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A R Cashmore

Publications and source records attributed to A R Cashmore.

At least 37 records · Page 2Linked to original sources
PubMed1996

The pef mutants of Arabidopsis thaliana define lesions early in the phytochrome signaling pathway.

In a screen for early-flowering mutants, a number of mutants that were early flowering under both short and long days were isolated. One such mutant, pef1, was selectively insensitive to both red and far-red light in the inhibition of hypocotyl elongation response; a classic phytochrome phenotype mediated by both PHYA and PHYB. The pef1 mutant seedlings could not be phenotypically rescued by biliverdin, a precursor of the phytochrome chromophore, nor did they fail to complement any previously identified elongated hypocotyl (hy) mutants. Difference spectra and Western blot analysis showed normal concentrations of PHYA photoreceptor apoprotein, which appeared photochemically active. It was concluded that the pef1 mutant is defective in both PHYA- and PHYB-mediated signaling pathways, and may represent a lesion in an early step of the phytochrome signal transduction pathway. Additional pef mutants deficient specifically in PHYB-mediated responses were also identified by this screen.

Arabidopsis↗
PubMed1995

Expression of an Arabidopsis cryptochrome gene in transgenic tobacco results in hypersensitivity to blue, UV-A, and green light.

The Arabidopsis HY4 gene, required for blue-light-induced inhibition of hypocotyl elongation, encodes a 75-kDa flavoprotein (CRY1) with characteristics of a blue-light photoreceptor. To investigate the mechanism by which this photoreceptor mediates blue-light responses in vivo, we have expressed the Arabidopsis HY4 gene in transgenic tobacco. The transgenic plants exhibited a short-hypocotyl phenotype under blue, UV-A, and green light, whereas they showed no difference from the wild-type plant under red/far-red light or in the dark. This phenotype was found to cosegregate with overexpression of the HY4 transgene and to be fluence dependent. We concluded that the short-hypocotyl phenotype of transgenic tobacco plants was due to hypersensitivity to blue, UV-A, and green light, resulting from over-expression of the photoreceptor. These observations are consistent with the broad action spectrum for responses mediated by this cryptochrome in Arabidopsis and indicate that the machinery for signal, transduction required by the CRY1 protein is conserved among different plant species. Furthermore, the level of these photoresponses is seen to be determined by the cellular concentration of this photoreceptor.

Arabidopsis↗
PubMed1995

Association of flavin adenine dinucleotide with the Arabidopsis blue light receptor CRY1.

The arabidopsis thaliana HY4 gene encodes CRY1, a 75-kilodalton flavoprotein mediating blue light-dependent regulation of seedling development. CRY1 is demonstrated here to noncovalently bind stoichiometric amounts of flavin adenine dinucleotide (FAD). The redox properties of FAD bound by CRY1 include an unexpected stability of the neutral radical flavosemiquinone (FADH.). The absorption properties of this flavosemiquinone provide a likely explanation for the additional sensitivity exhibited by CRY1-mediated responses in the green region of the visible spectrum. Despite the sequence homology to microbial DNA photolyases, CRY1 was found to have no detectable photolyase activity.

Arabidopsis↗
PubMed1995

Multifactorial resistance to 5,10-dideazatetrahydrofolic acid in cell lines derived from human lymphoblastic leukemia CCRF-CEM.

