Alteration of the exonuclease activities of DNA polymerase I by captan

DNA polymerase I is a multifaceted enzyme with one polymerizing and two exonuclease activities. Captan was previously shown to be an inhibitor of this enzyme's polymerizing activity and this report measures the effects of captan on the two exonuclease activities. When the holoenzyme was tested, captan enhanced the degradation of poly(dA-dT), T7 DNA and, to a significantly lesser extent, heat-denatured DNA. However, when the effects of captan were tested as a function of substrate concentration, the stimulatory influence was measured only at high substrate concentrations. At low concentrations of DNA, captan was inhibitory. Inhibition and enhancement each showed an ED50 of the same value (approx. 100 microM). By assaying the two exonuclease activities separately it was shown that the differential effect on the holoenzyme by captan was the result of a combined inhibition of the 3'----5' exonuclease and enhancement of the 5'----3' exonuclease. Klenow fragment with poly(dA-dT) as substrate was used to assay for 3'----5' exonuclease activity. Captan inhibited this exonuclease and the inhibition could be prevented by the addition of greater concentrations of substrate. Holoenzyme and poly(rA)-poly(dT) were used to assay for 5'----3' exonucleolysis, which was enhanced at higher concentrations of substrate in the presence of captan.     

Primary structure of the chromosomal protein HMb from the archaebacteria Methanosarcina barkeri

The amino acid sequence of the protein HMb, a protein of 93 residues (Mr 10757) which represents the major acid-soluble component of the Methanosarcina barkeri nucleoprotein complex, has been established from automated sequence analysis of the protein and from structural data provided by peptides derived from cleavage of the protein at aspartic acid, arginine and methionine residues. The protein HMb is mainly characterized by a high amount of charged residues (15% of acidic residues and 26.8% of basic residues) which are distributed all along the polypeptide chain. The amino acid sequence of the protein HMb is not homologous to any eubacterial, archaebacterial or eukaryotic chromosomal proteins known up to now.     

An intermediate polymer in the assembly of clathrin baskets

Clathrin (8 S) is known to polymerize into two varieties of basket structures (150 S or 300 S) under the normal buffer conditions [100 mM 2-(N-morpholino)ethanesulfonic acid (Mes), pH 5.9-6.7] used for the isolation of coated vesicles. However, it is now observed that under very low salt conditions (2 mM Mes, pH 5.9), it forms a homogeneous species with a sedimentation coefficient of 27 S. Increasing the salt concentration to 50 mM Mes completely converts all the 27S species into 150S baskets. Sedimentation equilibrium data show that this 27S species has a molecular weight that is 6 times that of the clathrin protomer and is the result of highly cooperative reversible self-association of the 8S protomer. Light-scattering studies show that the stabilities of 27S species and baskets (150 S or 300 S) are comparable. Fluorescent labeling of sulfhydryl groups with N-(1-anilinonaphthalenyl)maleimide indicates that the conformation of clathrin in 27S species and baskets (150 S or 300 S) is similar. Trypsin digestion reveals that in the 27S species clathrin has a conformation differing from that in both the 8S species and baskets.     

Interleukin 1 activates phospholipase A2 in rabbit chondrocytes: a possible signal for IL 1 action

We examined the effects of Interleukin 1 (IL 1) on rabbit articular chondrocytes with particular emphasis on arachidonic acid metabolism in these cells. Articular chondrocytes were isolated from the knee joints of normal New Zealand white rabbits and were cultured in vitro until confluent. Addition of 5 U/ml of purified IL 1 to chondrocytes led to an early increase in cell-associated phospholipase A2 (PLA2; measured by hydrolysis of [14C]arachidonic acid-labeled E. coli). Within 1 hr after IL 1 addition, cell-associated PLA2 activity was increased by more than threefold relative to basal PLA2 activity, and further increases in cellular enzyme activity were observed up to 48 hr of IL 1 treatment. IL 1 stimulation also led to a time- and dose-related release of extracellular PLA2 and PGE2, but IL 1-induced PLA2 and PGE2 secretion occurred after the initial burst of intracellular PLA2 activity. Similar PLA2 and PGE2 responses were also observed when purified human IL 1 or IL 1-containing conditioned medium from LPS-stimulated human monocytes were used, but recombinant IL 2 or IL 3 were inactive. IL 1-induced chondrocyte PLA2 did not release radiolabeled free fatty acid from phosphatidylethanolamine labeled at the C-1 position with [14C]stearic acid, confirming the identity of this enzyme as PLA2. These data, therefore, provide the first direct evidence that IL 1 activates cellular PLA2, and we propose that PLA2 activation may be an early signal that initiates the inflammatory actions of IL 1.     

