Genbank accession #: AF 124983

Plant Molecular Biology -     

GRASP55, a second mammalian GRASP protein involved in the stacking of Golgi cisternae in a cell-free system.

We have identified a 55 kDa protein, named GRASP55 (Golgi reassembly stacking protein of 55 kDa), as a component of the Golgi stacking machinery. GRASP55 is homologous to GRASP65, an N-ethylmaleimide-sensitive membrane protein required for the stacking of Golgi cisternae in a cell-free system. GRASP65 exists in a complex with the vesicle docking protein receptor GM130 to which it binds directly, and the membrane tethering protein p115, which also functions in the stacking of Golgi cisternae. GRASP55 binding to GM130, could not be detected using biochemical methods, although a weak interaction was detected with the yeast two-hybrid system. Cryo-electron microscopy revealed that GRASP65, like GM130, is present on the cis-Golgi, while GRASP55 is on the medial-Golgi. Recombinant GRASP55 and antibodies to the protein block the stacking of Golgi cisternae, which is similar to the observations made for GRASP65. These results demonstrate that GRASP55 and GRASP65 function in the stacking of Golgi cisternae.     

Rapid Consumption of Low Concentrations of Methyl Bromide by Soil Bacteria

A dynamic dilution system for producing low mixing ratios of methyl bromide (MeBr) and a sensitive analytical technique were used to study the uptake of MeBr by various soils. MeBr was removed within minutes from vials incubated with soils and ~10 parts per billion by volume of MeBr. Killed controls did not consume MeBr, and a mixture of the broad-spectrum antibiotics chloramphenicol and tetracycline inhibited MeBr uptake by 98%, indicating that all of the uptake of MeBr was biological and by bacteria. Temperature optima for MeBr uptake suggested a biological sink, yet soil moisture and temperature optima varied for different soils, implying that MeBr consumption activity by soil bacteria is diverse. The eucaryotic antibiotic cycloheximide had no effect on MeBr uptake, indicating that soil fungi were not involved in MeBr removal. MeBr consumption did not occur anaerobically. A dynamic flowthrough vial system was used to incubate soils at MeBr mixing ratios as low as those found in the remote atmosphere (5 to 15 parts per trillion by volume [pptv]). Soils consumed MeBr at all mixing ratios tested. Temperate forest and grassy lawn soils consumed MeBr most rapidly (rate constant [k] = 0.5 min⁻¹), yet sandy temperate, boreal, and tropical forest soils also readily consumed MeBr. Amendments of CH₄ up to 5% had no effect on MeBr uptake even at CH₄:MeBr ratios of 10⁷, and depth profiles of MeBr and CH₄ consumption exhibited very different vertical rate optima, suggesting that methanotrophic bacteria, like those presently in culture, do not utilize MeBr when it is at atmospheric mixing ratios. Data acquired with gas flux chambers in the field demonstrated the very rapid in situ consumption of MeBr by soils. Uptake of MeBr at mixing ratios found in the remote atmosphere occurs via aerobic bacterial activity, displays first-order kinetics at mixing ratios from 5 pptv to ~1 part per million per volume, and is rapid enough to account for 25% of the global annual loss of atmospheric MeBr.     

Reductive inactivation of soybean lipoxygenase 1 by catechols: a possible mechanism for regulation of lipoxygenase activity

Nordihydroguaiaretic acid (NDGA), one of the most efficient inhibitors of lipoxygenases, is shown, by electron paramagnetic resonance, circular dichroism, and fluorescence studies, to reduce the catalytically active ferric soybean lipoxygenase 1 (Eox) to the inactive ferrous form (Ered). In decreasing order of reactivity, the following also reduce Eox: catechol greater than hydroquinone greater than 2,6-di-tert-butyl-4-methylphenol greater than esculetin greater than caffeic acid approximately equal to alpha-tocopherol greater than norepinephrine greater than dithiothreitol. The reduction of Eox by NDGA (kappa = 8.1 X 10(6) M-1 S-1, pH 9.0, 25 degrees C) is almost as fast as the Eox-catalyzed conversion of linoleate (LH) to 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoate (LOOH) and the oxidation of Ered by LOOH to give Eox. Thus, NDGA can efficiently inhibit the Eox-catalyzed conversion of LH to LOOH by reducing Eox to the inactive Ered, thereby diminishing the turnover rate. Lipoxygenase catalyzes the oxidation of NDGA by LOOH at a rate that is consistent with the independently determined rate constant for the reduction of Eox by NDGA. All four reducing equivalents from the two catechol groups in NDGA can be utilized in the reduction of Eox, leading to the consumption of 4 mol of LOOH/mol of NDGA initially present. Because the catalytically inactive Ered is oxidized by fatty acid hydroperoxides (e.g., LOOH) to give the active Eox, reducing agents such as NDGA are most effective as lipoxygenase inhibitors at low hydroperoxide concentrations. Our results suggest that in vivo, where lipid hydroperoxides are maintained at low steady-state levels, reduction of lipoxygenase from the ferric to ferrous state may be important in the regulation of lipoxygenase activity and hence leukotriene biosynthesis.     

Tempo and mode of concerted evolution in the L1 repeat family of mice

A 300-bp DNA sequence has been determined for 30 (10 from each of three species of mice) random isolates of a subset of the long interspersed repeat family L1. From these data we conclude that members of the L1 family are evolving in concert at the DNA sequence level in Mus domesticus, Mus caroli, and Mus platythrix. The mechanism responsible for this phenomenon may be either duplicative transposition, gene conversion, or a combination of the two. The amount of intraspecies divergence averages 4.4%, although between species base substitutions accumulate at the rate of approximately 0.85%/Myr to a maximum divergence of 9.1% between M. platythrix and both M. domesticus and M. caroli. Parsimony analysis reveals that the M. platythrix L1 family has evolved into a distinct clade in the 10-12 Myr since M. platythrix last shared a common ancestor with M. domesticus and M. caroli. The parsimony tree also provides a means to derive the average half-life of L1 sequences in the genome. The rates of gain and loss of individual copies of L1 were estimated to be approximately equal, such that approximately one-half of them turn over every 3.3 Myr.