Jacques Bonnet

Primary structure of tRNAval2 from brewer's yeast

In order to study the interactions between valyltRNA synthetase of yeast and the corresponding tRNAs, we have isolated tRNAyal from brewer’s yeast [l] . We have studied its degradation products obtained by pancreatic and Ti ribonuclease digestions. We expected to find the monoand oligonucleotides described by Bayev et al. [2] after ribonuclease digestions of tRNAyal from baker’s yeast, but observed several differences, which could be explained by building a nucleotide sequence analogous to that described by Bayev et al., but differing at four points. tRNAya’ was further purified either by reversed phase chromatography [l] according to Kelmers et al. [6] or by chromatography on BD-cellulose at pH 3.5 according to Gillam et al. [7] (fig. 1). Structural analysis was accomplished by enzymatic fragmentation with subsequent resolution of the fragments by chromatography or electrophoresis. The methods for complete hydrolysis by T1 or pancreatic ribonucleases, subsequent separation of the oligo-

cDNA library construction from small amounts of unfractionated RNA: Association of cDNA synthesis with polymerase chain reaction amplification

We describe here a general and simple procedure for cDNA library construction making use of in vitro amplification of cDNA by polymerase chain reaction (PCR). The first-strand cDNA is synthesized from total RNA with a primer EcoRI-(dT)17 and oligo(dG) tailed. An oligonucleotide, EcoRI-BamHI-(dC)13, is used to prime the second-strand synthesis by the thermostable DNA polymerase of Thermus aquaticus. The double-stranded cDNA is then amplified directly by PCR. A study of the effect of the elongation time on the PCR products showed that a long extension time is necessary to overcome the size heterogeneity of the cDNA population. Starting from 1 microgram of total brain RNA, the products obtained ranged from 200 to more than 2000 bp. The presence of the myelin basic protein cDNA sequence was determined. A lambda gt10 library containing 2 x 10(6) clones was established with the amplified cDNA. No sequences originating from rRNA were detected by Southern blot analysis. The ability to produce representative cDNA libraries from minute amounts of total RNA by this protocol should have many applications to studies of gene expression in small amounts of tissues or cells.

Quantitative study of the ionic interactions between yeast tRNAVal and tRNAPhe and their cognate aminoacyl-tRNA ligases

A general characteristic of the interactions between the tRNAs and the aminoacyl-tRNA ligases is that they are lowered by ionic strength. One explanation is that ionic strength weakens ionic interactions existing between phosphate groups of the tRNA and positive charges of the enzyme. A quantitative study based on this assumption was undertaken by Loftfield [l] . Using the Debye-Hiickel law, he found 2 to 4 charges interacting with the same number of positive ones. Since the Debye-Htickel law is valid only for small ions, the validity of these conclusions is questionable. A second explanation for the salt effect was proposed by Yarus [2] who postulated that the interactions between tRNA and enzyme required a conformational change and that this change was inhibited by salt. We report here a study of the affinity of tRNAVa’ and tRNAPhe for their cognate aminoacyl-tRNA ligases as a function of ionic strength under conditions where conformational changes can be considered as negligeable. Our results agree with the model of interaction between nucleic acids and protein developed by Daune [3-51. Our data are consistent with the existence of 5-6 phosphate groups interacting with the same number of positive charges on the enzyme. The participation of ionic interactions in the binding of the tRNA by its cognate aminoacyl-tRNA ligase would be about 40 to 60% of the total energy of interaction under usual conditions. This model is also sufficient to explain the increase in tRNA affinity for its aminoacyl-tRNA ligase in the presence of methanol

Correlation between the morphology and the lipid and protein compositions in the peripheral nervous system of individual 8-day-old normal and trembler mice

The hereditary, hypertrophic interstitial neuropathy which afflicts the trembler mouse manifests itself about two weeks after birth. Consequently, the identification of these mutant mice was not possible before this age, except when double mutants were available. We show that the trembler mice can be easily distinguished from their normal littermates before the clinical symptoms appear by using an HPTLC/densitometry technique that allows the simple and rapid analysis of the polar lipids extracted from one sciatic nerve. The results presented in this paper demonstrate important differences between the polar lipid compositions of sciatic nerves from 8-day-old normal and trembler littermates, whose phenotypes were confirmed by the morphological analysis of the contralateral sciatic nerves. The small amount of material that is needed for this identification makes it possible to use the remaining nerve material for other studies. Furthermore, important differences between the sciatic nerve protein compositions of normal and trembler mice, identified according to their polar lipid composition, were also observed and these differences can, therefore, also be employed for the identification of the mutants before the manifestation of the clinical symptoms of the trembler neuropathy.

Developmental Expression of the P0 Glycoprotein and Basic Protein mRNAs in Normal and Trembler Mutant Mice

Mice affected by the autosomal dominant Trembler mutation exhibit a severe hypomyelinization of the PNS. Previous biochemical studies have shown that the accumulation of the major PNS myelin proteins, P0 and myelin basic protein (MBP), is strongly diminished in Trembler sciatic nerves during postnatal development. We performed Northern blots which showed that the size of mRNA species for P0 and MBP in normal and mutant mice are indistinguishable. Densitometric analysis of Northern blots showed that, in normal mice, the proportion of P0 mRNA increases up to the 12th day, then decreases slowly. At day 40, the proportion is 60% of the maximal value. In the mutant, the proportion of P0 mRNA increases up to the 12th day and then decreases much faster than in the control. At days 12 and 40, the P0 mRNA proportion measured in Trembler sciatic nerves rep resents only 40% and 7%, respectively, of the proportion measured in control littermates. The MBP mRNA proportion in the normal mice increases up to the 16th day, and then decreases to attain 45% of the maximum level at day 40. In the Trembler mouse, there is a maximum level at day 12, representing 25% of the normal level, but the MBP mRNA is barely detectable at days 8 or 40. Thus, these data seem to indicate that in the Trembler sciatic nerves, the proportions of P0 and MBP mRNAs are too small to allow the synthesis of normal levels of the corresponding proteins.

