Cis-Effects of Heterochromatin on Heterochromatic and Euchromatic Gene Activity in Drosophila Melanogaster

Chromosomal rearrangements that juxtapose heterochromatin and euchromatin can result in mosaic inactivation of heterochromatic and euchromatic genes. This phenomenon, position effect variegation (PEV), suggests that heterochromatic and euchromatic genes differ in their regulatory requirements. This report describes a novel method for mapping regions required for heterochromatic genes, and those that induce PEV of a euchromatic gene. P transposase mutagenesis was used to generate derivatives of a translocation that variegated for the light(+) (lt(+)) gene and carried the euchromatic white(+) (w(+)) gene on a transposon near the heterochromatin-euchromatin junction. Cytogenetic and genetic analyses of the derivatives showed that P mutagenesis resulted in deletions of several megabases of heterochromatin. Genetic and molecular studies showed that the derivatives shared a euchromatic breakpoint but differed in their heterochromatic breakpoint and their effects on seven heterochromatic genes and the w(+) gene. Heterochromatic genes differed in their response to deletions. The lt(+) gene was sensitive to the amount of heterochromatin at the breakpoint but the heterochromatic 40Fa gene was not. The severity of variegated w(+) phenotype did not depend on the amount of heterochromatin in cis, but varied with local heterochromatic environment. These data are relevant for considering mechanisms of PEV of both heterochromatic and euchromatic genes.     

Intragenomic distribution and stability of transposable elements in euchromatin and heterochromatin of drosophila melanogaster: non-LTR retrotransposon

The intragenomic location of the elements of the I, G, jockey, F, and Doc transposon families has been studied by the Southern blot analysis, in 12 laboratory Drosophila melanogaster stocks. Elements located in euchromatin, heterochromatin, and on the Y chromosome are identified, and their stability has been assessed by comparing the autoradiographs detected in different stocks and analysis of individual flies. Evidence is shown suggesting that preferential location in euchromatin or heterochromatin and the distribution within heterochromatin are distinctive of transposon families. Elements located in heterochromatin can be unstable. These results are discussed in the context of the relationship between transposable elements and the host genome. Received: 21 August 1996 / Accepted: 24 March 1997     

IN VITRO ALLELOPATHIC INHIBITION OF NITRIFICATION BY BALSAM POPLAR AND BALSAM FIR

A soil percolation apparatus was used to study the effect of plant extracts on nitrification. Foliar leachates, leaf extracts, and bud extracts of balsam fir (Abies balsamea) and balsam poplar (Populus balsamifera) were added to the ammonium percolation solution. Color tests and quantitative measurement of residual ammonium and formed nitrate were applied. Foliar leachates of balsam fir strongly inhibited nitrification; leachates of balsam poplar were slightly less inhibitory. Balsam fir needle and balsam poplar dormant bud extracts (5% w/v) completely prevented the oxidation of ammonium. The same estracts (2% w/v) still prevented the oxidation of ammonium to nitrate to a large extent. Once again balsam fir extracts inhibited the process more strongly than balsam poplar. No evidence was obtained for the presence of a specific inhibitor of nitrifier microorganisms.     

Cytogenetic Analysis of the Second Chromosome Heterochromatin of Drosophila Melanogaster

This paper reports the cytogenetic characterization of the second chromosome heterochromatin of Drosophila melanogaster. High resolution cytological analysis of a sample of translocations, inversions, deficiencies and free duplications involving the pericentric regions of the second chromosome was achieved by applying sequential Hoechst 33258 and N-chromosome banding techniques to larval neuroblast prometaphase chromosomes. Heterochromatic rearrangements were employed in a series of complementation assays and the genetic elements previously reported to be within or near the second chromosome heterochromatin were thus precisely assigned to specific heterochromatic bands. The results of this analysis reveal a nonhomogeneous distribution of loci along the second chromosome heterochromatin. The l(2)41Aa, l(2)41Ab, rolled (l(2)41Ac) and l(2)41Ad loci are located within the proximal heterochromatin of 2R, while the nine remaining loci in the left arm and two (l(2)41Ae and l(2)41Ah) in the right arm map to h35 and to h46, respectively, the most distal heterochromatic regions. In addition, a common feature of these loci revealed by the cytogenetic analysis is that they map to specific heterochromatic blocks but do not correspond to the blocks themselves, suggesting that they are not as large as the Y fertility factors or the Rsp locus. Mutations of the proximal most heterochromatic loci, l(2)41Aa and rolled, were also examined for their phenotypic effects. Extensive cell death during imaginal disc development was observed in individuals hemizygous for either the EMS 31 and rolled mutations, leading to a pattern of phenotypic defects of adult structures.     

Positive regulation in a eukaryote, a study of the uaY gene of Aspergillus nidulans

Summary In this publication we report the identification of a protein likely to be coded by uaY, a regulatory gene in the ascomycete Aspergillus nidulans. uaY is a positive control gene necessary for the expression of at least eight unlinked structural genes involved in purine uptake and degradation (Scazzocchio and Gorton 1977). The physiological effector of the uaY system is uric acid, while some of its thioanalogs serve as gratuitous inducers. Effector binding proteins were detected by binding to 2-thiouric acid after phosphocellulose column chromatography, or as uric acid binding fractions after DNA-cellulose column chromatography. Two binding peaks are present in mycelial extracts purified by either method. These are missing in a putative small deletion of the uaY gene. A leaky mutation, uaY 109 described in detail elsewhere (Scazzocchio et al. 1980) shows only one peak. The wild type peaks are eluted at 55 mM NaCl and at 720 mM NaCl while the peak present in uaY109 is eluted at 120 mM NaCl. This implies that at least one peak represents a protein coded by the uaY gene. The major peak was analysed by equilibrium dialysis experiments. These establish a K_diss.2×10^-7 and a minimum number of binding sites of 3×10^-14 moles/mg of soluble protein in a crude extract derived from protoplast lysis. An extract from a strain carrying the uaY207 deletion, purified blind, lacks any binding activity in the equilibrium dialysis cell.     

