Processive replication of single-stranded DNA templates by the herpes simplex virus-induced DNA polymerase

The DNA polymerase encoded by herpes simplex virus 1 consists of a single polypeptide of Mr 136,000 that has both DNA polymerase and 3'----5' exonuclease activities; it lacks a 5'----3' exonuclease. The herpes polymerase is exceptionally slow in extending a synthetic DNA primer annealed to circular single-stranded DNA (turnover number approximately 0.25 nucleotide). Nevertheless, it is highly processive because of its extremely tight binding to a primer terminus (Kd less than 1 nM). The single-stranded DNA-binding protein from Escherichia coli greatly stimulates the rate (turnover number approximately 4.5 nucleotides) by facilitating the efficient binding to and extension of the DNA primers. Synchronous replication by the polymerase of primed single-stranded DNA circles coated with the single-stranded DNA-binding protein proceeds to the last nucleotide of available 5.4-kilobase template without dissociation, despite the 20-30 min required to replicate the circle. Upon completion of synthesis, the polymerase is slow in cycling to other primed single-stranded DNA circles. ATP (or dATP) is not required to initiate or sustain highly processive synthesis. The 3'----5' exonuclease associated with the herpes DNA polymerase binds a 3' terminus tightly (Km less than 50 nM) and is as sensitive as the polymerase activity to inhibition by phosphonoacetic acid (Ki approximately 4 microM), suggesting close communication between the polymerase and exonuclease sites.     

DNA polymerase-primase from embryos of Drosophila melanogaster. The DNA polymerase subunit

The DNA polymerase-primase from Drosophila melanogaster has been separated into its constituent polymerase and primase subunits by sedimentation in glycerol gradients containing 50% ethylene glycol. Both activities have been obtained in good yield. The properties of the 182-kDa polymerase subunit are similar to those of the intact four-subunit enzyme. However, there are three significant differences. (i) The polymerase activity of the 182-kDa subunit shows an increased thermolability; (ii) the pause sites during replication of singly primed, single-stranded circular DNA by the 182-kDa subunit are altered; and (iii) unlike the intact enzyme, the 182-kDa subunit is highly processive in the presence of the single-stranded DNA-binding protein of Escherichia coli.     

Fluorescent measurements of intracellular free calcium in isolated toad urinary bladder epithelial cells

Summary Sodium-calcium exchange has been suggested to play a pivotal role in the regulation of cytosolic free calcium (Ca _f ) by epithelial cells. Using isolated epithelial cells from the toad urinary bladder, Ca _f has been measured using the intracellular Casensitive fluorescent dyes Fura 2 and Quin. 2. Dye loading did not alter cell viability as assessed by measurements of ATP and ADP content or cell oxygen consumption. When basal Ca _f was examined over a wide range of cell dye content (from 0.04 to 180 nmol dye/mg protein) an inverse relationship was observed. At low dye content, Ca _f was 300–380 nM and, as dye content was increased, Ca _f progressively fell to 60 nM. Using low dye content cells, in which minimal alteration in Ca steady state would be expected, the role for plasma membrane Na–Ca exchange was examined using either medium sodium substitution or ouabain. While medium sodium substitution increased Ca _f , prolonged treatment with ouabain had no effect on Ca _f despite a clear increase in cell sodium content. The lack of effect of ouabain suggests that Na–Ca exchange-mediated Ca efflux plays a minimal role in the regulation of basal Ca _f . However, exchange-mediated Ca efflux may play a role in Ca _f regulation when cytosolic calcium is elevated.     

Endosymbiotic origin and codon bias of the nuclear gene for chloroplast glyceraldehyde-3-phosphate dehydrogenase from maize

Summary The nuclei of plant cells harbor genes for two types of glyceraldehyde-3-phosphate dehydrogenases (GAPDH) displaying a sequence divergence corresponding to the prokaryote/eukaryote separation. This strongly supports the endosymbiotic theory of chloroplast evolution and in particular the gene transfer hypothesis suggesting that the gene for the chloroplast enzyme, initially located in the genome of the endosymbiotic chloroplast progenitor, was transferred during the course of evolution into the nuclear genome of the endosymbiotic host. Codon usage in the gene for chloroplast GAPDH of maize is radically different from that employed by present-day chloroplasts and from that of the cytosolic (glycolytic) enzyme from the same cell. This reveals the presence of subcellular selective pressures which appear to be involved in the optimization of gene expression in the economically important graminaceous monocots.     

