The first and fourth upstream open reading frames in GCN4 mRNA have similar initiation efficiencies but respond differently in translational control to change in length and sequence.

The third and fourth AUG codons in GCN4 mRNA efficiently repress translation of the GCN4-coding sequences under normal growth conditions. The first AUG codon is approximately 30-fold less inhibitory and is required under amino acid starvation conditions to override the repressing effects of AUG codons 3 and 4. lacZ fusions constructed to functional, elongated versions of the first and fourth upstream open reading frames (URFs) were used to show that AUG codons 1 and 4 function similarly as efficient translational start sites in vivo, raising the possibility that steps following initiation distinguish the regulatory properties of URFs 1 and 4. In accord with this idea, we observed different consequences of changing the length and termination site of URF1 versus changing those of URFs 3 and 4. The latter were lengthened considerably, with little or no effect on regulation. In fact, the function of URFs 3 and 4 was partially reconstituted with a completely heterologous URF. By contrast, certain mutations that lengthen URF1 impaired its positive regulatory function nearly as much as removing its AUG codon did. The same mutations also made URF1 a much more inhibitory element when it was present alone in the mRNA leader. These results strongly suggest that URFs 1 and 4 both function in regulation as translated coding sequences. To account for the phenotypes of the URF1 mutations, we suggest the most ribosomes normally translate URF1 and that the mutations reduce the number of ribosomes that are able to complete URF1 translation and resume scanning downstream. This effect would impair URF1 positive regulatory function if ribosomes must first translate URF1 in order to overcome the strong translational block at the 3'-proximal URFs. Because URF1-lacZ fusions were translated at the same rate under repressing and derepressing conditions, it appears that modulating initiation at URF1 is not the means that is used to restrict the regulatory consequences of URF1 translation to starvation conditions.     

Transmembrane orientation of the fibronectin receptor complex (integrin) demonstrated directly by a combination of immunocytochemical approaches

The avian 140-KD cell adhesion receptor or "integrin," a complex of three glycoproteins with molecular masses averaging 140 KD, interacts with extracellular fibronectin and forms a linkage complex that co-localizes with intracellular actin. To probe the molecular interactions involved in this linkage complex, we used monoclonal antibodies and a combination of immunolocalization approaches to determine whether any component was transmembrane. Immunoadsorption and immunoblotting experiments demonstrated that anti-120-KD Mabs recognized the band 3 component of integrin isolated from chicken embryo fibroblasts (CEF) by JG22E immunoaffinity chromatography, and they co-localize with anti-fibronectin and polyclonal anti-integrin at cell contact sites in double-labeling experiments. Immunofluorescence experiments involved comparisons of double-labeled intact cells or substrate-attached, ventral plasma membrane "rip-off" fragments, using anti-fibronectin and each of the anti-120-KD Mabs. The extracellular faces of living intact cells were strongly labeled by a majority (approximately 70%) of the anti-120-KD Mabs at fibronectin-membrane attachment sites. The remainder (approximately 30%) labeled intact cells weakly or not at all. However, although the anti-120-KD Mab ES186 did not stain living cells, it did demonstrate positive staining above substratum contact sites over entire isolated rip-off membranes. In contrast, Mabs directed against putative extracellular epitopes and anti-fibronectin antibodies did not label these sites at the center of rip-offs unless the membranes were detergent permeabilized. Proteolysis experiments suggested that the ES186 epitope was located at one end of the molecule, since removal of short fragments from integrin band 3 concomitantly removed or destroyed the ES186 epitope, whereas the extracellular epitopes still remained. These experiments directly demonstrate that integrin band 3 is a transmembrane polypeptide with at least one epitope recognized by anti-120-KD Mabs on the cytoplasmic side of the plasma membrane and at least one epitope on the extracellular cell surface.     

Regulation of chloroplast metabolism in leaves: Evidence that NADP-dependent glyceraldehydephosphate dehydrogenase,but not ferredoxin-NADP reductase,controls electron flow to phosphoglycerate in the dark-light transition

