PubMed Health⌕ Search

Biomedical subjects

J L Key

Publications and source records attributed to J L Key.

At least 73 records · Page 4Linked to original sources
PubMed1976

Analysis of plant RNA polymerase I transcript in chromatin and nuclei.

Chromatin isolated (pH 8.0) from soybean hypocotyl contains only RNA polymerase I activity as judged by its elution at low ionic strength (0.11 M ammonium sulfate) from DEAE-cellulose and DEAE-Sephadex, its total resistance to alpha-amanitin, and lack of preference for poly(dA-dT). The in vitro RNA product from this chromatin contains rRNA as a major component (36%) with little or no symmetry of transcription. The transcript from nuclei, where both RNA polymerases I and II are active, shows a dramatic increase in % rRNA (from 35 to 65%) when alpha-amanitin is present during synthesis. These observations suggest that plant RNA polymerase I is similar to animal RNA polymerase I in both its insensitivity to alpha-amanitin and preferential transcription of rRNA genes.

Amanitins↗
PubMed1976

Purification and characterization of RNA polymerase I from a higher plant.

RNA polymerase I was purified from chromatin isolated from auxin-treated soybean hypocotyl. Purification was achieved by using Agarose A-1.5m gel filtration, DEAE-cellulose, CM-sephadex, and phosphocellulose chromatography, and sucrose density gradient centrifugation. With denatured calf thymus DNA as template, the enzyme has a high specific activity (200-300 nmol/mg/30 min at 28 degrees C) which is comparable to other RNA polymerase I enzymes purified from animals and yeast. While the gel profiles indicate that purification to homogeneity (greater than 90%) may not have been achieved, the enzyme appears to be composed of possibly 7 subunits, several of which are similar to the subunits of yeast RNA polymerase I. The putative subunits and molar ratios are 183 000 (1), 136 000 (1), 50 000 (0.5), 46 000 (0.5), 40 000 (0.5), 33 000 (0.2), and 28 000 (2). The purified enzyme strongly prefers a completely denatured template such as poly(dC).

Amanitins↗
PubMed1976

Auxin-induced Changes in the Incorporation of H-Amino Acids into Soybean Ribosomal Proteins.

Auxin-induced activation of 80S ribosomes and polyribosome formation in mature soybean (Glycine max var. Hawkeye) hypocotyl (R. L. Travis, J. M. Anderson, and J. L. Key. 1973. Plant Physiol. 52: 608-612) in the presence of a mixture of radioactive amino acids correlates with an increased specific radioactivity of at least three ribosomal proteins; the labeling of one of these increased severalfold above the control level. Results of experiments with 5-fluorouracil and cycloheximide indicated that the proteins in question were synthesized in response to auxin and became associated with pre-existing ribosomes. Ribosome dissociation experiments indicated that these proteins were associated with the 60S ribosome subunit.

Journal Article↗
PubMed1976

Selective Modulation of RNA Polymerase I Activity during Growth Transitions in the Soybean Seedling.

RNA polymerase I and II activities were measured in tissues of the soybean (Glycina max, var. Wayne) hypocotyl where dramatic changes in the relative level of RNA synthesis are associated with normal and auxin-induced growth transitions. When assayed in isolated nuclei, the activity of RNA polymerase I changed much more than the activity of RNA polymerase II during these growth transitions. The activity of RNA polymerase I expressed in the nuclei generally showed a positive correlation with the relative level of RNA synthesis (i.e. accumulation) of that tissue. Following solubilization of the RNA polymerases from these isolated nuclei and fractionation of them on DEAE-cellulose, the activity of RNA polymerase I relative to that of RNA polymerase II showed smaller changes during these growth transitions than when assayed in the nuclei. Thus, these data indicate that the activity of RNA polymerase I is significantly modulated in the nucleus, up or down depending upon the growth state, during growth transitions in the soybean in addition to lesser changes which occur in the apparent level of the enzyme.

Journal Article↗
PubMed1975

Enhancement of soybean RNA polymerase I by auxin.

