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).     

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.     

Effect of sample transport systems on survival of bacteria in ground beef.

The effects of two transport systems and cryoprotective agents on the survival of bacteria in ground beef samples were evaluated. Survival of Clostridium perfringens in ground beef samples after simulated transport (72 h) was higher (about 99%) in Dry Ice than in Trans Temp shipping units (-3 degrees C). There were no significant differences between the two transport systems in survival of coliforms, Escherichia coli, Staphylococcus aureus, or aerobic bacteria. Mixing ground beef samples at a ratio of 1:1 (wt/vol) with 10, 20, or 30% buffered solutions of dimethyl sulfoxide or glycerol before freezing improved the survival of C. perfringens and coliforms in both transport systems. Recovery of E. coli was significantly higher with the addition of 10% dimethyl sulfoxide before Dry Ice transport. Addition of 10% dimethyl sulfoxide resulted in a 100% recovery of both S. aureus and aerobic bacteria from ground beef after simulated transport in Trans Temp shipping units. The use of cryoprotective agents can improve the survival of bacteria during transport of ground beef samples.     

An Auxin-Responsive Promoter Is Differentially Induced by Auxin Gradients during Tropisms

We constructed a chimeric gene consisting of a soybean small auxin up RNA (SAUR) promoter and leader sequence fused to an Escherichia coli [beta]-glucuronidase (GUS) open reading frame and a 3[prime] untranslated nopaline synthase sequence from Agrobacterium tumefaciens. This chimeric gene was used to transform tobacco by Agrobacterium-mediated transformation. In R2 etiolated transgenic tobacco seedlings, GUS expression occurred primarily in elongation regions of hypocotyls and roots. In green plants, GUS was expressed primarily in the epidermis and cortex of stems and petioles, as well as in elongation regions of anther filaments in developing flowers. GUS expression was responsive to exogenous auxin in the range of 10-8 to 10-3 M. During gravitropism and phototropism, the GUS activity became greater on the more rapidly elongating side of tobacco stems. Auxin transport inhibitors and other manipulations that blocked gravitropism also blocked the asymmetric distribution of GUS activity in gravistimulated stems. Light treatment of dark-grown seedlings resulted in a rapid decrease in GUS activity. Light-induced decay in GUS activity was fully reversed by application of auxin. Taken together, our results add support for the formation of an asymmetric distribution of auxin at sites of action during tropism.     

Auxin-induced changes in the population of translatable messenger RNA in elongating sections of soybean hypocotyl

In vitro translation products of polyadenylated RNA from untreated and auxin-treated elongating sections of soybean (Glycine max var. Wayne) hypocotyl were analyzed by two-dimensional polyacrylamide gel electrophoresis. The levels of translatable messenger RNA for at least ten in vitro translation products are increased by auxin treatment. The induction by auxin occurs rapidly (within 15 minutes), and the amounts of the induced in vitro translation products increase with time of auxin treatment. Indoleacetic acid has the same effect on the population of translatable messenger RNA as 2,4-dichlorophenoxyacetic acid. The auxin-induced in vitro translation products disappear rapidly when Actinomycin D is present during the last two hours of a three-hour auxin treatment.     

Auxin-related gene families in abiotic stress response in Sorghum bicolor

Sorghum, a C4 model plant, has been studied to develop an understanding of the molecular mechanism of resistance to stress. The auxin-response genes, auxin/indole-3-acetic acid (Aux/IAA), auxin-response factor (ARF), Gretchen Hagen3 (GH3), small auxin-up RNAs, and lateral organ boundaries (LBD), are involved in growth/development and stress/defense responses in Arabidopsis and rice, but they have not been studied in sorghum. In the present paper, the chromosome distribution, gene duplication, promoters, intron/exon, and phylogenic relationships of Aux/IAA, ARF, GH3, and LBD genes in sorghum are presented. Furthermore, real-time PCR analysis demonstrated these genes are differently expressed in leaf/root of sorghum and indicated the expression profile of these gene families under IAA, brassinosteroid (BR), salt, and drought treatments. The SbGH3 and SbLBD genes, expressed in low level under natural condition, were highly induced by salt and drought stress consistent with their products being involved in both abiotic stresses. Three genes, SbIAA1, SbGH3-13, and SbLBD32, were highly induced under all the four treatments, IAA, BR, salt, and drought. The analysis provided new evidence for role of auxin in stress response, implied there are cross talk between auxin, BR and abiotic stress signaling pathways.     

Integrity of White Matter is Compromised in Mice with Hyaluronan Deficiency

Neurochemical Research - Brain white matter is the means of efficient signal propagation in brain and its dysfunction is associated with many neurological disorders. We studied the effect of...