Double-strand break repair in yeast requires both leading and lagging strand DNA polymerases.
Mitotic double-strand break (DSB)-induced gene conversion at MAT in Saccharomyces cerevisiae was analyzed molecularly in mutant strains thermosensitive for essential replication factors. The processivity cofactors PCNA and RFC are essential even to synthesize as little as 30 nucleotides following strand invasion. Both PCNA-associated DNA polymerases delta and epsilon are important for gene conversion, though a temperature-sensitive Pol epsilon mutant is more severe than one in Pol delta. Surprisingly, mutants of lagging strand replication, DNA polymerase alpha (pol1-17), DNA primase (pri2-1), and Rad27p (rad27 delta) also greatly inhibit completion of DSB repair, even in G1-arrested cells. We propose a novel model for DSB-induced gene conversion in which a strand invasion creates a modified replication fork, involving leading and lagging strand synthesis from the donor template. Replication is terminated by capture of the second end of the DSB. (+info)
Gibberellic acid stabilises microtubules in maize suspension cells to cold and stimulates acetylation of alpha-tubulin.
Gibberellic acid is known to stabilise microtubules in plant organs against depolymerisation. We have now devised a simplified cell system for studying this. Pretreatment of a maize cell suspension with gibberellic acid for just 3 h stabilised protoplast microtubules against depolymerisation on ice. In other eukaryotes, acetylation of alpha-tubulin is known to correlate with microtubule stabilisation but this is not established in plants. By isolating the polymeric tubulin fraction from maize cytoskeletons and immunoblotting with the antibody 6-11B-1, we have demonstrated that gibberellic acid stimulates the acetylation of alpha-tubulin. This is the first demonstrated link between microtubule stabilisation and tubulin acetylation in higher plants. (+info)
Perturbation of mammalian cell division. III. The topography and kinetics of extrusion subdivision.
If mitotic-arrested, cold-stored HeLa cells are incubated at 37 degrees C a proportion of the population divides by an aberrant process which we have called subdivision by extrusion. This process has been studied by time-lapse photography and shown to differ from normal cleavage in several respects. The cell surface becomes more generally mobile and, instead of producing the precisely localized furrowing activity of cytokinesis, gives rise to multiple surface protrusions. These protrusions enlarge at the expense of the parent cell and develop into a cluster of small daughter cells (mini segregants). The surface structure of the cell, as seen by scanning electron microscopy, also changes; the microvilli characteristic of interphase, metaphase and cleaving HeLa cells are lost during extrusion and the cell surface becomes smooth. Extrusion activity is much more variable than division by cleavage in terms of both topography and kinetics, and in general takes longer to complete. Some cells in the cold-treated populations divide by mixtures of cleavage and extrusion or by cleavage alone. The relative numbers of cells dividing in different ways vary with the conditions of pretreatment and incubation of the mitotic cells. The greater the perturbation (e.g. longer cold storage), the greater the proportion of extruding rather than cleaving cells. Human diploid cells can also be induced to subdivide by extrusion. Possible mechanisms underlying the different types of division activity are discussed. (+info)
Low temperature and pressure stability of picornaviruses: implications for virus uncoating.
The family Picornaviridae includes several viruses of great economic and medical importance. Poliovirus replicates in the human digestive tract, causing disease that may range in severity from a mild infection to a fatal paralysis. The human rhinovirus is the most important etiologic agent of the common cold in adults and children. Foot-and-mouth disease virus (FMDV) causes one of the most economically important diseases in cattle. These viruses have in common a capsid structure composed of 60 copies of four different proteins, VP1 to VP4, and their 3D structures show similar general features. In this study we describe the differences in stability against high pressure and cold denaturation of these viruses. Both poliovirus and rhinovirus are stable to high pressure at room temperature, because pressures up to 2.4 kbar are not enough to promote viral disassembly and inactivation. Within the same pressure range, FMDV particles are dramatically affected by pressure, with a loss of infectivity of more than 4 log units observed. The dissociation of polio and rhino viruses can be observed only under pressure (2.4 kbar) at low temperatures in the presence of subdenaturing concentrations of urea (1-2 M). The pressure and low temperature data reveal clear differences in stability among the three picornaviruses, FMDV being the most sensitive, polio being the most resistant, and rhino having intermediate stability. Whereas rhino and poliovirus differ little in stability (less than 10 kcal/mol at 0 degrees C), the difference in free energy between these two viruses and FMDV was remarkable (more than 200 kcal/mol of particle). These differences are crucial to understanding the different factors that control the assembly and disassembly of the virus particles during their life cycle. The inactivation of these viruses by pressure (combined or not with low temperature) has potential as a method for producing vaccines. (+info)
CspI, the ninth member of the CspA family of Escherichia coli, is induced upon cold shock.
