Direct radioactive labelling of poly(ADP-ribose) in developingXenopus laevis embryos (original) (raw)
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Poly (Adenosine Diphosphate Ribose) Synthetase During Oogenesis of the Xenopus Laevis
Molecular Approaches to Gene Expression and Protein Structure, 1981
Poly(ADP-ribose) synthetase activity is found in nuclei of regenerating epithelial cells in the lower half of the crypts of guinea-pig small intestine. Nuclei from non-dividing but differentiating and maturinig cells in the upper crypts and on the villi contain no more than about 10% of the synthetase activity of lower-crypt cell nuclei. The product in the active nuclei is shown to be 80% poly(ADP-ribosylated) protein and 20% mono-(ADP-ribosylated) protein; 60% of the total labelled product was attached to acidsoluble proteins (including histones), and 40 % to acid-insoluble (non-histone) proteins. The average number of ADP-ribosyl units in the oligomeric chains of the poly(ADPribosylated) proteins was 15 but the range of sizes of (ADP-ribose) oligomers attached to nuclear proteins was smaller in villus than in crypt cell nuclei.
Biochemical Journal
Poly(ADP-ribose) polymerase (PARP) is an abundant nuclear enzyme that is dependent on DNA breaks and nicks for its enzyme activity. These DNA nicks and breaks function as allosteric effectors of the enzyme activity. This reaction is important for efficient DNA base excision repair, although it is not a component of the elementary repair pathway itself. The physiological relevance of this reaction might be to ensure correct and efficient DNA repair. We have examined the enzyme activity of PARP in oocytes and eggs of Xenopus laevis. Although both oocytes and eggs contain approximately the same amounts of enzyme protein, there is no detectable enzyme activity in the oocytes, whereas in the eggs the enzyme is active. Enzyme activity appears during oocyte maturation, approx. 4 h after induction by progesterone. This enzyme activation coincides with the appearance of active maturation-promoting factor. Enzyme activation is accompanied by a shift in the electrophoretic mobility of the poly...
Purification and Properties of Poly(ADP-ribose) Polymerase from Pig-Thymus Nuclei
European Journal of Biochemistry, 1978
The nuclear enzyme poly(ADP-ribose) polymerase has been purified about 9200-fold from pig thymus nuclei with a 46% yield. An aqueous organic solvent system was used for the isolation of the polymerase from nuclei and for its purification by chromatography at sub-zero temperatures. Electrophoretic analysis under both denaturing and non-denaturing conditions revealed a single protein band suggesting that the preparation was homogeneous and that the enzyme is composed of one polypeptide chain. The molecular weight estimated from sodium dodecyl sulphate-/polyacrylamide gel electrophoresis was 63 500 and from gel filtration through columns of Sephadex G-100, 58 000. The enzyme preparation was free from poly(ADP-ribose)-degrading enzymes and from DNA. The purified polymerase showed an absolute requirement for both DNA and histones. The maximal specific activity of the homogeneous preparation measured by the standardized assay, was 20.7 pmol NAD* incorporated x min-' x mg-' of protein at 37 "C. Amino-terminal group analysis with dansyl chloride did not reveal a terminal amino acid suggesting that the amino-terminal group may be blocked. In the presence of histones, the K,,, for NAD' was 23 pM.
NAD turnover during early development of Xenopus laevis
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1983
The NAD pools of Xenopus laevis oocytes and early embryos can be radioactively labelled by microinjection of [adenine-3H]NAD. This technique is used to study the metabolism of NAD in oocytes and during early development. The rate at which NAD is degraded in vivo has been monitored by determining the rate of transfer of adenine residues from the NAD pool into other nucleotides and polynucleotides. In oocytes, NAD turnover is extremely slow, with a half-life of about 400 h. NAD turnover increases dramatically after fertilisation, and the half-life of the compound decreases to 37 h in 5-h-old embryos and to I0 h in 40-h-old embryos. 2 mM 3-aminobenzamide, a specific inhibitor of poly(ADP-ribose) polymerase, reduces the NAD turnover rate by about 20%, whereas 5 mM isonicotinic acid hydrazide, a specific inhibitor of NAD glycohydrolase, produces no significant inhibition. This indicates that a significant fraction of the considerable NAD turnover observed involves poly(ADP-ribose) polymerase. Our results indicate that poly(ADP-ribose) polymerase is active during early development and suggest that this activity may be involved in one or more aspects of the nuclear metabolism of the embryo.
