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Biomedical subjects

G Ciliberto

Publications and source records attributed to G Ciliberto.

135 records · Page 8Linked to original sources
PubMed1982

Functional assay of tRNA molecules transcribed from a purified gene.

Purified tRNA genes are expressed when microinjected into the nucleus of X.laevis oocytes. In this paper we describe a method to assay the capacity to be aminoacylated of the tRNA transcribed in the frog oocytes. The method exploits the radiochemical purity of the transcript and relies on the binding of aminoacyl-tRNA but not of uncharged tRNA to purified elongation factor EF-Tu. We also present some preliminary results on several single point mutants of tRNAPro from Caenorhabditis elegans. We show that nucleotide 73 of tRNAPro can be substituted by any other nucleotide without loss of acceptor activity. A double mutant, causing transition from G45G46 to A45A46 has lost acceptor activity. Also inactive is a mutant carrying an insertion of a single base in the anticodon loop.

Amino Acyl-tRNA Synthetases↗
PubMed1982

Processing of eukaryotic tRNA precursors: secondary structure of the precursor specific sequences affects the rate but not the accuracy of processing reactions.

The primary transcriptional product of eukaryotic tRNA genes is a precursor molecule with extranucleotides at the 5' and at the 3' end. We show that the 5' and 3' sequences, uniquely present in the RNA precursor molecule do not play any role in the efficiency and accuracy of processing reactions. If, however, as a consequence of in vitro manipulation, these extranucleotides form a base-paired extension of the aminoacid acceptor stem, the rate of processing is slowed down. The rate of processing is brought back to normal in a single base-pair deletion mutant probably as a consequence of a destabilization of the base-paired extension of the aminoacid acceptor stem.

Base Composition↗
PubMed1982

A novel method for site-directed mutagenesis: its application to an eukaryotic tRNAPro gene promoter.

We present a novel general method for localized mutagenesis. The DNA segment to be mutagenized is inserted in the beta-galactosidase gene of a M13-lac vector, generally causing loss of beta-galactosidase function by generation of frameshifts or nonsense codons. Mutations in the inserted DNA which restore beta-galactosidase function are readily detected and analyzed. The application of this method to the promoter of an eukaryotic (Caenorhabditis elegans) tRNAPro gene has allowed the isolation of several mutants altered in transcription.

Animals↗
PubMed1982

A prokaryotic tRNATyr gene, inactive in Xenopus laevis oocytes, is activated by recombination with an eukaryotic tRNAPro gene.

Eukaryotic tDNA promoters are composed of two essential regions contained within the coding sequence (Box A and Box B). Due to the highly conserved structure of prokaryotic and eukaryotic tRNA, most prokaryotic tRNA genes are expected to be active templates in eukaryotic transcriptional systems. In this paper we show that Escherichia coli tDNATyr is not transcribed in the nucleus of Xenopus laevis oocytes. By in vitro construction of hybrid molecules between inactive prokaryotic tDNATyr from E. coli, and active eukaryotic tDNAPro from Caenorhabditis elegans, we show that tDNATyr can be made into an active gene if its first third, including the Box A region, is replaced by that of the eukaryotic tDNA . These results suggest that an improper Box A sequence is responsible for the inactivity of the E. coli tRNATyr gene, and argue against the role of secondary and tertiary DNA conformations in RNA polymerase III transcription.

Animals↗
PubMed1982

Promoter of a eukaryotic tRNAPro gene is composed of three noncontiguous regions.

The 71-base-pair coding sequences of the tRNAPro gene from Caenorhabditis elegans contains all of the information required for transcription and processing in the injected oocytes. Several subclones of the DNA coding for the tRNAPro were constructed, carrying deletions or insertions, or both. Their transcriptional properties lead to the hypothesis that the tRNAPro gene promoter is composed of three discontinuous regions within the coding sequence.

Animals↗
PubMed1982

Relationship between the two components of the split promoter of eukaryotic tRNA genes.

Plasmids containing eukaryotic tRNA genes are faithfully transcribed in the nucleus of Xenopus laevis oocytes [Cortese, R., Melton, D. A., Tranquilla, T. & Smith, J. D. (1978) Nucleic Acids Res. 5, 4593-4611]. It has been established that two separated regions within the coding sequence of a tRNA gene are essential and sufficient for promotion of transcription [Hofstetter, H., Kressmann, A. & Birnstiel, M. L. (1981) Cell 24, 573-585; Ciliberto, G., Castagnoli, L., Melton, D. A. & Cortese, R. (1982) Proc. Natl. Acad. Sci. USA 79, 1195-1199]. We have constructed a hybrid tRNA gene containing one essential region from tDNALeu and the other from tDNAPro, both from Caenorhabditis elegans. This hybrid gene is efficiently transcribed, thus showing that the essential regions are independent transcriptional signals regardless of the overall regularities of the structure of tRNA genes. We have also constructed mutants of the tRNAPro gene in which the distance between the two essential regions is changed; optimal transcription occurs when this distance is about 40-50 nucleotides.

Caenorhabditis↗
PubMed1978

Purification of pseudouridylate synthetase I from Salmonella typhimurium.

Pseudouridylate synthetase from Salmonella typhimurium has been purified 1,000 fold and is about 90% pure. The enzyme has a molecular weight of 50,000 daltons. In the presence of tRNA there is a change in molecular weight from 50.000 to 100.000. This change does not seem to be due to the formation of a tRNA-enzyme complex but rather to a tRNA induced dimerization. Other properties of the enzyme are described.

Amino Acid Sequence↗
PubMedDate not supplied

A Multimeric Synthetic Peptide Combinatorial Library.

We describe here a novel type of synthetic peptide library, named Multimeric Synthetic Peptide Combinatorial Library (M-SPCL), where multiple small peptide ligands are tied together in the same molecule. The advantage of using small peptides in the form of M-SPCL is two-fold: first, the high density assembly of the sequences on the branching scaffold leads to signal amplification, thereby effectively lowering the binding threshold for the selection of ligands; second, to interfere with protein-protein interactions, multimericity has been shown to be a desirable feature per se. The M-SPCL is prepared by solid-phase peptide synthesis, based on the structure of Multiple Antigen Peptides. When prepared in Positional Scanning format [C. Pinilla, J. Appel, P. Blanc and R.A. Houghten. 1992. BioTechniques 13: 901-905], selection is based on the amplified interaction of a single residue in a sequence-defined position. The usefulness of the new library was demonstrated by the selection of octameric peptides, which inhibit the binding of the cytokine human interleukin-6 to its receptor, with an apparent nanomolar affinity. Tetrameric, but not dimeric, branched peptides with the same sequences were also active with comparable affinity. The success of this approach is noteworthy, since screening of the corresponding monomeric pentapeptide SPCL did not lead to the selection of any inhibitory compound in the same system.

Amino Acid Sequence↗