Jan W. Szarkowski

Purification and properties of endonuclease from wheat chloroplasts, specific for single-stranded DNA

Abstract A nuclease, present in the chloroplast of wheat was purified about 150-fold. The enzyme was homogeneous on polyacrylamide gel. The optimal pH for denaturated DNA was 7.8 and for RNA 6.8, its M r was 29 000. The enzyme was an endonuclease degrading single-stranded DNA at a 15-fold higher rate than native DNA. The nuclease did not show an absolute requirement for added divalent cations. The enzyme degraded denatuiated DNA to oligonucleotides with a phosphomonoester bond at position 5′, and RNA to 5′-OH and 3′-phosphate-terminated fragments. The enzyme catalysed hydrolysis of synthetic polyribo- and polydeoxyribonucleotides in the following order: poly(A) > poly(U) > poly(C) > poly(G) and poly(dA) > poly(dT) > poly(dC) > poly(dG).

A single-strand-specific nuclease from a fraction of wheat chloroplast stromal protein

Abstract A single-strand-specific nuclease from the stroma fraction of wheat chloroplasts was purified to homogeneity. The nuclease exhibits an activity within a wide pH range of 5.0–7.5, its M r is 20 000. The enzyme degrades DNA and RNA endonucleolytically, producing fragments with 3′-OH and 5′-phosphate termini. The nuclease shows no absolute requirement for added divalent cations. The enzyme catalyses hydrolysis of synthetic polyribonucleotides in the following order: poly(U) > poly(A) > poly(C) > poly(G). Polydeoxynucleotides except poly(dA) and poly(dT) remain intact. Supercoiled, covalently closed circular OX174 DN A and plasmid pBR322 DNA are converted initially to the open-circular (relaxed) form and subsequently to the linear form.

Two plant nucleases as tools for structural analysis of rna

Abstract Two distinct plant nucleases: nuclease I from nucleoplasm of rye germs—Rn enzyme [1] and nuclease I from wheat chloroplasts—Wch enzyme [2] have been used to investigate the secondary structure of the small RNA molecule-CopA RNA in solution. Both nucleases are good tools for such studies: they can act on RNA within the physiological pH range and in commonly used TMN buffer. Rn nuclease is base non-specific and is capable of cleaving all four phosphonucleotide bonds in non-base-paired regions while double-stranded structures are completely resistant to the nucleolytic attack even with more extensive hydrolysis. The enzyme is very stable and can retain its activity for many years. Wch nuclease has a clear preference for pyrimidines in non-base-paired regions.

Application of a nuclease from rye nucleus for structural studies of plant ribonucleic acids

A new nuclease (Rn) isolated from rye nucleus was applied for the structural studies of methionine initiator transfer ribonucleic acid and ribosomal 5S rRNA from yellow lupin seeds. The enzyme shows high specificity for some regions of both RNAs. The dihydrouridine and ribothymidine loops which are supposed to be involved in the tertiary interactions of the methionine initiator tRNA were hydrolysed. The anticodon loop is not digested at all. 5S rRNA was digested in single stranded regions (loops). The cleavage pattern of the tRNA and 5S rRNA obtained with Rn enzyme, suggests not only the high specificity toward single stranded regions, but also some dependence on their tertiary structure.

An acid ribonuclease from rye germ cytosol

Abstract Acid ribonuclease from rye germ cytosol was purified 1200-fold. The enzyme is homogeneous on polyacrylamide gel. The optimum pH for ribonuclease activity is 5.8, its MW is 28 500. The enzyme is an endonuclease yielding in the first step of its activity oligonucleotides with a free —OH group in the 3′ position. The end products of RNA hydrolysis are cyclic purine and pyrimidine nucleoside phosphates and the corresponding nucleoside 3′-phosphates. This ribonuclease preferentially attacks sites close to the adenine base and shows a lag in the release of the cytosine base. Specificity tests on natural and synthetic substrates are in good agreement.

Thymidine kinase activity in rye chloroplasts

Thymidine kinase (ATP, thymidine 5-phosphotransferase, EC 2.7.1.75) activity has been studied in a variety of biological systems. However, neither the subcellular distribution of the enzyme, particularily in plants, nor its involvement in DNA synthesis has been fully elucidated [cf. l] . Here we report on [he occurrence of thymidine kinase in chloroplasts isolated from rye (Se&e cereule) seedlings.

Effect of rye embryo ribosome nuclease on double-stranded RNA

Abstract In extracts obtained by treating rye embryo ribosomes with 0.5 M NH 4 Cl, nuclease activity was noted towards double-stranded RNA from virus of Penicillium chrysogenum and towards synthetic poly (A)-poly (U) and poly (I)-poly (C) complexes.

Native bovine selenocysteine tRNAsec secondary structure as probed by two plant single-strand-specific nucleases

Two single-strand-specific nucleases, discovered in plants, have been used to investigate the secondary and tertiary structures of the native bovine liver selenocysteine tRNA(Sec). To check the possible influence of nucleotide modifications on these structures, we compared the results obtained with the fully modified tRNA to the unmodified transcript prepared by in vitro T7 transcription of the Xenopus laevis tRNA(Sec) gene. We found that the structures in solution of the native tRNA(Sec) and the transcript are very similar despite some differences in accessibility to the enzymatic probes. Indeed, the modified anticodon-loop of native bovine tRNA(Sec), containing 5-methylcarboxymethyluridine (mcm5U34) and N6-isopentenyladenosine (i6A37), is less accessible to Rn nuclease than that of the transcript: the intensity of bands representing cuts at A36 and A38 is much lower as compared to those of the transcript, whereas no cuts were found at the level of i6A37 in the anticodon loop of the native molecule. Surprisingly, the variable arm of the native molecule has been found to be more susceptible to single-strand-specific nuclease action, suggesting a looser structure of the variable arm in native bovine tRNA(Sec) than in the transcript.

Appearance of thymidine kinase activity in growing rye seedlings

Abstract 1. 1. The appearance of thymidine kinase activity in leaves of growing rye Secale cereale L. seedlings has been observed. 2. 2. Specific activities of the enzyme(s) associated with various subcellular fractions were compared, the highest value being found in that bound with mitochondria. This work was supported by the Polish Academy of Sciences within the project 09.3.1.

Ribosomal Nuclease with Activity Towards Double-Stranded RNA

Ribonucleolytic activity has been suggested to be associated with plant ribosomes, but its role has not been elucidated (Hsiao 1968, Farkas 1982). According to Wilson (1982) nucleolytic ribosomal enzymes represent only a small fraction of the total nucleolytic activity of plant tissues. In our earlier investigations ribonucleolytic activity has been observed in nuclei, plastids, mitochondria, postribosomal supernatant and cytoplasmic ribosomes of rye embryos and 24-h seedlings. Specific ribonucleolytic activity was highest in the ribosome fraction from dry rye embryos (Siwecka et al., 1971). We have further demonstrated that deoxyribonucleolytic activity is also associated with rye embryo ribosomes. Both these activities are strongly associated with ribosomes and their subunits but a part of activity may be released by washing with 0.5 M ammonium chloride or 4 M urea (Siwecka et al., 1979). The NH4Cl ribosomal wash seems to contain an enzyme hydrolyzing double-stranded RNA, namely, double-stranded viral RNA from Penicillium chrysbgenum and the double-stranded poly(A)*poly(U) complex (Siwecka et al., 1982). So far there have been no reports on isolation from higher plants of enzymes hydrolyzing double-stranded RNA.