5,10-dideaza-5,6,7,8-terrahydrofolic acid (DDATHF) is a potent antiproliferative agent in cell culture systems and in vivo in a number of murine and human xenograft tumors. In contrast to classical antifolates, which are dihydrofolate reductase inhibitors, DDATHF primarily inhibits GAR transformylase, the first folate-dependent enzyme along the pathway of de novo purine biosynthesis. The (6R) diastereomer of DDATHF (Lometrexol), currently undergoing clinical investigation, was used to develop CCRF-CEM human leukemia sublines resistant to increasing concentrations of the drug. Three cell lines were selected for ability to grow in medium containing 0.1 microM, 1.0 microM, and 10 microM of (6R)DDATHF, respectively. Impaired polyglutamylation was identified as a common mechanism of resistance in all three cell lines. A progressive decrease in the level of polyglutamylation was associated with diminished folylpolyglutamate synthetase activity and paralleled increasing levels of resistance to the drug. However, the expression of folylpolyglutamate synthetase RNA was not altered in the resistant cell lines compared to the parent cells. The most resistant cell subline also displayed an increased activity of gamma-glutamyl hydrolase. The sublines were scrutinized for other possible mechanisms of resistance. No alterations in drug transport or in purine economy were found. Modest increases were found in the activity of methylene tetrahydrofolate dehydrogenase but no alterations of other folate-dependent enzymes were observed. Increases in accumulation and conversion of folic acid to reduced forms, particularly 10-formyltetrahydrofolate, was also seen. The resistant cell lines were sensitive to dihydrofolate reductase inhibitors, methotrexate and trimetrexate, for a 72-h exposure period but showed cross-resistance to methotrexate for 4 and 24 h exposures. Cross-resistance was also shown toward other deazafolate analogues for both short- and long-term exposures.

5,10-Methylenetetrahydrofolate Reductase (FADH2)↗
PubMed1995

The G-box: a ubiquitous regulatory DNA element in plants bound by the GBF family of bZIP proteins.

The G-box (CACGTG) is a ubiquitous, cis-acting DNA regulatory element found in plant genomes. Proteins known as G-box factors (GBFs) bind to G-boxes in a context-specific manner, mediating a wide variety of gene expression patterns. We suggest that, as for many biological systems, different combinations of these common elements can lead to diversity and specificity in the regulation of plant gene expression.

Amino Acid Sequence↗
PubMed1995

Mutations throughout an Arabidopsis blue-light photoreceptor impair blue-light-responsive anthocyanin accumulation and inhibition of hypocotyl elongation.

This paper reports the characterization of novel mutations within the Arabidopsis thaliana HY4 gene, which has previously been shown to encode a protein (CRY1) with characteristics of a blue-light photoreceptor. Several point mutations were identified within the amino-terminal domain of CRY1--this region of CRY1 has high homology to photolyase and is likely to be involved in blue-light-mediated electron transfer. Mutations were found within the region of homology to the known chromophore binding domains of photolyase. Point mutations within the 200 amino acid carboxy-terminal extension distinguishing CRY1 from photolyase, likewise disrupt function of the protein. CRY1 was originally defined as the photoreceptor responsible for blue-light-mediated inhibition of hypocotyl elongation and we now report that anthocyanin accumulation in germinating seedlings is an additional phenotype under the control of this photoreceptor--this is shown to be mediated in part by modulation of mRNA levels of chalcone synthase, one of the anthocyanin biosynthetic enzymes. The effect of the novel mutations on both inhibition of hypocotyl elongation and anthocyanin biosynthesis have been evaluated, and it is demonstrated that mutations with less severe effects on hypocotyl elongation show a similarly reduced effect on anthocyanin biosynthesis. These results are consistent with the cryptochrome photoreceptor mediating multiple regulatory pathways by the same primary mode of action.

Amino Acid Sequence↗
PubMed1995

Multiple isoforms of Arabidopsis casein kinase I combine conserved catalytic domains with variable carboxyl-terminal extensions.

