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PubMedDate not supplied

[Metabolism of oligoadenylates in cell nuclei and regulation of protein ADP-ribosylation].

2',5'-Oligo(A)synthetase (2-5A) and 2-phosphodiesterase were found in the L cells nuclei. In the cell nuclei 2-5A is 10-30 times higher, than in the cytoplasm. It is induced by interferon and depends on the cell growth state. 2-Phosphodiesterase activity has two pH optima of hydrolysis of 2-5A, namely 7.1, and 7.9 and decreases after interferon treatment of cells. Thus, interferon treatment of cells leads to an increase of the 2-5A level in cell nuclei. One of the possible pathways for 2-5A action in cell nuclei is the regulation of (ADP-ribose)transferase activity. Treatment of L cells with 2-5A (A2pA2pA) leads to activation of ADP-ribosylation of proteins by a factor of 1.5 in a concentration range of 10(-9)-10(-7) M, but more higher concentrations of 2-5A inhibit this process up to 60%. Treatment of cells with actinomycin D has no influence on 2-5A induced changes in protein ADP-ribosylation. This result is indicative for a new pathway of interferon action and 2-5A mediated regulation of cell metabolism.

Adenine Nucleotides↗
PubMed1986

[Isolation and various properties of soluble and membrane-bound acid RNAse from rat brain lysosomes].

Preparations of soluble (I) and membrane-bound (II) acid RNAse with Mr 68,000 and 72,000 Da, respectively, and purified about 2000-fold were isolated from lysosome-rich fractions of rat brain large hemispheres. RNAase II differed from RNAase I by a lower temperature stability. The pH optimum (pH 5.8-6.1), temperature optimum and substrate specificity of RNAase I and II appeared to be identical. The Km values of RNAases I and II for poly(U) are 166 and 160 micrograms/ml; those for RNA--1200 and 1250 mu k/ml, respectively. RNAases I and II extensively hydrolyze soluble, polymeric RNA, rRNA from brain and yeast and poly(U) but do not influence poly(C), poly(A), poly(G), tRNA and DNA. Monovalent cations (K+, Na+, NH4+) activate both RNAase forms.

Animals↗
PubMedDate not supplied

[Regulation of the 2',5'-oligoadenylate level in mouse L cells].

A study of pH dependence for ppp5'A2'p5'A2'p5'A hydrolysis in interferon treated and untreated mouse L-cells extracts led to the detection of two types of the 2'-phosphodiesterase activities: interferon dependent and interferon resistant. Several pH-optima were observed for hydrolysis of ppp5'A2'p5'A2'p5'A in cell extracts after their treatment with non-ionic detergent NP-40 or their differential centrifugation. The 2'-phosphodiesterase activity was found in the membrane fraction as well as in the cytoplasmic one. The presence of several pH-optima for 2'-phosphodiesterase activity in L-cells and changes of the level of this activity depending on the growth stage of cells and time of their interferon treatment indicate the complicated character of the regulation of 2'-5'-oligoadenylate's concentration and localization. The results obtained suggest that in mouse L-cells several 2'-phosphodiesterases or one enzyme in different forms may be present.

Adenine Nucleotides↗
PubMed1981

[DNA-methylase from Arthrobacter luteus screens DNA from the action of site-specific endonuclease Alu I].

DNA-methylase was isolated from a cell extract of A. luteus and partially purified by chromatography on phosphocellulose. The purified enzyme methylates DNA of phage lambda and plasmids pBr322, thus making them resistant to a subsequent action of endonuclease Alu I. It has been shown that cytosine is the object of methylation within DNA. This modification does not screen DNA from the action of site-specific endonucleases Sal I, Bam HI, EcoR I, EcoR II, Xho I and Xho II. It has been experimentally demonstrated that the isolated methylase is site-specific and identifies in the DNA the nucleotide sequence 5'-AGCT-3', by methylating cytosine in the DNA.

Arthrobacter↗
PubMedDate not supplied

[Intracellular and extracellular ribonucleases of Bacillus intermedius].

Endocellular and exocellular ribonucleases were studied in Bacillus intermedius. Two fractions of ribonucleases (Rf 0.72 and 0.96) were found to be associated with the cellular surface and seven fractions (Rf 0.1, 0.17, 0.33, 0.45, 0.72, 0.82 and 0.96) were detected in the cytoplasm. RNAase with Rf 0,096 had the highest activity and was repressed by inorganic orthophosphate. This RNase accumulated in the cell during the stationary growth phase just as the free enzyme form did in the culture medium. The immunological characteristics of these enzymes were identical as was shown by immunochemical analysis.

