Differential activity staining: its use in characterization of guanylyl-specific ribonuclease in the genus Ustilago.

Abstract

Guanylyl-specific ribonuclease can be identified by a novel technique employing electrophoresis in polyacrylamide slabs followed by differential activity staining. The technique requires as little as 7 ng of enzyme which may be grossly admixed with contaminants, including other ribonucleases.Upon electrophoresis and activity staining, a variety of ribonucleases can be visualized as light or clear bands in a colored background formed by toluidine blue complexed with oligonucleotide substrate. Guanylyl-specific ribonuclease, which is detectable when using an oligonucleotide substrate of random base sequence, does not yield a band when using oligonucleotides bearing guanylyl residues at the 3'-termini only and containing, therefore, no susceptible internucleotide bonds; in contrast, a ribonuclease with a different base specificity or no base specificity yields a band with either substrate. This differential activity staining method for establishing guanylyl specificity permits estimation of the extent of nonspecific cleavage of internucleotide linkages by a putatively guanylyl-specific enzyme and is at least as sensitive as conventional procedures for determination of base specificity.With this new technique guanyloribonuclease has been identified in the unfractionated culture medium of 10 organisms belonging to the phytopathogenic fungal genus Ustilago. It is suggested that guanylyl-specific ribonuclease is widely distributed among Ustilago species; its electrophoretic properties may be revealing of phylogenetic relationships among these plant parasites and among their hosts.The general technique of differential activity staining, developed for determination of the base specificity of ribonucleases, may be widely applicable to analysis of enzymes catalyzing depolymerization reactions.Since discovery of the classic RNase T1, guanylyl-specific ribonucleases have been found in bacteria, in the three classes of fungi, and in a slime mold (1-5). Most of these enzymes are extracellular proteins of molecular weight about 11,000; the amino-acid composition of six (1-4) and the amino-acid sequence of two of them (1, 6) have been reported. Although they are most widely known as an analytical tool for determination of nucleotide sequence in RNA (1, 7), the guanylyl-specific ribonucleases are highly interesting in their own right. On-going studies of these enzymes include analysis of their mechanism of action (1) and comparison, at a basic level, with that of ribonuclease A (8); determination of structure-function relationships among the various G-specific RNases and the related, purine-preferring RNase U2 (9); and investigation of their distribution, biological function, and regulation (10, 11).Among known guanylyl-specific ribonucleases are two excreted by members of the phytopathogenic fungal genus Ustilago-RNase Ui from U. sphaerogena and an enzyme from the corn smut U. maydis (1, 10). We report here that extracellular guanylyl-specific ribonuclease is produced by Abbreviation: G-specific, guanylyl-specific. eight...

Studies in Heterobasidiomycetes. Part 24. On Ustilago violacea (Pers.) Rouss. from Saponaria officinalis L.

“…There are also differences m the relative electrophoretic mobilities of (8) aminopetidase (BRADFORD, JONES and GARBER 1975) and (9) urease (BAIRD and GARBER 1978). BLANK and DEKKER (1975) have shown RNAse activity in cultures of some Ustilago species from Poaceae. In the single Ustilago violacea strain they used, RNAase activity was not detectable.…”

Multiple ribonucleases of human urine

“…Electrophoresis at low pH in polyacrylamide gels was carried out as described by Blank (1978). Since RNA was not embedded in these latter gels, activity staining was performed by diffusing in an oligonucleotide substrate as described by Blank & Dekker (1975).…”

RNA Biology in Fungal Phytopathogens

“…Although this has so far only been shown for bacterial and oomycete phytopathogens [25], we predict that similar mechanisms will be discovered for fungal phytopathogens. For example, it has been known for many years that U. maydis secretes ribonucleases that might function as virulence factors in degrading host RNAs [26]. A prominent case for bacterial effectors is HopU1 from Pseudomonas syringae that is essential for virulence on Arabidopsis thaliana [27].…”