Three cDNA clones encoding isoforms of casein kinase I (CKI) were isolated from Arabidopsis thaliana. One full-length clone, designated CKI1, contained an open reading frame of 1371 bp encoding a protein of 51,949 D with an isoelectric point of 9.7. In addition to the highly conserved catalytic domain (of about 300 amino acids), the Arabidopsis CKI isoforms contain 150 to 180 amino acid carboxyl-terminal extensions, which show among themselves a lower level of sequence conservation. These extensions do not show any sequence similarity to nonplant CKI isoforms, such as rat testis CKI delta, which is their closest isolated homolog, or to yeast CKI isoforms. Three additional isoforms of Arabidopsis CKI were found in the data bases of expressed sequence tags and/or were isolated serendipitously in nonspecific screening procedures by others. One of them also shows a carboxyl-terminal extension, but of only 80 amino acids. Casein kinase activity was detected in the soluble fraction of Escherichia coli strains expressing the CKI1 protein. This activity showed the crucial properties of CKI, including the ability to phosphorylate the D4 peptide, a specific substrate of CKI, and inhibition by N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide, a specific CKI inhibitor. Like several recombinant CKI isoforms from yeast, CKI1 was able to phosphorylate tyrosine-containing acidic polymers.

Amino Acid Sequence↗
PubMed1995

Reconstitution of Arabidopsis casein kinase II from recombinant subunits and phosphorylation of transcription factor GBF1.

In contrast to the well-defined tetrameric structure of animal and yeast casein kinase II (CKII), plant CKII is found in two forms: a monomeric form and an oligomeric form whose subunit composition is not well defined. The Arabidopsis homologs of the catalytic subunit alpha (CKA1) and the regulatory subunit beta (CKB1) of CKII were expressed in Escherichia coli to examine their ability to form complexes, the effect of CKB1 on the catalytic activity, and the relationship of the recombinant enzymes to those isolated from plant material. Both subunits were found mainly in the inclusion body fraction in the bacterial expression strains, and they were solubilized and renatured with the recovery of catalytic (CKA1) and stimulatory (CKB1) activities. The combination of purified CKA1 and CKB1 proteins resulted in up to 100-fold stimulation of casein kinase activity compared with the CKA1 activity alone, showing that CKB1 has biochemical properties similar to those of the beta subunit from animals. CKA1 and CKB1 spontaneously assembled into a tetrameric complex, CKA1(2)CKB12, which had properties very similar to those of the oligomeric CKII form isolated from broccoli. However, the properties of the catalytic subunit CKA1 alone differed from those of the broccoli monomeric form of CKII-like activity. Phosphorylation of transcription factor GBF1 with the reconstituted CKA1(2)CKB1(2) enzyme resulted in stimulation of its DNA binding activity and retardation of the protein-DNA complex; these results are identical to those obtained previously with isolated nuclear CKII from broccoli.

Animals↗
PubMed1994

Isolation and characterization of a fourth Arabidopsis thaliana G-box-binding factor, which has similarities to Fos oncoprotein.

A fourth member of the Arabidopsis G-box-binding factor (GBF) family of bZIP proteins, GBF4, has been isolated and characterized. In a manner reminiscent of the Fos-related oncoproteins of mammalian systems, GBF4 cannot bind to DNA as a homodimer, although it contains a basic region capable of specifically recognizing the G-box and G-box-like elements. However, GBF4 can interact with GBF2 and GBF3 to bind DNA as heterodimers. Mutagenesis of the leucine zipper of GBF4 indicates that the mutation of a single amino acid confers upon the protein the ability to recognize the G-box as a homodimer, apparently by altering the charge distribution within the leucine zipper.

Amino Acid Sequence↗
PubMed1993

HY4 gene of A. thaliana encodes a protein with characteristics of a blue-light photoreceptor.

Specific responses to blue light are found throughout the biological kingdom. These responses--which in higher plants include phototropism, inhibition of hypocotyl elongation, and stomatal opening--are in many cases thought to be mediated by flavin-type photoreceptors. But no such blue-light photoreceptor has yet been identified or isolated, although blue-light responses in plants were reported by Darwin over a century ago, long before the discovery of the now relatively well characterized red/far-red light photoreceptor, phytochrome. Here we describe the isolation of a gene corresponding to the HY4 locus of Arabidopsis thaliana. The hy4 mutant is one of several mutants that are selectively insensitive to blue light during the blue-light-dependent inhibition of hypocotyl elongation response, which suggests that they lack an essential component of the cryptochrome-associated light-sensing pathway. The HY4 gene, isolated by gene tagging, was shown to encode a protein with significant homology to microbial DNA photolyases. As photolyases are a rare class of flavoprotein that catalyse blue-light-dependent reactions, the protein encoded by HY4 has a structure consistent with that of a flavin-type blue-light photoreceptor.

