Data-Driven Modeling of Intracellular Auxin Fluxes Indicates a Dominant Role of the ER in Controlling Nuclear Auxin Uptake

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Specific Fragments of φX174 Deoxyribonucleic Acid Produced by a Restriction Enzyme from Haemophilus aegyptius, Endonuclease Z

A restriction-like enzyme has been purified from Haemophilus aegyptius. This nuclease, endonuclease Z, produces a rapid decrease in the viscosity of native calf thymus and H. influenzae deoxyribonucleic acids (DNA), but does not degrade homologous DNA. The specificity of endonuclease Z is different from that of the similar endonuclease isolated from H. influenzae (endonuclease R). The purified enzyme cleaves the double-stranded replicative form DNA of bacteriophage phiX174 (phiX174 RF DNA) into at least 11 specific limit fragments whose molecular sizes have been estimated by gel electrophoresis. The position of these fragments with respect to the genetic map of phiX174 can be determined by using the genetic assay for small fragments of phiX174 DNA.     

The kinetic mechanism of 3-hydroxy-3-methylglutaryl-coenzyme A synthase from baker''s yeast

1. The effect of independent variation of both acetyl-CoA and acetoacetyl-CoA on the initial velocity at pH8.0 and pH8.9 gives results compatible with a sequential mechanism involving a modified enzyme tentatively identified as an acetyl-enzyme, resulting from the reaction with acetyl-CoA in the first step of a Ping Pong (Cleland, 1963a) reaction. 2. Acetoacetyl-CoA gives marked substrate inhibition that is competitive with acetyl-CoA. This suggests formation of a dead-end complex with the unacetylated enzyme and is in accord with the inhibition pattern given by 3-oxohexanoyl-CoA, an inactive analogue of acetoacetyl-CoA. 3. The inhibition pattern given by products of the reaction is compatible with the above mechanism. CoA gives mixed inhibition with respect to both substrates, whereas dl-3-hydroxy-3-methylglutaryl-CoA competes with acetyl-CoA but gives uncompetitive inhibition with respect to acetoacetyl-CoA. 4. 3-Hydroxy-3-methylglutaryl-CoA analogues lacking the 3-hydroxyl group are found to compete, like 3-hydroxy-3-methylglutaryl-CoA, with acetyl-CoA but have K(i) values ninefold higher, indicating the importance of the 3-hydroxyl group in the interaction. 5. A comparison of inhibition by CoA and desulpho-CoA at pH8.0 and pH8.9 shows that at the higher pH value a kinetically significant reversal of the formation of acetyl-enzyme can occur. 6. Acetyl-CoA homologues do not act as substrates and compete only with acetyl-CoA. A study of the variation of K(i) with acyl-chain length suggests the presence near the active centre of a hydrophobic region. 7. These results are discussed in terms of a kinetic mechanism in which there is only one CoA-binding site the specificity of which is altered by acetylation of the enzyme. 8. The rate of 3-hydroxy-3-methylglutaryl-CoA synthesis in yeast is calculated from the kinetic constants determined for purified 3-hydroxy-3-methylglutaryl-CoA synthase and from estimates of the physiological substrate concentrations. The rate of synthesis of 12nmol of 3-hydroxy-3-methylglutaryl-CoA/min per g wet wt. of yeast is still greater than the rate of utilization in spite of the extremely low (calculated) acetoacetyl-CoA concentration (1.8nm).     

SALMONELLA TYPHIMURIUM-ESCHERICHIA COLI Hybrids for the Tryptophan Region

Fifty-eight hybrids were analyzed for their phenotypic stability, presence and nature of cryptic trp alleles and by P22-mediated transduction to yield percent homologies. The hybrids fall into 5 distinguishable classes: a haploid class in which selected E. coli genes replace equivalent sites in the S. typhimurium chromosome; three merodiploid classes in which the selected E. coli genes are integrated at novel sites in the S. typhimurium chromosome-on the same transducing fragment as the female genes selected against, with or without cryptic damage to a nearby gene, or not on the same transducing fragment; and one class in which recombination has not taken place and the E. coli DNA is presumed to be an exogenote. The homology values are heterogeneous and do not permit an accurate determination of the relative frequency of incorporation of the integrated male genetic material. A further study of 20 hybrids indicates that genetic rearrangements can occur in the hybrids.     

Selective breeding as a tool to probe skeletal response to high voluntary locomotor activity in mice

We present a novel mouse-model for the study of skeletal structureand evolution, based on selective breeding for high levels ofvoluntary wheel running. Whereas traditional models (originallyinbred strains, more recently knockouts and transgenics) relyon the study of mutant or laboratory-manipulated phenotypes,we have studied changes in skeletal morphometrics resultingfrom many generations of artificial selection for high activityin the form of wheel running, in which mice engage voluntarily.Mice from the four replicate High Runner (HR) lines run nearlythree times as many revolutions during days 5 and 6 of a 6-dayexposure to wheels (1.12 m circumference). We have found significantchanges in skeletal dimensions of the hind limbs, includingdecreased directional asymmetry, larger femoral heads, and widerdistal femora. The latter two have been hypothesized as evolutionaryadaptations for long-distance locomotion in hominids. Exercise-trainingstudies involving experimental groups with and without accessto wheels have shown increased diameters of both femora andtibiafibulae, and suggest genetic effects on trainability (genotype-by-environmentinteractions). Reanalysis of previously published data on bonemasses of hind limbs revealed novel patterns of change in bonemass associated with access to wheels for 2 months. Withoutaccess to wheels, HR mice have significantly heavier tibiafibulaeand foot bones, whereas with chronic access to wheels, a significantincrease in foot bone mass that was linearly related to increasesin daily wheel running was observed. Mice exhibiting a recentlydiscovered small-muscle phenotype ("mini-muscle," [MM] causedby a Mendelian recessive gene), in which the mass of the tricepssurae muscle complex is 50% lower than in normal individuals,have significantly longer and thinner bones in the hind limb.We present new data for the ontogenetic development of musclemass in Control, HR, and MM phenotypes in mice of 1–7weeks postnatal age. Statistical comparisons reveal highly significantdifferences both in triceps surae mass and mass-corrected tricepssurae mass between normal and MM mice at all but the postnatalage of 1 week. Based on previously observed differences in distributionsof myosin isoforms in adult MM mice, we hypothesize that a reductionof myosin heavy-chain type-IIb isoforms with accounts for ourobserved ontogenetic changes in muscle mass.