Distribution Of Nucleotides Research Articles

Absence of long pyrimidine tracts in cytosine-rich repeating units present in Escherichia coli DNA

The distribution of pyrimidine nucleotides in cytosine-rich repeating sequences isolated from the DNA of Escherichia coli was obtained by means of chromatography on thin layers of polyethyleneimine-impregnated cellulose and on DEAE-cellulose columns. Nine nucleotides of different pyrimidine composition were detected, the longest of which contained four base residues.

N6-(delta2-isopentenyl)adenosine, an inhibitor of cellular transport of uridine and cytidine.

N6-(delta2-Isopentenyl)adenosine (IPAR) inhibited severely the incorporation of uridine and cytidine into S-180 cells in culture. When IPAR and the nucleosides were simultaneously present in the medium the inhibition was competitive (Ki 3.4 muM) and indicated inhibition of transport. However, the inhibition occurred even in the absence of extracellular IPAR if the cells had been preincubated with IPAR. Since 5'-IPAMP was the product which accumulated in large quantities in S-180 cells when incubated with IPAR, the effects of this AMP analog of the intracellular metabolism of uridine had to be considered. No direct correlation between the amount of intracellular IPAMP and the degree of inhibition of uridine utilization was observed and the relative distribution of uridine nucleotides in the acid soluble pool of the cells was unaltered in cells treated with IPAR. Also, IPAMP was not an inhibitor of uridine kinase in a cell free system nor was the activity of this enzyme affected by treatment of cells with IPAR. In addition, a profound inhibition of uridine utilization was also observed in a resistant subline of S-180 cells, which is unable to form IPAMP. These data suggest that IPAMP was not the inhibitory agent. Furthermore, the observation that the inhibition in both sensitive and resistant cells was caused even by a 15-second exposure to 100 muM IPAR, followed by rinsing, suggests that IPAR itself is the effective agent. It is concluded that IPAR exerts its inhibitory effect on uridine and cytidine utilization by becoming lodged in the cell membrane and thereby preventing the passage of these nucleosides into the cells. It is also shown that the inhibition of uridine and cytidine utilization by IPAR and by other potent nucleoside uptake inhibitors is unrelated to inhibition of growth or RNA-synthesis when the cells do not depend on an extracellular source of a nucleoside for growth.

The origin of the genetic code.

A new approach to the origin of the genetic code is proposed based on some regularities in the nucleotide distribution pattern of the code. The relative amounts of various amino acids in primitive proteins were possibly different from those in organisms living today. The primordial ratio was supposed to shift to the modern one guided by the action of primitive nucleotides. Each primitive tRNA had a discriminator site and, distinguished from it, an anticodon site. It also postulated that primordially each amino acid could correspond to a wide variety of codons. During the course of the evolutionary change, a selective mechanism worked among the protobionts so that less frequent nucleotides became associated with more abundant amino acids in the primordial conditions,thus finally leading to the present codon catalogue.

Fine structures in denaturation curves of bacteriophage lambda DNA. Their relation to the intramolecular heterogeneity in base composition

Precise recording of polyphasic optical melting curves was carried out for three kinds of bacteriophage lambda DNA differing in length ( λ cI857 S7 , λ cIb2 and λ cIb2 b5 ). Each of denaturation steps in melting profiles was characterized by two parameters, the melting temperature and the relative size. Any difference in fine structures in melting profiles was not recognized between the intact λ cI857 S7 DNA and the DNA fragmented into halves. The change in fine structures in melting profiles caused by the deletions of the b2 and b5 regions agreed qualitatively well with the prediction based on the physical and the genetical maps of phage lambda chromosome. The combined results indicate that, first, the well-known linear relationship between melting temperature and G+C content may apply also to each of denaturation steps in polyphasic melting curves due to heterogeneity of nucleotide distribution in a single DNA species, and, second, the effect of molecular ends on melting fine structures can be neglected at moderate salt concentration ( 0.01 M ⩽ Na + ⩽ 0.2 M ) for such a high molecular weight DNA. The heterogeneous distribution of nucleotides was derived for λDNA and for its b2 and b5 regions.

