radioimmunoassay, using antibody prepared against polygalacturonase 2 and 125I-labelled polygalacturonase 2. 4. The results from radioimmunoassay of extracts from green and ripening fruits suggest that the increase in polygalacturonase activity during ripening is due to net synthesis of protein.

Centrifugation, Density Gradient↗
PubMed1978

Isolation and characterisation of 14-S RNA from spinach chloroplasts.

14-S RNA was purified from spinach chloroplasts. It has a molecular weight of 0.43 . 10(6) and the following nucleotide composition: 20% CMP, 23.9% AMP, 24.2% GMP and 31.9% UMP. The accumulation of 14-S RNA in chloroplasts of cotyledons of dark-grown plants is stimulated by light. Conditions are described for the isolation of 14-S RNA in the absence of appreciable fragmentation of chloroplast 23-S rRNA and the evidence that it represents a distinct type of chloroplast RNA is discussed. Translation of 14-S RNA in a protein synthesising system from Escherichia coli gives rise to two polypeptides with molecular weights of 13 200 and 12 600 and the possible role of 14-S RNA as a chloroplast messenger is discussed.

Chloroplasts↗
PubMed1977

Ribonucleic acid from the higher plant Matthiola incana. Molecular weight measurements and DNA-RNA hybridisation studies.

The percentage of DNA from the crucifer Matthiola incana coding for different types of RNA was measured by filter saturation hybridisation experiments using RNA labelled in vivo. In addition, the melting curves of the various DNA - RNA hybrids formed and the buoyant densities of the DNA sequences complementary to different types of RNA were measured. 1. The RNA preparations used were 25, 18, and 5 S rRNA and 4 S RNA, purified by gel electrophoresis, and poly(A)-containing RNA purified by oligo-(dT)-cellulose chromatography. The molecular weights of the 25 S and 18 S rRNAs, calculated from the mobility in formamide-acrylamide gels relative to Escherichia coli RNA, are 1.25 - 10(6) and 0.64 - 10(6). The rRNA precursor has a molecular weight of approx. 2.1 - 10(6) and the average molecular weight of the poly(A)-containing RNA from both cotyledons and roots is 4 - 10(5). 2. The percentage of the genome, calculated on the basis of double-stranded DNA, coding for these RNAs and the estimated number of genes per haploid DNA amount are approximately 0.46% and 1100 for 25 S plus 18 S rRNA, 0.032% and 3600 for 5 S rRNA and 0.072% and 13 000 for 4 S RNA. In filter hybridisation experiments very little hybridisation of poly(A)-containing RNA was found. A rapidly-hybridising component is attributed to small amounts of contaminating rRNA. 3. M. incana DNA has a main band at 1.697 g - ml-1 in CsCl and a satellite constituting approximately 3% of the DNA, at 1.708 g - ml-1 - 25 and 18 S rRNA hybridise to DNA with a buoyant density of 1.701--2 g - ml-1. The buoyant density of 5 S DNA is slightly less at 1.700--1 g - ml-1. 4. S RNA hybridises to at least two separate regions, one within the main-band DNA and a second lighter component. None of the RNAs tested hybridised to the satellite DNA. The Tm of the DNA - RNA hybrids in 1 X SSC is 89 degrees C for 25 S rRNA, 85 degrees C for 5 S rRNA and 82 degrees C for 4 S RNA. 4. 5 and 4 S RNA preparations contain fragments which hybridise to sequences complementary to high-molecular-weight rRNA. This spurious hybridisation can be eliminated by competition with unlabelled high-molecular-weight RNA.

Base Sequence↗