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     

Computer controlled fed-batch fermentation of the methylotroph Pseudomonas AM1

Summary A simple on-line computer control strategy based on dissolved oxygen level readings has been developed to control methanol addition during a fermentation of the methylotroph Pseudomonas AM1. This strategy has led to significant and reproducible improvements in the performance of the fermentation.     

Direct freezing of cattle embryos after partial dehydration at room temperature

A new and simple method for freezing of bovine morulae and blastocysts was developed. Embryos were predehydrated at room temperature, frozen at -30 degrees C (cooling rate = 12 degrees C/min), and plunged into liquid nitrogen. This method was compared in vitro and in vivo to the slow freezing method (0.3 degrees C/min to -30 degrees C). Predehydration of the embryos in 1.5M glycerol was achieved by sucrose solution that makes the cells osmotically shrink. After the predehydrated morulae and blastocysts were frozen and thawed, 6 .4% (33 52 ) were developed in vitro for 48h and 44.2% (23 52 ) were hatched. Development obtained with slowly frozen embryos were 70.8% (17 24 ) and 58.3% (14 24 ) respectively. After transfer to recipient heifers, 33.3% (7 21 ) of the embryos frozen according this new method developed normally into viable foetuses or calves. This was the case for 48.5% (16 33 ) of the slowly frozen embryos.     

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.     

Mid51/Fis1 mitochondrial oligomerization complex drives lysosomal untethering and network dynamics

Lysosomes are highly dynamic organelles implicated in multiple diseases. Using live super-resolution microscopy, we found that lysosomal tethering events rarely undergo lysosomal fusion, but rather untether over time to reorganize the lysosomal network. Inter-lysosomal untethering events are driven by a mitochondrial Mid51/Fis1 complex that undergoes coupled oligomerization on the outer mitochondrial membrane. Importantly, Fis1 oligomerization mediates TBC1D15 (Rab7-GAP) mitochondrial recruitment to drive inter-lysosomal untethering via Rab7 GTP hydrolysis. Moreover, inhibiting Fis1 oligomerization by either mutant Fis1 or a Mid51 oligomerization mutant potentially associated with Parkinson’s disease prevents lysosomal untethering events, resulting in misregulated lysosomal network dynamics. In contrast, dominant optic atrophy–linked mutant Mid51, which does not inhibit Mid51/Fis1 coupled oligomerization, does not disrupt downstream lysosomal dynamics. As Fis1 conversely also regulates Mid51 oligomerization, our work further highlights an oligomeric Mid51/Fis1 mitochondrial complex that mechanistically couples together both Drp1 and Rab7 GTP hydrolysis machinery at mitochondria–lysosome contact sites. These findings have significant implications for organelle networks in cellular homeostasis and human disease.     

A simplified method for the reconstruction of fully competent mouse zygotes from adult somatic donor nuclei

Until now, full-term development of mouse embryos reconstructed from somatic nuclei has been convincingly achieved only when a piezoimpact pipette drive unit is used for the injection of a donor nucleus into an enucleated recipient oocyte. Here we describe a simplified method for mouse cloning that requires neither electrofusion nor a piezo device. Efficient rates of enucleation can be achieved without staining the chromosomes of the recipient oocyte and high survival rates are obtained after direct injection of the donor nucleus. Although a low proportion of reconstructed embryos could implant after their transfer into the oviducts of foster mothers (less than 5%), we show that at least some of them can develop into normal young.     

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.     

Characterization of backcross generations obtained under field conditions from oilseed rape-wild radish F1 interspecific hybrids: an assessment of transgene dispersal

Gene flow from glufosinate-resistant transgenic oilseed rape to wild radish was studied over two backcross generations. Under field conditions,?seed production from oilseed rape-wild radish F₁ hybrids due to pollination by wild radish was always low: on average 0.12 and 0.78 seeds per 100 flowers and per plant, respectively. The cytogenetics of the resulting «BC₁» plants can be explained in the main by three different genomic constitutions: either ACRrRr, 2n=37, ACRr, 2n=28 (the same chromosome number as the mother plant), or by the amphidiploid AACCRrRr, 2n=56. The probability of gene exchange through chromosome pairing was high only in plants with 2n=28 or 37 chromosomes. Due to the viability of unreduced or partially reduced female gametes, most of the «BC₁» plants (81.9%) were Basta resistant whereas the analysis of oilseed rape specific loci indicated that their transmission varied with the locus. In spite of low male fertility (8.7%), an improvement of the female fertility over the F₁ hybrids was observed with an average production of 1.4 and 11 seeds per 100 flowers and per plant, respectively. At the following «BC₂» generation, the bar gene transmission (57.2% of Basta-resistant plants) decreased as did the chromosome number, with a majority of plants having between 24 and 27 chromosomes, with 10.5% similar to wild radish (2n=18). The lower the chromosome number, the better the fertility of the «BC₂» plants. On average, 7.9 and 229.3 seeds per 100 flowers and per plant were produced. Gene-flow assessment is discussed based on these data.     

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.