When etiolated soybean seedlings are treated with the synthetic auxin, 2,4-dichlorophenoxy-acetic acid, cells of the mature hypocotyl become swollen and proliferate abnormally. This abnormal growth induced by auxin coincides with a 5- to 8-fold increase in the alpha-amanitin-insensitive RNA polymerase associated with isolated chromatin or nuclei. The alpha-amanitin-sensitive RNA polymerase activity of the auxin-treated hypocotyl was similar to that of control tissue. The increase in RNA polymerase I activity of chromatin and nuclei was maintained after solubilization and fractionation on DEAE-cellulose. Auxin thus appears to enhance RNA synthetic activity (i.e., ribosomal RNA) in mature soybean tissue by altering RNA polymerase I directly rather than by altering RNA polymerase I directly rather than by altering the chromatin template.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1975

Studies on Pea Ribosomal Proteins: Conformational and Biological Activity Changes of Ribosomal Subunits Derived by NH(4)Cl Dissociation.

Ribosomal subunits prepared by NH(4)Cl dissociation (0.5 m) of the monomeric ribosomes were much less active in in vitro protein synthesis than those prepared by KCl dissociation. The decrease in activity correlated with a detachment of some proteins (L(2) and L(9) as shown by gel electrophoresis) within the 60S ribosomal subunits. Subunits prepared with 0.3 m NH(4)Cl retained L(2) and L(9), but the activity remained low. Incubation of these 60S subunits in TKM buffer (50 mm tris [pH 7.5], 20 mm KCl, and 5 mm MgCl(2)) for 20 min at 37 C restored the activity almost to the level of those obtained by KCl dissociation. Treatment of the 0.3 m NH(4)Cl-derived 60S subunits with a protein reagent, Procion brilliant blue, prior to extraction of the ribosomal proteins resulted in the loss of L(2) and L(9), showing that these proteins were made accessible for dye binding. These observations suggest that a considerable degree of unfolding of the 60S subunit occurs at 0.3 m NH(4)Cl (this apparently leads to a preferential detachment of L(2) and L(9) at 0.5 m NH(4)Cl) and that the activity of the purified subunits depends not only on the presence of L(2) and L(9) but also on the organization of these proteins within the 60S subunits.

Journal Article↗
PubMed1975

Isolation and Properties of Nuclei from Control and Auxin-treated Soybean Hypocotyl.

A quick procedure for the isolation of nuclei with good yield from soybean hypocotyl (Glycine max var. Wayne) was developed. The isolated nuclei appeared to retain their structural integrity. They were typically ellipsoidal with minima and maxima diameter of about 6 and 8 to 10 micrometers. While the nuclei were similar in size, the nucleoli were significantly larger in nuclei from auxin-treated tissue. The DNA content per nucleus was 4 +/- 1 picograms for both untreated and auxin-treated tissues. The DNA: RNA: protein ratio of isolated nuclei in untreated and auxin-treated tissues was 1: 3.1: 11 and 1: 5.4: 21.7, respectively. The purified nuclei were active in RNA synthesis; the level of RNA polymerase II activity expressed in the nuclei from untreated tissue was 50 to 60% higher than RNA polymerase. I. The nuclei from auxin-treated tissues contained about 2.5 times as much RNA polymerase I activity as nuclei from untreated tissue. The purified nuclei from both untreated and auxin-treated tissues were also active in the incorporation of (3)H-TTP into DNA.

Journal Article↗
PubMed1975

The occurrence and distribution of poly(a) ribonucleic Acid in soybean.

The occurrence and distribution of poly(A) sequences in the RNA of soybean (Glycine max var. Wayne) have been studied. Only one of the two species of AMP-rich RNA contains poly(A). D-RNA does not contain detectable poly(A) sequences. The TB-RNA is the poly(A) RNA in this system. At least a part (up to 50% or more) of the mRNA in polyribosomes contains a poly(A) sequence. The poly(A) RNA is heterodisperse in size but has a mean size of approximately 18S (2,000 nucleotides) in urea and formamide gels. The poly(A) fragment resulting from ribonuclease A and T(1) digestion migrates as a broad band overlapping the 4 to 5.8S regions of the gels with a mean size of somewhat greater than 5S. No evidence was found for the occurrence of a discrete oligo(A) fragment in the poly(A) RNA; however, oligonucleotides which migrate faster than the poly(A) fraction were observed in preparations which were not bound to oligo(dT) cellulose prior to electrophoresis. This oligonucleotide region was enriched in AMP (up to about 65%) as would be expected after ribonuclease A and T(1) digestion.

