Thymus nuclei

Zaitsev, S.V., Haberland, A., Otto, A., Vorob ev, V.I., Haller, H., Bottger, M. (1997). HI and HMG17 extracted from calf thymus nuclei are efficient DNA carriers in gene transfer. Gene Ther., 4, 586-592. 

Beullens M, Van Eynde A, Stalmans W et al (1992) The isolation of novel inhibitory polypeptides of protein phosphatase 1 from bovine thymus nuclei. J Biol Chem 267 16538-16544... 

Three histone-specific acetyltransferases have been partially purified and characterized from rat thymus nuclei (225). The enzymes were extracted from rat thymus nuclei by sonication in the presence of 1M ammonium sulfate and separated into two active fractions (A and B) by DEAE-cellulose chromatography. Fraction B was further separated into two active fractions (Bi and B2) by gel filtration on Sephadex G-200. Each fraction was then purified further by chromatography on hydroxyapatite. The molecular weights, determined by Sephadex G-200 and by sucrose density gradient centrifugation, were 99,000, 110,000, and 92,000 for enzymes A, Bi, and B2, respectively. All three enzymes required acetyl CoA as acetate donor, and the activity of the enzymes was inhibited by p-chloromercuribenzoate. Acetyltransferase A preferentially acetylated histone I (FI) and also poly-L-lysine. Acetyltransferase Bi and B2 preferred histone H4 (other names IV, F2al) and did not acet-ylate poly-L-lysine and histone H3 (III, F3). In addition to c-N-acetyl-lysine, two other unidentified amino acid derivatives were obtained from a digest of histone H4 acetylated by the two B enzymes. 

Bromobenzamide 3- Hydroxy benazmide Pig thymus nuclei 3-Methoxybenzamide 3-N itrobenzamide Nicotinamide 3-Acetamidobenzamide Inhibition 172... 

Tsopanakis C, Leeson E, Tsopanakis A, Shall S (1978) Purification and properties of poly-(ADP-ribose) polymerase from pig-thymus nuclei. Eur J Biochem 90 337-345... 

Fig. 4. (right) Possible cleavage sites of poly(ADP-ribose) synthetase in calf thymus nuclei. 

Nuclear oxidative phosphorylation is difficult to quantify. Although oxygen uptake in the nucleus can be measured, no exact P/O ratio is available. This is because only the AMP already present in the nuclear preparation can be converted to ATP any AMP added to the nuclei remains unaltered. An intriguing observation is the effect of DNase on the phosphorylation of AMP. (Allfrey has proposed that DNase blocks ATP synthesis in the nucleus indirectly namely, by inhibition of the nuclei by the histones, which after DNA extraction are no longer associated with DNA by salt linkages [35].) The enzymic extraction of 55% of the DNA in the nucleus leads to the loss of nuclear phosphorylation properties, which can be restored by adding DNA to the system. The effect of DNA is not specific because DNA can be replaced by RNA, polyadenylic acid, heparin, chondroitin sulfate, and polyethylene sulfate. Oxidative phosphorylation in thymus nuclear preparation has been confirmed in two laboratories. Whole body doses of ionizing radiation inhibited oxidative phosphorylation in thymus nuclei. 

Stern and Timonen [37] demonstrated that thymus nuclei contain little cytochrome oxidase, but this finding is not in agreement with observations made on liver, spleen, or pancreas nuclei prepared in 0.25 m sucrose. Although the difference between the effect of inhibitors on nuclear and mitochondrial oxidative phosphorylation excludes the possibility of contamination of nuclei by intact mitochondria, it does not eliminate the possibility of contamination by fragmented mitochondria. Inhibitors do not affect the phosphorylating particle and the intact mitochondria the same way. 

Klouwen and his associates [38, 39] have further investigated the occurrence of oxidative phosphorylation in thymus nuclei they acknowledge contamination of the nuclear pellet with as much as 10% intact cell. Yet they believe that the small levels of oxidative phosphorylation measured in nuclei (P/O ratio 0.6-1.0) do not result from contamination by intact or fragmented mitochondria. Further, they claim that the nuclear oxidative phosphorylation is sensitive to various inhibitors of the electron transport chain. Using low-temperature spectophotometry, it was established that a nuclear preparation free of cytoplasmic contamination contained cytochrome b, c, and aa3 and that as much as 40% of cytochrome aa3 may be found in association with calf thymus nuclei. Other researchers have also found cytochrome b and c, but not cytochrome aa3, in calf thymus nuclear preparations. 

When whole thymus nuclei are washed with increasing concentrations of sodium chloride, different types of histones are extracted for example, lysine-rich histone is extracted with the lowest concentration of sodium chloride. 

An energy-dependent histone phosphorylation was discovered in calf and rat thymus nuclei [68, 69], A histone phosphokinase has been purified from liver, and some of its properties have been studied. The kinase phosphorylates only the serine residues of histones or protamines. It is inactive on other proteins and therefore different from the typical phosphopro-tein kinase. In vitro the enzyme phosphorylates all histone fractions, but FI appears to be a preferred substrate. 

Chloramphenicol has been reported to have no effect on amino acid activation or incorporation of amino acids into sRNA. In high concentration, however, it inhibits nearly completely the incorporation of labelled amino acid into the protein of calf thymus nuclei . The antibiotic thus appears to interfere at a late stage in the process of protein synthesis. It is of interest that its L-erythro isomer has bttle effect on protein synthesis but inhibits the synthesis of a D-glutamyl pol5q>eptide by Bacillus suhtilis. ... 