Amino Acid Sequence↗
PubMed1993

Purification and characterization of casein kinase I from broccoli.

Casein kinase I from broccoli was purified approximately 65,000-fold by chromatography on phosphocellulose, phenyl-Sepharose, CM-Sephacel, and affinity chromatography on N-(2-aminoethyl)-5-chloroisoquinolone-8-sulphonamide (CKI-7)-Sepharose. The catalytic subunit of casein kinase I was identified as a 36-38 kDa polypeptide doublet by using the technique of activity gel assay after SDS/PAGE with casein as a gel-incorporated substrate. A silver-stained polypeptide doublet of the same molecular mass constituted at least 95% of the protein in the final preparation, corresponding to a specific activity of approximately 1800 nmol/min per mg of protein. The enzyme was found to be a monomer by gel filtration and glycerol gradient sedimentation; the native molecular mass was calculated to be 34.2 kDa. These characteristics, as well as other essential features of plant casein kinase I activity, such as substrate specificity and sensitivity to inhibitors, were found to be similar to those established for animal casein kinase I. Broccoli casein kinase I showed weak immunological cross-reactivity with antibodies raised against bovine casein kinase I.

Casein Kinases↗
PubMed1993

5,10-Dideazatetrahydrofolic acid (DDATHF) transport in CCRF-CEM and MA104 cell lines.

5,10-Dideazatetrahydrolic acid (DDATHF) is representative of a new class of antifolates acting through inhibition of de novo purine synthesis. We report here the transport characteristics of the diastereomers of DDATHF, which differ in configuration at C6, and comparison studies with other folate and antifolate analogs. (6R)-DDATHF showed high affinity for the influx system of CCRF-CEM cells with a Km of 1.07 microM and an influx Vmax of 4.04 pmol/min/10(7) cells. Comparative studies with methotrexate yielded an influx Km of 4.98 microM and a Vmax of 6.64 pmol/min/10(7) cells, and with 5-formyltetrahydrofolate an influx Km of 2.18 microM and a Vmax of 6.84 pmol/min/10(7) cells. Uptake of (6R)-DDATHF was competitively inhibited by (6S)-DDATHF, methotrexate (MTX), and 5-formyltetrahydrofolate, all with Ki values similar to their influx Km. The (6S)-DDATHF diastereomer had an influx Km of 1.04 microM, similar to that of (6R)-DDATHF; however, the Vmax of 1.72 pmol/min/10(7) cells was 2.3-fold lower than for (6R)-DDATHF. The transport properties of DDATHF were also studied in a mutant cell line (CEM/MTX), resistant to MTX based on impaired drug transport. In this system (6R)-DDATHF showed an influx Km of 1.49 microM and a decreased influx Vmax of 0.60 pmol/min/10(7) cells. A similar effect was shown for MTX (Km of 7.48 microM, Vmax of 1.02 pmol/min/10(7) cells). The number of binding sites in CCRF-CEM cells was similar for (6R)-DDATHF, (6S)-DDATHF, and MTX, 0.74, 0.71, and 0.76 pmol/10(7) cells, respectively. These values were slightly higher in the CEM/MTX cell line (1.07 and 1.09 pmol/10(7) cells for (6R)-DDATHF and MTX, respectively). Treatment of CCRF-CEM cells with either the N-hydroxysuccinimide ester of MTX or the corresponding N-hydroxysuccinimide ester of (6R)-DDATHF caused substantial inhibition (> 90%) of the influx of (6R)-[3H]DDATHF and [3H]MTX, respectively. These results suggest strongly that DDATHF and MTX share a common influx mechanism through the reduced folate transport system. The internalization of DDATHF by monkey kidney epithelial MA104 cells, which express a high affinity folate receptor, was also studied. Competitive binding studies using purified folate receptor and radiolabeled 5-methyltetrahydrofolate showed that (6S)- and (6R)-DDATHF both had I50 values lower than 5-methyltetrahydrofolate (12 nM). Further studies indicate that both DDATHF isomers are actively intracellularly concentrated through this route and are also rapidly converted to high chain length polyglutamates. Transport via this system was inhibited in folate-depleted cells by 10 nM folic acid.(ABSTRACT TRUNCATED AT 400 WORDS)

