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)     

Recycling of platelet phosphorylation and cytoskeletal assembly

The shape change and aggregation of washed platelets induced by 10 microM arachidonic acid (AA) can be reversed by 20 ng/ml prostacyclin (PGI2), but these platelets can be reactivated by treatment with 30 microM epinephrine and subsequent addition of 10 microM AA mixture. These events may be modulated by cAMP since 2 mM dibutyryl cAMP also reversed activation without reactivation by epinephrine and AA. We examined protein phosphorylation and formation of cytoskeletal cores resistant to 1% Triton X-100 extraction of these platelets and correlated these processes with aggregation, fibrinogen binding, and changes in ultrastructure. Unactivated platelet cores contained less than 15% of the total actin and no detectable myosin or actin-binding protein. AA-induced cytoskeletal cores, which contained 60-80% of the total actin, myosin, and actin-binding protein as the major components, were disassembled back to unactivated levels by PGI2 and then fully reassembled by epinephrine and AA. Phosphorylation of myosin light chain and a 40,000-dalton protein triggered by AA (two- to fivefold) was reversed to basal levels by PGI2 but was completely restored to peak levels upon addition of the epinephrine and AA mixture. The reversibility of actin-binding protein phosphorylation could not be established clearly because both PGI2 and dibutyryl cAMP caused its phosphorylation independent of activation. With this possible exception, cytoskeletal assembly with associated protein phosphorylation, aggregation, fibrinogen binding, and changes in ultrastructure triggered by activation are readily and concertedly recyclable.     

Regulation of proneural gene expression and cell fate during neuroblast segregation in the Drosophila embryo.

The Drosophila embryonic central nervous system develops from sets of progenitor neuroblasts which segregate from the neuroectoderm during early embryogenesis. Cells within this region can follow either the neural or epidermal developmental pathway, a decision guided by two opposing classes of genes. The proneural genes, including the members of the achaete-scute complex (AS-C), promote neurogenesis, while the neurogenic genes prevent neurogenesis and facilitate epidermal development. To understand the role that proneural gene expression and regulation play in the choice between neurogenesis and epidermogenesis, we examined the temporal and spatial expression pattern of the achaete (ac) regulatory protein in normal and neurogenic mutant embryos. The ac protein is first expressed in a repeating pattern of four ectodermal cell clusters per hemisegment. Even though 5-7 cells initially express ac in each cluster, only one, the neuroblast, continues to express ac. The repression of ac in the remaining cells of the cluster requires zygotic neurogenic gene function. In embryos lacking any one of five genes, the restriction of ac expression to single cells does not occur; instead, all cells of each cluster continue to express ac, enlarge, delaminate and become neuroblasts. It appears that one key function of the neurogenic genes is to silence proneural gene expression within the nonsegregating cells of the initial ectodermal clusters, thereby permitting epidermal development.     

Seemingly unrelated measurement error models, with application to nutritional epidemiology

Motivated by an important biomarker study in nutritional epidemiology, we consider the combination of the linear mixed measurement error model and the linear seemingly unrelated regression model, hence Seemingly Unrelated Measurement Error Models. In our context, we have data on protein intake and energy (caloric) intake from both a food frequency questionnaire (FFQ) and a biomarker, and wish to understand the measurement error properties of the FFQ for each nutrient. Our idea is to develop separate marginal mixed measurement error models for each nutrient, and then combine them into a larger multivariate measurement error model: the two measurement error models are seemingly unrelated because they concern different nutrients, but aspects of each model are highly correlated. As in any seemingly unrelated regression context, the hope is to achieve gains in statistical efficiency compared to fitting each model separately. We show that if we employ a "full" model (fully parameterized), the combination of the two measurement error models leads to no gain over considering each model separately. However, there is also a scientifically motivated "reduced" model that sets certain parameters in the "full" model equal to zero, and for which the combination of the two measurement error models leads to considerable gain over considering each model separately, e.g., 40% decrease in standard errors. We use the Akaike information criterion to distinguish between the two possibilities, and show that the resulting estimates achieve major gains in efficiency. We also describe theoretical and serious practical problems with the Bayes information criterion in this context.     

Tunable, monochromatic X-rays: an enabling technology for molecular/cellular imaging and therapy

Pulsed, tunable, monochromatic X-rays hold great potential as a cellular and molecular probe. These beams can be tuned to the binding energy of orbital electrons in atoms, making them extremely useful in diagnostic k-edge imaging and Auger cascade radiotherapy. Their wide tunability makes them ideal for the performance of various techniques as disparate as protein crystallography and three-dimensional, compressionless, monochromatic mammography. Since only the frequency best suited to the task at hand is used, radiation exposure to patients or animals is exceedingly low when compared to standard X-ray techniques.