Ataxia telangiectasia: the effects of chemical mutagens and x-rays on sister chromatid exchanges in blood lymphocytes

It is now possible to examine in detail exchanges between sister chromatids (SCEs) and to attempt to investigate the relationships of such exchanges to aberration formation and DNA-repair mechanisms. The frequency of SCEs is dramatically increased by chemical mutagens and may reflect the level of DNA damage. Lymphocytes from patients with ataxia telangiectasis (AT) show high levels of spontaneous chromosome damage and are hypersentive to ionising radiations and it was of interest to examine the levels of SCE induced in these cells by various mutagens. The frequencies of SCE after treatment with X=rays or three chemical mutagens were equivalent to those in normal cells. The effects of fluorodeoxyuridine and deoxycytidine on SCE frequencies were also tested.     

Enzymatic O-glycosylation of kappa-caseinomacropeptide by ovine mammary Golgi membranes

The results of subcellular fractionation of sheep mammary gland membranes indicate that N-acetylgalactosaminyl polypeptide transferase and galactosyl-N-acetylgalactosaminyl transferase, which are involved in the assembly of disaccharide units of kappa-casein, are localized chiefly in Golgi membranes. The glycosyltransferase activities incorporating N-acetyl [1-14C] galactosamine and [U-14C] galactose from uridine diphosphate N-acetyl [1-14C] galactosamine and uridine diphosphate [U-14C] galactose, respectively, were measured after membrane solubilization with Triton X-100 either with unglycosylated caseinomacropeptide, or with this polypeptide containing the N-acetylgalactosamine side chain residues (desialylated and degalactosylated caseinomacropeptide). Radioactive N-acetylgalactosamine was incorporated in the unglycosylated acceptor peptide, and the glycosidic bonds in the product were alkali labile, suggesting that they were linked to the hydroxyamino acid residues. In addition radioactive N-acetylgalactosamine was released after alpha N-acetyl-D-galactosaminidase treatment of labelled caseinomacropeptide. [U-14C] galactose was incorporated in the desialylated and degalactosylated acceptor peptide. Reductive alkaline treatment of [U-14C] galactose peptide resulted in the release of a major product, the chromatographic properties of which in TLC were identical with authentic galactosyl (1 leads to 3) N-acetylgalactosaminitol. The structure of the labelled disacchariditol determined after periodate oxidation (two equivalents) by gas liquid chromatography-mass spectrometry revealed that the [U-14C] galactose was linked to position C-3 on the N-acetylgalactosaminyl-residue. The anomery of the galactose, as determined by a chemical method, indicates unambiguously a beta configuration.     

Inhibition of isoproterenol-induced DNA and RNA synthesis in rat parotid and pancreas following removal of submandibular-sublingual glands

Summary [³H] thymidine incorporation into DNA of the parotid (PA) gland of adult and 20-day-old rats and into DNA of the pancreas (PANC) of 20-day-old rats was increased markedly following a 2-day regimen of isoproterenol (ISO) administration. However, when the submandibular-sublingual (SM-SL) glands had been removed just prior to initiation of the ISO injections, the [³H] thymidine incorporation into PA and PANC was inhibited, and cpm/mg protein of these organs was even lower than that of organs of untreated rats with SM-SL glands present. Removal of the PA glands just prior to initiation of the ISO regimen had no effect on the ISO-induced [³H] thymidine incorporation into DNA of PANC but partially inhibited that of the submandibular (SM) gland. It is suggested that the inhibitory effects on DNA and RNA synthesis that follow removal of SM-SL glands are attributable to the growth factors (epidermal growth factor and nerve growth factor) found in the rat SM gland. These factors appear to regulate normal DNA synthetic activity of exocrine glands as well as ₁-adrenoceptor mediated DNA synthesis. Cellular hypertrophy induced by the ISO was less markedly affected by absence of the SM glands, but a partial inhibition of [³H] uridine incorporation into RNA of PA of adult rats also occurred when SM-SL glands were removed prior to initiation of the ISO-regimen.     

Ammonium ions prevent methylation of uridine to ribothymidine inAzotobacter vinelandii tRNA

It was shown that tRNA fromAzotobacter vinelandii grown in the presence of ammonium chloride lacks ribothymidine while that grown in the absence of the ammonium salt contains this modified nucleoside. [³²P]-Labelled tRNA from this organism grown in a medium containing the ammonium salt was digested with RNase T1 and the pseudouridinecontaining tetranucleotide, common to all tRNAs was isolated and analysed for the nucleoside replacing the ribothymidine. It was found to be uridine. Cells previously labelled with [³²P]-phosphate in the ammonium salt medium were washed and incubated in the ammonium saltfree medium to test whether ribothymidine would be formed upon removal of the ammonium ions. Methylation of the uridine did not take place.     