Species specificity of bacterial palindromic units

We described previously a family of dispersed palindromic sequences highly repeated in Escherichia coli and Salmonella typhimurium genomes. These sequences, called PU (palindromic units), are located outside structural genes. We report here observations suggesting that PU may have a role in bacterial speciation.     

Sterol Composition and Ecdysteroid Content of Eggs of the Root-knot Nematodes Meloidogyne incognita and M. arenaria

Free and esterified sterols of eggs of the root-knot nematodes Meloidogyne incognita races 2 and 3 and M. arenaria race 1 were isolated and identified by gas-liquid chromatography-mass spectrometry. The major sterols of eggs of each race were 24-ethylcholesterol (33.4-38.8% of total sterol), 24-ethylcholestanol (18.3-25.3%), 24-methylcholesterol (8.6-11.7%), 24-methylcholestanol (7.7-12.5%), and cholesterol (4.6-11.6%). Consequently, the major metabolic transformation performed by Meloidogyne females or eggs upon host sterols appeared to be saturation of the sterol nucleus. The free and esterified sterols of the same race did not differ appreciably, except for a slight enrichment of the steryl esters in cholesterol. Although the sterol composition of Meloidogyne eggs differed from that of other life stages of other genera of plant-parasitic nematodes, the three Meloidogyne races could not be distinguished from each other by their egg sterols. Ecdysteroids, compounds with hormonal function in insects, were not detected by radioimmunoassay in the Meloidogyne eggs either as free ecdysteroids or as polar conjugates.     

d-Aspartate in Human Brain

The presence of the biologically uncommon D-aspartic acid (D-aspartate) in human brain white matter has been previously reported. The earlier study has now been expanded to include D/L-aspartate ratios from 67 normal brains. The data show that the D-aspartate content increases rapidly from 1 year to approximately 35 years of age, levels off in middle age, and then appears to decrease somewhat. The D-aspartate content in gray matter remains at a consistently low level (half of that found in white matter) throughout the human life span. Within the limitations of current analytical methods, there was no detectable difference in D/L-aspartate ratios in white and gray matter of brains with Alzheimer's disease and several other pathologies when compared with brains of normal subjects. However, the presence of a significant D-aspartate level in white matter during the adult life span may lead to changes in protein configuration related to dysfunctions associated with the aging brain.     

Ultraviolet irradiances of various lamps used in animal husbandry

A variety of lamps are used in animal husbandry, but data with respect to ultraviolet irradiances from these lamps, which may be important for alleviating or preventing metabolic bone disease, are generally unavailable. This paper reports irradiances from representative lamps as well as transmission and reflective properties of various materials.     

Betaine Transport Imparts Osmotolerance on a Strain of Lactobacillus acidophilus

Unlike most Lactobacillus acidophilus strains, a specific strain, L. acidophilus IFO 3532, was found to grow in rich medium containing 1 M sodium acetate, KCl, or NaCl. This strain could also grow with up to 1.8 M NaCl or 3 M nonelectrolytes (fructose, xylose, or sorbitol) added. Thus, this strain was tolerant to osmotic pressures up to 2.8 osM. A search for an intracellular solute which conferred osmoprotection led to the identification of glycine betaine (betaine). Betaine was accumulated to high concentrations in cells growing in MRS medium supplemented with 1 M KCl or NaCl. Uptake of [¹⁴C]betaine by L. acidophilus 3532 cells suspended in buffer was stimulated by increasing the medium osmotic pressure with 1 M KCl or NaCl. The accumulated betaine was not metabolized further; transport was relatively specific for betaine and was dependent on an energy source. Other lactobacilli, more osmosensitive than strain 3532, including L. acidophilus strain E4356, L. bulgaricus 8144, and L. delbrueckii 9649, showed lower betaine transport rates in response to an osmotic challenge than L. acidophilus 3532. Experiments with chloramphenicol-treated L. acidophilus 3532 cells indicated that the transport system was not induced but appeared to be activated by an increase in osmotic pressure.