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P M Chandler

Publications and source records attributed to P M Chandler.

At least 19 recordsLinked to original sources
PubMed2001

The CYP88A cytochrome P450, ent-kaurenoic acid oxidase, catalyzes three steps of the gibberellin biosynthesis pathway.

We have shown that ent-kaurenoic acid oxidase, a member of the CYP88A subfamily of cytochrome P450 enzymes, catalyzes the three steps of the gibberellin biosynthetic pathway from ent-kaurenoic acid to GA(12). A gibberellin-responsive barley mutant, grd5, accumulates ent-kaurenoic acid in developing grains. Three independent grd5 mutants contain mutations in a gene encoding a member of the CYP88A subfamily of cytochrome P450 enzymes, defined by the maize Dwarf3 protein. Mutation of the Dwarf3 gene gives rise to a gibberellin-responsive dwarf phenotype, but the lesion in the gibberellin biosynthesis pathway has not been identified. Arabidopsis thaliana has two CYP88A genes, both of which are expressed. Yeast strains expressing cDNAs encoding each of the two Arabidopsis and the barley CYP88A enzymes catalyze the three steps of the GA biosynthesis pathway from ent-kaurenoic acid to GA(12). Sequence comparison suggests that the maize Dwarf3 locus also encodes ent-kaurenoic acid oxidase.

Arabidopsis↗
PubMed1998

Identification of a negative regulator of gibberellin action, HvSPY, in barley.

To broaden our understanding of the molecular mechanisms of gibberellin (GA) action, we isolated a spindly clone (HvSPY) from barley cultivar Himalaya and tested whether the HvSPY protein would modulate GA action in barley aleurone. The HvSPY cDNA showed high sequence identity to Arabidopsis SPY along its entire length, and the barley protein functionally complemented the spy-3 mutation. HvSPY and SPY proteins showed sequence relatedness with animal O-linked N-acetylglucosamine transferases (OGTs), suggesting that they may also have OGT activity. HvSPY has a locus distinct from that of Sln, a mutation that causes the constitutive GA responses of slender barley, which phenotypically resembles Arabidopsis spy mutants. The possibility that the HvSPY gene encodes a negative regulator of GA action was tested by expressing HvSPY in a barley aleurone transient assay system. HvSPY coexpression largely abolished GA3-induced activity of an alpha-amylase promoter. Surprisingly, HvSPY coexpression increased reporter gene activity from an abscisic acid (ABA)-inducible gene promoter (dehydrin), even in the absence of exogenous ABA. These results show that HvSPY modulates the transcriptional activities of two hormonally regulated promoters: negatively for a GA-induced promoter and positively for an ABA-induced promoter.

Amino Acid Sequence↗
PubMed1994

A dehydrin cognate protein from pea (Pisum sativum L.) with an atypical pattern of expression.

Dehydrins are a family of proteins characterised by conserved amino acid motifs, and induced in plants by dehydration or treatment with ABA. An antiserum was raised against a synthetic oligopeptide based on the most highly conserved dehydrin amino acid motif, the lysine-rich (core sequence KIKEK-LPG). This antiserum detected a novel M(r) 40,000 polypeptide and enabled isolation of a corresponding cDNA clone, pPsB61 (B61). The deduced amino acid sequence contained two lysine-rich blocks, however the remainder of the sequenced differed markedly from other pea dehydrins. Surprisingly, the sequence contained a stretch of serine residues, a characteristic common to dehydrins from many plant species but which is missing in pea dehydrin. The expression patterns of B61 mRNA and polypeptide were distinctively different from those of the pea dehydrins during seed development, germination and in young seedlings exposed to dehydration stress or treated with ABA. In particular, dehydration stress led to slightly reduced levels of B61 RNA, and ABA application to young seedlings had no marked effect on its abundance. The M(r) 40,000 polypeptide is thus related to pea dehydrin by the presence of the most highly conserved amino acid sequence motifs, but lacks the characteristic expression pattern of dehydrin. By analogy with heat shock cognate proteins we refer to this protein as a dehydrin cognate.