Regulation of uridine kinase activity in BLAB/C-3T3 cells by serum components

Summary After the stimulation of quiescent density-inhibited BALB/c-3T3 cells with fresh bovine calf serum, uridine kinase activity measured in cellular extracts increased between hours 3 and 6 of incubation and remained elevated through 12 h after stimulation. The addition of either partially purified platelet-derived growth factor (PDGF) or platelet-poor plasma (PPP) also caused increased uridine kinase activity by 6 h, but the increased activity was not maintained and the activity returned to the prestimulated level by 12 h. However, when PDGF and PPP were added in combination an increased level of uridine kinase activity was maintained in a manner similar to that seen after the addition of serum. The components of PPP eluted in the void volume from Sephadex G-50 chromatography did not induce uridine kinase activity when present alone, although they did act synergistically with PDGF to allow the maintenance of elevated levels of uridine kinase activity over the period from 6 to 12 h after stimulation. Thymidine kinase activity was not induced by the addition of either PDGF or PPP alone, although either serum or the combination of PDGF and PPP did produce an induction of thymidine kinase activity in late G₁. This work was supported by NCI Grants CA24913 and CA16084. W. W. was supported by NIH Postdoctoral Fellowship AM 1477. W. J. P. was supported by JFRA32 from the American Cancer Society. A preliminary report of this research was given at the Eighth International Cell Cycle Conference held at Research Triangle Park, NC, May 15–16, 1980.     

Uridine transport in basolateral plasma membrane vesicles from rat liver

Summary The characteristics of uridine transport were studied in basolateral plasma membrane vesicles isolated from rat liver. Uridine was not metabolized under transport measurement conditions and was taken up into an osmotically active space with no significant binding of uridine to the membrane vesicles. Uridine uptake was sodium dependent, showing no significant stimulation by other monovalent cations. Kinetic analysis of the sodium-dependent component showed a single system with Michaelis-Menten kinetics. Parameter values were K _M 8.9 m and V _max 0.57 pmol/mg prot/sec. Uridine transport proved to be electrogenic, since, firstly, the Hill plot of the kinetic data suggested a 1 uridine: 1 Na⁺ stoichiometry, secondly, valinomycin enhanced basal uridine uptake rates and, thirdly, the permeant nature of the Na⁺ counterions determined uridine transport rates (SCN^– > NO ₃ ^– > Cl^– > SO ₄ ^2– ). Other purines and pyrimidines cis-inhibited and trans-stimulated uridine uptake.This work has been partially supported by grant PM90-0162 from D.G.I.C.Y.T. (Ministerio de Educación y Ciencia, Spain). B.R.-M. is a research fellow supported by the Nestlé Nutrition Research Grant Programme.     

Pseudouridine residues in the 5′-terminus of uridine-rich nuclear RNA I (U1 RNA)

The primary nucleotide sequence was reported earlier for U1 RNA (Reddy et al, (1974) J. Biol. Chem. $\text{249}$, 6486–6494), an snRNA implicated in splicing of HnRNAs. In view of the presence of homologous pseudouridine (ψ) residues in 5′-ends of several highly conserved U-snRNAs and the recent report of modified bases in the U1 RNA structure (Branlant et al, (1980) Nucleic Acids Res. $\text{8}$, 4143–4154) a study was made for the presence of ψ and other modified nucleotides in the 5′-end of the U1 RNA. Identification of ψ residues at positions 6 and 7, shows the 5′-sequence of U1 RNA is: m₃², 2,7 GpppAm-Um-A-C-ψ-ψ-A-C-C-U-G-G-C-A-G-G-G-G-A-G-A-U-A-C. The ψ residues in place of U at positions 6 and 7 may affect the binding of U1 RNA at intron-exon splice junctions.     

Multiple regulation of nucleoside catabolizing enzymes in Escherichia coli: effects of 3:5'' cyclic AMP and CRP protein.

Summary The regulation of the synthesis of nucleoside metabolizing enzymes has been studied in cya and crp mutant strains of Escherichia coli.The synthesis of the cyt-enzymes, cytidine deaminase and uridine phosphorylase regulated by the cytR gene product, is activated by the cAMP-CRP complex. On the other hand the synthesis of the deoenzymes: deoxyriboaldolase, thymidine phosphorylase, phosphodeoxyribomutase and purine nucleoside phosphorylase, appears to be increased if an active cAMP-CRP complex cannot be formed.It also seems that nucleosides serve as poor carbon sources for cya and crp mutants; this could not solely be explained by low levels of nucleoside metabolizing enzymes nor by a deficiency in nucleoside uptake. Addition of casamino acids stimulated the growth of cya and crp mutants, with nucleosides as carbon sources. When grown on glucose and casamino acids growth could be stimulated by adenine and hypoxanthine nucleosides; these results suggest an impaired nitrogen metabolism in cya and crp mutants.Abbreviations and Symbols cAMP cyclic adenosine 3:5-monophosphate - CRP cAMP receptor protein. Genes coding for: adenyl cyclase - cya cAMP receptor protein - crp cytidine deaminase - cdd uridine phosphorylase - udp thymidine phosphorylase - tpp purine nucleoside phosphorylase - pup; cytR regulatory gene for cdd, udp, dra, tpp, drm, and pup - deoR regulatory gene for dra, tpp, drm, and pup     

Change of substrate specificity by polyamines of ribonucleases which hydrolyze ribonucleic acid at linkages attached to pyrimidine nucleotides.

The activities of ribonucleases (RNase HS and RNase A), which hydrolyze ribonucleic acid at linkages attached to pyrimidine nucleotides were stimulated by polyamines, while the activities of ribonucleases (RNase T₁ and RNase M), which attack ribonucleic acid at linkages attached to purine nucleotides were not influenced by polyamines. In the presence of polyamines, the cleavage of C_5′-O-P linkages adjacent to cytosine nucleotide was stimulated, while the cleavage of C_5′-O-P linkages adjacent to uracil nucleotides was inhibited slightly. The effect of polyamines on the activities of ribonucleases occured through the binding of the polyamines to nucleic acid.