Purification and characterization of two new high molecular weight forms of DNA polymerase delta

Two high molecular weight DNA polymerases, which we have designated delta I and delta II, have been purified from calf thymus tissue. Using Bio Rex-70, DEAE-Sephadex A-25, and DNA affinity resin chromatography followed by sucrose gradient sedimentation, we purified DNA polymerase delta I 1400-fold to a specific activity of 10 000 nmol of nucleotide incorporated h-1 mg-1, and DNA polymerase delta II was purified 4100-fold to a final specific activity of 30 000 nmol of nucleotide incorporated h-1 mg-1. The native molecular weights of DNA polymerase delta I and DNA polymerase delta II are 240 000 and 290 000, respectively. Both enzymes have similarities to other purified delta-polymerases previously reported in their ability to degrade single-stranded DNA in a 3' to 5' direction, affinity for an AMP-hexane-agarose matrix, high activity on poly(dA) X oligo(dT) template, and relative resistance to the polymerase alpha inhibitors N2-(p-n-butylphenyl)dATP and N2-(p-n-butylphenyl)dGTP. These two forms of DNA polymerase delta also share several common features with alpha-type DNA polymerases. Both calf DNA polymerase delta I and DNA polymerase delta II are similar to calf DNA polymerase alpha in molecular weight, are inhibited by the alpha-polymerase inhibitors N-ethylmaleimide and aphidicolin, contain an active DNA-dependent RNA polymerase or primase activity, display a similar extent of processive DNA synthesis, and are stimulated by millimolar concentrations of ATP. We propose that calf DNA polymerase delta I, which also has a template specificity essentially identical with that of calf DNA polymerase alpha, could be an exonuclease-containing form of a DNA replicative enzyme.     

Cell cycle associated changes in histamine release from rat basophilic leukemia cells separated by counterflow centrifugal elutriation

This study evaluated histamine release from cells at different stages of the cell cycle. Cells from the cloned rat basophilic leukemia subline (RBL-2H3) were fractionated by counterflow elutriation according to size and density. The smallest cells were predominantly in the G1 phase of the cell cycle. These cells contained the least histamine and after IgE-mediated triggering released the lowest fraction of their total histamine. In contrast cells in the S, G2, and M stages were larger, contained more histamine and released more of their histamine after activation. When G1 stage cells were recultured, there was an increase in cell size, in histamine content and histamine release. Therefore, there is heterogeneity in the capacity of cells for IgE-mediated triggering at different stages in the cell cycle, with optimal release from the more mature cells.     

Co-amplification of rRNA genes with CAD genes in N-(phosphonacetyl)-L-aspartate-resistant Syrian hamster cells.

The amplified CAD genes in N-(phosphonacetyl)-L-aspartate (PALA)-resistant Syrian hamster cells are located in an expanded chromosomal region emanating from the site of the wild-type gene at the tip of the short arm of chromosome B-9. The terminus of B-9 in PALA-sensitive cells contains a cluster of rRNA genes (i.e., a nucleolus organizer, rDNA). We have used a molecular clone containing sequences complementary to Syrian hamster 28S rRNA to investigate whether rDNA is coamplified with CAD genes in the PALA-resistant mutants. In situ hybridization of this probe to metaphase chromosomes demonstrates that rDNA and CAD genes do coamplify in two independently isolated PALA-resistant mutants. The tight linkage of CAD and rDNA genes was demonstrated by their coordinate translocation from B-9 to the end of the long arm of chromosome C-11 in one mutant. Blot hybridization studies substantiate the in situ hybridization results. Both types of analysis indicate that only one or two rDNA genes, on the average, are coamplified per CAD gene. The data are consistent with the model that unequal exchanges between rDNA genes mediate the amplification of CAD genes in the Syrian hamster mutants that were analyzed.     

Production of Alanine by Fusarium moniliforme

Fusarium moniliforme grown in a chemically defined medium in submerged culture accumulated amino acids extracellularly. Alanine and glutamic acid were present in greatest amounts, with traces of glycine, lysine, threonine, and valine detectable. Increasing the glucose and urea concentrations of the medium increased yields of alanine. Further increases in alanine production occurred with elevated levels of mineral salts in the medium, whereas the addition of a vitamin mixture proved to be inhibitory. Chemical changes resulting from the growth of F. moniliforme in the final fermentation medium disclosed maximal alanine production, mycelial weight, and glucose consumption after 72 hr of incubation at 28.5 C. Total soluble nitrogen, by contrast, was minimal at the same time period. The pH remained in the alkaline range throughout the fermentation.     

