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T J Guilfoyle

Publications and source records attributed to T J Guilfoyle.

At least 37 records · Page 2Linked to original sources
PubMed1994

Soybean GH3 promoter contains multiple auxin-inducible elements.

The soybean GH3 gene is transcriptionally induced in a wide variety of tissues and organs within minutes after auxin application. To determine the sequence elements that confer auxin inducibility to the GH3 promoter, we used gel mobility shift assays, methylation interference, deletion analysis, linker scanning, site-directed mutagenesis, and gain-of-function analysis with a minimal cauliflower mosaic virus 35S promoter. We identified at least three sequence elements within the GH3 promoter that are auxin inducible and can function independently of one another. Two of these elements are found in a 76-bp fragment, and these consist of two independent 25- and 32-bp auxin-inducible elements. Both of these 25- and 32-bp auxin-inducible elements contain the sequence TGTCTC just upstream of an AATAAG. An additional auxin-inducible element was found upstream of the 76-bp auxin-inducible fragment; this can function independently of the 76-bp fragment. Two TGA-box or Hex-like elements (TGACGTAA and TGACGTGGC) in the promoter, which are strong binding sites for proteins in plant nuclear extracts, may also elevate the level of auxin inducibility of the GH3 promoter. The multiple auxin-inducible elements within the GH3 promoter contribute incrementally to the overall level of auxin induction observed with this promoter.

Agrobacterium tumefaciens↗
PubMed1992

Sequence of the fifth largest subunit of RNA polymerase II from plants.

An affinity-purified antibody raised against the fifth largest subunit of cauliflower (Brassica oleracea) RNA polymerase II was used to screen an expression library and isolate an Arabidopsis thaliana cDNA clone. This cDNA clone was used to isolate a soybean (Glycine max) cDNA clone, and both clones were sequenced. The open reading frames contain 176 amino acids and predict polypeptides of 19.5 and 19.6 kDa for Arabidopsis and soybean, respectively. The amino acid sequences of the Arabidopsis and soybean polypeptides are 91.5% identical. The fifth largest subunit in plant RNA polymerase II is present at unit stoichiometry in purified enzyme and does not dissociate from the holoenzyme during nondenaturing polyacrylamide gel electrophoresis. The gene encoding the 19.5-kDa subunit has been isolated and sequenced from Arabidopsis. The gene is single copy and contains five introns. The size of the mRNA encoding this RNA polymerase II subunit in Arabidopsis and soybean is approximately 1 kilobase. None of the published yeast or animal RNA polymerase subunit sequences show similarity to the fifth largest subunit in plants.

Amino Acid Sequence↗
PubMed1992

Altered morphology in transgenic tobacco plants that overproduce cytokinins in specific tissues and organs.

An auxin-inducible bidirectional promoter from the soybean SAUR gene locus was fused to a reporter gene in one direction and a cytokinin biosynthetic gene in the opposite direction and the expression of these fused genes was examined in transgenic tobacco. The Escherichia coli uidA gene, which encodes the enzyme beta-glucuronidase (GUS), was used as the reporter gene and the Agrobacterium tumefaciens ipt gene, which encodes the enzyme isopentenyl transferase, was used as the cytokinin biosynthetic gene. These constructs allowed the overproduction of cytokinins in tobacco in a tissue- and organ-specific manner. Localized overproduction of cytokinins was monitored using the GUS reporter gene and measured by an ELISA assay. The tissue- and organ-specific overproduction of cytokinins produced a number of morphological and physiological changes, including stunting, loss of apical dominance, reduction in root initiation and growth, either acceleration or prolonged delayed senescence in leaves depending on the growth conditions, adventitious shoot formation from unwounded leaf veins and petioles, altered nutrient distribution, and abnormal tissue development in stems. While some of these morphological changes result directly from the localized overproduction of cytokinins, other changes probably result from the mobilization of plant nutrients to tissues rich in cytokinins.

Amino Acids↗
PubMed1991

Auxin-induced expression of the soybean GH3 promoter in transgenic tobacco plants.

