Purification of calf thymus adenosine diphosphate ribose polymerase]

Calf thymus poly ADPR polymerase has been purified to electrophoretic homogenity. The enzyme has a molecular weight of 120,000 +/- 10,000 dalton. The substrate affinity is very high (apparent Km 82.5 micrometer). The presence of exogenous DNA does not appear to be a requisite for enzymatic activity of the purified enzyme.     

Degradation of ribosomal RNA from Escherichia coli by ribonuclease U2 and in vitro transcription of "RNA-fragments" into complementary DNA]

Under well defined conditions ribosomal RNAs purified from Escherichia coli can be degraded by ribonuclease U2 giving rise to RNA fragments of 60--70 nucleotides. In vitro, these fragments are efficiently transcribed into a complementary DNA by DNA polymerase RNA dependent, partially purified from extracts of E. coli. In vivo, "RNA-fragments-U2" inhibit the development of plant tumors.     

Purification of RNA from animal cells using diethyl-pyrocarbonate

Summary Extraction of RNA from animal cells by a method using diethyl-pyrocarbonate yielded 50–60% of the total RNA. RNA purified by a hot phenol-SDS method from adenovirus 2 infected cells showed about 9% homology with adenovirus DNA, and RNA purified by diethyl-pyrocarbonate-SDS showed over 7% hybridization. Profiles of RNA prepared by both methods were identical when studied by polyacrylamide gel electrophoresis.Acknowledgment. This investigation was supported by U.S. Public Health Services research grant No. 5R01-CA10724-03 from the National Cancer Institute.     

Detection and localization of a ribonuclease activity associated with vaccinia virus]

A ribonuclease associated with vaccinia virus can be detected when reduced concentrations of nucleotides are used for an in vitro RNA synthesis assay. The non-viral origin of this ribonuclease may be inferred from its external location and from its variable activity on different purified virus stocks. The detection of this ribonuclease activity on purified virus grown without foetal Calf serum may suggest that this enzyme is of cellular origin.     

RNA editing in plant organelles: machinery, physiological function and evolution

In plants, RNA editing is a process for converting a specific nucleotide of RNA from C to U and less frequently from U to C in mitochondria and plastids. To specify the site of editing, the cis-element adjacent to the editing site functions as a binding site for the trans-acting factor. Genetic approaches using Arabidopsis thaliana have clarified that a member of the protein family with pentatricopeptide repeat (PPR) motifs is essential for RNA editing to generate a translational initiation codon of the chloroplast ndhD gene. The PPR motif is a highly degenerate unit of 35 amino acids and appears as tandem repeats in proteins that are involved in RNA maturation steps in mitochondria and plastids. The Arabidopsis genome encodes approximately 450 members of the PPR family, some of which possibly function as trans-acting factors binding the cis-elements of the RNA editing sites to facilitate access of an unidentified RNA editing enzyme. Based on this breakthrough in the research on plant RNA editing, I would like to discuss the possible steps of co-evolution of RNA editing events and PPR proteins. Received 30 September 2005; received after revision 5 November 2005; accepted 28 November 2005     

Calf thymus ribonuclease H IIa activity lacks ribonuclease H specificity.

Less purified fractions of ribonuclease H IIa activity of calf thymus display divalent cation-dependent ribonuclease H activity and divalent cation-independent ribonuclease activity. Because the ratio of the two enzyme activities does not change during successive chromatographic procedures, we suggest that ribonuclease H IIa activity is indeed able to degrade both ssRNA and the RNA moiety of RNA.DNA-hybrids. Ribonuclease H IIa activity can therefore be differentiated from calf thymus ribonuclease H I and H IIb by its lack of ribonuclease H specificity. The native molecular mass of ribonuclease H IIa activity is between 23 and 28 kDa. Under denaturing conditions a 23 kDa-protein band copurifies with the enzyme activity suggesting that this enzyme is monomeric.     

Serum-induced stimulation of snRNA synthesis in mouse 3T3 fibroblasts

Small nuclear RNAs (snRNAs) from quiescent and serum-stimulated 3T3 cultures, labeled with [3H]uridine [( 3H]U), were electrophoresed in polyacrylamide-urea slab gels and revealed by staining with ethidium bromide and by fluorography. Judged by labeling with [3H]U, synthesis of 7S and U1-U6 RNAs was very low or absent in quiescent cultures. The serum-induced transition of 3T3 cells from a resting to a growing state was accompanied by an early, apparently sequential stimulation of snRNA synthesis; stimulated synthesis of 7S, U1, U2, U3, U4 and U6 RNAs coincided in time with serum-induced stimulation of 45S pre-ribosomal RNA (pre-rRNA) and heterogeneous nuclear RNA (hnRNA) synthesis.     

