Pierre Guillemaut

Isolation of plant DNA: A fast, inexpensive, and reliable method

We describe here a simple method to isolate DNA of high molecular weight from a wide variety of plant materials, such as trees, herbaceous plants, cell suspension cultures, calli, seeds, dried embryos, ferns and lichens. The crucial step of the extraction is the use of an acidic extraction medium. When necessary, the sample was separated on a fast RPC-5 column providing us with highly purified DNA suitable not only for restriction endonuclease analyses but also for PCR experiments, RLFP analyses, or detection of adducts.

APhaseolus vulgaris mitochondrial tRNALeu is identical to its cytoplasmic counterpart: sequencing andin vivo transcription of the gene corresponding to the cytoplasmic tRNALeu

We report here that the sequence ofP. vulgaris mitochondrial and cytoplasmic tRNALeu (NAA) are identical except for a post-transcriptional modification. There is an unidentified modification at the “wobble” position which, from the sequence of the nuclear tRNALeu gene, we identify as a derivative of C. We also show that thisP. vulgaris nuclear gene is functional by demonstrating its transcription in anin vivo eukaryotic transcription system.

Codon recognition mechanisms in plant chloroplasts

In chloroplasts, all 61 sense codons are found in chloroplast (cp) DNA sequences coding for proteins. However among the sequenced cp tRNAs or tRNA genes, tRNAs with anticodons complementary to codons CUU/C (Leu), CCU/C (Pro), GCU/C (Ala) and CGC/A/G (Arg) [or CGC/A (Arg) in Marchantia] have not been found. In this paper we show that cp tRNAAla(U*GC), cp tRNAPro(U*GG) and cp tRNAArg(ICG) are able to decode the corresponding four-codon family. In the case of leucine codons CUU/C, we show that ‘U:U and U:C wobble’ mechanisms can operate to allow the reading of these codons by cp tRNALeu (UAm7G).

Structure of bean mitochondrial tRNAPhe and localization of the tRNAPhe gene on the mitochondrial genomes of maize and wheat

Bean mitochondrial tRNAPhe, purified by RPC‐5 chromatography and two‐dimensional gel electrophoresis, has been sequenced using in vitro post‐labeling techniques. It is the first plant mitochondrial tRNA sequenced. It shows 76% homology with bean chloroplast tRNAPhe and has many features characteristic of prokaryotic tRNAsPhe. It was used as a probe to localize the tRNAPhe gene on the mitochondrial genomes of maize and wheat.

Primary structure of bean chloroplastic tRNAPhe Comparison with euglena chloroplastic tRNAPhe

The nucleotide sequence of Euglena gracilis chloroplast (chls) tRNA Phe has been published [ 1 ]. This is the first organellar tRNA so far sequenced. To date, in higher plants, only cytoplasmic tRNAs Phe had been sequenced, and it was shown that the tRNAs Phe from wheat germ [2], pea germ [3] and a minor species (20%) of lupine seeds [4] have identical primary structures. It was therefore of great interest to compare the algal chls tRNA Phe (Euglena gracilis), to the major chls tRNA Phe from a higher plant Phaseolus vulgaris, which has been purified and whose overall nucleoside composition has been established recently [51 In this work we report the analyses of the exhaustive T1 RNAase and pancreatic RNAase digestion products. We also report the study of the primary structure of two large fragments (1/3 and 2/3) derived from chemical cleavage of the tRNA Phe at mTG. The primary structure is deduced and discussed.

A powerful but simple technique to prepare polysaccharide-free DNA quickly and without phenol extraction

A protocol for the rapid isolation of polysaccharide-free DNA without phenol extraction is described. Total nucleic acids are first extracted according to Guillemaut and Maréchal-Drouard (1992) and then bound to an ion exchanger while contaminants not bound are washed away. The entire procedure can be carried out in Eppendorf tubes. The highly purified DNA obtained is suitable for restriction by the common endonucleases and serves as a substrate for PCR.

Sequences of two bean mitochondria tRNAsTyr which differ in the level of post-transcriptional modification and have a prokaryotic-like large extra-loop

SummaryTwo bean mitochondrial tRNAsTyr purified by RPC-5 chromatography and two-dimensional gel electrophoresis have been sequenced using post-labeling techniques. These two tRNAs only differ by three post-transcriptional modifications in the D-loop. They have a large variable loop and therefore resemble prokaryotic tRNAsTyr rather than eukaryotic cytoplasmic tRNAsTyr.

Sequences of initiator and elongator methionine tRNAs in bean mitochondria : Localization of the corresponding genes on maize and wheat mitochondrial genomes.

SummaryTwo bean mitochondria methionine transfer RNAs, purified by RPC-5 chromatography and two-dimensional gel electrophoresis, have been sequenced usingin vitro post-labeling techniques.One of these tRNAsMet has been identified by formylation using anE. coli enzyme as the mitochondrial tRNAFMet. It displays strong structural homologies with prokaryotic and chloroplast tRNAFMet sequences (70.1–83.1%) and with putative initiator tRNAmMet genes described for wheat, maize andOenothera mitochondrial genomes (88.3–89.6%).The other tRNAMet, which is the mitochondrial elongator tRNAFMet, shows a high degree of sequence homology (93.3–96%& with chloroplast tRNAmMet, but a weak homology (40.7%) with a sequenced maize mitochondrial putative elongator tRNAmMet gene.Bean mitochondrial tRNAFMet and tRNAmMet were hybridized to Southern blots of the mitochondrial genomes of wheat and maize, whose maps have been recently published (15, 22), in order to locate the position of their genes.

Comparison of the nucleotide sequences of chloroplast tRNAsPhe and tRNAs3Leu from spinach and bean

Abstract The nucleotide sequences of spinach chloroplast tRNAPhe and tRNA3Leu were determined using post-labelling techniques. Comparison of the primary structures of spinach chloroplast tRNAPhe and tRNA3Leu with their bean chloroplast counterparts shows, in each case, only one base difference. In addition, there are two differences in post-transcriptional modifications between bean and spinach chloroplast tRNAsPhe.

Preparation of a tRNA-dependent wheat germ protein-synthesizing system.

A method is described for the preparation of a tRNA-dependent wheat germ protein-synthesizing system. This system can be supplied with exogenous tRNAs of eukaryotic or prokaryotic origin. In order to obtain maximal aminoacylation of the added tRNAs, the translation assays can be supplemented with homologous aminoacyl-tRNA synthetase preparations. Such a tRNA-dependent wheat germ protein-synthesizing system, which is easy to prepare, can be used not only to translate plant cytoplasmic mRNAs in the presence of added cytoplasmic tRNAs, but also to determine the translation activity of exogenous tRNAs from various sources in the presence of either natural or in vitro synthesized mRNAs.