Specific transcription of eukaryotic tRNA genes in Xenopus germinal vesicle extracts.

Abstract

Cloned tRNA genes from Drosophila and from yeast have been transcribed faithfully in extracts prepared from Xenopus germinal vesicles. The newly formed RNA is composed of precursor tRNAs (of 5S RNA size and of tRNAs. The plasmid pCIT12 carries genes for Drosophila tRNALYS, tRNAg, and tRNA1sn. Nucleotide analysis of one RNA species transcribed from pCIT12 DNA showed it to be identical to Drosophila tRNALYS; it even contained some of the modified nucleotides expected for this tRNA. This RNA species is formed in the germinal vesicle extract via a larger precursor tRNA molecule that does not contain nucleotide modifications. This simple transcription system should aid studies aimed at defining the regulatory DNA regions responsible for eukaryotic gene transcription. In addition, it may provide tRNA precursors that are neeiled for detailed investigations of eukaryotic tRNA biosynthesis. Our understanding of the process of RNA biosynthesis in eukaryotic cells and of its regulation is limited (1), due in part to the lack of recognized mutants affecting tRNA biosynthesis, even in genetically well-studied eukaryotic organisms such as yeast and Drosophila. The development of techniques for molecular cloning of DNA (reviewed in ref.2) and for rapid determination of nucleotide sequences (3-5) has given some insight into the organization of tRNA genes in the genome (6) and allowed elucidation of the actual DNA sequence of some tRNA genes (7,8). However, these findings have not provided an experimental strategy for defining the detailed sequence of enzymatic steps leading to mature tRNA, nor have they shed light on the importance of the intervening sequences found in some tRNA genes (7,8). But it is clear that novel enzymes must be involved to exclude such nucleotide sequences from the mature tRNA (9).The availability of tRNA genes emphasized the need for routine tests of their biological function. In particular, an in vitro transcription system is desirable in which cloned eukaryotic tRNA genes can be easily transcribed. This should help to define the presumptive sites of initiation and termination of transcription and also to make available precursor tRNAs for studies of their enzymatic conversion to mature tRNA. The Xenopus oocyte is a promising system for such studies. DNA injected into the oocyte nucleus can be transcribed in vivo (10, 11). Cloned Xenopus genes for 5S RNA (12) and for the initiator tRNA (13) have been faithfully transcribed into their mature products in this fashion. Brown and his colleagues (14) have greatly simplified this procedure by their demonstration that cloned Xenopus 5S DNA can be efficiently transcribed in vitro in an extract prepared from germinal vesicles.In this paper we show that cloned tRNA genes from Drosophila and from yeast can be faithfully transcribed in extracts from Xenopus germinal vesicles. The major products of transcription are tRNA precursors which can be processed to mature tRNA in the germinal vesicle extract. (1500), and tRNALYS(1900). For hybridization 32P-5'-end...

Identification of multiple RNases in Xenopus laevis oocytes and their possible role in tRNA processing.

“…Of the alcohol-soluble radioactivity released by the cytoplasmic enzyme, 90% was in the form of a mononucleotide since it could be converted to uridine by alkaline phosphatase. In contrast, only 22 and 10% of the radioactivity released by the high-and low-molecular-weight nuclear enzymes, respectively, were mononucleotide. These results suggest that the cytoplasmic activity is primarily exonucleolytic and the nuclear activities primarily endonucleolytic, although the higher-molecular-weight nuclear enzyme is probably contaminated with some exonuclease.…”

Characterisation of a Dictyostelium discoideum DNA fragment coding for a putative tRNAValGUU gene.

“…To find out if our gene could be actively transcribed by RNA polymerase I11 in vitro, we performed transcription experiments using a nuclear extract prepared from germinal vesicles isolated from Xenopus laevis oocytes [39] and cytoplasmic extracts prepared from HeLa cells [40]. While the HeLa cell extract cannot initiate transcription of our plasmid, transcription products can be observed using a germinal vesicle extract (Fig.…”

“…Transcription in vitro 0.5 pg plasmid DNA was used as a template for RNApolymerase-111-directed transcription either in extracts prepared from Xenopus laevis germinal vesicles [39] or from HeLa cells [40]. Transcription reactions, using a germinal vesicle extract, contained per 20 pl: 0.5 mM ATP, CTP and UTP, 0.05 mM GTP, 3 pCi [ce3'P]GTP (635 Ci/mmol), 0.5 mM dithiothreitol, 70mM KC1, 7 mM MgCI2, 10 mM Hepes pH 7.4 and extract prepared from 10 germinal vesicles.…”

The nucleotide sequence of lysine tRNA2from Drosophila

“…Investigations into the expression of tRNA genes of yeast (10,11), Xenopus (12), and DrosophiZa (13) have demonstrated that in vivo and in vitro analysis of eukaryotic precursor tRNAs is now feasible. These studies require the knowledge of the nucleotide sequence of the specific mature tRNAs.…”