P₇₀₀ is rapidly, but only transiently photooxidized upon illuminating dark-adapted leaves. Initial oxidation is followed by a reductive phase even under far-red illumination which excites predominantly photosystem (PS) I. In this phase, oxidized P₇₀₀ is reduced by electrons coming from PSII. Charge separation in the reaction center of PSI is prevented by the unavailability of electron acceptors on the reducing side of PSI. It is subsequently made possible by the opening of an electron gate which is situated between PSI and the electron acceptor phosphoglycerate. Electron acceptors immediately available for reduction while the gate is closed corresponded to 10 nmol · (mg chlorophyll)^–1 electrons in geranium leaves, 16 nmol · (mg chlorophyll)^–1 in sunflower and 22 nmol · (mg chlorophyll)^–1 in oleander. Reduction of NADP during the initial phase of P₇₀₀ oxidation showed that the electron gate was not represented by ferredoxin-NADP reductase. Availability of ATP indicated that electron flow was not hindered by deactivation of the thylakoid ATP synthetase. It is concluded that NADP-dependent glyceraldehydephosphate dehydrogenase is completely deactivated in the dark and activated in the light. The rate of activation depends on the length of the preceding dark period. As chloroplasts contain both NAD- and NADP-dependent glyceraldehydephosphate dehydrogenases, deactivation of the NADP-dependent enzyme disconnects chloroplast NAD and NADP systems and prevents phosphoglycerate reduction in the dark at the expense of NADPH and ATP which are generated by glucose-6-phosphate oxidation and glycolytic starch breakdown, respectively.Abbreviations Chl chlorophyll - P₇₀₀ electron donor pigment in the reaction center of photosystem I Cooperation of the Institute of Botany of the University of Würzburg with the Institute of Astrophysics and Atmospheric Physics of the Estonian Academy of Sciences in Tartu was supported by the Deutsche Forschungsgemeinschaft and the Estonian Academy of Sciences. This work was performed within the Sonderforschungsbereich 251 of the University of Würzburg.     

Oscillations in photosynthesis are initiated and supported by imbalances in the supply of ATP and NADPH to the Calvin cycle

Oscillations in the rate of photosynthesis of sunflower (Helianthus annuus L.) leaves were induced by subjecting leaves, whose photosynthetic apparatus had been activated, to a sudden transition from darkness or low light to high-intensity illumination, or by transfering them in the light from air to an atmosphere containing saturating CO₂. It was found that at the first maximum, light-and CO₂-saturated photosynthesis can be much faster than steady-state photosynthesis. Both Q_A in the reaction center of PS II and P₇₀₀ in the reaction center of PS I of the chloroplast electron-transport chain were more oxidized during the maxima of photosynthesis than during the minima. Maxima of P₇₀₀ oxidation slightly preceded maxima in photosynthesis. During a transition from low to high irradiance, the assimilatory force F_A, which was calculated from ratios of dihydroxyacetone phosphate to phosphoglycerate under the assumption that the reactions catalyzed by NADP-dependent glyceraldehydephosphate dehydrogenase, phosphoglycerate kinase and triosephosphate isomerase are close to equilibrium, oscillated in parallel with photosynthesis. However, only one of its components, the calculated phosphorylation potential (ATP)/(ADP)(P_i), paralleled photosynthesis, whereas calculated NADPH/NADP ratios exhibited antiparallel behaviour. When photosynthetic oscillations were initiated by a transition from low to high CO₂, the assimilatory force F_A declined, was very low at the first minimum of photosynthesis and increased as photosynthesis rose to its second maximum. The observations indicate that the minima in photosynthesis are caused by lack of ATP. This leads to overreduction of the electron-transport chain which is indicated by the reduction of P₇₀₀. During photosynthetic oscillations the chloroplast thylakoid system is unable to adjust the supply of ATP and NADPH rapidly to demand at the stoichiometric relationship required by the carbonreduction cycle.Abbreviations PGA 3-phosphoglycerate - DHAP dihydroxyacetone phosphate - P₇₀₀ electron-donor pigment in the reaction enter of PS I - Q_A quinone acceptor in the reaction center of PS II This work received support from the Estonian Academy of Sciences, the Bavarian Ministry of Science and Art and the Sonderforschungsbereich 251 of the University of Würzburg. We are grateful for criticism by D.A. Walker, Robert Hill Institute, University of Sheffield, U.K. and by Mark Stitt, Institute of Botany, University of Heidelberg, FRG.     

Stereospecific microbial reduction of 4,5-dihydro-4-(4-methoxyphenyl)-6-(trifluoromethyl-1H-1)-benzazepin+ ++-2-o ne.

A key intermediate, (3R-cis)-1,3,4,5-tetrahydro-3-hydroxy-4-(4-methoxyphenyl)-6-(trifluorome thyl)- 2H-1-benzazepin-2-one (compound II or SQ32191), with high optical purity was made by the stereoselective microbial reduction of the parent ketone 1. Several strains of bacterial and yeast cultures were screened for the ability to catalyse the stereoselective reduction of 4,5-dihydro-4-(4-methoxyphenyl)-6-(trifluoromethyl)-1H-1-benzazepin++ +-2,3-dione [compound I or SQ32425]. Microorganisms from the genera Nocardia, Rhodococcus, Alkaligenes, Corynebacterium, Arthrobacter, Hansenula, and Candida reduced compound I to compound II with 60-70% conversion yield. In contrast, microorganisms from the genera Pseudomonas and Acinetobacter reduced compound I stereospecifically to (trans)-1,3,4,5-tetrahydro-3-hydroxy-4-(4-methoxyphenyl)-6-(trifluoromet hyl-2H- 1-benzazepin-2-one (compound III or SQ32408). Among various cultures evaluated, N. salmonicolor SC6310 effectively catalysed the transformation of compound I to compound II with 96% conversion yield at 1.5-2.0 gl-1 concentration. Compound II was isolated and identified by NMR analysis, mass spectrometry, and comparison to an authentic sample. Preparative scale fermentation process and transformation process were developed using cell suspensions of N. salmonicolor SC6310 to catalyse the transformation of compound I to compound II. The isolated compound II had a melting point of 222 degrees C (reference 221-223 degrees C), optical rotation of +130.4 (reference +128 degrees C), and optical purity of greater than 99.9% as analyzed by NMR and chiral HPLC.     

