Studies on the metabolic role of peptidyl-tRNA hydrolase

Summary A mutant strain of Eschrichia coli that is temperature-sensitive for growth stopped protein biosynthesis at 43° C after a brief lag (Fig. 1). Cell-free extracts from the strain showed no specific defect in aminoacyl-tRNA synthetases, binding initiator tRNA to ribosomes (Table 1), protein chain elongation (Tables 2, 5) or protein chain termination (Tables 3, 4) at high temperature.The partially purified enzyme peptidyl-tRNA hydrolase, however, was temperature-sensitive (Table 6); the mutant hydrolase was inactivated rapidly at 43° C (Table 7). Mixing experiments ruled out the presence, in the mutant enzyme preparation, of an inhibitor and also demonstrated, on the mutant enzyme, a protective effect by wild type enzyme that was not shown by general coli proteins (Tables 8, 9).Interrupted mating allowed the temperature-sensitive growth phenotype to be mapped near to and before trp (Figs. 4, 5). Co-transsduction, mediated by bacteriophage P1, with trp ⁺ (frequency 7.5%) located the marker at 24 min on the coli map. All transductants for temperature-sensitive growth also had temperature-sensitive peptidyl-tRNA hydrolase activity in crude sonicates (Table 10). We provisionally conclude that the temperature-sensitive protein synthesis and growth are caused by a single genetic change in the structural gene (pth) for peptidyl-tRNA hydrolase.After shift to 43° C the polysomes of the mutant cells broke down into 70S particles (Figs. 2, 3). A defect in protein biosynthesis thus appeared to be located after termination and before reformation of new polysomes.The metabolic role of peptidyl-tRNA hydrolase is discussed in the light of these experiments.Journal paper No. J-7465 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, project no. 1747.     

Agar-Screen Specimen Carrier for Bulk Processing of Biopsy Materials for Electron Microscopy

A specimen carrier for processing large numbers of biopsy materials for epoxy embedding and electron microscopy is described. Commercially available 18-mesh stainless steel or 16-mesh aluminum wire screening is used. The screening is cut into 1 × 3-inch strips. One corner is snipped off for orientation purposes. Four drops of warm 4% agar is placed on a prewarmed standard microscopic glass slide. A thin agar support film is formed on the bottom side of the horizontally held wire screen by lightly running it against the agar. Tissue blocks trimmed to 1 mm³ are blotted on filter paper and placed in a prearranged order on the top surface of the support film. A thin top coating of agar is applied on the specimen by touching it with the tip of a pasteur pipette containing warm 4% agar. The agar-screen unit with the mounted specimens is stabilized in 4% buffered formalin and rinsed with Sorenson's phosphate buffer, pH 7.4, with 6.8% sucrose. It is then processed as a unit through routine osmium tetroxide postfixation, alcohol dehydration, and Epon 812 infiltration. The tissue blocks are plucked off the agar support film with fine-tipped tweezers and embedded in individual capsules. No difficulty in thin sectioning was encountered and examination of the sections under the electron microscope showed good infiltration by the epoxy resin.     

Filopodium-derived vesicles produced by MIM enhance the migration of recipient cells

1. Download : Download high-res image (144KB)
2. Download : Download full-size image