Deoxyribonucleic acid polymerases of Euglena gracilis. Purification and properties of two distinct deoxyribonucleic acid polymerases of high molecular weight.

Two DNA polymerases of high molecular weight, pol A (mol.wt. 190 000) and pol B (mol.wt. 240 ooo), have been purified 6300-fold and 1600-fold respectively from an extramitochondrial supernatant of a bleached strain of Euglena gracilis. They have very similar requirements when assayed with an 'activated'-DNA primer-template [the optimum conditions of pH and ionic (K+ and Mn2+) composition being 7.2, 25 mM and 0.2 mM respectively]. 0.2 mM-Mn2+ was about 1.5-2-fold as effective as 2 mM-Mg2+, owing to substrate activation by deoxyribonucleoside 5'-triphosphates in the presence of Mn2+. Km values for the triphosphates in the absence of activation were about 10(-6)M with Mn2+ and 8 X 10(-6) M with Mg2+ for both enzymes. They were inhibited to the same extent by N-ethylmaleimide, novobiocin and o-phenanthroline, but differed in their chromatographic behaviour on DEAE-cellulose and in their electrophoretic mobilities on polyacrylamide gel. No evidence was found for the existence in these cells of a DNA polymerase of low molecular weight, but there were indications that a third enzyme of high molecular weight might exist.     

Amino acids and the synaptic pharmacology of granule cells in the dentate gyrus of the rat

Granule cells in the dentate gyrus in the hippocampi of anaesthetized rats were excited by stimulation of the contralateral hippocampus (the commissural input) and the ipsilateral entorhinal cortex (the perforant path). The cells were also activated by the electrophoretic administration of various amino acids. A selective antagonism of glutamate and perforant path excitations was obtained with glutamic acid diethylester, and of aspartate and other amino acid induced and commissural excitations with D- or DL-alpha-aminoadipate. An excitatory effect of alpha-aminoadipate which was sometimes observed was prevented by the gamma-aminobutyric acid antagonist bicuculline, and may be a disinhibitory phenomenon. The results lend support to the proposition that the transmitter of the perforant path is glutamate while that of the commissural fibres is aspartate.     

Recruitment, genetic counseling, and BRCA testing for underserved women at a public hospital

Genetic counseling and testing for heritable susceptibility to breast cancer caused by mutations in BRCA genes are largely unavailable to underserved women in the United States. Starting in 2002 the UCSF Cancer Risk Program offered this service free of charge to poor and medically indigent women at San Francisco General Hospital (SFGH). One recruitment strategy was a single-page questionnaire in four languages administered to women waiting for mammograms at SFGH. This report analyzes our first 3 years of experience with the recruitment questionnaire and compares the patient demographics and BRCA test results at SFGH with a more typical population undergoing genetic counseling and testing at UCSF's Mt. Zion Hospital (MZH). To our knowledge this is the first comprehensive clinical service for hereditary breast cancer in a U.S. public hospital. The ethnic mix of all 350 patients counseled was Caucasian 49% (approximately 20% of Caucasians reported Ashkenazi Jewish ancestry), Latina, 26%; African American, 13%; and Asian/other, 12%. Compared to the MZH population, SFGH patients were more ethnically diverse, less educated and more likely to be unemployed. Of 72 patients tested for BRCA mutations, 51 (71%) were negative, 5 were BRCA1 positive, and 12 were BRCA2 positive. Four (1 Caucasian, 1 Latina, 2 African American) had a total of 13 BRCA variants of unknown significance (VUS). The ratio of BRCA1/BRCA2 positive SFGH patients (5/12) was reversed compared to MZH (119/91). We evaluated 4573 recruitment questionnaires and 280 (6%) were judged to represent a high risk of heritable cancer. After additional screening and referral negotiation, 74 were scheduled for counseling. We judged the recruitment questionnaire to be a feasible, efficient, and reasonably cost-effective way to identify women at high risk of hereditary cancer in a traditionally underserved population. Underserved populations present special challenges for genetic counselors because of large, geographically dispersed families, cultural taboos about cancer diagnoses, and social marginalization. Despite these complexities, the clinical service at SFGH has been well accepted by patients and staff. Our successful venture can serve as a model for other public hospitals contemplating this clinical service.     