The gene encoding the auxin-responsive GH3 mRNA (G. Hagen, A. Kleinschmidt, TJ. Guilfoyle, Planta 162: 147-153 (1984] from soybean was cloned, and its sequence and transcription initiation site were determined. The promoter of the GH3 gene has been fused to the open reading frame of the Escherichia coli uidA gene which encodes beta-glucuronidase (GUS). This fusion gene was introduced into tobacco via Agrobacterium tumefaciens-mediated transformation, and the expression of the gene was examined by fluorometric assay and histochemical staining of young R1 tobacco seedlings and mature plants. In transgenic tobacco plants that have not been exposed to exogenous auxin, expression of the fusion gene is largely restricted to roots of young green plants and developing floral organs, including ovules, developing seeds, and pollen, of mature plants. Application of exogenous auxin to tobacco seedlings or plant organs results in a greater than 50-fold increase in expression of GUS. Auxin-induced GUS expression is greatest in vascular tissue, but not restricted to this tissue. The auxin-deduced GUS expression was characterized for kinetics, auxin specificity and dose response.

Amino Acid Sequence↗
PubMed1991

Tissue-specific and organ-specific expression of soybean auxin-responsive transcripts GH3 and SAURs.

We used in situ hybridization to localize two classes of auxin-regulated transcripts, GH3 and SAURs, within organs and tissues of soybean seedlings and flowers. GH3 transcripts occurred in the inner cortex and protoxylem ridges of roots and were expressed transiently during flower and pod development. SAUR transcripts were expressed in the epidermis, cortex, and starch sheath of epicotyls and immature hypocotyls. SAUR transcripts became more abundant on the bottom side of hypocotyls that were undergoing gravitropic curvature. SAURs were also expressed in developing xylem elements of the hypocotyl hook. When soybean organ sections were treated with 50 micromolar 2,4-dichlorophenoxyacetic acid (2,4-D), GH3 transcripts became more abundant in the vascular regions of all organs analyzed. High levels of GH3 transcripts were also found in developing palisade mesophyll cells of leaves, cotyledons, and flowers treated with 2,4-D. SAUR transcripts became more abundant in the epidermis, cortex, starch sheath, and pith of epicotyls and hypocotyls after 2,4-D treatment. Our results showed that a variety of tissues and cell types express auxin-responsive transcripts and that different tissues respond rapidly to exogenous auxin by expressing different hormone-responsive genes.

Gene Expression Regulation↗
PubMed1990

Induction and superinduction of auxin-responsive mRNAs with auxin and protein synthesis inhibitors.

We have identified a class of small mRNAs (approximately 0.5 kilobases), referred to as small auxin-up RNAs (SAURs), that increases in abundance within minutes after auxin application to excised elongating hypocotyl sections of soybean. In this study, we present evidence that SAURs accumulate in the absence of auxin when protein synthesis is inhibited. Superinduction of SAURs occurs if the synthetic auxin 2,4-dichlorophenoxyacetic acid is added under conditions where protein synthesis is inhibited. Transcription run-on experiments with isolated nuclei show that, unlike 2,4-dichlorophenoxyacetic acid, protein synthesis inhibitors do not activate transcription of the SAUR genes. These results suggest that protein synthesis inhibitors act by stabilizing SAURs and that some labile protein(s) are involved in the rapid turnover of SAURs. This stabilization is not observed with GH3, another auxin-inducible mRNA. Whether induced with 2,4-dichlorophenoxyacetic acid or cycloheximide, SAURs are primarily expressed in epidermal and cortical cells of elongating hypocotyl sections, with little or no expression in vascular tissue. Unlike 2,4-dichlorophenoxyacetic acid-induced SAUR accumulation, the increase in abundance of SAURs induced by cycloheximide is transient, with a peak approximately 1 h after inhibitor addition. Complete inhibition of protein synthesis is not required for SAUR accumulation in the presence of protein synthesis inhibitors.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1990

Analysis of the genes encoding the largest subunit of RNA polymerase II in Arabidopsis and soybean.

We have cloned and sequenced the gene encoding the largest subunit of RNA polymerase II (RPB1) from Arabidopsis thaliana and partially sequenced genes from soybean (Glycine max). We have also determined the nucleotide sequence for a number of cDNA clones which encode the carboxyl terminal domains (CTDs) of RNA polymerase II from both soybean and Arabidopsis. The Arabidopsis RPB1 gene encodes a polypeptide of approximately 205 kDa, consists of 12 exons, and encompasses more than 8 kb. Predicted amino acid sequence shows eight regions of similarity with the largest subunit of other prokaryotic and eukaryotic RNA polymerases, as well as a highly conserved CTD unique to RNA polymerase II. The CTDs in plants, like those in most other eukaryotes, consist of tandem heptapeptide repeats with the consensus amino acid sequence PTSPSYS. The portion of RPB1 which encodes the CTD in plants differs from that of RPB1 of animals and lower eukaryotes. All the plant genes examined contain 2-3 introns within the CTD encoding regions, and at least two plant genes contain an alternatively spliced intron in the 3' untranslated region. Several clustered amino acid substitutions in the CTD are conserved in the two plant species examined, but are not found in other eukaryotes. RPB1 is encoded by a multigene family in soybean, but a single gene encodes this subunit in Arabidopsis and most other eukaryotes.