Regional distribution of adenine nucleotides, glycogen, glucose and lactate in the adult rat brain.

Le cervelet contient des quantites plus grandes d'ATP, d'ADP et de nucleotides totaux que le lobe frontal du cerveau ou la moelle cervicale. Ces differences ne sont pas trouvees avec l'AMP. Le contenu en glycogene, en glucose et en lactate est plus grand dans la moelle cervicale que dans le cervelet et dans le lobe frontal.

The distribution of pyridine nucleotides between nucleus and cytoplasm.

AbstractFollowing enucleation of a portion of human culture cells containing 3H‐pyridine nucleotides, autoradiography revealed no difference in the grain density over enucleated and whole cells. These results provide evidence that the concentration of pydine nucleotides does not differ by more than threefold between nucleus and cytoplasm and probably does not vary at all.

Distribution of adenine nucleotides between the inner and outer spaces of the mitochondrion as a determinant of phosphorylation pattern.

Mitochondrial preparations incubated with 32P1 and 3H-ADP were subjected to rapid filtration through a Millipore filter to study the intramitochondrial distribution of labelled nucleotides. The atractyloside-induced inhibition of the distribution of internally-labelled nucleotides and of externally-added 3H-ADP revealed that the nucleotides in the matrix space are successfully separated by this means from those outside the inner membrane. The addition of Mg2+ to the incubation medium has no effect on the labelling pattern in the matrix space but results in a rapid interconversion of adenine nucleotides outside the inner membrane through the activation of adenylate kinase [EC 2.7.4.3] and nucleosidediphosphate kinase [EC 2.7.4.6], which do not function in an Mg2+-free medium. The localization of GTP-AMP phosphotransferase [EC 2.7.4.10] outside the membrane is questionable, but was not definitely excluded. The translocation of internal ATP outwards in exchange for external ATP or ADP induced by the addition of ATP or ADP, of hexokinase plus glucose or of myokinase (muscle adenylate kinase) plus AMP into the incubation medium appears to be a significant factor in promoting the labelling of ATP, reflecting oxidative phosphorylation. 32P1-Labelling of ADP dependent on substrate-level phosphorylation is greatly suppressed under these conditions. This apparent suppression of substrate-level phosphorylation is at least partly accounted for in terms of the lowered specific radioactivity of the P1 compartment selectively supporting AMP phosphorylation as compared to the specific radioactivity of the major P1 pool serving as the substrate for oxidative phosphorylation. A possible interaction of these two phosphorylation reactions in rat-liver mitochondria is also discussed.

Pyridine Nucleotide Metabolism in Escherichia coli: II. NIACIN STARVATION

Abstract The effect of niacin starvation has been studied in a niacin-requiring auxotroph of Escherichia coli. If a culture is totally deprived of niacin, cells continue to divide until the total pyridine nucleotide content has fallen from 1.9 x 106 to 1.2 x 105 molecules per cell. During starvation, the relative proportion of the pyridine nucleotides changes greatly: the TPN: DPN ratio increases from 0.30 to over 2.0 and nicotinic acid mononucleotide accumulates until it is present at concentrations comparable to DPN. The changes in the distribution of the pyridine nucleotides suggest that DPN is turning over during niacin starvation and that the normally observed breakdown of TPN to DPN during exponential growth is inhibited. If cells are starved for niacin by balanced growth under limiting concentrations of niacin, less disproportion in the TPN: DPN ratio is observed, and growth occurs with a pyridine nucleotide content as low as 4 x 104 molecules per cell.

DNA base composition in mycoplasma, bacteria and yeast.