Nutrients from salmon parents alter selection pressures on their offspring

Organisms can modify their surrounding environment, but whether these changes are large enough to feed back and alter their evolutionary trajectories is not well understood, particularly in wild populations. Here we show that nutrient pulses from decomposing Atlantic salmon (Salmo salar) parents alter selection pressures on their offspring with important consequences for their phenotypic and genetic diversity. We found a strong survival advantage to larger eggs and faster juvenile metabolic rates in streams lacking carcasses but not in streams containing this parental nutrient input. Differences in selection intensities led to significant phenotypic divergence in these two traits among stream types. Stronger selection in streams with low parental nutrient input also decreased the number of surviving families compared to streams with high parental nutrient levels. Observed effects of parent‐derived nutrients on selection pressures provide experimental evidence for key components of eco‐evolutionary feedbacks in wild populations.     

Cell-to-cell and phloem-mediated transport of potato virus X. The role of virions

Movement-deficient potato virus X (PVX) mutants tagged with the green fluorescent protein were used to investigate the role of the coat protein (CP) and triple gene block (TGB) proteins in virus movement. Mutants lacking either a functional CP or TGB were restricted to single epidermal cells. Microinjection of dextran probes into cells infected with the mutants showed that an increase in the plasmodesmal size exclusion limit was dependent on one or more of the TGB proteins and was independent of CP. Fluorescently labeled CP that was injected into epidermal cells was confined to the injected cells, showing that the CP lacks an intrinsic transport function. In additional experiments, transgenic plants expressing the PVX CP were used as rootstocks and grafted with nontransformed scions. Inoculation of the PVX CP mutants to the transgenic rootstocks resulted in cell-to-cell and systemic movement within the transgenic tissue. Translocation of the CP mutants into sink leaves of the nontransgenic scions was also observed, but infection was restricted to cells close to major veins. These results indicate that the PVX CP is transported through the phloem, unloads into the vascular tissue, and subsequently is transported between cells during the course of infection. Evidence is presented that PVX uses a novel strategy for cell-to-cell movement involving the transport of filamentous virions through plasmodesmata.     

A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells

Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed a microtubule reporter gene by fusing the microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene with the green fluorescent protein (GFP) gene, and transient expression of the recombinant protein in epidermal cells of fava bean was induced. The reporter protein decorates microtubules in vivo and binds to microtubules in vitro. Confocal microscopy and time-course analysis of labeled cortical arrays along the outer epidermal wall revealed the lengthening, shortening, and movement of microtubules; localized microtubule reorientations; and global microtubule reorganizations. The global microtubule orientation in some cells fluctuates about the transverse axis and may be a result of a cyclic self-correcting mechanism to maintain a net transverse orientation during cellular elongation.     

Characterization of the bis(5''-nucleosidyl) tetraphosphate pyrophosphohydrolase from encysted embryos of the brine shrimp Artemia.

The P1P4-bis(5'-nucleosidyl) tetraphosphate asymmetrical-pyrophosphohydrolase from encysted embryos of the brine shrimp Artemia has been purified over 11,000-fold to homogeneity. Anion-exchange chromatography resolves two major species with very similar properties. The enzyme is a single polypeptide of Mr 17,600 and is maximally active at pH 8.4 and 2 mM-Mg2+. It is inhibited by Ca2+ (IC50 = 0.9 mM with 2 mM-Mg2+) but not by Zn2+ ions. It preferentially hydrolyses P1P4-bis(5'-nucleosidyl) tetraphosphates, e.g. P1P4-bis(5'-adenosyl) tetraphosphate (Ap4A) (kcat. = 12.7 s-1; Km = 33 microM) and P1P4-bis(5'-guanosyl) tetraphosphate (Gp4G) (kcat. = 6.2 s-1; Km = 5 microM). With adenosine 5'-P1-tetraphospho-P4-5"'-guanosine (Ap4G) as substrate, there is a 4.5-fold preference for AMP and GTP as products and biphasic reaction kinetics are observed giving Km values of 4.7 microM and 34 microM, and corresponding rate constants of 6.5 s-1 and 11.9 s-1. The net rate constant for Ap4G hydrolysis is 7.6 s-1. The enzyme will also hydrolyse nucleotides with more than four phosphate groups, e.g. Ap5G, Ap6A and Gp5G are hydrolysed at 25%, 18% and 10% of the rate of Ap4A respectively. An NTP is always one of the products. Ap2A and Gp2G are not hydrolysed, while Ap3A and Gp3G are very poor substrates. When the enzyme is partially purified from embryos and larvae at different stages of development by sedimentation through a sucrose density gradient, its activity increases 3-fold during the first 12 h of pre-emergence development. This is followed by a slow decline during subsequent larval development. The similarity of this enzyme to other asymmetrical-pyrophosphohydrolases suggests that it did not evolve specifically to degrade the large yolk platelet store of Gp4G which is found in Artemia embryos, but that it probably serves the same general function in bis(5'-nucleosidyl) oligophosphate metabolism as in other cells.