Abscisic Acid↗
PubMed1992

Pea dehydrins: identification, characterisation and expression.

An antiserum raised against dehydrin from maize (Zea mays) recognised several polypeptides in extracts of pea (Pisum sativum) cotyledons. A cDNA expression library was prepared from mRNA of developing cotyledons, screened with the antiserum and positive clones were purified and characterised. The nucleotide sequence of one such clone, pPsB12, contained an open reading frame which would encode a polypeptide with regions of significant amino acid sequence similarity to dehydrins from other plant species. The deduced amino acid sequence of the pea dehydrin encoded by B12 is 197 amino acids in length, has a high glycine content (25.9%), lacks tryptophan and is highly hydrophilic. The polypeptide has an estimated molecular mass of 20.4 kDa and pI = 6.4. An in vitro synthesised product from the clone comigrates with one of the in vivo proteins recognised by the antiserum. A comparison of the pea dehydrin sequence with sequences from other species revealed conserved amino acid regions: an N-terminal DEYGNP and a lysine-rich block (KIKEKLPG), both of which are present in two copies. Unexpectedly, pea dehydrin lacks a stretch of serine residues which is conserved in other dehydrins. B12 mRNA and dehydrin proteins accumulated in dehydration-stressed seedlings, associated with elevated levels of endogenous abscisic acid (ABA). Applied ABA induced expression of dehydrins in unstressed seedlings. Dehydrin expression was rapidly reversed when seedlings were removed from the stress or from treatment with ABA and placed in water. During pea cotyledon development, dehydrin mRNA and proteins accumulated in mid to late embryogenesis. Dehydrin proteins were some of the most actively synthesised at about the time of maximum fresh weight and represent about 2% of protein in mature cotyledons.

Abscisic Acid↗
PubMed1992

Expression of "Dehydrin-Like" Proteins in Embryos and Seedlings of Zizania palustris and Oryza sativa during Dehydration.

Proteins inducible by dehydration and abscisic acid (ABA), termed dehydrins or RAB (Responsive to ABA) proteins, have been identified in a number of species and have been suggested to play a role in desiccation tolerance, particularly during seed development. Seeds (caryopses) of North American wild rice (Zizania palustris var interior [Fassett] Dore) are tolerant of dehydration to <10% moisture content (fresh weight basis) only under restricted dehydration and rehydration conditions. In comparison, seeds of paddy rice (Oryza sativa L.) readily tolerate desiccation to <5% water content. Expression of "dehydrin-like" proteins in Zizania and Oryza seedlings and embryos was examined to investigate the relationship between the presence of such proteins and desiccation tolerance. [(35)S]Methionine labeling of newly synthesized proteins showed that seedlings (first leaf stage) of both Zizania and Oryza synthesized a novel "heat-stable" protein of apparent molecular weight = 20,000 when dehydrated to <75% of their initial fresh weight. ABA (100 micromolar) induced synthesis of a protein with similar electrophoretic mobility in both species. Western blots using antiserum raised against maize (Zea mays L.) dehydrin detected a protein band from dehydrated Zizania shoots and mature embryonic axes that comigrated with the labeled 20-kilodalton polypeptide. Northern blots using a cDNA for an ABA-responsive protein from Oryza (rab 16a) showed that both seedlings and excised embryonic axes of Zizania accumulated RNA similar in sequence to rab 16a in response to water loss. Zizania seedlings and embryonic axes were also capable of ABA accumulation during dehydration. The intolerance of Zizania seeds to dehydration at low temperature is apparently not due to an absence of dehydrin-like proteins or an inability to accumulate ABA.

Journal Article↗
PubMed1991

Primer extension studies on alpha-amylase mRNAs in barley aleurone. I. Characterization and quantification of the transcripts.

