Tom J. Guilfoyle

Auxin-regulated gene expression in intact soybean hypocotyl and excised hypocotyl sections.

A library of complementary DNA (cDNA) clones has been prepared from polyadenylated RNA (poly(A)+RNA) from auxin (2,4-dichlorophenoxyacetic acid)-treated soybean (Glycine max (L.) Merr. cv. Wayne) seedlings. Using differential hybridization, four clones were selected as auxin-responsive, and characterized. The levels of the RNA sequences homologous to the cDNA clones were examined in the hypocotyl of the intact seedling and in excised hypocotyl sections before and after auxin treatment, using RNA blot hybridization analysis. RNA levels are rapidly increased (within 0.25–0.5 h) following auxin treatment and the response in the hypocotyl of the intact seedling is transient, reaching maximum RNA levels 2–4 h after auxin application. Increases in RNA levels were also observed with the auxins indole 3-acetic acid and 2,4,5-trichlorophenoxyacetic acid, but not with the ethylene-producing compound, Ethephon (2-chloroethylphosphonic acid). Hybridization analysis of in-vitro transcription products made in nuclei isolated from untreated and auxin-treated soybean primary leaves and excised hypocotyl sections indicates that, for the two cDNA clones analyzed, the increased RNA levels in auxin-treated organs are at least partially the result of increased transcriptional activity of specific DNA sequences.

Auxin‐regulated gene expression in higher plants

The naturally occurring auxin, indole‐3‐acetic acid, is thought to regulate normal growth and developmental processes in higher plants, including cell extension, cell division, and cell differentiation. Applied auxins, including synthetic and natural auxins, can dramatically alter normal growth and developmental patterns, and applied auxins have been shown to modify expression of genes transcribed by all three classes of nuclear RNA polymerase (i.e., RNA polymerase I, II, and III). The regulation of specific genes by auxin may occur over long (e.g., several hours to days) or short (e.g., minutes) time spans after hormone application. The more interesting responses to applied auxins are the short‐term responses since these are more likely primary responses to the hormone. Several experimental approaches have been used to demonstrate that applied auxins rapidly alter gene expression, and these include in vivo labeling of polypeptides, in vitro translation of mRNAs, and cDNA cloning of auxin‐regulated mRNAs ...

A transcriptionally active, covalently closed minichromosome of cauliflower mosaic virus DNA isolated from infected turnip leaves

Purified nuclei from turnip leaves infected by cauliflower mosaic virus (CaMV) have been shown to contain a fraction of CaMV DNA that consists of covalently closed circular molecules; possesses a nucleosome structure, based on sensitivity to micrococcal nuclease; and contains nuclear RNA polymerase II that selectively transcribes the coding strand of CaMV DNA in vitro. Our results suggest that the transcriptionally active CaMV DNA is in the form of a minichromosome and that this DNA does not contain the site-specific discontinuities characteristic of the virion.

Transcription of the cauliflower mosaic virus genome in isolated nuclei from turnip leaves.

Nuclei isolated from turnip (Brassica rapa L. c.v. Just Right) leaves infected with cauliflower mosaic virus synthesize RNA in vitro which hybridizes to purified cauliflower mosaic virus DNA. Nuclei isolated from uninfected leaves do not produce these viral transcripts in vitro. Viral-specific transcription in isolated nuclei is catalyzed by endogenous DNA-dependent RNA polymerase 11 based on sensitivity to alpha-amanitin and ionic strength optima. Only one strand of the viral genome is transcribed in vitro in isolated nuclei. The RNA synthesized in vitro hybridizes to the same strand and EcoRI restriction fragments of cauliflower mosaic virus DNA as the viral-specific RNA that accumulates in vivo in infected turnip leaves.