Biological Transport↗
PubMed1993

HAT3.1, a novel Arabidopsis homeodomain protein containing a conserved cysteine-rich region.

Homeodomain proteins have been shown to play a major role in the development of various organisms. A novel Arabidopsis homeodomain protein has been isolated based on its capability to interact with a DNA motif derived from the light-induced cab-E promoter of Nicotiana plumbaginifolia. The homeodomain of this protein, designated HAT3.1, differs substantially from those in other plant homeobox proteins identified so far. Furthermore, HAT3.1 is unique among other Arabidopsis proteins in that it does not contain a leucine zipper motif following the homeodomain. HAT3.1 is further characterized by an N-terminal region that shares substantial sequence similarity with the maize homeodomain protein Zmhox1a. Within this conserved region, the presence of eight regularly spaced cysteine/histidine residues was observed reminiscent of other metal-binding domains. Based on the strong evolutionary conservation of this domain, it is proposed that this region represents a novel protein-motif which is denoted PHD-finger (plant homeodomain-finger). In vitro DNA binding studies demonstrated that HAT3.1 is capable of interacting with any DNA fragment larger than 100 bp. Interestingly, a deletion of the N-terminal PHD-finger domain completely abolished DNA binding, suggesting that this region may play an important functional role in protein-protein or protein-DNA interaction. HAT3.1 mRNA was primarily detected in root tissue, implying a regulatory function of this protein in root development.

Amino Acid Sequence↗
PubMed1992

Isolation and characterization of a cDNA clone encoding a cognate 70-kDa heat shock protein of the chloroplast envelope.

The translocation of proteins into the endoplasmic reticulum, the mitochondrion, and the chloroplast has recently been shown to involve homologues of the highly conserved 70-kDa heat shock protein (HSP70) family. In this study, we have isolated and sequenced a full-length cDNA clone encoding a cognate 70-kDa heat shock protein of the spinach chloroplast envelope (SCE70). The cDNA insert is 2,535 base pairs long and codes for 653 amino acid residues of a protein with a predicted molecular mass of 71,731 daltons. The deduced amino acid sequence shows a high degree of homology with HSP70 proteins from other organisms. Southern genomic and RNA analyses reveal different hybridization patterns than that observed for a heat-inducible 70-kDa protein gene. The protein synthesized from the SCE70 cDNA insert co-migrates with a 70-kDa polypeptide of the chloroplast envelope following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis and import studies indicate that SCE70 is associated with the chloroplast outer envelope. The import data suggest that SCE70 is targeted to the envelope membrane via a pathway different from other plastidic precursors but similar to that recently reported for outer envelope proteins SOE1 and OM14.

Amino Acid Sequence↗
PubMed1992

Heterodimerization between light-regulated and ubiquitously expressed Arabidopsis GBF bZIP proteins.