Knowledge of the base composition of DNA in microorganisms has increased rapidly over the last decade. The main incentive for the efforts made has been the usefulness of the base composition values in microbial taxonomy. Many general features have emerged which may be important for an understanding of the evolution of DNA composition. One of the main characteristics is the broad distribution of base composition among the various organisms. The question is whether this distribution is mainly random or caused by some selective pressures. Another characteristic feature is the heterogeneity in distribution of nucleotides within the individual DNA molecules. The compositional heterogeneity, which may also include differences in base composition between the two complementary strands of the DNA molecules, may be caused by conserved sequences, such as ribosomal RNA cistrons and sequences related to the regulation of replication and transcription. The specific amino acid sequences of the invariable parts of the proteins also create a possibility for heterogeneity in base composition between the DNA corresponding to different genetic markers. The present review gives a brief discussion of some of these problems and attempts to give a complete key to the literature on DNA base composition in mycoplasmas, bacteria and yeasts up to the end of 1971. The methods and problems related to determinations of base compositions are also discussed.KeywordsBase CompositionDeoxyribonucleic AcidBuoyant DensityDenaturation CurveCompositional DistributionThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Unusual Features of the Structure of the Major RNA Component of the Larger Subribosomal Particle of the Rabbit Reticulocyte

AbstractThe major RNA moiety of the larger subribosomal particle of the rabbit reticulocytes was subjected to partial hydrolysis by RNase T1. The fragments were separated first by gel filtration and then by zonal centrifugation. The fractions so obtained were analysed for molecular weight, nucleotide composition and for “melting” properties (E vs. temperature). The largest fragments (ca. 400,000 daltons) that were obtained had a high G + C content (ca. 78%). Fragments of this size appear to account for most of ca. 78% G·C basepairs which comprises ca. 40% of the intact molecule. Melting properties of reticulocyte RNA were compared with those of rRNA of the larger subribosomal particle Escherichia coli, Bacillus stearothermophilus and Xenopus laevis. The possible evolutionary significance of the unusual distribution of nucleotides within the polynucleotide chain of the RNA moiety of the larger subribosomal particle of reticulocytes is discussed.

Adenylate energy charge: Relation to guanylate energy charge and the adenylate kinase equilibrium constant

There is no functional cascade regulatory step between the adenylate energy charge and the guanylate energy charge in rat brain in vivo. In fact, the guanylate energy charge changes slower than the adenylate energy charge in phenobarbital, ether, ammonia, and insulin coma, in ischemia and in metrazol shock. It parallels the adenylate energy charge in fasting. The adenylate kinase equilibrium appears to be a good approximation to the normal in vivo distribution of the adenosine nucleotides and is thus useful in model studies.

Methylation pattern of lambda deoxyribonucleic acid.

Deoxyribonucleic acid (DNA) extracted from phage lambda grown on Escherichia coli K-12 strain W4032 had 113 +/- 10 5-methylcytosine residues and 215 +/- 20 6-methyl adenine residues per genome, as determined by three independent methods. These methylated nucleotides were distributed equally among the two strands of lambda DNA. Shearing of double-stranded DNA to half-length fragments revealed a slight deficiency of 5-methyl cytosine in the 55% guanine plus cytosine half. Shearing the DNA to fragments of smaller length showed that the distribution of methylated nucleotides along the double helix was uniform with the exception of an undermethylated fragment arising from the center of the lambda DNA molecule. The implication of these results for the function of methylated nucleotides in the lambda DNA molecule is discussed.

Comparisons of the Distribution of Nucleotides and Common Sequences in Deoxyribonucleic Acid from Selected Bacteriophages

Results from comparisons of deoxyribonucleic acid (DNA) from several classes of bacteriophages suggest that most phage chromosomes contain either a homogeneous distribution of nucleotides or are made up of a few, rather large segments of different quanine plus cytosine (G + C) contents which are internally homogeneous. Among those temperate phages tested, most contained segmented DNA. Comparisons of sequence similarities among segments from lambdoid phage DNA species revealed the following order in relatedness to lambda: 82 (and 434) > 21 > 424 > phi80. Most common sequences are found in the highest G + C segments, which in lambda contain head and tail genes. Hybridization tests with lambda and 186 or P2 DNA species verified that the lambdoids and 186 and P2 belong to two distinct groups. There are fewer homologous sequences between the DNA species of coliphages lambda and P2 or 186 than there are between the DNA species of coliphage lambda and salmonella phage P22.