Journal Article↗
PubMed1975

Isolation of nucleoli and localization of ribonucleic Acid polymerase I from soybean hypocotyl.

An effective method for the isolation of nucleoli from auxin-treated soybean (Glycine max, var. Wayne) hypocotyl was developed by polytron homogenization and sucrose gradient centrifugation. The nucleoli expressed only the alpha-amanitin-insensitive RNA synthetic activity. This activity chromatographed as RNA polymerase I on DEAE-cellulose. It appears that the plant nucleolus, like the animal nucleolus, is the site of localization for RNA polymerase I.

Journal Article↗
PubMed1974

Activation of 80S Maize Ribosomes by Red Light Treatment of Dark-grown Seedlings.

The in vitro protein synthetic activity of 80S ribosomes from leaves of dark-grown corn seedlings was enhanced (at low Mg(2+) levels) by a 5-minute red light treatment applied 2 hours prior to tissue harvest. The effect was completely reversed by an immediate brief far red treatment, suggesting that ribosome activation is controlled by the phytochrome system. Experiments in which the interval between light treatment and tissue harvest was shortened indicate that the response was quite rapid. The initial increase in activity was detected within 30 minutes, followed by a rapid increase during the next 1.5 hours. No further increase occurred after 2 to 3 hours.The change in ribosome activity relates, at least in part, to an increase in the level of peptidyl-tRNA associated with ribosomes. Removal of peptidyl-tRNA from light-treated ribosomes also completely reversed the red light effect. Activation of ribosomes by 2 to 3 hours continuous white light (as previously reported) differs from red light activation in that reversal of this response requires salt washing of the ribosomes in addition to removal of peptidyl-tRNA.

Journal Article↗
PubMed1974

Distribution of ribosomal deoxyribonucleic Acid in subcellular fractions of higher plants.

Methods are described by which ribosomal DNA can be enriched in subcellular fractions of carrot and soybean. With both carrot and cucumber it was possible to obtain a distinct satellite DNA which contained the rDNA. Hybridization values greater than 0.49% were necessary before a satellite component was observed. Saturation hybridization values for soybean, carrot, and cucumber DNA were 0.2, 0.49, and 1.14%, respectively. These values were increased 1.6- and 2-fold in soybean and carrot, respectively, but enrichment was not obtained for cucumber.

Journal Article↗
PubMed1973

Changes in certain aminoacyl transfer ribonucleic Acid synthetase activities in developing pea roots.

Tyrosyl-, arginyl-, leucyl-, and phenylalanyl-tRNA synthetase activities were measured in extracts from three root sections of 3-day-old pea seedlings. The sections 0 to 2, 3 to 7, and 8 to 22 millimeters from the root tip were chosen to represent the regions of cell division, elongation, and maturation, respectively. The specific activity for each aminoacyl-tRNA synthetase was highest in the 0- to 2-millimeter section and lowest in the 8 to 22 millimeter section. The changes in specific activity between the sections, however, varied with the particular synthetase. Tyrosyl-tRNA synthetase from each section was fractionated into two activity regions on a diethylaminoethyl cellulose column. Approximately 10, 22, and 44% of the total tyrosyl-tRNA synthetase activity in the 0 to 2, 3 to 7, and 8- to 22-millimeter sections, respectively, were associated with the first tyrosyl-tRNA synthetase region; the remaining activity was located in the second tyrosyl-tRNA synthetase region. Only one activity region for arginyl-tRNA synthetase was detected by diethylaminoethyl cellulose column fractionation.

Journal Article↗