Mechanism of DNA replication fidelity for three mutants of DNA polymerase I: Klenow fragment KF(exo+), KF(polA5), and KF(exo-)

Inhibition of the pre-steady-state burst of nucleotide incorporation by a single incorrect nucleotide (nucleotide discrimination) was measured with the Klenow fragment of DNA polymerase I [KF(exo+)]. For the eight mispairs studied on three DNA sequences, only low levels of discrimination ranging from none to 23-fold were found. The kinetics of dNTP incorporation into the 9/20-mer at low nucleotide concentrations was also determined. A limit of greater than or equal to 250 s-1 was placed on the nucleotide off-rate from the KF(exo+)-9/20-dTTP complex in accord with nucleotide binding being at equilibrium in the overall kinetic sequence. The influence of the relatively short length of the 9/20-mer on the mechanism of DNA replication fidelity was determined by remeasuring important kinetic parameters on a 30/M13-mer with high homology to the 9/20-mer. Pre-steady-state data on the nucleotide turnover rates, the dATP(alpha S) elemental effect, and the burst of dAMP misincorporation into the 30/M13-mer demonstrated that the kinetics were not affected by the length of the DNA primer/template. The effects on fidelity of two site-specific mutations, KF(polA5) and KF(exo-), were also examined. KF(polA5) showed an increased rate of DNA dissociation and a decreased rate of polymerization resulting in less processive DNA synthesis. Nevertheless, with at least one misincorporation event, that of dAMP into the 9/20-mer, KF(polA5) displays an increased replication fidelity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The initial characterization of the iron environment in lipoxygenase by M?ssbauer spectroscopy

The incorporation of 57Fe into two lipoxygenase isoenzymes from soybeans has been achieved making possible the first observations of the iron environment in these proteins using M?ssbauer spectroscopy. Immature soybean seeds were grown in tissue culture medium supplied with 57Fe. The iron in the active lipoxygenases that were isolated from the cultured seeds was readily detected in M?ssbauer measurements. It was unequivocally demonstrated that the native enzyme contains high-spin Fe(II). Based on the sign of the electric field gradient, the most likely ligand sphere for the iron in native lipoxygenase consists of oxygen and nitrogen ligands in a roughly octahedral field of symmetry. It was possible to detect M?ssbauer signals in highly concentrated samples of native lipoxygenases containing 57Fe at natural abundance. The spectra obtained for enriched and natural abundance native enzyme had the same high-spin Fe(II) M?ssbauer parameters. This confirmed that the environment of the iron in enzymes isolated from cultured seeds and dry soybeans were the same. The M?ssbauer spectra (4.2-250 K) for samples of both isoenzymes after oxidation of the iron in native enzyme by the product of lipoxygenase catalysis were extremely broad (20 mm/s) with no obvious narrow resonance lines. This was the result of the existence of paramagnetically broadened spectra for such samples even at relatively high temperature as evidenced by the appropriate EPR signal. A small molecule containing an iron site sharing many of these M?ssbauer and electron paramagnetic resonance properties with lipoxygenase was identified: Fe(II)/(III).diethylenetriaminepentaacetic acid.     

Exercise hyperventilation in patients with McArdle's disease

This study was undertaken to determine if patients who lack muscle phosphorylase (i.e., McArdle's disease), and therefore the ability to produce lactic acid during exercise, demonstrate a normal hyperventilatory response during progressive incremental exercise. As expected these patients did not increase their blood lactate above resting levels, whereas the blood lactate levels of normal subjects increased 8- to 10-fold during maximal exercise. The venous pH of the normal subjects decreased markedly during exercise that resulted in hyperventilation. The patients demonstrated a distinct increase in ventilation with respect to O2 consumption similar to that seen in normal individuals during submaximal exercise. However their hyperventilation resulted in an increase in pH because there was no underlying metabolic acidosis. End-tidal partial pressures of O2 and CO2 also reflected a distinct hyperventilation in both groups at approximately 70-85% maximal O2 consumption. These data show that hyperventilation occurs during intense exercise, even when there is no increase in plasma [H+]. Since arterial CO2 levels were decreasing and O2 levels were increasing during the hyperventilation, it is possible that nonhumoral stimuli originating in the active muscles or in the brain elicit the hyperventilation observed during intense exercise.     