The promoters of a variety of plant genes are characterized by the presence of a G-box (CCACGTGG) or closely related DNA motifs. These genes often exhibit quite diverse expression characteristics and in many cases the G-box sequence has been demonstrated to be essential for expression. The G-box of the Arabidopsis rbcS-1A gene is bound by a protein, GBF, identified in plant nuclear extracts. Here we report the isolation of three Arabidopsis thaliana cDNA clones encoding GBF proteins referred to as GBF1, GBF2 and GBF3. GBF1 and GBF2 mRNA is present in light and dark grown leaves as well as in roots. In contrast, GBF3 mRNA is found mainly in dark grown leaves and in roots. The deduced amino acid sequences of the three cDNAs indicate that each encodes a basic/leucine zipper protein. In addition, all three proteins are characterized by an N-terminal proline-rich domain. Homodimers of the three proteins specifically recognize the G-box motif, with GBF1 and GBF3 binding symmetrically to this palindromic sequence. In contrast, GBF2 binds to the symmetrical G-box sequence in such a way that the juxtaposition of the protein and the DNA element is clearly asymmetric and hence distinct from that observed for the other two proteins. The fact that GBF1, GBF2 and GBF3 possess both distinct DNA binding properties and expression characteristics prompt us to entertain the notion that these proteins may individually mediate distinct subclasses of expression properties assigned to the G-box. Furthermore, we demonstrate that GBF1, GBF2 and GBF3 heterodimerize and these heterodimers also interact with the G-box, suggesting a potential mechanism for generating additional diversity from these GBF proteins.

Amino Acid Sequence↗
PubMed1992

DNA binding site preferences and transcriptional activation properties of the Arabidopsis transcription factor GBF1.

The G-box is a cis-acting element found within the promoters of many plant genes where it mediates expression in response to a variety of different stimuli. This palindromic DNA motif (CCACGTGG) is composed of two identical half sites, the base pairs of which we have numbered -4 to +4 (numbering from 5' to 3'). Both half sites are involved in the binding of the bZIP protein GBF1, a member of the GBF family of Arabidopsis thaliana. Here we demonstrate using the random binding site selection method that GBF1 interacts with, in addition to the palindromic G-box, other DNA motifs that fall into seven distinct groups. All groups share the ACGT core sequence, common to most DNA motifs bound by plant bZIP proteins so far characterized. Our studies demonstrate that a high affinity GBF1 binding site is further defined by the following two parameters: first, all sites contain a G residue at position +3 (as in ACGTG) and secondly, only certain base pair combinations are allowed at positions -4, -3 and +4. Two of the identified groups (TGACGTGG and TGACGTGT) contain the base pairs TG at positions -4 and -3 and hence resemble the binding sites of another class of plant bZIP proteins (TGACGT/C binding proteins). However, GBF1 only interacts with the TGACGT sequence if the two 3' distal nucleotides (positions +3 and +4) are occupied by GG or GT. These data define the differences between a G-box binding protein and TGACGT/C binding proteins. The N-terminal domain of GBF1 is defined by a high proline content. Such regions were also identified in proteins related to GBF1. We demonstrate that this N-terminal proline-rich domain of GBF1, when fused to a heterologous DNA binding domain, stimulates transcription in both plant protoplasts and mammalian cells. These extensive DNA binding studies and the characterization of the GBF1 activation domain will facilitate both the identification of regulatory elements and the in vivo function of GBF1.

3T3 Cells↗
PubMed1992

Nuclear factors binding to the extensin promoter exhibit differential activity in carrot protoplasts and cells.

The expression of the cell wall protein extensin, a hydroxyproline-rich glycoprotein, is induced by several different stimuli, including wounding. The process of protoplast preparation mimics the wounding effect and results in the induction of extensin. Using transient expression in protoplasts we analyzed several deletions of the extensin promoter. We identified an important transcriptional regulatory element located between the two TATA boxes that characterize the extensin promoter. Other regulatory elements, located further upstream between -719 to -658, are necessary for maximum level of expression. Employing electrophoretic mobility shift assays and methylation interference experiments, we demonstrate the interaction of nuclear factors with these upstream regulatory elements. In addition to the previously identified factors EGBF-1 and EGBF-2, which are mainly present in unwounded cells, we identified an additional novel DNA-binding activity that is present in extracts prepared from protoplasts but not in extracts from unwounded cells. This factor, designated EBF (extensin-binding protein), binds to a DNA fragment which when deleted results in a 48% reduction in expression.

Base Sequence↗