Distribution of nucleotides in Escherichia coli DNA from λ transducing phages

We have analyzed the region in the chromosome of Escherichia coli adjacent to the λ insertion site by examining DNA of selected lines of transducing phages λ- gal and λ- bio. We found that bacterial DNA for a distance of at least 0.32 fractional length of λ, to the left of the insertion site, contains a fairly uniform distribution of nucleotides and is about 51% in guanine + cytosine content. Bacterial DNA for about 0.22 fractional distance to the right of the insertion site contains a less uniform distribution of nucleotides with average composition about 54% G + C. The over-all distribution of nucleotides in the gal-bio region of E. coli does not resemble that in the λ DNA molecule. DNA-DNA hybridization tests suggest that the gal region contains more sequence similarities with λ DNA than does unfractionated E. coli DNA, but the gal-homologous sequences seem equally distributed in both the left and right arms of the λ molecule. Analysis of a defective bio-transducing phage which, in addition to the bio-A-marker, contains a section of about 0.25 fractional length of DNA from an unknown location(s) on the bacterial chromosome gave contrasting results: the bacterial component of this phage DNA contains at least three distinct segments with nucleotide distributions similar to those in the right arm of λ. From left to right, the first, which includes the bio-A marker, is about 0.10 fractional length and contains about 43% G + C; the next is about 0.05 fractional length and 37% G + C; and the third is about 0.08 fractional length and 45% G + C. These are followed by a region (of uncertain length) that contains 43% G + C. About half of the nucleotide sequences in this section of bacterial DNA are similar to those in λ's right arm. Our analyses place the phage attachment site in a 43% G + C segment, about 0.57 fractional length from the left end of λ DNA (Skalka, Burgi & Hershey, 1968). The λ insertion site appears to be at or near the left edge of a very short segment in the bacterial DNA which also contains 43% G + C.

Distribution of nucleotides in Escherichia coli DNA from φ80 transducing bacteriophage

Escherichia coli DNA in the neighborhood of the φ80 attachment site, including the tryptophan operon, an ochre suppressor gene ( su c ) and F lac DNA transposed into the same region, was analyzed. DNA isolated from the transducing phages φ80 pt 1, φ80 pt 0, φ80 dsu c and φ80 d 1 lac was fragmented by shear and fractionated with respect to buoyant density of mercury complexes in cesium sulphate. Bacterial and episomal DNA components were identified by hybridization experiments. A segment containing the tryptophan operon consists of DNA ranging in G + C content from 43 to 57%, and the operator proximal end is richer in G + C. The composition of a segment between su c and att 80 varies in the same range, with an average G + C content of 51%. A segment containing lactose genes is more homogeneous, with an average G + C content of 54%. A portion of F factor, recovered from φ80 d 1 lac, ranges in G + C content from 37 to 52%.

Quantitative comparisons of certain nucleotides in the hepatic tissues of some aquatic animals

1. 1. The distribution of certain nucleotides in the hepatic tissues of some aquatic animals, shark, kokanee salmon, carp, rainbow trout, crab, surf clam, abalone, and squid, was studied. 2. 2. In the hepatic tissues the content of nucleotides was comparatively low as compared with that in the muscles. 3. 3. Adenine nucleotides, accounting for more than 90 per cent of the total amount of nucleotides in the muscle, showed a small proportion of 50–60 per cent. 4. 4. In fish liver UDP- N-acetylhexosamines, UDP-hexoses, and UDP-uronic acids were found accounting for 20–30 per cent of the total uracil nucleotides, whereas in other hepatic tissues examined UDP- N-acetylhexosamines, as major uracil nucleotides, few UDP-hexoses, and no UDP-uronic acid were found.

Chemical and genetic analysis of 16S ribosomal RNA in Escherichia coli.

Comparative chemical analyses of oligonucleotides arising from pancreatic RNase digestions of 16S ribosomal RNAs from Escherichia coli strains K12 and B(H) showed that a decanucleotide fragment, (5Ap,4Gp)Cp, could be detected exclusively in strain K12 but not in strain B(H), in spite of gross similarity of nucleotide distributions between the two strains.

The trapping of uridine phosphates by D-galactosamine. D-glucosamine, and 2-deoxy-D-galactose. A study on the mechanism of galactosamine hepatitis.