Amino Acid Sequence↗
PubMed1989

Transcription, organization, and sequence of an auxin-regulated gene cluster in soybean.

We have characterized a soybean gene cluster that encodes a group of auxin-regulated RNAs (small auxin up RNAs). DNA sequencing of a portion of the locus reveals five homologous genes, spaced at intervals of about 1.25 kilobases and transcribed in alternate directions. At least three of the genes are transcriptionally regulated by auxin. An increase in the rate of transcription is detected 10 min after application of auxin to soybean elongating hypocotyl sections. Each of the genes contains an open reading frame that could encode a protein of 9 kilodaltons to 10.5 kilodaltons. Sequence comparisons among the five genes reveal several areas of high homology. Two regions of high homology begin about 250 base pairs upstream of the open reading frames and two regions of homology have been identified in sequences downstream of the open reading frames. One of the latter sequences occurs in the 3'-untranslated region of the RNAs. The other occurs far downstream, 618 base pairs to 741 base pairs from the stop codon. Conservation of these sequences among the five different genes suggests that they may be important for the regulation of expression of the genes.

Amino Acid Sequence↗
PubMed1989

A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase II.

A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase IIA has been partially purified and characterized. The kinase has a native molecular weight of about 200 kilodaltons. This kinase utilizes Mg2+ and ATP and transfers about 20 phosphates to the heptapeptide repeats Pro-Thr-Ser-Pro-Ser-Tyr-Ser in the carboxyl-terminal domain of the 220-kilodalton subunit of soybean RNA polymerase II. This phosphorylation results in a mobility shift of the 220-kilodalton subunits of a variety of eukaryotic RNA polymerases to polypeptides ranging in size from greater than 220 kilodaltons to 240 kilodaltons on sodium dodecyl sulfate-polyacrylamide gels. The phosphorylation is highly specific to the heptapeptide repeats since a degraded subunit polypeptide of 180 kilodaltons that lacks the heptapeptide repeats is poorly phosphorylated. Synthetic heptapeptide repeat multimers inhibit the phosphorylation of the 220-kilodalton subunit.

Adenosine Triphosphate↗
PubMed1988

Regulation of expression of an auxin-induced soybean sequence by cadmium.

An auxin-regulated soybean sequence has been characterized and shown to be induced by the heavy metals cadmium, silver, and copper. Cadmium induces the accumulation of two size classes of mRNA: a 1-kilobase (kb) RNA class, which is the same size as the RNA class induced by auxin, silver, and copper, and a 1.4-kb RNA class. DNA sequence analysis of cDNA clones and a soybean genomic fragment has shown the presence of an intron in this gene. A restriction fragment probe isolated from the intron segment hybridizes specifically to the 1.4-kb mRNA. The transcription rate of this sequence is rapidly increased following exposure of soybean primary leaves to cadmium, as assayed by nuclear run-off transcription experiments. These results suggest that cadmium not only induces the transcription of a specific soybean sequence, but interferes with the processing of the precursor mRNA, resulting in the accumulation of the 1.4-kb mRNA precursor species.

Amino Acid Sequence↗
PubMed1985

Rapid induction of selective transcription by auxins.

