Nucleotide composition of RNA by polyacrylamide gel electrophoresis

A procedure using polyacrylamide gel electrophoresis has been devised for determining the nucleotide composititon of small amounts (50 μg) of RNA. The procedure permits analysis of the separated nucleotides in the gel matrix, thereby avoiding sample manipulation and allowing for greater reproducibility.     

Preparative two-dimensional polyacrylamide gel electrophoresis of 32 P-labeled RNA

Complex mixtures of RNA molecules may be separated by two-dimensional electrophoresis on polyacrylamide gel slabs. The first dimension of the separation is carried out on acid gels in the presence of a high urea concentration, the second on more concentrated gels buffered at pH 8. The method has been applied to the complete separation of RNA fractions obtained after a preliminary gel electrophoresis of partial enzymic digests of ³²P-labeled bacteriophage RNA. Another application is the fractionation of partial digests as obtained in sequence determination of RNA molecules. Spots are detected by autoradiography and extracted by a simple micro procedure which yields the material in a concentrated form suitable for sequence analysis by fingerprinting.     

High-resolution polyacrylamide gradient gel electrophoresis (PGGE) of isoenzymes from five Naegleria species

High-resolution polyacrylamide gradient gel electrophoresis (PGGE) was used to separate isoenzymes of 12 Naegleria strains: one N. australiensis, two N. lovaniensis, one N. jadini, two N. gruberi isolated from environmental samples, and six N. fowleri strains isolated from patients with primary amoebic meningoencephalitis. Of the eight enzymes studied, seven showed zymograms with interspecific variation that identified all the species tested. Although the six N. fowleri strains were biochemically the most homogeneous, they showed intraspecific isoenzyme variation that allowed them to be grouped into four zymodemes. The PGGE technique, which separates isoenzymes by their molecular shape, is both sensitive and economical. It offers an addition or an attractive alternative to isoelectric focusing which has commonly been used to aid species identification of Naegleria by separating isoenzymes by their isoelectric point.     

The comparison of seed protein patterns within the genusArachis by polyacrylamide gel electrophoresis

The seed protein patterns of 12Arachis species were compared by polyacrylamide gel electrophoresis (PAGE), similarities between patterns were measured by the Jaccard index. Results obtained confirm the close relationships established between members of the genus on morphological grounds and support the more recent classification schemes.A. villosa andA. correntina could well be regarded as distinct species on grounds of protein differences whileA. macedoi andA. villosulicarpa (although members of the same section, Extranervosae) show considerable differentiation of their protein patterns. Surprisingly, the formA. ×batizogaea showed less similarity in protein pattern to those of its parental species than might have been expected. The principle value of seed protein pattern data appears to be in distinguishing species within sections.     

Characterization of a novel eukaryal nick-sealing RNA ligase from Naegleria gruberi

The proteome of the amoebo-flagellate protozoan Naegleria gruberi is rich in candidate RNA repair enzymes, including 15 putative RNA ligases, one of which, NgrRnl, is a eukaryal homolog of Deinococcus radiodurans RNA ligase, DraRnl. Here we report that purified recombinant NgrRnl seals nicked 3′-OH/5′-PO₄ duplexes in which the 3′-OH strand is RNA. It does so via the “classic” ligase pathway, entailing reaction with ATP to form a covalent NgrRnl–AMP intermediate, transfer of AMP to the nick 5′-PO₄, and attack of the RNA 3′-OH on the adenylylated nick to form a 3′–5′ phosphodiester. Unlike members of the four known families of ATP-dependent RNA ligases, NgrRnl lacks a carboxy-terminal appendage to its nucleotidyltransferase domain. Instead, it contains a defining amino-terminal domain that we show is important for 3′-OH/5′-PO₄ nick-sealing and ligase adenylylation, but dispensable for phosphodiester synthesis at a preadenylylated nick. We propose that NgrRnl, DraRnl, and their homologs from diverse bacteria, viruses, and unicellular eukarya comprise a new “Rnl5 family” of nick-sealing ligases with a signature domain organization.