Nucleolar necklaces in chick embryo fibroblast cells. II. Microscope observations of the effect of adenosine analogues on nucleolar necklace formation. (41/752)

The round nucleoli of chick embryo fibroblast cells, when exposed to adenosine (2 mM)or to a number of adenosine analogues, lose material and unravel over a period of several hours to become beaded strands, 20 mu M in length, termed nucleolar necklaces (NN). Light microscope observations on this process are described. Biochemical experiments have revealed that most of these analogues interfere with both messenger RNA synthesis and ribosome synthesis, causing extensive degradation of the preribosome species containing 32S RNA although most of the preribosomes containing 18S RNA survive. We suggest that it is the depletion from the nucleolus of the adhesive 32S and 28S RNA preribosomes which allows the remaining nucleolar apparatus to spread apart into the NN configuration. Also required for the maintenance of the NN structure is the synthesis of some ribosomal RNA (rRNA) possibly present as rRNA "feathers" on the DNA. The addition of inhibitors of rRNA synthesis such as actinomycin D to the NN-containing cells causes loss of rRNA. Then a contraction and collapse of the NN structure into small dense spheres is observed.  (+info)

Mutagenesis induced by a single 1,N6-ethenodeoxyadenosine adduct in human cells. (42/752)

To study the genotoxic properties of 1,N6-ethenodeoxyadenosine (epsilondA) in human cells, a novel site-specific mutagenesis approach was developed, in which a single DNA adduct was uniquely placed in either strand of a shuttle plasmid vector. The analysis of progeny plasmid derived from the modified strand shows that epsilondA, when incorporated into the position of the second A of 5'-CAA (codon 61 of the ras gene), is mutagenic in human cells, inducing A-->T, A-->G, and A-->C mutations. The efficient induction of A-->T transversions in experiments using modified double- and singlestranded DNA substrates supports the hypothesis that A:T-->T:A transversions in human and animal tumors induced by vinyl compounds reflect misinsertion of dAMP opposite this adduct. Mutagenic events were similar when the adduct was incorporated into either the leading or the lagging strand. EpsilondA was more mutagenic than 8-oxodeoxyguanosine, which induced targeted G-->T transversions in HeLa cells. In Escherichia coli, epsilondA did not significantly miscode (<0.27%) even in the presence of induced SOS functions.  (+info)

The N(8)-(2'-deoxyribofuranoside) of 8-aza-7-deazaadenine: a universal nucleoside forming specific hydrogen bonds with the four canonical DNA constituents. (43/752)

The 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin-4-amine) N(8)-(2'-deoxyribonucleoside) (2) which has an unusual glycosylation position was introduced as a universal nucleoside in oligonucleotide duplexes. These oligonucleotides were prepared by solid-phase synthesis employing phosphoramidite chemistry. Oligonucleotides incorporating the universal nucleoside 2 are capable of forming base pairs with the four normal DNA nucleosides without significant structural discrimination. The thermal stabilities of those duplexes are very similar and are only moderately reduced compared to those with regular Watson-Crick base pairs. The universal nucleoside 2 belongs to a new class of compounds that form bidentate base pairs with all four natural DNA constituents through hydrogen bonding. The base pair motifs follow the Watson-Crick or the Hoogsteen mode. Also an uncommon motif is suggested for the base pair of 2 and dG. All of the new base pairs have a different shape compared to those of the natural DNA but fit well into the DNA duplex as the distance of the anomeric carbons approximates those of the common DNA base pairs.  (+info)

2'-Deoxyadenosine and A23187 as agents for inducing synchrony in the budding yeast, Kluyveromyces fragilis. (44/752)

The effects of induction synchrony by 2'-deoxyadenosine and the ionophore A23187 were compared with selection synchrony methods in Kluyveromyces fragilis. 2'-Deoxyadenosine synchronizes nuclear division and this is followed by synchronous cell division, but DNA synthesis is not synchronized. The ionophore A23187 also induces synchronous nuclear division and synchronous cell division, but likewise does not synchronize DNA synthesis.  (+info)

Misincorporation of (TP during transcription of poly dAT-dAT and poly dABU-dABU. (45/752)