Bacillus↗
PubMedDate not supplied

[Effect of chloramphenicol and actinomycin D on extracellular alkaline RNAse biosynthesis by Bacillus intermedius].

By means of chloramphenicol it was found that biosynthesis of alkaline exocellular RNAase was repressed in Bacillus intermedius by inorganic phosphate. Actinomycin D at a low concentration stimulates RNAase biosynthesis in a medium with a minimal phosphorus concentration in model experiments with washed cells and in the batch culture. As a result, the activity of RNAase rises 2-4 times. The stimulating effect of actinomycin D decreases when phosphorus concentration in the medium is increased The effect of actinomycin D is maximal if the antibiotic is added to the medium when the specific growth rate of the bacterium falls down and the rate of RNAase biosynthesis rises.

Bacillus↗
PubMed1994

The activity of the Saccharomyces cerevisiae strand exchange protein 1 intrinsic exonuclease during joint molecule formation.

Strand exchange protein 1 (Sep1) from Saccharomyces cerevisiae catalyzes the formation of heteroduplex DNA from single-stranded and homologous linear duplex DNA. The initial pairing reaction requires limited exonucleolytic digestion of the double-stranded DNA (dsDNA) by the intrinsic 5' to 3' exonuclease of Sep1 or by an exogenous exonuclease. Subsequent strand exchange proceeds without the need for exonuclease activity. Sep1 degrades linear dsDNA at a rate of 20 nucleotides/min with an average processivity of 45 nucleotides. During strand exchange reactions joint molecules are first observed after 1-2 min, suggesting that only limited digestion is necessary for pairing. The linear dsDNA was found to pair with single-stranded DNA (ssDNA) when it was resected by only 22 nucleotides, and linear dsDNA digested by more than 22 nucleotides was observed only in joint molecules. Approximately 20 nucleotides were also the minimum extent of digestion that could support pairing. In the absence of exonuclease activity, Sep1-promoted pairing requires dsDNA molecules with single-stranded tails homologous to the circular ssDNA. These results suggest that Sep1-promoted strand exchange requires a single-strand annealing event prior to the strand displacement phase of the reaction.

Bacteriophage M13↗
PubMed1994

Characterization of the interaction of Saccharomyces cerevisiae strand exchange protein 1 with DNA.

We have analyzed in greater detail the interaction of strand exchange protein 1 (Sep1) from Saccharomyces cerevisiae with DNA. The binding site size of Sep1 on single-stranded DNA (ssDNA) was determined to be 70 nucleotides per protein monomer using a fluorescence assay and 100 nucleotides using an exonuclease titration technique. The amount of Sep1 required for maximum aggregation of ssDNA was the amount needed to saturate the DNA. When double-stranded DNA (dsDNA) and ssDNA were both present, the duplex DNA was efficiently aggregated only at protein concentrations above that required for saturation of ssDNA. Strand exchange reactions with blunt-ended linear dsDNA and homologous ssDNA substrates required saturation of the ssDNA with Sep1 since free Sep1 is needed for exonuclease activity to initiate pairing with the dsDNA substrate. Preincubation of Sep1 with resected duplex DNA before adding ssDNA allowed joint molecule formation to occur at protein concentrations at least 10-fold below that required for saturation of the ssDNA. However, preincubation of Sep1 with ssDNA before the addition of resected duplex DNA required saturating amounts of Sep1 for joint molecule formation to occur. These results suggest that pairing requires Sep1 on both the ssDNA and the resected ends of the dsDNA.

Base Sequence↗
PubMed1994

Gene required for normal MHC class II expression and function is localized to approximately 45 kb of DNA in the class II region of the MHC.

In certain mutant human B cell lines, MHC-encoded class II molecules displayed at the cell surface have an abnormal conformation and are unstable in the presence of SDS. The mutants cannot present exogenous protein Ags to T cells but elicit responses with exogenous antigenic peptides; thus, formation of intracellular complexes between antigenic peptides and class II molecules is impaired. Previous analysis of LCL deletion mutants, .82, .174, and 5.2.4, showed that genes needed for this function must be present in approximately 230 kb of DNA in the class II region of the MHC. We now describe a new deletion mutant, .61, which has normal class II-mediated Ag processing/presentation. The TAP1, TAP2, LMP2, and LMP7 genes are deleted from .61, demonstrating that those genes are not needed for normal formation of intracellular class II/peptide complexes. The genes in question must be located in DNA that is present in .61 and .82 (both normal) and absent from .174 and 5.2.4. (both defective). Mapping of the deletion breakpoints indicates that genes needed for normal class II-associated Ag processing/presentation are either: 1) in an approximately 40 kb L DNA segment located between the DMB and LMP2 loci or 2) in an R region between the DQA2 and DQB1 loci and are completely included on a 5.1-kb fragment formed by joining of DNA that flanks the deletion in .61. The evidence favors location of the genes in the L DNA segment.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Substrate-specific regulation of RNA deadenylation in Xenopus embryo and activated egg extracts.

