Zygosaccharomyces lentus sp. nov., a new member of the yeast genus Zygosaccharomyces Barker.
(17/37801)
Unusual growth characteristics of a spoilage yeast, originally isolated from spoiled whole-orange drink and previously identified as Zygosaccharomyces bailii, prompted careful re-examination of its taxonomic position. Small-subunit rRNA gene sequences were determined for this strain and for four other strains also originally described as Z. bailii but which, in contrast to other strains of this species, grew poorly or not at all under aerobic conditions with agitation, failed to grow in the presence of 1% acetic acid and failed to grow at 30 degrees C. Comparative sequence analysis revealed that these strains represented a phylogenetically distinct taxon closely related to, but distinct from, Z. bailii and Zygosaccharomyces bisporus. Furthermore, sequence analysis of the internal transcribed spacer (ITS) region showed that, while all five strains had identical ITS2 sequences, they could be subdivided into two groups based on ITS1 sequences. Despite such minor inter-strain sequence variation, these yeasts could readily be distinguished from all other currently described Zygosaccharomyces species by using ITS sequences. On the basis of the phylogenetic results presented, a new species comprising the five strains, Zygosaccharomyces lentus sp. nov., is described and supporting physiological data are discussed, including a demonstration that growth of this species is particularly sensitive to the presence of oxygen. The type strain of Z. lentus is NCYC D2627T. (+info)
Heat shock protein 70 (Hsp70) protects postimplantation murine embryos from the embryolethal effects of hyperthermia.
(18/37801)
Previous work has shown that there is a positive correlation between the induction of Hsp70 and its transient nuclear localization and the acquisition and loss of induced thermotolerance in postimplantation rat embryos. To determine whether Hsp70 is sufficient to induce thermotolerance in postimplantation mammalian embryos, we used a transgenic mouse in which the normally strictly inducible Hsp70 is constitutively expressed in the embryo under the control of a beta-actin promoter. Day 8.0 mouse embryos heterozygous for the Hsp70 transgene were not protected from the embryotoxic effects of hyperthermia (43 degrees C); however, homozygous embryos, expressing approximately twice as much Hsp70 as heterozygous embryos, were partially protected (increased embryo viability) from the embryolethal effects of hyperthermia. Although the viability of transgenic embryos was significantly increased compared with that of nontransgenic embryos, this protection did not extend to embryo growth and development. To determine whether the failure to achieve a more robust protection was related to the expression of insufficient Hsp70 in transgenic embryos, we undertook experiments to determine whether the level of Hsp70 correlated with the level of thermotolerance induced by various lengths of a 41 degrees C heat shock. A 41 degrees C, 5-minute heat shock failed to induce Hsp70 or thermotolerance, a 41 degrees C, 15-minute heat shock induced Hsp70 and a significant level of thermotolerance, while a 41 degrees C, 60-minute heat shock induced an even higher level of Hsp70 as well as a higher level of thermotolerance. Quantitation of Hsp70 levels indicated that thermotolerance was associated with levels of Hsp70 of 820 pg/microg embryo protein or greater. Subsequent quantitation of the amount of Hsp70 expressed in homozygous transgenic embryos indicated a level of 577 pg/microg embryo protein, that is, a level below that associated with induced thermotolerance. Overall, results presented indicate that Hsp70 does play a direct role in the induction of thermotolerance in postimplantation mouse embryos; however, the level of thermotolerance is dependent on the level of Hsp70 expressed. (+info)
Long-term effects of prior heat shock on neuronal potassium currents recorded in a novel insect ganglion slice preparation.
(19/37801)
Brief exposure to high temperatures (heat shock) induces long-lasting adaptive changes in the molecular biology of protein interactions and behavior of poikilotherms. However, little is known about heat shock effects on neuronal properties. To investigate how heat shock affects neuronal properties we developed an insect ganglion slice from locusts. The functional integrity of neuronal circuits in slices was demonstrated by recordings from rhythmically active respiratory neurons and by the ability to induce rhythmic population activity with octopamine. Under these "functional" in vitro conditions we recorded outward potassium currents from neurons of the ventral midline of the A1 metathoracic neuromere. In control neurons, voltage steps to 40 mV from a holding potential of -60 mV evoked in control neurons potassium currents with a peak current of 10.0 +/- 2.5 nA and a large steady state current of 8.5 +/- 2.6 nA, which was still activated from a holding potential of -40 mV. After heat shock most of the outward current inactivated rapidly (peak amplitude: 8.4 +/- 2.4 nA; steady state: 3.6 +/- 2.0 nA). This current was inactivated at a holding potential of -40 mV. The response to temperature changes was also significantly different. After changing the temperature from 38 to 42 degrees C the amplitude of the peak and steady-state current was significantly lower in neurons obtained from heat-shocked animals than those obtained from controls. Our study indicates that not only heat shock can alter neuronal properties, but also that it is possible to investigate ion currents in insect ganglion slices. (+info)
A glial-neuronal signaling pathway revealed by mutations in a neurexin-related protein.
(20/37801)
In the nervous system, glial cells greatly outnumber neurons but the full extent of their role in determining neural activity remains unknown. Here the axotactin (axo) gene of Drosophila was shown to encode a member of the neurexin protein superfamily secreted by glia and subsequently localized to axonal tracts. Null mutations of axo caused temperature-sensitive paralysis and a corresponding blockade of axonal conduction. Thus, the AXO protein appears to be a component of a glial-neuronal signaling mechanism that helps to determine the membrane electrical properties of target axons. (+info)
Channeling of carbamoyl phosphate to the pyrimidine and arginine biosynthetic pathways in the deep sea hyperthermophilic archaeon Pyrococcus abyssi.