The synthetic DNA alternating copolymers poly dAT-dAT and dABU-dABU have been transcribed with E. coli RNA polymerase to measure the level of BrdU-induced misincorporation of guanine during transcription. GTP is found to be misincorporated into both copolymers at a frequency of 1 per 1000-2000 nucleotides polymerized. Using alpha-32P-GTP, the nearest neighbors to GMP are found to be UMP (approximately 63%), GMP (approximately 25%) and AMP (approximately 17%), with no apparent difference between the two templates. These results suggest that BrdU-substitution in DNA does not necessarily increase the potential for base mispairing during transcription, and hence, promote the production of faulty RNA molecules.  (+info)

Reverse methionine biosynthesis from S-adenosylmethionine in eukaryotic cells. (46/752)

The intracellular ratio between methionine and its activated form S-adenosylmethionine (AdoMet) is of crucial importance for the one-carbon metabolism. AdoMet recycling into methionine was believed to be largely achieved through the methyl and the thiomethyladenosine cycles. We show here that in yeast, AdoMet recycling actually occurs mainly through the direct AdoMet-dependent remethylation of homocysteine. Compelling evidences supporting this result were obtained owing to the identification and functional characterization of two new genes, SAM4 and MHT1, that encode the yeast AdoMet-homocysteine methyltransferase and S-methylmethionine-homocysteine methyltransferase, respectively. Homologs of the Sam4 and Mht1 proteins exist in other eucaryotes, indicating that such enzymes would be universal and not restricted to the bacterial or fungal kingdoms. New pathways for AdoMet or S-methylmethionine-dependent methionine synthesis are presented.  (+info)

Amino acids are not all initially attached to the same position on transfer RNA molecules. (47/752)

Escherichia coli tRNA has been modified by replacement of the 3'-terminal AMP with either 3'-amino-3'-deoxy AMP of 2'-amino-2'-deoxy AMP. These tRNA analogs have enabled us to determine the initial site of enzyme-catalyzed aminoacylation of different tRNAs by the formation of aminoacyl-tRNA molecules in which the amino acid is linked to the 3'-terminal ribose through a stable amide bond. The tRNA species specific for glutamic acid, glutamine, leucine, phenylalanine, tyrosine, and valine are all aminoacylated on the 2'-hydroxyl group. The tRNA species specific for alanine, asparagine, aspartic acid, glycine, histidine, lysine, and threonine are aminoacylated on the 3'-hydroxyl group. The amino acids arginine, isoleucine, methionine, proline, serine, and tryptophan form stable amide bonds with both amino tRNA analogs. This might suggest that the synthetases for these amino acids can acylate both the 2'- and 3'-hydroxyl groups, but it is more likely that these enzymes can acylate both hydroxyl and amino groups at either the 2' or 3'-position of the tRNA. These results clearly illustrate a fundamental heterogeneity which is apparent in the mechanism of action of aminoacyl-tRNA synthetases.  (+info)

Transport of purines and deoxyadenosine in Escherichia coli. (48/752)

The characteristics of adenine, guanine, hypoxanthine, xanthine, and uracil uptake in Escherichia coli B show that each base is transported by a specific system. The data support the concept that the transport of guanine, hypoxanthine, xanthine, and uracil function without direct involvement of the respective purine or pyrimidine phosphoribosyltransferase enzymes. Uracil phosphoribosyltransferase is not demonstrable in E. coli B, and large differences are observed in the inhibitory effects of heterologous purines on the uptake of guanine, hypoxanthine, and xanthine as compared to the corresponding inhibitory effects reported for the soluble purine phosphoribosyltransferase enzymes of E. coli B. Additional evidence is provided by the low Km values determined for the transport of adenine, guanine, hypoxanthine, and xanthine relative to the corresponding Km values for the phosphoribosyltransferase enzymes. Data are presented indicating that adenine may be transported without participation of adenine phosphoribosyltransferase. The stimulatory effect of glucose, the inhibitory effect of KCN, and the high intracellular to extracellular concentration gradients of the bases produced in the presence of glucose provide evidence that the transport processes are energy-dependent. The Km values for transport of the purines and uracil range from 10(-7) M to 5 X 10(-7) M. Characteristics of adenine and uracil uptake are similar in E. coli B, E. coli K-12, and a showdomycin-resistant mutant of E. coli B. Adenosine and deoxyadenosine are transported in E. coli B by independent transport systems. Adenine or hypoxanthine does not share the adenosine or deoxyadenosine transport systems as evidence by the mutual lack of competition of free bases and nucleosides on transport. The transport systems for deoxyadenosine and adenosine are defective in the mutant.  (+info)