Hydrolysis of the nucleohistones with trypsin caused a sharp increase in the rate of synthesis of RNA and proteins in thymus nuclei. In interphase nuclei of thymus lymphocytes the repressed nucleohistone zones were concentrated mainly in condensed heterochromatin masses, whereas active interphase DNA was found in euchromatin fibrils (Frenster et al., 1963). A large part of the fast-labeled nuclear RNA was found in these fibrils. 

Fig. 7 Identification of novel posttranslational modifications of histones H2A and H2B isolated from calf thymus nuclei by FT-ICR MS analysis of proteolytic peptides generated by the digestion of histone H2A with pepsin (a) or trypsin (b), and of histone H2B with trypsin (c) and V8DE (d). Reproduced from [375]...

Evidence has accumulated in recent years supporting the proposal of Stedman and Stedman (1950) that complexing with histones suppresses activity of the genes. Primarily this evidence is derived from experiments showing inhibition of the stimulating action of DNA on protein synthesis by thymus nuclei (Allfrey, 1961 Allfrey and Mirsky,... 

As previously indicated, the primary cells involved in the immune response are lymphocytes which have a centrally located round nucleus, lack specific granules, and have a basophilic cytoplasm containing free ribosomes. The (thymus-dependent) T-lyniphocytes are involved in cell mediated reactions and also interact with B-Iymphocytes (see later) to regulate the production of antibody, The B cells differentiate into the antibody-producing plasma cells. There is growing evidence that neither T... 

Poly A polyinerase an enzyme which spedfically catalyses the synthesis of poly A sequences, a process which does not require DNA. P. from calf thymus has M, about 150,000. It is localized in the nucleus, and is responsible for the covalent attachment of poly A sequences to RNA (see Polyadenylic acid). 

Poly(ADP-ribosyl)ation at 200 p,M NAD+ as substrate with an excess of whole thymus histones as additional poly(ADP-ribose) acceptors, probably simulating conditions prevailing in chromatin, yielded a less complicated kinetics (Fig. 2). In this system duplex C WAS the best synthetic coenzyme, whereas the activation curve of a sDNA remained sigmoidal. In the cell nucleus, the concentration of DNA is far greater than that of the poly(ADP-ribose) transferase, therefore the observed inhibition in the in vitro model... 

Poly A does not exist in an isolated form in the nucleus and must, therefore, be added to preexisting polynucleotide chains. Thus, when hnRNA molecules have been transcribed, they serve as primer for the biosynthesis of their poly A tail at the 3 -end. Inasmuch as they are no poly (dT) or poly (dA) regions in the DNA, it seems clear that poly A is not synthesized by the traditional transcription mechanisms. Moreover, the enzymes that synthesize poly A are sensitive to cordycepin, which has no or little effect on mRNA synthesis. The enzymes responsible for poly A synthesis have been purified to gel electrophoretic homogeneity from calf thymus in Mary Edmonds laboratory [196]. 

Betel, I., Klouwen, H.M. Oxidative phosphorylation in nuclei isolated from rat thymus. In The cell nucleus-metabolism and radiosensitivity. Proc. Int. Symp., p. 281-293. London Taylor and Francis 1966... 

The mutagenic properties of LSD-25 are now demonstrated and physicochemical studies by circular dichroism have shown direct interactions of LSD with calf-thymus DNA. Fluorescence studies also bring supporting evidence that indole-alkylamines can interact with DNA. Converging data are therefore at hand to support the provocative hypothesis of a direct competition of hallucinogens for tryptaminergic receptors located on DNA molecules in the nucleus or on RNAs located in the plasma membrane. 

Initially discovered in extracts of calf thymus as a contaminating enzyme during the purification of RNA polymerase (1,2), RNase H has since been found almost ubiquitously in lower and higher eukaryotes as well as in prokaryotes. A single cell type may have one or more species of RNases H residing in the cell nucleus and/or cytosol. Some RNases H have a single ribonucleolytic reaction specificity, whereas others are associated with DNA polymerase activities. Cellular RNases H, for example, from E. coli and calf thymus, are endonucleases the retroviral reverse transcriptase-associated RNases H can function as both exo- and endoribo-nucleases depending on the type of substrates available. 

The TDTase activity in calf thymus is localized in both cytoplasm and nucleus (1). In immature thymocytes and neoplastic cells, TDTase is observed almost exclusively in the nucleus (54). The structural gene for calf TDTase has been cloned and efficiently expressed from a plasmid expression vector in COS7 (monkey fibroblast) cells (43). 

In 1929, Jones, Phillips, Larsell, and Nokes (65) reported the hematopoietic effect of nuclear extractives in human anemias. They believed that nuclear extractives from various organ sources—considered to be nucleoproteins and the sodium salts of nucleic acids—contained an unknown hematopoietic stimulant. Oral administration of such extractives, prepared from chicken corpuscles, beef spleen, beef liver, beef kidney, beef heart muscle, salmon liver, beef thymus and beef pancreas, in J g. doses, yielded satisfactory results in pernicious as well as other anemias. They thus tentatively concluded that the hematopoietic stimulant is an integral part of the cell nucleus. 

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