HIV-1 and its envelope glycoprotein down-regulate chemotactic ligand receptors and chemotactic function of peripheral blood monocytes

Peripheral blood monocytes from AIDS patients exhibit defective migratory responses to chemotactic stimuli in vitro and to inflammatory sites in vivo. In studies presented here, normal monocytes were infected with the HIV-1/HTLV-IIIBa-L isolate in vitro and evaluated for chemotactic responsiveness. Within 2 days after viral exposure, but before evidence of virus production in the monocytes, chemotactic activity was significantly impaired. Decreased chemotactic activity was associated with modulation of receptors for the chemotactic ligands, C5a and FMLP, on the monocyte cell surface. In addition to HIV-1, monocytes treated with purified HIV-1 envelope glycoprotein gp120 demonstrated a comparable modulation of chemotactic ligand receptors and migratory function. In addition, the HIV-1 or HIV-1 gp120-treated monocytes were induced to undergo differentiation as monitored by HLA-DR expression. Immunoprecipitation of the gp120 with a specific antibody reversed its effects on monocyte chemotaxis and HLA-DR expression. Taken together, these data indicate that the initial interaction of HIV-1 with the monocyte is not passive, but that the binding of HIV-1 and/or HIV-1 gp120 to the CD4R on monocytes transduces a signal leading to transient monocyte activation.     

Tyrosine phosphorylation of phospholipase C-II in vitro by the epidermal growth factor receptor

In a number of cell lines, epidermal growth factor (EGF) rapidly stimulates the breakdown of inositol phospholipids. Phosphatidylinositol-specific phospholipase C (PLC), therefore, plays an important role in this biological response to EGF, but the mechanism by which EGF-receptor complexes modulate the activation of PLC is not understood. We have previously suggested that tyrosine phosphorylation of PLC or an unknown PLC-associated protein by the EGF receptor is involved in the activation process (Wahl, M. I., Daniel, T. O., and Carpenter, G. (1988) Science 241, 968-970) and have recently shown by immunoprecipitation that the addition of EGF to 32P-labeled cells increases tyrosine and serine phosphorylation of PLC-II (Wahl, M. I., Nishibe, S., Suh, P.-G., Rhee, S. G., and Carpenter, G. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 1568-1572). In this communication we demonstrate that PLC-II (Mr = 145,000) purified from bovine brain can be phosphorylated in vitro in an EGF-dependent manner by the tyrosine kinase activity of the purified EGF receptor. While PLC-II is an efficient phosphorylation substrate for the purified EGF receptor, PLC-I is a poor substrate and PLC-III is not phosphorylated to any detectable extent. Though all three PLC isozymes possess typical tyrosine phosphorylation sequences, the EGF receptor is surprisingly selective in vitro for the phosphorylation of PLC-II. High performance liquid chromatography comparison of tryptic phosphotyrosyl peptides from PLC-II phosphorylated in vivo and in vitro indicated a similar pattern of multiple tyrosine phosphorylation sites. These findings show that the EGF receptor can directly phosphorylate PLC-II in an efficient and selective manner.     

Dual role of the CD44 molecule in T cell adhesion and activation

Studies of T cell adhesion and activation reveal two new functions of the CD44 molecule, a molecule now recognized to be identical to three molecules of functional interest: Pgp-1, Hermes, and extracellular matrix receptor type III (ECMRIII). By screening for mAb which inhibit T cell adhesion to E, we have identified a functionally unique CD44-specific mAb, NIH44-1, which partially inhibits T cell rosetting by binding to CD44 on the E. NIH44-1, which immunoprecipitates a protein of 85 to 110 kDa with broad tissue distribution, was determined to be specific for CD44 based on comparison of its tissue distribution with multiple CD44-specific reference mAb and sequential immunoprecipitation with such mAb. Anticipating a role for many adhesion molecules in signal transduction, we studied the effect of CD44 mAb on T cell activation and observed that CD44 mAb dramatically augments T cell proliferation induced by CD3- and CD2-receptor-mediated activation. The augmentation of the response to immobilized CD3 mAb by exhaustively monocyte-depleted T cells indicates that augmentation can be mediated by binding to the T cell. Thus, our studies demonstrate specific new roles for CD44 in T cell adhesion and activation. Furthermore, we suggest that: 1) CD44 has a role in adhesion of cells of multiple lineages; and 2) CD44 may participate in adhesion not (only) by functioning as an adhesion receptor but rather by serving as an anchorage site for other adhesion molecules.     

Intracellular Ca2+ measurement with Indo-1 in substrate-attached cells: advantages and special considerations

The dual emission, Ca2+ sensitive fluorescent dye, Indo-1, offers several potential advantages over its dual excitation analogue, Fura-2. Most notable among these advantages are increased speed of measurement using dual wavelength photometry and the absence of a requirement for special quartz optics. Despite these potential advantages, only a tiny fraction of the microscopic studies of intracellular free calcium ([Ca2+]i) on substrate-attached cells has employed Indo-1. Among the reasons for the infrequent use of Indo-1 are the fact that it exhibits somewhat different spectral properties in the cytosol than it does in extracellular buffers, and the notion that it is much more sensitive to photobleaching than Fura-2. We report here that under our experimental conditions, Indo-1 photobleaching is small and does not noticeably affect the measurement of free Ca2+, even after 30 minutes of continuous illumination. We also report a new method for creating in situ standard curves that is easy, reproducible, and yields values for [Ca2+]i that are identical to those obtained with Fura-2. In addition, we have found that Indo-1 is less subject than Fura-2 to compartmentalization within subcellular organelles. These results provide baseline data to take advantage of the significant improvement afforded by Indo-1 in the measurement of rapid [Ca2+]i responses and the avoidance of compartmentalization artifacts during experiments of long duration.