Tryptophanyl-tRNA synthetase is a major soluble protein species in bovine pancreas

Besides their central role in protein synthesis, aminoacyl-tRNA synthetases have been found or thought to be involved in other processes. We present here a study showing that tryptophanyl-tRNA synthetase has a surprising tissular distribution. Indeed, immunochemical determinations showed that in several bovine organs such as liver, kidney and heart, tryptophanyl-tRNA synthetase constitutes, as expected, about 0.02% of soluble proteins. In spleen, brain cortex, stomach, cerebellum or duodenum, this amount is about 10-times higher, and in pancreas it is 100-fold. There is no correlation between these amounts and the RNA content of the organs. Moreover, the concentration of another aminoacyl-tRNA synthetase (methionyl-tRNA synthetase) is higher in liver than in pancreas, while the amount of tRNATrp is not higher in pancreas than in liver as compared to other tRNAs. Among several interpretations, it is possible that tryptophanyl-tRNA synthetase is involved in a function other than tRNA aminoacylation. This unknown function would be specific to the differentiated organs, since fetal cerebellum and fetal pancreas contain the same amount of tryptophanyl-tRNA synthetase as adult liver.

Expression of the PMP-22 Gene in Trembler Mutant Mice: Comparison with the Other Myelin Protein Genes

The Trembler mouse suffers from a dominantly inherited autosomal mutation affecting the Schwann cell activities, which results in an abnormal myelination of the peripheral nervous system. Very recently, it has been shown that the mutation is in the PMP-22 gene. However, the level of expression of the mutated gene in trembler mice has not been studied. Therefore, we measured the steady-state levels of mRNA encoding PMP-22 in the sciatic nerve of normal and trembler mice, and we compared these results with the steady-state levels of mRNAs encoding the major peripheral nervous system myelin proteins. Our results show that all the myelin protein genes studied are affected by the trembler mutation but to a different extent, and that the PMP-22 gene is expressed at very low levels in the trembler nerve. This suggests that regulation of the expression of the PMP-22 gene is altered in the Trembler mutant.

Myelin P0 glycoprotein : identification of the site phosphorylated in vitro and in vivo by endogenous protein kinases

Abstract: Myelin membrane prepared from mouse sciatic nerve possesses both kinase and substrates to incorporate [32P]PO43− from [γ‐32P]ATP into protein constituents. Among these, P0 glycoprotein is the major phosphorylated species. To identify the phosphorylated sites, P0 protein was in vitro phosphorylated, purified, and cleaved by CNBr. Two 32P‐phosphopeptides were isolated by HPLC. The exact localization of the sequences around the phosphorylated sites was determined. The comparison with rat P0 sequence revealed, besides a Lys172 to Arg substitution, that in the first peptide, two serine residues (Ser176 and Ser181) were phosphorylated, Ser176 appearing to be modified subsequently to Ser181. In the second peptide, Ser197, Ser199, and Ser204 were phosphorylated. All these serines are clustered in the C‐terminal region of P0 protein. This in vitro study served as the basis for the identification of the in vivo phosphorylation sites of the C terminal region of P0. We found that, in vivo, Ser181 and Ser176 are not phosphorylated, whereas Ser197, Ser199, Ser204, Ser208, and Ser214 are modified to various extents. Our results strongly suggest that the phosphorylation of these serine residues alters the secondary structure of this domain. Such a structural perturbation could play an important role in myelin compaction at the dense line level.

Isolation and characterization of the yeast aspartyl-tRNA synthetase gene ☆

A yeast genomic library in Escherichia coli, constructed by insertion of Sau3A restriction fragments into the hybrid Saccharomyces cerevisiae-E. coli plasmid pFL1, was screened by a radioimmunoassay (RIA) for colonies expressing yeast aspartyl-tRNA synthetase (AspRS). Four clones were isolated by this technique. Data obtained by Southern and restriction analysis of the inserts showed a common 3.8-kb BamHI restriction fragment which, when inserted into the plasmid pFL1, gave a positive RIA. Several controls showed that this 3.8-kb insert codes for the entire AspRS: (i) S. cerevisiae transformed by the PFL1 plasmid carrying the 3.8-kb fragment overproduces AspRS activity by a factor of ten compared to the wild-type yeast strain; and (ii) a new protein with electrophoretic behaviour similar to AspRS and immuno-reactive toward anti-AspRS appears in crude extracts of transformed yeast and E. coli.

IMPROVED MICROPLATE IMMUNOENZYMATIC ASSAY OF PCR PRODUCTS FOR RAPID DETECTION OF MYCOPLASMA PNEUMONIAE

We developed a microtitre hybridization assay for the detection of polymerase chain reaction (PCR) amplified sequences. For this, cloned Mycoplasma pneumoniae DNA containing a sequence complementary to the PCR products is first covalently bound to microtitre wells. These coated microplates can be stored for several months. Then, an aliquot of the PCR product, labelled with digoxigenin-dUTP during its synthesis is hybridized to the immobilized DNA. The use of a rapid hybridization buffer makes this step very short (5 min). Finally, the hybridization signal is detected by an anti-digoxigenin antibody conjugated with alkaline phosphatase. Compared to Southern or other microplate hybridization techniques, this method is cheaper, involved fewer steps and allows easy handling of a large number of samples. This method was used for detection of M. pneumoniae in a series of clinical specimens.