Primer extension was used to characterize alpha-amylase mRNAs from aleurone tissue of barley (Hordeum vulgare L. cv. Himalaya) grains. Two synthetic oligonucleotides, specific for the low-pI and high-pI alpha-amylase groups, were used as primers for synthesis of cDNA from total RNA preparations. Between them, these two oligonucleotides appear to account for all major alpha-amylase mRNAs as judged by hybrid-arrested translation of alpha-amylase mRNAs in a cell-free system. Reconstruction experiments indicated that the levels of extended primers (determined by scintillation counting) were directly proportional to the level of input mRNA over a wide range. This indicates that the technique is suitable for quantification of relative levels of individual alpha-amylase from approximately 2% to 100% of maximal levels. The nucleotide sequences of extended primers defined two different alpha-amylase mRNAs in each of the low-pI and high-pI groups, and possibly a third mRNA in the high-pI group.

Base Sequence↗
PubMed1991

Primer extension studies on alpha-amylase mRNAs in barley aleurone. II. Hormonal regulation of expression.

Relative levels of different alpha-amylase mRNAs were assessed by primer extension experiments using RNA prepared from aleurone of barley (Hordeum vulgare L. cv. Himalaya). Three different aleurone systems were studied: protoplasts prepared from aleurone layers, isolated aleurone layers, and aleurone from germinated grain. Oligonucleotide primers specific for the low-pI and high-pI alpha-amylase groups allowed the levels of different alpha-amylase mRNAs to be assessed both within and between the two groups. In all aleurone systems the same set of alpha-amylase mRNAs was produced in response to either applied gibberellic acid (aleurone protoplasts, isolated aleurone layers) or, presumably, native gibberellin(s) (germinated grain). This result indicates that the same set of genes is being expressed in each case. Differences were observed between the different aleurone systems in regulation of levels of alpha-amylase mRNAs. In particular, the regulation of alpha-amylase mRNA levels in aleurone of germinated grain has unique features which are not adequately explained by the response of isolated aleurone layers to gibberellic acid.

Gibberellins↗
PubMed1989

A cDNA-based comparison of dehydration-induced proteins (dehydrins) in barley and corn.

Several cDNAs related to an ABA-induced cDNA from barley aleurone were isolated from barley and corn seedlings that were undergoing dehydration. Four different barley polypeptides with sizes of 22.6, 16.2, 14.4 and 14.2 kDa and a single corn polypeptide with a size of 17.0 kDa were predicted from the nucleotide sequences of the cDNAs. These dehydration-induced proteins (dehydrins) are very similar to each other and to a previously identified rice protein induced by ABA and salt, and have at least some similarity to a previously identified cotton embryo protein. Each dehydrin is extremely hydrophilic, glycine-rich, cysteine- and tryptophan-free and contains repeated units in a conserved linear order. A lysine-rich repeating unit occurs twice in each protein, once at the carboxy terminus and once partway through the polypeptide, adjacent to a succession of serines. This repeating unit and the adjacent flanking run of serines are conserved with minimal variation among all dehydrins. Another repeating unit is flanked by the two copies of the lysine-rich unit, and varies in number from one to five copies. This latter repeating unit is less conserved than the former, varying even within a singly dehydrin. The messenger RNAs corresponding to each cDNA are abundant in dehydrating, but not in well-watered seedlings. The amino acid sequence of tryptic peptides from purified dehydration-induced proteins of corn established that the corn cDNAs correspond to a protein that is produced in abundance during the response of corn seedlings to dehydration.

Amino Acid Sequence↗
PubMed1986

Gene structure, protein structure, and regulation of the synthesis of a sulfur-rich protein in pea seeds.

Two low molecular weight pea seed albumins (Mr approximately 6000 and approximately 4000) have been characterized by protein, cDNA, and gene sequencing. Both proteins are encoded by separate regions of the same mRNA species. The initial translation product is a preproprotein from which a signal sequence is removed co-translationally. The resultant proprotein (PA1) is then cleaved post-translationally to yield the mature form of the two albumins (PA1a and PA1b). Comparison of cDNA and protein sequences suggests that at least four different PA1 genes are expressed in the pea genome. Both PA1a and PA1b have an unusually high cysteine content (7.5 and 16.2%, respectively). Pea seeds developing under suboptimal levels of sulfur nutrient supply contain reduced levels of PA1 mRNA and accumulate greatly reduced levels of PA1a and PA1b in the mature seed. In vitro transcription studies showed that this reduced level of PA1 mRNA resulted from reduced post-transcriptional stability rather than an altered rate of transcription of the PA1 gene. In contrast, during normal seed development, the level of PA1 mRNA seems to be under transcriptional control. Sequence comparisons reveal some homology between PA1 and a number of low molecular weight proteins from seeds of a wide range of mono- and dicotyledonous plants.