RNA uridylyltransferases

The terminal RNA uridylyltransferases (TUTases) catalyze transfer of UMP residues to the 3' hydroxyl group of RNA. These activities are widespread among eukaryotes and appear to be involved in a variety of RNA-processing pathways. Recent studies of RNA editing in trypanosomatids have provided the first insights into the biological functions of RNA uridylyltransferases, which had eluded biochemical identification despite 30-year-old evidence of such activities in mammals and plants. Comparative sequence analysis of trypanosomal TUTases and their homologs revealed by large-scale genomic projects demonstrates a significant level of biochemical and structural diversity between putative uridylyltransferases. The conserved catalytic domain has acquired additional protein modules and appears to have adapted to perform functionally distinct tasks of guided U-insertion into mRNA and constrained addition of an oligo[U] tail to guide RNAs. Here I discuss the current knowledge of this novel enzyme family and possible roles of RNA uridylylation in the regulation of gene expression.     

Studies on stable and during early imbibition phases synthesized poly(A)-RNA of Agrostemma githago embryos (author's transl)]

RNA isolated from dry embryos of Agrostemma githago seeds contains poly(A)-sequences, but in very small amounts. In the early phase of imbibition, an intensive synthesis of poly(A)-containing RNA is brought about. The importance of this synthesis of poly(A)-RNA is discussed.     

Serum-induced stimulation of snRNA synthesis in mouse 3T3 fibroblasts

Summary Small nuclear RNAs (snRNAs) from quiescent and serum-stimulated 3T3 cultures, labeled with [³H]uridine ([³H]U), were electrophoresed in polyacrylamide-urea slab gels and revealed by staining with ethidium bromide and by fluorography, Judged by labeling with [³H]U, synthesis of 7S and U1-U6 RNAs was very low or absent in quiescent cultures. The serum-induced transition of 3T3 cells from a resting to a growing state was accompanied by an early, apparently sequential stimulation of snRNA synthesis; stimulated synthesis of 7S, U1, U2, U3, U4 and U6 RNAs coincided in time with serum-induced stimulation of 45S pre-ribosomal RNA (pre-rRNA) and heterogeneous nuclear RNA (hnRNA) synthesis.     

Calf thymus ribonuclease H IIa activity lacks ribonuclease H specificity

Summary Less purified fractions of ribonuclease H IIa activity of calf thymus display divalent cation-dependent ribonuclease H activity and divalent cation-independent ribonuclease activity. Because the ratio of the two enzyme activities does not change during successive chromatographic procedures, we suggest that ribonuclease H IIa activity is indeed able to degrade both ssRNA and the RNA moiety of RNA·DNA-hybrids. Ribonuclease H IIa activity can therefore be differentiated from calf thymus ribonuclease H I and H IIb by its lack of ribonuclease H specificity. The native molecular mass of ribonuclease H IIa activity is between 23 and 28 kDa. Under denaturing conditions a 23 kDa-protein band copurifies with the enzyme activity suggesting that this enzyme is monomeric.     

The effect of sulfhydryl reagents upon the activity of 40S ribosomal subunits

Summary p-Chloromercuribenzoate inhibited the poly (U)-dependent binding of Phe-tRNA to the 40S ribosomal subunit but displayed no inhibitory effect on the binding of poly (U) to the ribosome. Other sulfhydryl reagents tested, likeN-ethylmaleimide and iodoacetamide, did not affect the binding of Phe-tRNA to the small ribosomal subunit.     

2’-5’ Oligoadenylate synthetase shares active site architecture with the archaeal CCA-adding enzyme

The 2'-5' oligoadenylate synthetases (OAS) are interferon-induced antiviral enzymes that recognise virally produced dsRNA and initiate an RNA destabilisation within the infected cell. We compared the structure of OAS to that of poly adenosine polymerase (PAP) and the class I CCA-adding enzyme from Archeoglobus fulgidus (AfCCA). This comparison revealed a strong structural homology between the three enzyme families. In particular, the active sites of OAS and CCA class I enzymes are highly conserved. We conducted an extensive mutagenesis of amino acid residues within the putative active site in OAS, thereby identifying enzymatically important residues and confirming the common active site architecture for OAS and the AfCCA. Our findings also have profound implications for our understanding of the evolutionary origin of the OAS enzymes and suggest that the OAS proteins diverged from a common 3'-specific ancestor at the beginning of metazoan evolution.     