Escherichia coli contains the CspA family, consisting of nine proteins (CspA to CspI), in which CspA, CspB, and CspG have been shown to be cold shock inducible and CspD has been shown to be stationary-phase inducible. The cspI gene is located at 35.2 min on the E. coli chromosome map, and CspI shows 70, 70, and 79% identity to CspA, CspB, and CspG, respectively. Analyses of cspI-lacZ fusion constructs and the cspI mRNA revealed that cspI is cold shock inducible. The 5'-untranslated region of the cspI mRNA consists of 145 bases and causes a negative effect on cspI expression at 37 degrees C. The cspI mRNA was very unstable at 37 degrees C but was stabilized upon cold shock. Analyses of the CspI protein on two-dimensional gel electrophoresis revealed that CspI production is maximal at or below 15 degrees C. Taking these results together, E. coli possesses a total of four cold shock-inducible proteins in the CspA family. Interestingly, the optimal temperature ranges for their induction are different: CspA induction occurs over the broadest temperature range (30 to 10 degrees C), CspI induction occurs over the narrowest and lowest temperature range (15 to 10 degrees C), and CspB and CspG occurs at temperatures between the above extremes (20 to 10 degrees C). (+info)
A cold-active glucanase from the ruminal bacterium Fibrobacter succinogenes S85.
We previously characterized two endoglucanases, CelG and EGD, from the mesophilic ruminal anaerobe Fibrobacter succinogenes S85. Further comparative experiments have shown that CelG is a cold-active enzyme whose catalytic properties are superior to those of several other intensively studied cold-active enzymes. It has a lower temperature optimum, of 25 degrees C, and retains about 70% of its maximum activity at 0 degrees C, while EGD has a temperature optimum of 35 degrees C and retains only about 18% of its maximal activity at 0 degrees C. When assayed at 4 degrees C, CelG exhibits a 33-fold-higher kcat value and a 73-fold-higher physiological efficiency (kcat/Km) than EGD. CelG has a low thermal stability, as indicated by the effect of temperature on its activity and secondary structure. The presence of small amino acids around the putative catalytic residues may add to the flexibility of the enzyme, thereby increasing its activity at cold temperatures. Its activity is modulated by sodium chloride, with an increase of over 1.8-fold at an ionic strength of 0.03. Possible explanations for the presence of a cold-active enzyme in a mesophile are that cold-active enzymes are more broadly distributed than previously expected, that lateral transfer of the gene from a psychrophile occurred, or that F. succinogenes originated from the marine environment. (+info)
The role of the sympathetic nervous system in the regulation of leptin synthesis in C57BL/6 mice.
The objectives of this study were to determine whether leptin synthesis is regulated by the sympathetic nervous system and if so whether beta-adrenergic receptors mediate this effect. We show that sympathetic blockade by reserpine increases leptin mRNA levels in brown but not white adipose tissue, while acute cold-exposure decreases leptin expression 10-fold in brown adipose tissue and 2-fold in white adipose tissue. The cold-induced reduction in leptin mRNA can be prevented by a combination of propranolol and SR 59230A but not by either antagonist alone, indicating that beta3-adrenergic receptors and classical beta1/beta2-adrenergic receptors both mediate responses to sympathetic stimulation. Circulating leptin levels reflect synthesis in white adipose tissue but not in brown adipose tissue. (+info)
Differential regulation of uncoupling protein-1, -2 and -3 gene expression by sympathetic innervation in brown adipose tissue of thermoneutral or cold-exposed rats.
The control of uncoupling protein-1, -2 and -3 (UCP-1, UCP-2, UCP-3) mRNA levels by sympathetic innervation in rats was investigated by specific and sensitive RT-PCR assays. In rats reared at thermoneutrality (25 degrees C), unilateral surgical sympathetic denervation of interscapular brown adipose tissue (BAT) markedly reduced the UCP-1 mRNA level (-38%) as compared with the contralateral innervated BAT pad, but was without significant effect on UCP-2 and -3 mRNA levels. Cold exposure (7 days, 4 degrees C) markedly increased UCP-1 (+180%), UCP-2 (+115%) and UCP-3 (+195%) mRNA levels in interscapular BAT. Unilateral sympathetic denervation prevented the cold-induced rise in BAT UCP-1 and UCP-2 mRNAs, but not that in BAT UCP-3 mRNA. Results were confirmed by Northern blot analysis. These data indicate a differential endocrine control of UCP-1, UCP-2 and UCP-3 gene expression in rat BAT both at thermoneutrality and during prolonged cold exposure. (+info)