Tubulin pseudogenes as markers for hominoid divergence

Processed pseudogenes arise via unimolecular events that result in the integration of nonfunctional (and therefore non-selected) regions of DNA into the germ line. The sequence of such pseudogenes can be used as a novel form of evolutionary clock: the older a particular pseudogene, the more mutations it has acquired relative to the selectively constrained functional gene from which it was originally derived. We have used specific beta-tubulin gene probes to assay for the presence of fully sequenced processed pseudogenes in genomic DNA from various hominoid species. The data suggest that orangutan is more closely related to human, chimpanzee and gorilla than is generally believed.     

Differential turnover of phosphate groups on neurofilament subunits in mammalian neurons in vivo

The phosphorylation and dephosphorylation of specific proteins was demonstrated directly in the intact vertebrate nervous system in vivo. By exploiting the neurons' ability to segregate a select group of cytoskeletal proteins from most other phosphorylated constituents of the cell by axoplasmic transport, we were able to examine the dynamics of phosphate turnover on neurofilament proteins in mouse retinal ganglion cell neurons simultaneously labeled with [32P]orthophosphate and [3H]proline in vivo. Three [3H]proline-labeled neurofilament protein (NFP) subunits, designated H (160-200 kDa), M (135-145 kDa), and L (68-70 kDa), entered optic axons in a mole:mole ratio similar to that of isolated axonal neurofilaments, supporting the notion that newly synthesized NFPs are transported along axons as assembled neurofilaments. NFP subunits incorporated high levels of 32P before reaching axonal sites at the level of the optic nerve. As neurofilaments were transported along axons, however, many initially incorporated [32P]phosphate groups were removed. Loss of these phosphate groups occurred to a different extent on each subunit. A minimum of 50-60 and 35-40% of the labeled phosphate groups was removed in a 5-day period from the L and M subunits, respectively. By contrast, the H subunit exhibited relatively little or no phosphate turnover during the same period. Dephosphorylation of L in axons is accompanied by a decrease in its net state of phosphorylation; changes in the phosphorylation state of H and M, however, also reflect ongoing addition of phosphates to these polypeptides during axonal transport (Nixon, R.A., Lewis, S.E., and Marotta, C.A. (1986) J. Neurosci., in press). The possibility is raised that dynamic rearrangements of phosphate topography on NFPs represent a mechanism to coordinate interactions of neurofilaments with other proteins as these elements are transported and incorporated into the stationary cytoskeleton along retinal ganglion cell axons.     

Lateral regionalization and diffusion of a maturation-dependent antigen in the ram sperm plasma membrane

We have used a monoclonal antibody ESA 152 in fluorescence recovery after photobleaching (FPR) studies of a maturation-dependent surface antigen of ram sperm. The antibody is an immunoglobulin G secreted by a hybridoma derived from NS1 mouse myeloma cells. The ESA 152 antigen is not detectable in testicular sperm. It is localized on the surface of ejaculated sperm where it is present on all regions of the surface, but tends to be concentrated on the posterior region of the head. The ESA 152 antigen can be extracted by detergents or chloroform-methanol. The extracted antigen is sensitive to proteases and migrates with an apparent Mr approximately 30,000 in SDS-containing 10-20% polyacrylamide gradient gels. FPR measurements of ESA 152 lateral mobility in the membrane yield diffusion coefficients in the range 10(-9)-10(-8) cm2/s, values typical of lipids but observed for proteins only at the fluid dynamic limit where diffusion is controlled by lipid fluidity. Immobile fractions, typical of membrane proteins, are observed on all regions. When the antigen is stained by a fluoresceinated Fab fragment of the ESA 152 antibody, the diffusibility is highly regionalized, with particularly low, but rapid, recovery on the midpiece. Cross-linking of the antigen with the intact ESA 152 antibody induces a redistribution in which the antigen is excluded from the posterior head region. This cross-linking is accompanied by increases in ESA 152 diffusibility on both the anterior head and the midpiece.     