Rapid alteration of the phosphoproteome in the moss Physcomitrella patens after cytokinin treatment

Cytokinin hormones are crucial regulators of a large number of processes in plant development. Recently, significant progress has been made toward the elucidation of the molecular details of cytokinin that has led to a model for signal transduction involving a phosphorylation cascade. However, the current knowledge of cytokinin action remains largely unknown and does not explain the different roles of this hormone. To gain further insights into this aspect of cytokinin action and the inducible phosphorelay, we have produced the first large-scale map of a phosphoproteome in the moss Physcomitrella patens. Using a protocol that we recently published (Heintz, D.; et al. Electrophoresis 2004, 25, 1149-1159) that combines IMAC, MALDI-TOF-MS, and LC-MS/MS, a total of 172 phosphopeptide sequences were obtained by a peptide de novo sequencing strategy. Specific P. patens EST and raw genomic databases were interrogated, and protein homology searches resulted in the identification of 112 proteins that were then classified into functional categories. In addition, the temporal dynamics of the phosphoproteome in response to cytokinin stimulation was studied at 2, 4, 6, and 15 min after hormone addition. We identified 13 proteins that were not previously known targets of cytokinin action. Among the responsive proteins, some were involved in metabolism, and several proteins of unknown function were also identified. We have mapped the time course of their activation in response to cytokinin and discussed their hypothetical biological significance. Deciphering these early induced phosphorylation events has shown that the cytokinin effect can be rapid (few minutes), and the duration of this effect can be variable. Also phosphorylation events can be differentially regulated. Taken together our proteomic study provides an enriched look of the multistep phosphorelay system mediating cytokinin response and suggests the existence of a multidirectional interaction between cytokinin and numerous other pathways.     

Metabolic Stability of Hippocampal Slice Preparations During Prolonged Incubation

Abstract: Hippocampal slices were prepared under three conditions: (1) in medium containing glucose and oxygen at 4°C; (2) as in (1), but at 37°C; (3) in medium devoid of glucose and oxygen at 37°C. The rates of recovery to roughly steady-state levels and through 8 h of incubation were monitored for energy metabolite levels and related parameters. In vitro stable values are compared with in situ hippocampal levels. Regardless of the conditions under which slices were prepared, metabolite levels required up to 3 h to stabilize, and these levels were maintained or improved through 8 h of incubation. Further, the maximal concentrations of metabolites were independent of the conditions of slice preparation. Total adenylates and total creatine levels reached 55% of those in vivo. Lactate decreased from the decapitation-induced high levels, but stabilized at concentrations about twice those in rapidly frozen brain. Cyclic AMP and cyclic GMP exhibited peak levels at 30 min of incubation, and cyclic GMP remained elevated for 3 h. Although all three methods of slice preparation resulted in similar metabolite profiles on incubation, the initial decreases in high energy phosphates were delayed by chilling. Most striking, the slices prepared in the absence of glucose and oxygen exhibited much smaller orthodromic evoked potentials in the dentate gyrus. The presence of glucose and oxygen during preparation of the slices appears to be critical to the electrophysiological response of the tissue.     

Mn/Fe superoxide dismutase interaction fingerprints and prediction of oligomerization and metal cofactor from sequence

Fe- and Mn-containing superoxide dismutase (sod) enzymes are closely related and similar in both amino acid sequence and structure, but differ in their mode of oligomerization and in their specificity for the Fe or Mn cofactor. The goal of the present work is to identify and analyze the sequence and structure characteristics that ensure the cofactor specificities and the oligomerization modes. For that purpose, 374 sod sequences and 17 sod crystal structures were collected and aligned. These alignments were searched for residues and inter-residue interactions that are conserved within the whole sod family, or alternatively, that are specific to a given sod subfamily sharing common characteristics. This led us to define key residues and inter-residue interaction fingerprints in each subfamily. The comparison of these fingerprints allows, on a rational basis, the design of mutants likely to modulate the activity and/or specificity of the target sod, in good agreement with the available experimental results on known mutants. The key residues and interaction fingerprints are furthermore used to predict if a novel sequence corresponds to a sod enzyme, and if so, what type of sod it is. The predictions of this fingerprint method reach much higher scores and present much more discriminative power than the commonly used method that uses pairwise sequence comparisons.     

Evolutionary temperature compensation of carbon fixation in marine phytoplankton

The efficiency of carbon sequestration by the biological pump could decline in the coming decades because respiration tends to increase more with temperature than photosynthesis. Despite these differences in the short‐term temperature sensitivities of photosynthesis and respiration, it remains unknown whether the long‐term impacts of global warming on metabolic rates of phytoplankton can be modulated by evolutionary adaptation. We found that respiration was consistently more temperature dependent than photosynthesis across 18 diverse marine phytoplankton, resulting in universal declines in the rate of carbon fixation with short‐term increases in temperature. Long‐term experimental evolution under high temperature reversed the short‐term stimulation of metabolic rates, resulting in increased rates of carbon fixation. Our findings suggest that thermal adaptation may therefore have an ameliorating impact on the efficiency of phytoplankton as primary mediators of the biological carbon pump.