Dynamics of a hydrophobic peptide in membrane bilayers by solid-state nuclear magnetic resonance

Solid-state NMR studies of the dynamics of a synthetic hydrophobic peptide, tert-butyloxycarbonylleucylphenylalanine methyl ester (Boc-Leu-Phe-OMe), in phospholipid bilayers are described. Motionally averaged powder pattern line shapes from 2H- and 15N-labeled backbone and side-chain sites of the peptide in phospholipid bilayers demonstrate the presence of both overall and internal reorientations of substantial amplitude. The peptide motions are shown to be strongly influenced by the motions of the lipids.     

The role of erythropoietin in the production of principal erythrocyte proteins other than hemoglobin during terminal erythroid differentiation

Erythropoietin (EP) controls the terminal phase of differentiation in which proerythroblasts and their precursors, the colony forming units-erythroid (CFU-e), develop into erythrocytes. Biochemical studies of this hormone-directed terminal differentiation have been hindered by the lack of a homogeneous population of erythroid cells at the developmental stages of CFU-e and proerythroblasts that will synchronously differentiate in response to EP. Such a population of cells can be prepared from the spleens of mice with the acute erythroblastosis resulting from infection with anemia-inducing Friend virus (FVA). Using these FVA-infected erythroid cells, which were induced to differentiate with EP, four proteins other than hemoglobin that have key functions in mature erythrocytes were monitored during the 48-hour period of terminal differentiation. Synthesis of spectrin and membrane band 3 proteins were determined by immunoprecipitation and SDS-polyacrylamide gel electrophoresis; accumulation of the cytoskeletal protein band 4.1 was monitored by immunoblotting; carbonic anhydrase activity was measured electrometrically. Band 3 synthesis and band 4.1 accumulation could be detected only after exposure of the cells to EP. Spectrin synthesis was ongoing prior to culture with EP, but it did increase after exposure to the hormone. Carbonic anhydrase-specific activity changed very little throughout the terminal differentiation process. These results reveal at least three patterns of production of principal erythrocyte proteins during EP-mediated terminal differentiation of FVA-infected erythroid cells. Depending on the specific protein examined, de novo synthesis can be induced by EP, an ongoing production can be enhanced by EP, or the production of a protein can be completed at a developmental stage prior to EP-mediated differentiation in these cells.     

Phenylacetic acid in an anaerobic swine manure digester

Summary The presence of phenylacetic acid (PAA) in an anaerobic swine manure digester was determined by gas chromatography of the butyl ester and confirmed by mass spectroscopy. PAA concentration increased during start-up of a digester and with low carbon, high nitrogen loading. Unlike acetate, propionate and butyrate, the concentration of PAA varied little through the day in a stable digester loaded once per day. The laboratory scale digester was loaded at 4 g of swine manure solids/liter digester volume per day. The retention time and temperature were 15 days and 37°C. PAA is a microbial intermediate which is produced by one group of anaerobic bacteria and converted to methane by other members of the bacterial community in the digester. As such, it may be a useful indicator of the relative metabolic activity of the bacterial groups and thus of the overall stability of the anaerobic process.     

Phosphorylation of histone H1 through the cell cycle of Physarum polycephalum. 24 sites of phosphorylation at metaphase

H1 phosphorylation has been studied through the precise nuclear division cycle of Physarum polycephalum. The number of sites of phosphorylation of Physarum H1 is very much larger than the number of sites reported for mammalian H1 molecules which is consistent with the larger molecular weight of Physarum H1. At metaphase all of the Physarum H1 molecules contain 20-24 phosphates. Immediately following metaphase, these metaphase-phosphorylated H1 molecules undergo rapid dephosphorylation to give an intermediate S phase set of phosphorylated H1 molecules containing 9-16 phosphates. Progressing into S phase newly synthesized H1 is phosphorylated and eventually merges with the old dephosphorylated H1 to give a ladder of bands 1-20. By the end of S phase or early G2 phase, there is a ladder of bands 1-16 all of which undergo phosphate turnover. Further into G2 phase the bands move to higher states of phosphorylation, and by prophase all of the H1 molecules contain 15-24 phosphates which increases to 20-24 phosphates at metaphase. These results support the proposals that H1 phosphorylation is an important factor in the process of chromosome condensation through G2 phase, prophase to metaphase.