Albumins↗
PubMed1984

Influence of Sulfur Nutrition on Developmental Patterns of Some Major Pea Seed Proteins and Their mRNAs.

In addition to the marked reduction in legumin synthesis and legumin mRNA levels reported earlier (Chandler, Higgins, Randall, Spencer 1983 Plant Physiol 71: 47-54), pulse labeling of S-deficient Pisum sativum L. seeds showed that a high relative level of total vicilin (vicilin plus convicilin) synthesis was maintained throughout the entire phase of protein accumulation, whereas in nondeficient seeds vicilin synthesis is largely confined to the first half of this phase. Fractionation of pulse-labeled proteins on Na-dodecylsulfate-polyacrylamide gels showed that the synthesis of the M(r) 50,000 family of vicilin polypeptides was increased and greatly extended in S-deficient seeds whereas that of convicilin was slightly reduced. Other changes apparent from pulse-labeling experiments include a depression, to different degrees, in the synthesis of three major albumin polypeptides.The level of the mRNAs for seven major seed proteins was followed throughout development of control and sulfur-deficient seeds. In all cases, the changes in each mRNA closely reflected the pattern of synthesis of its corresponding polypeptide seen by pulse labeling. S-deficient seeds showed an elevated level of M(r) 50,000 vicilin mRNA which remained high throughout seed formation, whereas legumin mRNA levels were greatly reduced at all stages of development.When S-deficient plants were given an adequate supply of sulfate midway through seed development, there was a shift toward the protein synthesis profile characteristic of healthy plants. The synthesis of legumin and two albumins rapidly increased and the synthesis of M(r) 50,000 vicilin declined more slowly. Similar responses were seen in detached, S-deficient seeds supplied directly with adequate sulfate.

Journal Article↗
PubMed1983

The biosynthesis and primary structure of pea seed lectin.

Isolation of pea lectin from immature seeds, together with in vivo pulse-chase labeling and in vitro translation of RNA from such seeds, all gave results which indicated strongly that pea lectin is initially synthesized as a large precursor (pre-pro-form) (Mr = 25,000) which is cleaved both co- and post-translationally to yield the alpha and beta subunits of mature lectin. Two overlapping cDNA plasmids complementary to lectin mRNA were sequenced. They coded for both beta and alpha subunits of lectin, in the orientation beta leads to alpha from the NH2 terminus, together with a hydrophobic NH2-terminal leader sequence and included a 3' untranslated region of 124 nucleotides. In the appropriate reading frame, no stop codon was found between the coding sequences for the beta and alpha subunits, confirming the synthesis of pea lectin via a pre-pro-form. The entire pea lectin sequence showed a high degree of homology with the published amino acid sequences of lectins from lentil, broad bean, and, to a lesser extent, with concanavalin A from jack bean. Sequence variation occurred mainly in those regions thought not to be involved in metal- and sugar-binding or in the formation of beta-pleated sheets.

Amino Acid Sequence↗
PubMed1983

Intracellular sites of synthesis and processing of lectin in developing pea cotyledons.