The poly(A) tail of mRNAs plays an important role in translational control. In Xenopus laevis matured oocytes, maternal mRNAs that contain a cytoplasmic polyadenylation element (CPE) are polyadenylated, whereas CPE deficient mRNAs are deadenylated by a default process. Eg mRNAs are maternal transcripts that are poly(A)+ in matured oocytes and rapidly deadenylated after fertilization. This post-fertilization deadenylation of Eg mRNAs requires specific sequence information. Such a deadenylation element has been identified previously in the 3'UTR of Eg2 mRNA. In this study, we show that cell-free extracts made from embryos or activated eggs contain two kinetically distinct deadenylation activities, with different substrate specificities. One, responsible for the slow deadenylation of RNAs that are devoid of a functional CPE, has the characteristics of a default PAN activity. The other effectuates the rapid deadenylation of RNAs containing a deadenylation element. The in vitro system described here will allow the characterization of factors controlling the deadenylation of Eg mRNAs in embryos.

Animals↗
PubMed1996

Overexpression of poly(A) binding protein prevents maturation-specific deadenylation and translational inactivation in Xenopus oocytes.

The translational regulation of maternal mRNAs is the primary mechanism by which stage-specific programs of protein synthesis are executed during early development. Translation of a variety of maternal mRNAs requires either the maintenance or cytoplasmic elongation of a 3' poly(A) tail. Conversely, deadenylation results in translational inactivation. Although its precise function remains to be elucidated, the highly conserved poly(A) binding protein I (PABP) mediates poly(A)-dependent events in translation initiation and mRNA stability. Xenopus oocytes contain less than one PABP per poly(A) binding site suggesting that the translation of maternal mRNAs could be either limited by or independent of PABP. In this report, we have analyzed the effects of overexpressing PABP on the regulation of mRNAs during Xenopus oocyte maturation. Increased levels of PABP prevent the maturation-specific deadenylation and translational inactivation of maternal mRNAS that lack cytoplasmic polyadenylation elements. Overexpression of PABP does not interfere with maturation-specific polyadenylation, but reduces the recruitment of some mRNAs onto polysomes. Deletion of the C-terminal basic region and a single RNP motif from PABP significantly reduces both its binding to polyadenylated RNA in vivo and its ability to prevent deadenylation. In contrast to a yeast PABP-dependent poly(A) nuclease, PABP inhibits Xenopus oocyte deadenylase in vitro. These results indicate that maturation-specific deadenylation in Xenopus oocytes is facilitated by a low level of PABP consistent with a primary function for PABP to confer poly(A) stability.

Amino Acid Sequence↗
PubMed1995

Comparison of features of the RNase activity of 5'-exonuclease-1 and 5'-exonuclease-2 of Saccharomyces cerevisiae.

Features of the catalytic specificities of 5'-exonuclease-1 (Xrn1) and 5'-exonuclease-2 of Saccharomyces cerevisiae have been compared. For analysis of in vitro properties, 5'-exonuclease-2 has been highly purified, and data show that it is present in yeast cells at about 5-10% of the level of Xrn1. The basic features of the exonuclease activity of the two enzymes, i.e., their activities with RNA and ssDNA and their mode of action, are similar. We have initiated an analysis of structural elements (artificial and natural) that stall the exonucleolytic hydrolysis by the enzymes. A (G)18 artificial sequence in MFA2 mRNA stalls the hydrolysis by both enzymes, yielding a 3' stall fragment. The specific structural element(s) involved is being investigated. To access a similar in vivo specificity of the two enzymes, the effect of overexpression of the essential HKE1 gene encoding exonuclease-2 on several of the phenotypes of xrn1 cells has been examined. The results show that the slow growth rate is partially overcome and that the level of accumulation of specific cytoplasmic RNAs (fragments of the internal transcribed spacer 1 of pre-rRNA and poly (A)-deficient mRNAs) is reduced to 30-40% of the value found in the xrn1 cells.

Exoribonucleases↗