Giardia lamblia: Isolation of RNA

Abstract

In this study we report the isolation of RNA from two strains of the parasitic protozoan Giardia lamblia: Pl and WB. In order to detect all rRNA populations present in this parasite, we used three different methods to isolate total RNA. These included phenolchloroform extraction (Brawerman 1974), guanidine isothiocyanate extraction (Chirgwin et al. 1979), and a modified method of hydroxylapatite chromatography (Markow and Ivanov 1974). This latter one consisted of the use of 0.19 M sodium phosphate solution, pH 6.8, to elute total RNA. The yields of total RNA by the three methods used were 143, 21, and 51 kg/l X IO8 trophozoites, respectively. Among the methods used, phenol chloroform extraction was the most efficient procedure to obtain G. lamblia RNA. Gel electrophorcsis analysis, under nondenaturing conditions, of total RNA obtained from the Pl and WB strains by the three different methods, revealed the presence of two prominent rRNA bands which correspond to the large (LSrRNA) and small (SSrRNA) rRNA species of G. lamblia, as has also been reported by Boothroyd et al. (1987) and Edlind and Chakraborty (1987). The electrophoretic mobility of these species is shown in Fig. 1. A difference was observed in the pattern of total RNA extracted by guanidine isothiocyanate in that low molecular weight RNAs were hardly detected (lane 2). The RNA samples from Pl and WB strains were also analyzed under strong denaturing conditions. In this, RNA preparations were heated in a solution of formamideformaldehyde at 60°C followed by electrophoresis in agarose gels containing formaldehyde. This analysis revealed only two populations of 2390 and 1420 nucleotides. According to their intensity after staining with ethidium bromide, these RNA populations may be present in similar amounts in this parasite (rat brain and Escherichia coli rRNAs were used as standards). None of the methods used revealed a large labile rRNA as has been described for some protozoan (Albath et al. 1984; Castro er al. 1981). RNA purified by phenol extraction or by hydroxylapatite chromatography was centrifuged through sucrose gradients under nondenaturing conditions. The profiles of the optical density measurements from the collected fractions revealed the presence of two peaks which corresponded to the two rRNA species of G. lamb& (Pl strain) (Fig. 2). Sedimentation values of large and small rRNAs calculated using rat brain rRNAs as standards were 21s and 15S, respectively. The same sedimentation values were obtained for rRNAs from WB strain (data not shown). In order to determine the abundance of low molecular weight RNA species, RNA preparations from Pl and WB strains obtained by the three methods were fractionated by denaturing polyacrylamide gel electrophoresis. Two major RNA populations and three less abundant species of lower molecular weight were observed after isolation by phenol extraction and hydroxylapatite chromatography (Pl strain) (Fig. 3). The sizes of the two prominent RNA molecules were determined relative to denatured rat brain and yeast 5.8s and 5s RNA standards, and corresponded to approximately 130 and 118 bases. RNA isolated by guanidine isothiocyanate extraction contains only one band which corresponds to the 130 bases specie; other populations were not observed. This may be due, in part, to the low recovery of smaller RNA populations by this method. Integrity of the isolated RNA by phenolchloroform extraction, guanidine isothiocyanate extraction, and hydroxylapatite chromatography was confirmed by in vitro translation experiments in which a broad spectrum of polypeptides were obtained. Interestingly, all RNA species identified from both strains of G. fumblia are smaller than most of the prokaryotic and eukaryotic rRNAs described (Noller 1984). These results, together with the data obtained for other protozoa, show a considerable diversity in eukaryotic rRNAs, their genes, and the processing mechanisms in these organisms. The complete characterization of rRNA as well as their genes will contrib-