(21/37801)
The kinetics of the coupled reactions between carbamoyl-phosphate synthetase (CPSase) and both aspartate transcarbamoylase (ATCase) and ornithine transcarbamoylase (OTCase) from the deep sea hyperthermophilic archaeon Pyrococcus abyssi demonstrate the existence of carbamoyl phosphate channeling in both the pyrimidine and arginine biosynthetic pathways. Isotopic dilution experiments and coupled reaction kinetics analyzed within the context of the formalism proposed by Ovadi et al. (Ovadi, J., Tompa, P., Vertessy, B., Orosz, F., Keleti, T., and Welch, G. R. (1989) Biochem. J. 257, 187-190) are consistent with a partial channeling of the intermediate at 37 degrees C, but channeling efficiency increases dramatically at elevated temperatures. There is no preferential partitioning of carbamoyl phosphate between the arginine and pyrimidine biosynthetic pathways. Gel filtration chromatography at high and low temperature and in the presence and absence of substrates did not reveal stable complexes between P. abyssi CPSase and either ATCase or OTCase. Thus, channeling must occur during the dynamic association of coupled enzymes pairs. The interaction of CPSase-ATCase was further demonstrated by the unexpectedly weak inhibition of the coupled reaction by the bisubstrate analog, N-(phosphonacetyl)-L-aspartate (PALA). The anomalous effect of PALA suggests that, in the coupled reaction, the effective concentration of carbamoyl phosphate in the vicinity of the ATCase active site is 96-fold higher than the concentration in the bulk phase. Channeling probably plays an essential role in protecting this very unstable intermediate of metabolic pathways performing at extreme temperatures. (+info)
The yeast dynamin-like protein, Mgm1p, functions on the mitochondrial outer membrane to mediate mitochondrial inheritance.
(22/37801)
The mdm17 mutation causes temperature-dependent defects in mitochondrial inheritance, mitochondrial morphology, and the maintenance of mitochondrial DNA in the yeast Saccharomyces cerevisiae. Defects in mitochondrial transmission to daughter buds and changes in mitochondrial morphology were apparent within 30 min after shifting cells to 37 degrees C, while loss of the mitochondrial genome occurred after 4-24 h at the elevated temperature. The mdm17 lesion mapped to MGM1, a gene encoding a dynamin-like GTPase previously implicated in mitochondrial genome maintenance, and the cloned MGM1 gene complements all of the mdm17 mutant phenotypes. Cells with an mgm1-null mutation displayed aberrant mitochondrial inheritance and morphology. A version of mgm1 mutated in a conserved residue in the putative GTP-binding site was unable to complement any of the mutant defects. It also caused aberrant mitochondrial distribution and morphology when expressed at high levels in cells that also contained a wild-type copy of the gene. Mgm1p was localized to the mitochondrial outer membrane and fractionated as a component of a high molecular weight complex. These results indicate that Mgm1p is a mitochondrial inheritance and morphology component that functions on the mitochondrial surface. (+info)
All 16 centromere DNAs from Saccharomyces cerevisiae show DNA curvature.
(23/37801)
All 16 centromere DNA regions of Saccharomyces cerevisiae including 90 bp framing sequences on either side were cloned. These 300 bp long centromere regions were analysed by native polyacrylamide gel electrophoresis and found to display a reduced mobility indicative of DNA curvature. The degree of curvature is centromere dependent. The experimental data were confirmed by computer analysis of the 3-dimensional structure of the CEN DNAs. Altogether these data provide further evidence for a model for budding yeast centromeres in which CEN DNA structure could be important for the assembly, activity and/or regulation of the centromere protein-DNA complex. (+info)
Biophysical characterization of a designed TMV coat protein mutant, R46G, that elicits a moderate hypersensitivity response in Nicotiana sylvestris.
(24/37801)
The hypersensitivity resistance response directed by the N' gene in Nicotiana sylvestris is elicited by the tobacco mosaic virus (TMV) coat protein R46G, but not by the U1 wild-type TMV coat protein. In this study, the structural and hydrodynamic properties of R46G and wild-type coat proteins were compared for variations that may explain N' gene elicitation. Circular dichroism spectroscopy reveals no significant secondary or tertiary structural differences between the elicitor and nonelicitor coat proteins. Analytical ultracentrifugation studies, however, do show different concentration dependencies of the weight average sedimentation coefficients at 4 degrees C. Viral reconstitution kinetics at 20 degrees C were used to determine viral assembly rates and as an initial assay of the rate of 20S formation, the obligate species for viral reconstitution. These kinetic results reveal a decreased lag time for reconstitution performed with R46G that initially lack the 20S aggregate. However, experiments performed with 20S initially present reveal no detectable differences indicating that the mechanism of viral assembly is similar for the two coat protein species. Therefore, an increased rate of 20S formation from R46G subunits may explain the differences in the viral reconstitution lag times. The inferred increase in the rate of 20S formation is verified by direct measurement of the 20S boundary as a function of time at 20 degrees C using velocity sedimentation analysis. These results are consistent with the interpretation that there may be an altered size distribution and/or lifetime of the small coat protein aggregates in elicitors that allows N. sylvestris to recognize the invading virus. (+info)