Purification and characterization of a gamma-like DNA polymerase from Chlamydomonas reinhardtii

A crude in vitro system which initiates chloroplast DNA synthesis near the D-loop site mapped by electron microscopy [Wu, M., Lou, J. K., Chang, D. Y., Chang, C. H., & Nie, Z. Q. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6761-6765] consists of soluble proteins and proteins extracted from purified thylakoid membrane. In this paper, a DNA polymerase activity was purified to near homogeneity from the soluble protein fraction of this in vitro system by sequential chromatographic separations on heparin-agarose, DEAE-cellulose, and single-stranded DNA-agarose columns and sedimentation in a glycerol gradient. In the glycerol gradient, the enzyme activity sedimented at a position corresponding to a 110-kDa protein. Electrophoretic analysis of the highly purified fraction on SDS-polyacrylamide gel revealed a major polypeptide band with an apparent molecular mass of approximately 116 kDa. In situ DNA polymerase activity assay shows that the DNA polymerization function is associated with the 116-kDa band and an 80-kDa band which could be a subunit of the enzyme. Polymerization activity is inhibited by N-ethylmaleimide, ethidium bromide, and dideoxycytosine triphosphate and is relatively resistant to aphidicolin. Poly(dA).(dT)10 and gapped double-stranded DNA are preferred templates. The purified enzyme contains no exonuclease activity and can initiate DNA replication in a supercoiled plasmid DNA template containing the chloroplast DNA replication origin.     

Estimating equations for parameters in means and covariances of multivariate discrete and continuous responses.

Generalized estimating equations are introduced in an ad hoc fashion for the covariance matrix of a multivariate response. These equations are to be solved jointly with score equations from a generalized linear model for mean parameters. A class of quadratic exponential models is used to develop joint estimating equations for mean and covariance parameters in a more systematic fashion, and proposals for the use of such equations are developed. Comments on the relative merits of the ad hoc and model-based approaches to estimation are given and a regression illustration with a bivariate response is provided.     

Binding of peptides with basic residues to membranes containing acidic phospholipids.

There are clusters of basic amino acids on many cytoplasmic proteins that bind transiently to membranes (e.g., protein kinase C) as well as on the cytoplasmic domain of many intrinsic membrane proteins (e.g., glycophorin). To explore the possibility that these basic residues bind electrostatically to monovalent acidic lipids, we studied the binding of the peptides Lysn and Argn (n = 1-5) to bilayer membranes containing phosphatidylserine (PS) or phosphatidylglycerol (PG). We made electrophoretic mobility measurements using multilamellar vesicles, fluorescence and equilibrium binding measurements using large unilamellar vesicles, and surface potential measurements using monolayers. None of the peptides bound to vesicles formed from the zwitterionic lipid phosphatidylcholine (PC) but all bound to vesicles formed from PC/PS or PC/PG mixtures. None of the peptides exhibited specificity between PS and PG. Each lysine residue that was added to Lys2 decreased by one order of magnitude the concentration of peptide required to reverse the charge on the vesicle; equivalently it increased by one order of magnitude the binding affinity of the peptides for the PS vesicles. The simplest explanation is that each added lysine binds independently to a separate PS with a microscopic association constant of 10 M-1 or a free energy of approximately 1.4 kcal/mol. Similar, but not identical, results were obtained with the Argn peptides. A simple theoretical model combines the Gouy-Chapman theory (which accounts for the nonspecific electrostatic accumulation of the peptides in the aqueous diffuse double layer adjacent to the membrane) with mass action equations (which account for the binding of the peptides to greater than 1 PS). This model can account qualitatively for the dependence of binding on both the number of basic residues in the peptides and the mole fraction of PS in the membrane.     

漆树乳汁道分泌细胞的超微结构及其与生漆产生和分泌关系的研究

漆树(Rhus verniciflua)乳汁道分泌细胞含有丰富的质体、内质网和嗜锇物质。电子显微镜的现察结果表明,嗜锇的生漆成分合成的可能场所是质体和内质网,并且通过内质网分子和小泡群与质膜相互接触并融合以及质膜内褶包被等三种形式释放到质膜和细胞壁之间的间隙中;再经过细胞壁中乳汁道腔形成时断裂了的胞间连丝通道和扩散渗透两条途径,越过细胞壁分泌到乳汁道腔中。细胞核、线粒体、高尔基体以及细胞质基质或多或少也参与了上述过程。     

Promotive effect of chrysanthemic acids on adventitious root formation in some plants

Adventitious root formation in excised cucumber (Cucumis sativus L.) cotyledons was significantly promoted by (±)-cis-chrysanthemic acid at 0.006–1.8 mM. The effect of (±)-cis-chrysanthemic acid on indole-3-acetic acid (IAA)-induced rooting was additive. Rooting in excised cucumber cotyledons was significantly promoted by several isomers of chrysanthemic acid and sodium (±)-cis-chrysanthemate at 0.18 mM. Rooting in mung bean (Phaseolus radiatus L.) hypocotyls was also stimulated by the sodium salt at 0.06–0.6 mM. Rooting of kidney bean (Phaseolus vulgaris L.) hypocotyls was also clearly enhanced by sodium (±)-cis-chrysanthemate at 0.18–6 mM.     

Extrachromosomal human immunodeficiency virus type-1 DNA can initiate a spreading infection of HL-60 cells

In this report, we describe a human immunodeficiency virus type-1 (HIV-1)-infected promyelocytic cell line, OM, derived from HL-60 cells. Although the OM cell line was biologically cloned twice, the pattern of HIV-1 expression during culture appeared analogous to a classical acute spreading infection and was inhibited by both azidothymidine and recombinant soluble CD4 treatment. The number of OM cells actually expressing HIV-1 at the beginning of culture was 0%, reached a peak of nearly 100% at 6 weeks, and then fell to less than 10% HIV-1+ cells by 10 weeks. Clonal analysis of the surviving cells verified that stable HIV-1+ OM cells resulted from the spreading infection. Southern analysis confirmed the transmission of HIV-1 through these OM cultures and the occurrence of stable clones which resulted. The initial percentage of OM cells actually harboring the HIV-1 genome was less than 0.1%, indicating nonfaithful transmission of an unintegrated HIV-1 genome during clonal expansion. These results demonstrate that extrachromosomal HIV-1 DNA can contribute to the spread of HIV-1 infection and give rise to cells which have stably integrated HIV-1 provirus.     

Validation of the morphologic end point of necrosis in rat hepatocytes subjected to oxyradical damage.

We have used phase-contrast microscopy to determine a necrotic end point of the order of minutes in primary hepatocytes exposed to oxyradicals generated with xanthine oxidase plus hypoxanthine. This study examines whether the morphologic end point thus determined agrees with other criteria of cell necrosis. When 95-100% of the cells were shown to be necrotic by our morphologic assay, transmission electron microscopy confirmed definitive subcellular evidence of cell death, trypan blue exclusion revealed a 92% loss in the ability of cells to exclude the dye, and there was a 47% specific release of 51Cr (versus a 50% theoretical value). In contrast, the appearance of extracellular aspartate aminotransferase activity was relatively slow and did not corroborate the morphologic end point. In summary, we have validated the morphologic end point in our cell-based assay of oxyradical damage.