Phe-tRNAPhe species modified on the 3'-terminal ribose residue were investigated for their ability to participate in individual steps of the elongation cycle using eukaryotic ribosomes from reticulocytes. None of the Phe-tRNAs used, namely Phe-tRNAPhe-C-C-3'dA, Phe-tRNAPhe-C-C-3'-NH2A, and Phe-tRNAPhe-C-C-Aoxi-red, can function in the overall process. All modified Phe-tRNAPhe species can be bound nonenzymatically to ribosomes. Phe-tRNAPhe-C-C-3'NH2A exhibits exceptionally high binding at low Mg2+ concentration compared with Phe-tRNAPhe-C-C-A binding. Ac-Phe-tRNAPhe species prepared from the three modified tRNAs, when bound to the donor site, were devoid of donor activity. The enzymatic binding of both Phe-tRNAPhe-C-C-3'dA and Phe-tRNAPhe-C-C-3'NH2A is less efficient than that of Phe-tRNAPhe-C-C-A but these Phe-tRNAPhe species have acceptor activity. Phe-tRNAPhe-C-C--Aoxi-red is not a substrate for the EF-I promoted binding reaction and has no acceptor activity. The nonaminoacylated species, tRNAPhe-C-C-A, tRNAPhe-C-C-3'dA, and tRNAPhe-C-C-3'NH2A, bind to the ribosome to a larger extent than the corresponding aminoacylated tRNAs, both in the presence and in the absence of poly(U). Peptidyl-tRNAPhe-C-C-3'dA bound to the donor site cannot activate the acceptor site for EF-I promoted binding of Phe-tRNAPhe as does peptidyl-tRNAPhe-C-C-A. Further, it was observed that a correct codon-anticodon interaction influences the recognition of the 3' terminus of tRNA. Specificity of eukaryotic ribosomes for the 2'- and/or 3'-aminoacylated tRNA species is discussed and compared with the properties of Escherichia coli system.

H19 is expressed in a large percentage of bladder tumors, but not expressed in healthy bladder tissue. The aim of this study is to define H19 optimal transcriptional regulatory sequences in tumor cells, which can potentially be used to control expression of a toxin gene in constructs to be used in bladder cancer gene therapy trials in mice and human. Transient expression assays revealed that elements responsible for promoter activity are contained within the 85 bp upstream region. The transcriptional activity of this region was strongly inhibited by the methylation of the Hpa II sites. A modest cell specificity is conferred by the upstream sequences. The human and murine promoter activities were significantly increased by the human H19 4.1 kb enhancer sequence. The 85 bp H19 upstream region contains all the elements to interact with the enhancer. We showed that the human H19 promoter is highly active in a murine bladder carcinoma cell line, justifying its use to drive the expression of a cytotoxin gene in gene therapy trials in mice.

Rough endoplasmic reticulum was reconstituted from free polyribosomes and rough membrane stripped from its ribosomes by KCl and puromycin. The reconstituted rough membrane resembled the native rough membrane in the following aspects: RNA/protein ratio, buoyant density in a continuous sucrose gradient, amino acid incorporation capacity and sensitivity towards protein synthesis inhibitors. When the reconstitution was done with double labelled polyribosomes ([32-P] polyribosomes, [3-H] leucine labelling of nascent peptide chain before or after the attachment of the polyribosomes to the membrane) both labels banded together with the reconstituted rough membrane band. Hybrid rough membrane could be formed from rat liver stripped rough membrane and wheat germ ribosomes. This hybrid membrane could translate globin mRNA.

The isolation of rough and smooth endoplasmic reticulum from rat parotid salivary gland is described. The rough membrane was stripped of its bound ribosomes using the KCl-puromycin method. Rough endoplasmic reticulum was reconstituted from stripped-rough membrane and polyribosomes. The reconstituted rough membrane resembled the native rough membrane in the following aspects: RNA/protein ratio, buoyant density in a continuous sucrose gradient and amino acid incorporation capacity. The in vitro synthesis of alpha-amylase by both rough and in vitro reconstituted rough membrane was demonstrated using SDS polyacrylamide gel electrophoresis. The reconstituted rough membrane could be restripped by KCl-puromycin. The in vitro synthesized alpha-amylase remained associated with the rough or the in vitro reconstituted rough membrane, even after these membranes were stripped of their bound ribosomes.