The biosynthesis and processing of pea lectin was studied in developing pea cotyledons by a combination of pulse-chase experiments using 14C-aminoacids and subcellular fractionation of the homogenates. Lectin polypeptides were isolated on immunoaffinity gels and fractionated on sodium dodecyl sulfate-polyacrylamide gels followed by fluorography. Whether made in vivo or in an in vitro chain completion system containing polysomes still attached to microsomal membranes, newly synthesized lectin had an Mr of 23,000. In vivo labeling showed that radioactive lectin first associates with the rough endoplasmic reticulum and is then sequestered into the lumen of the endoplasmic reticulum. Pulse-chase experiments showed that it is chased out of the endoplasmic reticulum considerably more slowly than the storage proteins and accumulates in the protein bodies, initially as a polypeptide of Mr = 23,000. This pro-lectin is processed in the protein bodies to its mature form with polypeptides of Mr = 17,000 and 6,000. Pea lectin is synthesized throughout the protein accumulation phase of seed formation with maximum levels of synthesis and of mRNA approximately half-way through this period.

Endoplasmic Reticulum↗
PubMed1983

Regulation of Legumin Levels in Developing Pea Seeds under Conditions of Sulfur Deficiency: Rates of Legumin Synthesis and Levels of Legumin mRNA.

It was shown previously that when peas (Pisum sativum L.) are grown with suboptimal sulfur supply the level of legumin (the more S-rich of the two major seed storage proteins) in the mature seed is selectively reduced (Randall, Thomson, Schroeder, 1979 Aust J Plant Physiol 6: 11-24). This paper reports a study of the cellular mechanisms involved in regulating legumin synthesis under these conditions. Pulse and pulse-chase labeling experiments were carried out with excised, immature cotyledons from normal and S-deficient plants. Legumin was isolated from cotyledon extracts by immunochromatography, and the proportion of legumin synthesis relative to total protein synthesis was determined. Results showed that reduced legumin accumulation could largely be accounted for by a greatly reduced level of legumin synthesis (80-88% reduction) rather than by a major increase in legumin breakdown.Legumin mRNA levels were assayed by two methods. In vitro translation of polysomal RNA from cotyledons of normal and S-deficient plants indicated a reduction of 60 to 70% in synthesis of legumin-related products by preparations from S-deficient plants. A legumin cDNA clone was constructed, characterized, and used to measure the levels of legumin mRNA in polysomal and total RNA preparations from developing cotyledons. Legumin mRNA levels were reduced by 90% in preparations from S-deficient plants.When restored to an adequate S supply, S-deficient plants (or pods taken from such plants) recovered normal levels of legumin synthesis (in vivo and in vitro) and of legumin mRNA. These results indicate that reduced legumin accumulation under conditions of S deficiency is primarily a consequence of reduced levels of legumin mRNA.

Journal Article↗
PubMed1983

Autoimmune haemolytic disease in mice after exposure to a methylating carcinogen.

N-Methyl-N-nitrosourea (MNU) but not methyl methanesulphonate (MMS) induced both autoimmune haemolytic anaemia and thymic lymphoma in susceptible strains of mice, particularly the C57BL/6. These effects could be positively correlated with the formation of O6-methylguanine in target DNA. All murine lymphoid cells showed lack of ability to remove O6-methylguanine from their DNA, therefore the variation of responses between different mouse strains indicated that other host factors, probably genetic, must be involved. The results do indicate however that a potent pre-mutagenic DNA base modification can initiate the events leading to autoimmune disease in susceptible mice.

Anemia, Hemolytic, Autoimmune↗
PubMed1977

Characterization of Pseudomonas aeruginosa derepressed R-plasmids.

A genetic study of conjugal transmissibility of two R-plasmids was undertaken in Pseudomonas aeruginosa. Conjugally derepressed mutants of the R-plasmids were isolated, and examination of 11 independent mutants revealed that 10 were recessive to the wild-type transfer repressor, whereas 1 mutant was cis dominant. Cross-repression was observed between the two R-plasmids, suggesting that they have functionally equivalent systems for regulating the expression of tra loci. The derepressed R-plasmid mutants exhibited several characteristics, in addition to derepressed transfer, that were not expressed by the parental plasmids. These included sensitivity to certain donor-specific phages, inhibition of multiplication of a transducing phage, and, in the one case examined, a high degree of entry exclusion. The coexpression of these different functions suggests that their respective genetic loci are controlled by the same regulatory system as that of tra, or else that they are part of the tra complex.

Bacteriophages↗