Molecular Biology Reports, 1988
Poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase activities were both investigated in chicken erythroblasts transformed by Avian Erythroblastosis Virus. Respectively 21% and 58% of these activities were found to be present in the post-mitochondrial supernatant (PMS). Fractionation of the PMS on sucrose gradients and poly(A § mRNA detection by hybridization to [3H] poly(U) show that cytoplasmic poly(ADPribose) polymerase is exclusively localized in free mRNP. The glycohydrolase activity sedimented mostly in the 6 S region but 1/3 of the activity was in the free mRNP zone. Seven poly(ADP-ribose) protein acceptors were identified in the PMS in the Mr 21000-120000 range. The Mr 120000 protein corresponds to automodifled poly(ADP-ribose) polymerase. A Mr 21000 protein acceptor is abundant in PMS and a Mr 34000 is exclusively associated with ribosomes and ribosomal subunits. The existence of both poly(ADP-ribose) polymerase and glycohydrolase activities in free mRNP argues in favour of a role of poly(ADP-ribosylation) in mRNP metabolism. A possible involvement of this post translational modification in the mechanisms of repressionderepression of mRNA is discussed.
Nucleic Acids Research, 1992
We have analyzed several randomly selected mRNAs, of the relatively abundant category, on the basis of maternal or zygotlc origin and translational efficiency at different developmental stages. For this purpose, clones from a Xenopus embryo cDNA library were hybridized with cDNA probes prepared with poly(A)-t-RNA from polysomes and from mRNPs of embryos at different stages. The results obtained indicate that the majority of the relatively abundant mRNAs (38 out of 61) is subject to some kind of translational regulation during embryogenesis. Moreover, 30 clones have been selected as corresponding to mRNAs that behave, from the point of view of transcrlptional and translational regulation, similarly to previously studied ribosomal protein (rprotein) mRNAs. Sequence analysis of 20 of these selected cDNAs has shown that half of them are in fact homologous to already sequenced r-proteln mRNAs. Unexpectedly we have found that also the mRNA for a-cardiac actin and another mRNA homologous to creatine kinase M mRNA have a similar translational regulation during embryogenesis.
Biochimica et Biophysica Acta (BBA) - General Subjects, 1999
Poly(ADP-ribose) polymerase is a DNA break detecting enzyme playing a role in the surveillance of genome integrity. Poly(ADP-ribose) is synthesized rapidly and transiently from L-NAD in response to DNA damaging agents. In order to study the physiological significance of poly(ADP-ribose) metabolism, we have developed immunological methods which enable us to study endogenous poly(ADP-ribose) without interfering with cell metabolism and integrity. For this purpose, we produced a highly specific polyclonal anti-poly(ADP-ribose) antibody which immunoreacts with polymers and oligomers. In addition to the immunodot blot method recently described by us (Affar et al., Anal. Biochem. 259 (1998) 280^283), other applications were investigated in cells: (i) detection of poly(ADP-ribose) by ELISA; (ii) characterization of poly(ADPribose) size using high resolution gel electrophoresis of polymers, followed by its transfer onto a positively charged membrane and detection with anti-poly(ADP-ribose) antibody ; (iii) immunocytochemistry and flow cytometry analyses allowing poly(ADP-ribose) study at the level of individual cells. ß 0304-4165 / 99 / $^see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 -4 1 6 5 ( 9 9 ) 0 0 0 5 4 -9
Small nuclear RNA transcription and ribonucleoprotein assembly in early Xenopus development
The Journal of Cell Biology
The Xenopus egg and embryo, throughout the transcriptionally inactive early cleavage period, were found to contain a store of approximately 8 x 108 molecules of the small nuclear RNA (snRNA) U 1, sufficient for 4,000-8,000 nuclei. In addition, when transcription is activated at the twelfth cleavage (4,000 cell-stage), the snRNAs U1, U2, U4, U5, and U6 are major RNA polymerase II products. From the twelfth cleavage to gastrulation, U1 RNA increases sevenfold in 4 h, paralleling a similar increase in nuclear number. This level of snRNA transcription is much greater than that typical of somatic cells, implying a higher rate of U1 transcription or a greater number of U1 genes active in the embryo. The Xenopus egg also contains snRNP proteins, since it has the capacity to package exogenously added snRNA into immunoprecipitable snRNP particles, which resemble endogenous particles in both sedimentation coefficient and T1 RNase digestibility. SnRNP proteins may recognize conserved secondary structure of U1 snRNA since efficient packaging of both mouse and Drosophila U1 RNAs, differing 30% in sequence, occurs. The Xenopus egg and embryo can be used to pose a number of interesting questions about the transcription, assembly, and function of snRNA.