The effects of d‐galactosamine, d‐glucosamine, and 2‐deoxy‐d‐galactose on rat liver uracil nucleotides were studied in vivo. Enzymic and isotope dilution analyses of the UDP‐sugars and of uridine phosphates revealed three major, related changes: an accumulation of the respective UDP‐sugar derivatives, a marked decrease of UTP, UDP, and UMP, and a subsequent increase of the sum of hepatic uracil nucleotides. The decrease of uridine phosphates was accompanied by diminished contents of UDPG and UDP‐galactose. UMP of total liver RNA was not altered significantly. Inhibition of uridylate synthesis by use of 6‐azauridine resulted in a suppression of the d‐galactosamine‐induced stimulation of uridine phosphate synthesis and of the increase in total acid soluble uracil nucleotides. The trapping of uridine phosphates by formation of UDP‐sugar derivatives was most pronounced and most prolonged after administration of d‐galactosamine. The uridine phosphate content was lowered to less than 10% of normal within three hours, while the sum of uracil nucleotides increased by 0.35 μmole x g liver−1 x hour−1, from an initial value of 1.24 μmole/g. The quantitative analysis of the time dependent changes of UDP‐hexosamines, UDP‐N‐acetylhexosamines, UDPG, and UDP‐galactose revealed a pronounced alteration by d‐galactosamine of the UDP‐sugar pattern.Corresponding changes in the distribution of liver uracil nucleotides were obtained after administration of d‐glucosamine and 2‐deoxy‐d‐galactose. Both, however, are ineffective in provoking hepatitis. In contrast to d‐galactosamine, d‐glucosamine and 2‐deoxy‐d‐galactose do not lead to the formation of UDP‐hexosamines; furthermore they are less efficient in trapping uridine phosphates in vivo. These observations contribute to an understanding of the orotate‐mediated prevention of the galactosamine‐induced liver damage, and of the role of pyrimidine nucleotides in this experimental hepatitis.

Kinetic parameters and renal clearances of plasma adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate in man.

Kinetic parameters and the renal clearances of plasma adenosine 3',5'-monophosphate (cyclic AMP) and guanosine 3',5'-monophosphate (cyclic GMP) were evaluated in normal subjects using tritium-labeled cyclic nucleotides. Each tracer was administered both by single, rapid intravenous injection and by constant intravenous infusion, and the specific activities of the cyclic nucleotides in plasma and urine were determined. Both cyclic AMP and cyclic GMP were cleared from plasma by glomerular filtration. The kidney was found to add a variable quantity of endogenous cyclic AMP to the tubular urine, amounting to an average of approximately one-third of the total level of cyclic AMP excreted. Plasma was the source of virtually all of the cyclic GMP excreted. Plasma levels of the cyclic nucleotides appeared to be in dynamic steady state. The apparent volumes of distribution of both nucleotides exceeded extracellular fluid volume, averaging 27 and 38% of body weight for cyclic AMP and cyclic GMP, respectively. Plasma production rates ranged from 9 to 17 nmoles/min for cyclic AMP and from 7 to 13 nmoles/min for cyclic GMP. Plasma clearance rates averaged 668 ml/min for cyclic AMP and 855 ml/min for cyclic GMP. Approximately 85% of the elimination of the cyclic nucleotides from the circulation was due to extrarenal clearance.

Effects of ischemia on the concentration of adenine nucleotides in the kidney of anesthetized dogs.

SummaryIschemia causes a rapid decline in the concentration of ATP in the kidney cortex of anesthetized dogs. Within 8 sec of ischemia distribution of adenine nucleotides is determined by activity of adenylate kinase. Nucleotide metabolism of the cortex of dog is distinguished from that of rat by the observation that concentrations are lower and by the evidence of activity of adenylate kinase. We estimate that blood-flow dependent ATP turnover in the cortex is 27% of that calculated on the basis of renal O2 consumption. The medulla, in contrast to the cortex, maintains its ATP concentration during 60 sec of interruption of blood flow. Studies of nucleotide metabolism in the kidney must include consideration of differences in adenylate kinase activity between cortex and medulla and between different mammalian species.