Genetic mapping of the N. crassa pho-5 gene

We have recently cloned and disrupted (by RIP) the N. crassa pho-5 gene which encodes a phosphate-repressible, high-affinity phosphate permease (Versaw 1995 Gene 153:135-139). RFLP analysis indicates that pho-5 maps to Linkage group IV, near pyr-1. We wished to determine the map position of pho-5 more accurately. pho-5 null mutants have no obvious phenotype. Therefore, we constructed parental mapping strains which also contained a null allele of pho-4, a previously isolated high-affinity phosphate permease encoding gene (Mann et al. 1988 Mol. Cell. Biol. 8:1376-1379). pho-5 mutants in a pho-4 background are easily scored by their failure to grow on high pH, low phosphate medium like that used to score nuc-1 and nuc-2 mutants (Metzenberg and Chia 1979 Genetics 93:625-643). Results of the genetic mapping are shown in Table 1. The gene order on Linkage group IV is pyr-1, pho-5, cot-1

Study of methane fuel for subsonic transport aircraft

The cost and performance were defined for commercial transport using liquid methane including its fuel system and the ground facility complex required for the processing and storage of methane. A cost and performance comparison was made with Jet A and hydrogen powered aircraft of the same payload and range capability. Extensive design work was done on cryogenic fuel tanks, insulation systems as well as the fuel system itself. Three candidate fuel tank locations were evaluated, i.e., fuselage tanks, wing tanks or external pylon tanks

Ion homeostasis in the Chloroplast

peer reviewedThe chloroplast is an organelle of high demand for macro- and micro-nutrient ions, which are required for the maintenance of the photosynthetic process. To avoid deficiency while preventing excess, homeostasis mechanisms must be tightly regulated. Here, we describe the needs for nutrient ions in the chloroplast and briefly highlight their functions in the chloroplastidial metabolism. We further discuss the impact of nutrient deficiency on chloroplasts and the acclimation mechanisms that evolved to preserve the photosynthetic apparatus. We finally present what is known about import and export mechanisms for these ions. Whenever possible, a comparison between cyanobacteria, algae and plants is provided to add an evolutionary perspective to the description of ion homeostasis mechanisms in photosynthesis

Characterization of PitA and PitB from Escherichia coli

Escherichia coli contains two major systems for transporting inorganic phosphate (P(i)). The low-affinity P(i) transporter (pitA) is expressed constitutively and is dependent on the proton motive force, while the high-affinity Pst system (pstSCAB) is induced at low external P(i) concentrations by the pho regulon and is an ABC transporter. We isolated a third putative P(i) transport gene, pitB, from E. coli K-12 and present evidence that pitB encodes a functional P(i) transporter that may be repressed at low P(i) levels by the pho regulon. While a pitB(+) cosmid clone allowed growth on medium containing 500 μM P(i), E. coli with wild-type genomic pitB (pitA ΔpstC345 double mutant) was unable to grow under these conditions, making it indistinguishable from a pitA pitB ΔpstC345 triple mutant. The mutation ΔpstC345 constitutively activates the pho regulon, which is normally induced by phosphate starvation. Removal of pho regulation by deleting the phoB-phoR operon allowed the pitB(+) pitA ΔpstC345 strain to utilize P(i), with P(i) uptake rates significantly higher than background levels. In addition, the apparent K(m) of PitB decreased with increased levels of protein expression, suggesting that there is also regulation of the PitB protein. Strain K-10 contains a nonfunctional pitA gene and lacks Pit activity when the Pst system is mutated. The pitA mutation was identified as a single base change, causing an aspartic acid to replace glycine 220. This mutation greatly decreased the amount of PitA protein present in cell membranes, indicating that the aspartic acid substitution disrupts protein structure

Phosphorylation of MafA Is Essential for Its Transcriptional and Biological Properties

We previously described the identification of quail MafA, a novel transcription factor of the Maf bZIP (basic region leucine zipper) family, expressed in the differentiating neuroretina (NR). In the present study, we provide the first evidence that MafA is phosphorylated and that its biological properties strongly rely upon phosphorylation of serines 14 and 65, two residues located in the transcriptional activating domain within a consensus for phosphorylation by mitogen-activated protein kinases and which are conserved among Maf proteins. These residues are phosphorylated by ERK2 but not by p38, JNK, and ERK5 in vitro. However, the contribution of the MEK/ERK pathway to MafA phosphorylation in vivo appears to be moderate, implicating another kinase. The integrity of serine 14 and serine 65 residues is required for transcriptional activity, since their mutation into alanine severely impairs MafA capacity to activate transcription. Furthermore, we show that the MafA S14A/S65A mutant displays reduced capacity to induce expression of QR1, an NR-specific target of Maf proteins. Likewise, the integrity of serines 14 and 65 is essential for the MafA ability to stimulate expression of crystallin genes in NR cells and to induce NR-to-lens transdifferentiation. Thus, the MafA capacity to induce differentiation programs is dependent on its phosphorylation