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R Cortese

Publications and source records attributed to R Cortese.

At least 163 records · Page 9Linked to original sources
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

Site-directed mutagenesis of a tRNA gene: base alterations in the coding region affect transcription.

Point mutations have been introduced in vitro in a cloned nematode tRNAPro gene. Four different mutant clones altered in the DNA that codes for tRNAPro have been isolated. Studies on the expression of the mutant genes by microinjection into Xenopus oocyte nuclei reveal that their activities as transcription templates are reduced. The results show that DNA sequences that code for the extra arm and the stem of the T-psi-C-G arm of the tRNAPro have an important role in the initiation of tRNA gene transcription.

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↗
PubMed1982

Selective destruction of the outer hair cells in the chinchilla.

In the study of cochlear function an animal model in which apical outer hair cells could be destroyed with retention of inner hair cells would be valuable. Our object was to attempt to reproduce a reported method in a scientific manner. Behavioural response thresholds to pure tones were recorded in 7 monaural chinchillas. In 5, Kanamycin was administered in ototoxic dosage until the thresholds deteriorated. The other 2 served as controls. Cochlear histology was assessed from embedded surface preparations. In only one of the 5 animals was the aim achieved. In this animal outer hair cells were absent virtually throughout the cochlea and inner hair cell retention was basal. Ths thresholds were elevated on average 40 dB throughout the frequency range. This is in keeping with current concepts of cochlear function.

Animals↗
PubMed1980

Selective 32P-labelling of individual species in a total tRNA population.

A simple procedure to label individual tRNA species in a total tRNA preparation has been developed. The principle of the method is as follows: total crude tRNA (from E. coli) is incubated in the presence of a crude aminoacyl-tRNA synthetase preparation, containing most aminoacyl-tRNA synthetases and only one specific amino acid corresponding to the tRNA species which is intended to be labelled. This achieves the purpose of charging the desired tRNA species thereby protecting its 3'OH-terminus; obviously all the other tRNA species will have a free 3'OH group. Periodate oxidation, followed by beta-elimination, destroys any free 3'OH. After deacylation of the specific aminoacylated tRNA at pH 8.8 the only free 3'OH group will be the one of the desired tRNA species. High specific activity (32P)-pCp is ligated to this 3'OH by means of T4-RNA ligase. Two-dimensional polyacrylamide gel electrophoresis (2D-PGE) and sequence analysis of the isolated tRNA show that the method is very specific. Individually labelled tRNA species can be used as probes for cloning tRNA genes.

Base Sequence↗
PubMed1980

Order and intracellular location of the events involved in the maturation of a spliced tRNA.

Microinjected frog oocytes were used to analyse the RNA processing steps which lead to the appearance of a mature cytoplasmic tRNAtyr molecule. The results show that removal of the intervening sequence from within a yeast tRNAtyr precursor, excision of extra 3' and 5' nucleotides, addition of a 3'-terminal CCA and modification of at least seven ribonucleotides all occur in the nucleus before the tRNAtyr is transported to the cytoplasm. Moreover, we find that the ribonucleotide modifications occur in a strict order which precisely correlates with the size alterations of the tRNAtyr precursor.

Animals↗
PubMed1980

Transcription of tRNA genes in vivo: single-stranded compared to double-stranded templates.

The expression of cloned tRNA genes has been studied by injecting single-stranded and double-stranded DNA templates into Xenopus oocyte nuclei. In both forms the genes are faithfully transcribed after injection. Some single-stranded DNA is converted into double-stranded DNA in the oocyte nucleus. This conversion is necessary for the expression of the injected tRNA gene: no tRNA transcription is observed when DNA synthesis is inhibited. We conclude that single-stranded DNA does not serve as a template for faithful transcription of this gene in injected oocytes.

Coliphages↗
PubMed1979

Transcription of cloned tRNA genes and the nuclear partitioning of a tRNA precursor.

The transcription of transfer RNA genes (tDNAs) and processing of the transcripts have been studied by injecting cloned tDNAs into Xenopus oocyte nuclei. Three main conclusions can be drawn. First, eucaryotic nuclear tRNA genes, but neither procaryotic nor mitochondrial tRNA genes, are expressed in injected oocytes. While both nematode and yeast tDNAS direct the synthesis of authentic tRNAs, neither E. coli tDNA nor human mitochondrial tDNAs support the synthesis of defined tRNAs when injected into oocytes. Second, competition experiments with co-injected 5S genes and inhibition experiments with alpha-amanitin show that injected tDNAs are transcribed by RNA polymerase III. Third, oocytes injected with a nematode tDNA synthesize a tRNA precursor which is processed post-transcriptionally by removal of a 5' leader sequence. This precursor is found exclusively in the nucleus and is processed in the nucleus before the mature tRNA enters the cytoplasm.

Animals↗
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↗
PubMed1978

Characterization of a Salmonella typhimurium hisU mutant defective in tRNA precursor processing.

The DA11 mutant of Salmonella typhimurium, originally isolated as derepressed for the histidine operon, carries a temperature-dependent alteration in a nucleolytic enzyme specifically involved in the maturation of tRNA. As a consequence of this alteration, no detectable synthesis of any mature tRNA species occurs in DA11 upon shift at 43 degrees C, whereas many tRNA precursors, whose sizes range between 80 and 750 nucleotides, do accumulate. Kinetic studies on the synthesis and processing of these maturation intermediates show that these molecules represent different stages in the maturation pathway, most of them being the products of previous nucleolytic events. These RNA molecules are in vivo substrates of methylation and thiolation enzymes and can be cleaved in vitro to 4S RNA by wild-type but not by DA11 cell-free extract. Evidence is presented that DA11 is very probably a ribonuclease P mutant.

Histidine↗
PubMed1978

Mutagenicity of diallate, sulfallate, and triallate and relationship between structure and mutagenic effects of carbamates used widely in agriculture.

In an investigation of the mutagenic properties of 20 carbamate herbicides and fungicides by use of the Salmonella/microsome mutagenicity test as developed by Ames et al. (Mutation Res., 31: 347-364, 1975), we have found that three thiocarbamate compounds, diallate, sulfallate and triallate, are mutagenic in the presence of a liver microsomal fraction on strains TA1535 and TA100. This indicates that the metabolic products of these thiocarbamates are causing base-pair substitutions. Since the 2-chloro-allyl group is common to the three mutagenic compounds but is not common to the 17 nonmutagenic compounds, a metabolic derivative of this group is probably responsible for the mutagenic activity.

Animals↗
PubMed1978

Cloning of nematode tRNA genes and their expression in the frog oocyte.

Transfer RNA genes of the nematode Caenorhabditis elegans have been cloned in E. coli using the plasmid Col E1 as vector. The tRNAs coded by 3 hybrid plasmids were purified by hybridisation of labelled nematode tRNA with the plasmid DNAs. Each plasmid appears to code for a single distinct tRNA species. The expression of the cloned DNAs was analysed in vivo by injection into nuclei of Xenopus laevis oocytes. Evidence is presented which suggests that these nematode tRNA genes are accurately transcribed and processed in frog oocytes. Analysis of one hybrid plasmid shows that a 300 base pair DNA fragment contains both the structural gene and those regions required for its transcription in vivo. The results show that cloned eukaryotic DNAs from a heterologous source can be tested for functional gene activity in X. laevis oocytes.

Animals↗