Identification and characterization of cauliflower mosaic virus replication complexes ― analogy to hepatitis B viruses

Putative replication complexes of cauliflower mosaic virus (CaMV) which resemble hepatitis B virus replication complexes in several respects have been identified. The CaMV replication complexes contain a heterogeneous population of minus-strand DNA ranging in size from less than 600 nucleotides to full genome length of 8000 nucleotides. The minus-strand DNA of these replication complexes is largely single stranded or in a DNA/RNA hybrid structure. All of the plus-strand DNA within the complexes is in a DNA duplex which contains regions of single-stranded minus-strand DNA. These DNAs appear to be replication intermediates arising from reverse transcription of the CaMV 35 S RNA and to be encapsidated into virions or virion-like particles. The same fractions from sucrose density gradients that contain the putative replication intermediates also contain an enzymatic activity that incorporates deoxyribonucleotide triphosphates in both minus- and plus-strand CaMV DNA. This replication activity is consistent with a reverse transcription model for CaMV replication based on results which demonstrate that incorporation of deoxyribonucleotides into minus-strand DNA is refractory to actinomycin D while incorporation into plus-strand DNA is inhibited by the drug.

Cauliflower mosaic virus replication intermediates are encapsidated into virion-like particles

We have identified putative cauliflower mosaic virus (CaMV) replication intermediates of first (minus)-strand DNA synthesis which are associated with particles having properties similar to virions. The virion-like particles containing the putative replication intermediates sediment with CaMV virions on sucrose density gradients (L. Marsh, A. Kuzj, and T. Guilfoyle (1985) Virology 143, 212-223), band close to virions on CsCl density gradients, and are precipitable with antibody raised against the CaMV 42-kDa coat protein. The structures of the presumptive intermediates of DNA synthesis are compatible with a reverse transcription model for CaMV. Most or all of the observed minus-strand replication complexes appear to exist as RNA/DNA hybrids. Following RNase A treatment at low salt concentrations, the putative replication intermediates show characteristics of single-stranded DNA in selectively blotting and hybridizing to minus-strand-specific probes without prior denaturation of the blots.

The amounts, subunit structures, and template-engaged activities of RNA polymerases in germinating soybean axes☆

Abstract During the first 24 hr of soybean axis imbibition and growth, there is about a 25-fold increase in RNA polymerase activity associated with isolated nuclei. Within this same period, there is no increase in the amount of RNA polymerase I or II protein in soybean axes. There is no alteration in subunit structure of RNA polymerase II during germination and growth, with the possible exception of conversion of the 215,000 subunit to a 180,000 polypeptide, and no alteration in phosphorylation pattern of RNA polymerase II subunits. These results suggest that the rates of RNA synthesis during imbibition, germination, and growth of soybean axes are not regulated by altering the amount or subunit structure or by posttranslational modification of RNA polymerase II subunits.

Synthesis of 5S rRNA and putative precursor tRNAs in nuclei isolated from wheat embryos.

SummaryNuclei isolated from wheat embryos synthesize 4.5S precursor tRNAs and 5S rRNA in vitro. The precursor tRNAs can be processed to 4S tRNAs. Transcription of the tRNA and 5S rRNA genes is carried out by endogenous RNA polymerase III, and addition of exogenous wheat RNA polymerase III results in increased transcription on these genes. A number of experimental results suggest that proper initiation and accurate transcription of the tRNA and 5S rRNA genes occurs with isolated wheat nuclei.

A new method for determining the number of RNA polymerases active in chromatin transcription

A new method is described for determining the number of RNA chains which are being actively propagated in an invitro transcription system. This method employs [32P]-α-cordycepin triphosphate which terminates RNA chain propagation upon incorporation of the nucleotide analogue into an RNA transcript. This method is quicker and subject to fewer problems than end group analysis using [3H]nucleoside triphosphates. The auxin-induced increase in soybean RNA polymerase I activity was examined using this method. At 12, 24, and 48 hours after auxin treatment, the increase in chromatin-bound RNA polymerase I activity is predominantly due to a greater rate of RNA chain elongation rather than to an increase in the number of elongating RNA chains.

RNA Polymerases and Transcription During Developmental Transitions in Soybean

We have chosen two developmental transitions in soybean (Glycine max), (l) germination of embryonic axes and (2) auxin-induced cellular proliferation in mature hypocotyl, to investigate the effects of these transitions on DNA-dependent RNA polymerases and nuclear transcription. In both of these developmental transitions, relatively quiescent tissues are induced to proliferate. Our experimental approach has been to examine the levels and possible structural modification of RNA polymerases and attempt to correlate these with the rate of transcription in isolated nuclei and chromatin during various stages of growth and development in soybean.