Thermotropic phase behavior of model membranes composed of phosphatidylcholines containing omega-cyclohexyl fatty acids. Differential scanning calorimetric and 31P NMR spectroscopic studies

The thermotropic phase behavior of aqueous dispersions of 10 phosphatidylcholines containing omega-cyclohexyl-substituted acyl chains was studied by differential scanning calorimetry and 31P nuclear magnetic resonance spectroscopy. The presence of the omega-cyclohexyl group has a profound effect on the thermotropic phase behavior of these compounds in a manner dependent on whether the fatty acyl chains have odd- or even-numbered linear carbon segments. The thermotropic phase behavior of the odd-numbered phosphatidylcholines is characterized by a single heating endotherm that was shown to be a superposition of at least two structural events by calorimetric cooling experiments. 31P NMR spectroscopy also showed that the single endotherm of the odd-chain compounds is the structural equivalent of a concomitant gel-gel and gel to liquid-crystalline phase transition. The calorimetric behavior of the even-numbered phosphatidylcholines is characterized by a complex array of gel-state phenomena, in addition to the chain-melting transition, in both the heating and cooling modes. The gel states of these even-numbered compounds are characterized by a relatively greater mobility of the phosphate head group as seen by 31P NMR spectroscopy. The differences between the odd-numbered and even-numbered compounds are reflected in a pronounced odd-even alternation in the characteristic transition temperatures and enthalpies and in differences in their responses to changes in the composition of the bulk aqueous phase. Moreover, both the odd-numbered and even-numbered omega-cyclohexylphosphatidylcholines exhibit significantly lower chain-melting transition temperatures and enthalpies than do linear saturated phosphatidylcholines of comparable chain length.(ABSTRACT TRUNCATED AT 250 WORDS)     

Choroideremia is linked to the restriction fragment length polymorphism DXYS1 at XQ13-21.

Choroideremia (McK30310), an X-linked hereditary retinal dystrophy, causes night-blindness, progressive peripheral visual field loss, and, ultimately, central blindness in affected males. The location of choroideremia on the X chromosome is unknown. We have used restriction fragment length polymorphisms from the X chromosome to determine the regional localization of choroideremia by linkage analysis in families with this disease. One such polymorphic locus, DXYS1, located on the long arm (Xq) within bands q13-q21, shows no recombination with choroideremia at lod = 5.78. Therefore, with 90% probability, choroideremia maps within 9 centiMorgans (cM) of DXYS1. Another polymorphic locus, DXS11, located within Xq24-q26, also shows no recombination with choroideremia, although at a smaller lod score of 1.54 (90% probability limit theta less than 30 cM). This linkage with DXS11, a marker that is distal to DXYS1, suggests that the locus for choroideremia is also distal to DXYS1 and lies between these two markers in the region Xq13-q24. These results provide regional mapping for the disease that may be useful for prenatal diagnosis and, perhaps ultimately, for isolating the gene locus for choroideremia.     

Deuterium oxide and temperature effects on the properties of endplate channels at the frog neuromuscular junction

The effects of deuterium oxide (D2O) and temperature on the properties of endplate channels were studied in voltage-clamped muscle fibers from the frog Rana pipiens. Studies were performed at temperatures of 8, 12, 16, and 20 degrees C. The single channel conductance (gamma) and mean channel lifetime (tau) were calculated from fluctuation analysis of the acetylcholine-induced end-plate currents. The reversal potential was determined by interpolation of the acetylcholine-induced current-voltage relation. The mean reversal potential was slightly more negative in D2O Ringer's (-7.9 +/- 0.1 mV [+/- SEM]) compared with H2O Ringer's (-5.2 +/- 0.6 mV, P less than 0.01). The single channel conductance was decreased in D2O. This decrease was greater than could be accounted for by the increased viscosity of D2O solutions, and the amount of the decrease was greater at higher temperatures. For example, gamma was 38.4 +/- 1.3 pS (+/- SEM) in H2O Ringer's and 25.7 +/- 1.0 pS in D2O Ringer's for a holding potential of -70 mV at 12 degrees C. The mean channel lifetime was significantly shorter in D2O, and the effect was greater at lower temperatures. There was not a strong effect of solvent on the temperature dependence of gamma. On the other hand, the temperature dependence of the reciprocal mean channel lifetime, alpha (where alpha = 1/tau), was strongly dependent upon the solvent. The single channel conductances showed no demonstrable voltage dependence over the range of -90 to -50 mV in both solvents. The reciprocal mean channel lifetime showed a voltage dependence, which could be described by the relation alpha = B exp(AV). The slope A was not strongly affected by either temperature or the solvent. On the other hand, the intercept B was a strong function of temperature and was weakly dependent upon the solvent, with most values greater in D2O. The D2O effects on alpha were what would be expected if they were due to the properties of D2O as a solvent (solvent isotope effects), while the D2O effects on gamma must also include the exchange of D for H in the vicinity of the selectivity filter (primary and/or secondary kinetic isotope effects).