Inhibitory effect of flavonoids on DNA-dependent DNA and RNA polymerases

Flavonoids, (-)-epigallocatechin (1), myricetin (2) and quercetin (3), were investigated for inhibitory effects on E. coli DNA polymerase I and T7 bacteriophage RNA polymerase. In both DNA and RNA synthesis, 1 and 3 inhibited enzyme reactions by non-competitive and mixed type inhibition respectively, with regard to template DNAs. Myricetin (2) inhibited DNA and RNA polymerase reactions by mixed type and competitive type inhibition, respectively, with template DNAs. It was suggested that 2 interacts with covalently closed circular DNA.     

Compared expression of murine plasmacytoma associated virus in tumor cells and in mouse embryo cells]

A DNA complementary to the viral genome of C-type particles produced by a Mouse myeloma derived cell line (MF2 cell line) was synthesized. This cDNA was used as a probe to study the viral genome expression among the total RNA and the poly (A)-rich RNA extracted from the MF2 and Balb/c embryonic cells. As evidenced by molecular hybridization experiments, the presence of at least one endogenous Balb/c virus in the MF2 virus stocks is suggested. In the productive cells, the viral RNA sequences are expressed in the poly (A)-rich RNA fraction.     

Partial purification and characterization of acid phosphatase from sporulated oocysts of Eimeria tenella

Acid phosphatase of Eimeria tenella oocysts (Peak II) was purified 77-fold with a recovery of 26% using protamine sulfate precipitation, DEAE-cellulose chromatography and Sephadex G-200 gel filtration. This enzyme occurs in multiple forms as indicated by two peaks which can be separated by DEAE-cellulose chromatography and polyacrylamide gel electrophoresis. The partially purified enzyme has optimal activity at pH 4.5. With p-nitrophenyl phosphate the Km and Vmax values for (Peak II) were 25 mM and 1.57 mumol/min/mg protein, respectively. The enzyme (Peak II) is strongly inhibited by Hg++, Cu++, iodoacetamide, fluoride and molybdate. Tartrate and other divalent metal ions have no effect on enzyme activity. The partially purified Peak II phosphatase is not a glycoprotein as it is not absorbed on concanavalin-A Sepharose and its treatment with bacterial neuraminidase does not alter its elution profile through DEAE cellulose.     

Structural diversity of trypsin from different mosquito species feeding on vertebrate blood

Mosquito trypsin was purified using a combination of ion exchange and affinity chromatography with the ligand soybean trypsin inhibitor. Three Aedes and three Anopheles species were tested, all of which are specialized in the digestion of vertebrate blood. Amino-terminal sequences of HPLC-purified trypsins from Aedes aegypti and Anopheles quadrimaculatus revealed homologies of 30-40% with vertebrate and other invertebrate proteases previously identified as serine-proteases. The purified mosquito trypsins have molecular masses between 25 kDa and 36 kDa, as determined by denaturing polyacrylamide electrophoresis, and are heterogeneous in size and number in the various species. The number of SDS-bands varies between 3 and 6 in Aedes and between 1 and 3 in Anopheles. The specific activities, determined with the substrate TAME, range from 240 U/mg in Aedes aegypti to 1065 U/mg in Anopheles quadrimaculatus. All mosquito trypsins tested have acidic isoelectric points between pH 3.5 and pH 5.4. No alkaline proteases were detected. Polyclonal antisera against Aedes aegypti and Anopheles albimanus trypsin do not cross-react with bovine trypsin. Cross-reactivity of the two sera with trypsin from six mosquito species suggests the presence of at least 2 enzyme families.     

Untersuchungen über stabile und während der frühen Quellungsstadien neu synthetisierte Poly(A)-RNA vonAgrostemma-Samen

Summary RNA isolated from dry embryos ofAgrostemma githago seeds contains poly(A)-sequences, but in very small amounts. In the early phase of imbibition, an intensive synthesis of poly(A)-containing RNA is brought about. The importance of this synthesis of poly(A)-RNA is discussed.

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Herrn Dr.E. Serfling (Gatersleben, DDR) danke ich für methodische Hinweise und für die Überlassung einiger Chemikalien.     

Characterization of a soluble form of dipeptidyl peptidase IV from pig liver

Soluble dipeptidyl peptidase IV (EC 3.4.14.5) was purified from the 100,000 X g supernatant fraction of pig liver homogenate. The purified enzyme had the same properties as, and immunological identity with, the membrane-bound enzyme which was described previously. However, the purified enzyme had a pattern of molecular heterogeneity different from the membrane-bound enzyme; this was shown by isoelectric focusing. Carbohydrate analysis revealed that the soluble enzyme contained glucose, which is not found in the membrane-bound one, and less fucose, mannose, and sialic acid than the latter. From these results, we conclude that the soluble form of dipeptidyl peptidase IV in pig liver is closely related to the membrane-bound enzyme, but is not simply a proteolytically solubilized product of it.