Claude Van de Walle

Method for extraction of proteins from green plant tissues for two-dimensional polyacrylamide gel electrophoresis☆

A method has been developed for the extraction of proteins from green plant tissues to be used in two-dimensional polyacrylamide gel electrophoresis. Three purification steps were necessary to overcome the problems due to streaking, charge heterogeneity, and other artifacts: After it was ground in liquid nitrogen, the powdered material was stirred in the presence of insoluble polyvinylpyrrolidone for binding phenols, and sodium ascorbate for binding quinones; proteins were precipitated with ammonium sulfate, and the sample was dialyzed. Hundreds of proteins could be detected after Coomassie blue staining using 200 micrograms of proteins from apical buds of Sinapis alba L.

Changes in mRNA level rhythmicity in the leaves ofSinapis alba during a lengthening of the photoperiod which induces flowering

A previous study has shown that mRNAs exhibit complex patterns of diurnal rhythms in their quantity in the leaves ofSinapis alba during an 8 h light/16 h dark short day (SD). In order to determine whether this situation is rapidly modified in plants subjected to an extended light treatment, we have usedin vitro translation and two-dimensional polyacrylamide gel electrophoresis, together with a strict gel comparison procedure giving aP=0.03 certitude level, to analyse the mRNA complement at different times during a 22 h light/2 h dark long day (LD).During this LD, complex changes affected about 10% of the mRNAs. Thirty-four different patterns were observed. Some diurnal rhythms present in SD are not modified by the lengthening of the light period, but most are affected. Moreover, we have shown that some mRNAs presenting a constant quantity under a SD regime show an increase or a decrease during the first hours of the photoperiod lengthening.InSinapis, this LD also induces flowering. All the changes in mRNA quantity detected thus parallel the photoperiodic induction of flowering in the leaves and are quantitative; no mRNA was shown to appear or to disappear.

Presence of heterodisperse nuclear RNA in a plant: Zea mays

Heterodisperse nuclear RNA (hnRNA) is a rapidly labelled RNA species discovered in the cell nucleus of HeLa cells [l-3] and duck erythrocytes. [4,5]. It consists of very large molecules ranging in size from 20 to 80 S [2,3]. On the basis of its low guanine + cy tosine content [3-51, it was shown to be different from the rRNA precursor, the other rapidly labelled RNA species present in the nucleus. Despite the fact that most of the hnRNA is degraded within the nucleus [2,5], the hypothesis of a precursor product relationship between this RNA and cytoplasmic mRNA was soon proposed [2-41. Evidence for such a precursor rble came from the presence of viral mRNA sequence in hnRNA of mouse cells transformed by SV40 virus [6]. Recently, the use of an anti-messenger DNA prepared from purified globin mRNA of duck erythrocytes allowed to demonstrate the presence of the same nucleotide sequence in hnRNA and mRNA [7]. This suggests that the cleavage of a large hnRNA molecule occurs during a maturation process of the mRNA as it happens for the rRNA. However, as pointed out by Imaizumi et al., no proof for a direct physical precursor-product relationship has been given and the presence of intermediate stages in the transfer of information cannot be definitively excluded [7]. Apart from its presence in mammalian and bird cells, hnRNA was shown to be present in an insect [8], an echinoderm [9] and an amphibia [lo]. The presence of a rapidly labelled, non-ribosomal, heterodisperse RNA species has also been shown in two unicellular eukaryotes Tetrah_~mena pyriformis [ 1 l] and the cellular slime mold Dictyostelium discoideum [ 121 ; but the heterodisperse RNA molecules present

Modification of the Radiation Sensitivity of Bacteriophage T7 by O2 and N2O

SummaryThe radiation sensitivity of T7 suspensions is strongly dependent on chemical additives. We have compared the response to x-irradiation in the presence of 100 per cent N2, 100 per cent O2 (eaq− and H· scavenger) and 100 per cent N2O an eaq−(scavenger that increases ·OH concentration), in the presence of a 10−2 dilution of medium and a 10−5 dilution (equivalent to medium free).Survival curves are frequently multicomponent, with sharp break points, and radiation sensitivity must be measured at varying dosage levels. In the 10−3 dilution, the order of radiation sensitivity at low doses is, kL = N2 > N2O > O2, and at high doses, kH = O2 ≅ N2O > N2. In the 10−5 dilution, or medium free, the order at low doses is kL = N2O > N2 ≅ O2 with kH difficult to define.It is concluded: (1) the OH radical is an important damaging species for T7, and (2) ‘O2 protection’ is an experimental artifact associated with impurities in the suspension.

Floral organs of the same type but at different positions within the flower of Sinapis alba (Brassicaceae) display identical protein patterns.

Summary Different interpretations of the flower of the Brassicaceae exist, suggesting the presence of either 4, 5 or 6 whorls of floral organs. In these models, the 2 short and 4 long stamens account for either 1or 2 whorls, as do the lateral and median sepals. We have searched for inside-whorl differences in gene expression in the Brassicaceae through 2D-PAGE analysis of the total protein pattern of short and long stamens, as well as lateral and median sepals, in Sinapis alba . No detected polypeptide is specific to one type of stamen, none is specific to one type of sepal. On the other hand, about 10% of the polypeptides are specific of one stage of development when long stamens from 4-mm flower buds are compared with long stamens sampled at anthesis. These results suggest that at the molecular level only 4 whorls are detectable and that subtle quantitative variations may account for inside-whorl differences in morphology.