Nuclei isolated from excised soybean plumules that were treated with 2,4-dichlorophenoxyacetic acid (2,4-D) were active in transcription of four auxin-regulated genes or DNA sequences, which have been described previously (G. Hagen, A. Kleinschmidt, and T. Guilfoyle, Planta 162:147-153, 1984). The rates of transcription of the auxin-responsive sequences were 10- to 100-fold greater with nuclei isolated from auxin-treated plumules than with those from untreated plumules. The transcriptional response was also observed with hypocotyls of intact soybean seedlings and hypocotyl sections, as well as with green bean and mung bean plumules that were treated with 2,4-D. Other auxins, including 2,4,5-trichlorophenoxyacetic acid, alpha-naphthaleneacetic acid, and indole-3-acetic acid, also induced the transcriptional response. Increased transcription rates were observed within 5 min after application of auxins to excised plumules, and half-maximal to maximal transcription rates were achieved by 15 min after application of auxins. As little as 10(-7) to 10(-8) M 2,4-D induced a transcriptional response, but maximal transcription rates were achieved at 10(-3) M 2,4-D. Brief treatment with the protein synthesis inhibitor cycloheximide did not inhibit the induction of transcription by auxins. These results clearly demonstrated that auxin-regulated gene expression is under rapid transcriptional control.

2,4,5-Trichlorophenoxyacetic Acid↗
PubMed1984

Size heterogeneity of the largest subunit of nuclear RNA polymerase II. An immunological analysis.

Antibodies raised against the 180-kDa subunit of cauliflower RNA polymerase II bind selectively to the largest subunit of RNA polymerase II purified from a variety of plant species. The selective binding of this antibody to the largest RNA polymerase II subunit has allowed us to probe for the size of this subunit in crude cell extracts, in fractions containing partially purified RNA polymerase II, and in isolated nuclei. Fractions containing RNA polymerase II were subjected to electrophoresis in the presence of sodium dodecyl sulfate, blotted onto nitrocellulose, and blots were probed with antibody. Immunoglobulin complexes were revealed with 125I-Protein A. Published purification procedures result in rapid conversion of a 220-kDa subunit to a 180-kDa polypeptide, but purification at high pH (pH 9.0) retards this proteolysis. RNA polymerase II associated with isolated nuclei is largely protected from proteolytic degradation, and a 240-kDa polypeptide as well as a 220-kDa polypeptide can be detected. These results suggest that the 180-kDa subunit of RNA polymerase II arises artificially during cell lysis and enzyme purification, and that even the 220-kDa polypeptide may be a degradation product of a 240-kDa polypeptide in plants.

Animals↗
PubMed1984

Immunological studies on plant DNA-dependent RNA polymerases with antibodies raised against individual subunits.

Antibodies were raised against native soybean RNA polymerase II and individual subunits of RNA polymerase II purified from soybean, cauliflower, and wheat. These antibodies were used to study the immunological relationships of plant RNA polymerases I, II, and III at the subunit level. RNA polymerases I and II from soybean, I, II, and III from cauliflower and wheat, and II from turnip were purified to homogeneity, and enzyme subunits were separated on polyacrylamide gels containing sodium dodecyl sulfate. Separated RNA polymerase subunits were electrophoretically transferred to nitrocellulose, reacted with antibodies, and the immunoglobulin complexes were revealed by reaction with 125I-labeled Protein A. Antibodies directed against native soybean RNA polymerase II bind to all or most of the subunits of soybean and wheat RNA polymerase II and to the putative common subunits in RNA polymerases I and III. Antibodies directed against individual subunits of RNA polymerase II enzymes react specifically with the subunit to which the antibody was raised and to related or identical subunits in RNA polymerases I and III. These studies confirm the presence of common subunits in the three classes of nuclear RNA polymerase and provide information on analogous or related subunits within each class of enzyme purified from different plant species.

Animals↗
PubMed1983

Nuclei purified from cauliflower mosaic virus-infected turnip leaves contain subgenomic, covalently closed circular cauliflower mosaic virus DNAs.

Nuclei isolated from cauliflower mosaic virus (CaMV) infected turnip leaves contain subgenomic CaMV DNA species in addition to the genome length CaMV DNA. These subgenomic CaMV DNA species are present as covalently closed circles (form I), relaxed circles (form II) and linear (form III) molecules. The subgenomic form I DNA species range in size from about 10% of genome length to nearly genome length. These subgenomic DNA species appear in tissue infected with cloned CaMV DNA, indicating that they arise rapidly and have not accumulated in the virus population from serial propagation of CaMV. No specific region of the CaMV genome appears to be preferentially deleted to form the subgenomic CaMV DNA species. At least three distinct subgenomic species appear to accumulate preferentially in nuclei isolated from infected tissue. Two of these abundant subgenomic CaMV DNA species are form I and the other one is form III. Some of the subgenomic CaMV DNA species appear to be minichromosomes.

Brassica↗