Properties of a dolichol phosphokinase activity associated with rat liver microsomes

Rat liver microsomes show a capacity to synthesize [1-3H]dolichyl phosphate from [1-3H]-dolichol. Formation of [1-3H]dolichyl phosphate increased continuously over 15 min although the reaction rate was never completely linear. Product formation was directly proportional to microsomal protein concentration between 1.1 mg/mL and the highest concentration tested, 5.5 mg/mL. The reaction rate was linear with respect to the dolichol content of the assay mixture to a saturation point (120 microM). An apparent Km of 50 microM was established for dolichol. The normal phosphate donor for the reaction is CTP and not ATP. The optimum concentration of CTP was 10 mM, and an apparent Km of 4 mM was calculated for this nucleoside triphosphate. The reaction was totally dependent on divalent metal ion, magnesium being more effective than calcium. The optimum concentration of magnesium ion and CTP were the same (10 mM), suggesting that MgCTP2- is utilized as the normal enzyme substrate. Activity measured in the absence of Triton X-100 was only 5% of the activity observed at the optimum (0.5% w/v) detergent concentration. The measurable levels of dolichol phosphokinase could be doubled by the inclusion of 10-15 mM NaF as phosphatase inhibitor. Optimal enzymatic activity was obtained between pH 7.0 and pH 7.5 and could be inhibited by EDTA. The sulfhydryl reagent DTT was slightly stimulatory while the product of the reaction, dolichyl phosphate, was noninhibitory at the highest concentration tested (13.8 microM). The second reaction product (CDP) inhibits the enzymatic phosphorylation of dolichol.     

Changes in the histones of the sea urchin Strongylocentrotus purpuratus at fertilization

Both sperm and eggs of the sea urchin Strongylocentrotus purpuratus contain specific histones in place of some of the histones found during later development. Whether these specific histones are lost upon fertilization or are retained is not known. Therefore, we have examined the histones present in the zygote nucleus to determine the fate of the gamete histones. Nuclei of zygotes which have completed DNA replication in preparation for the first mitosis were isolated by sucrose density gradient centrifugation. Histones were extracted from the isolated nuclei, and were analyzed by acid-urea and SDS polyacrylamide gel electrophoreses, and by two-dimensional electrophoresis in which both gel electrophoresis systems were combined. Electrophoretic patterns of the zygote histones were compared with those of sperm, unfertilized eggs and embryos. The results show that the zygote histone pattern is identical with the unfertilized egg histone pattern. Neither the sperm histones H1, H2A, or H2B, nor the embryonic H1, H2A, or H2B, are present in the zygote pattern. The egg and the zygote do contain a unique H2A and H2B, but not an H1. After fertilization, sperm specific histones are not present on the DNA. Egg histones become associated with both the sperm DNA and the newly replicated DNA. The association of the embryonic histones with the DNA, therefore, occurs sometime later in development.     

Lévi-Strauss on the Oedipus Myth: A Reconsideration

The first half of the article presents a critique of Lévi-Strauss' well-known analysis of the Oedipus myth. A consideration of Greek beliefs suggests that Lévi-Strauss is incorrect in tying certain events in that myth to the "overvaluation of blood relations" and in asserting that the myth is concerned with the "affirmation/denial of man's autochthonous origins." The second half of the article presents a different structural analysis of the entire Theban Saga, of which the Oedipus myth is but a part. It concludes (1) that Lévi-Strauss is correct in identifying a series of events in the myth which indicate the devaluation of blood relations, but that these events specifically refer to the devaluation of patrilineal kin ties and that the range of events indicating the devaluation of patrilineal ties is broader than his analysis would suggest, and (2) there is another series of repeated events (unmentioned by Lévi-Strauss) which indicates the affirmation of patrilineal kin ties. The final hypothesis— that the opposition between the devaluation/affirmation of patrilineal kin ties underlies the Theban Saga— "makes sense" in terms of Greek history, as the period in which the Olympian myths look their present form is also the period in which the Greeks moved from a society organized along patrilineal kin ties to one organized around allegiance to the polis. [structuralism, Oedipus myth, myth and social structure, Lévi-Strauss, anthropological theory]     

The cell-wall phosphatase of cotton (Gossypium) is inhibited by kelthane.

Kelthane [4,4'-dichloro-alpha-(trichloromethyl)benzhydrol] was previously shown to decrease the limited tolerance of susceptible varieties of cotton (Gossypium) to Verticillium wilt. Kelthane was shown in the present study to inhibit the cell-wall p-nitrophenyl phosphatase of cotton. In view of information already establishing the cell wall as a primary site of action of Verticillium wilt, the data are interpreted as suggesting an as yet undefined interaction between Kelthane, cell-wall phosphatase and verticillium-resistance mechanisms of the cell wall.     

Adjacent repeating units of xenopus laevis 5S DNA can be heterogeneous in length

The distribution of length heterogeneity in adjacent repeating units of X. laevis 5S DNA has been examined by “cloning” 5S DNA in bacteria. Fragments of 5S DNA produced by partial digestion with Hind III and containing 1, 4, and 5 repeating units have been inserted at the single Hind III site of the tetracycline-resistance plasmid, pSC101, and the hybrid plasmids cloned in E. coli. Adjacent 5S DNA repeats in the cloned multi-repeat fragments can differ in length. This finding rules out some mechanisms which have been proposed to account for the parallel evolution of tandem repeated DNAs. The results are consistent with an unequal crossing-over mechanism and place some constraints on the molecular processes in this recombinatory event.