Alternative splicing of protein 4.1R exon 16: ordered excision of flanking introns ensures proper splice site choice.

Alternative splicing plays a major role in regulating tissue-specific expression of cytoskeletal protein 4.1R isoforms. In particular, expression of the protein's functionally critical spectrin-actin binding domain, essential for maintenance of red cell membrane mechanical properties, is governed by a developmentally regulated splicing switch involving alternative exon 16. Using a model 3-exon 4.1R pre-messenger RNA (pre-mRNA), we explored the sequence requirements for excision of the introns flanking exon 16. These studies revealed that splicing of this alternative exon occurs preferentially in an ordered fashion. The first step is excision of the downstream intron to join exons 16 and 17, followed by excision of the upstream intron. Constructs designed to test the converse pathway were spliced less efficiently and with less fidelity, in part due to activation of a cryptic 5' splice site in exon 16. This downstream-first model for ordered splicing is consistent with the hypothesis that regulated alternative splicing requires cooperation between multiple exonic and/or intronic regulatory elements whose spatial organization is critical for recruitment of appropriate splicing factors. Our results predict that exon 16 splicing is regulated at the first step-excision of the downstream intron-and that cells unable to catalyze this step will exhibit exon 16 skipping. In cells that include exon 16, adherence to an ordered pathway is important for efficient and accurate production of mature 4.1R mRNA encoding an intact spectrin-actin binding domain. (Blood. 2000;95:692-699)

Mouse erythroid cells express multiple putative RNA helicase genes exhibiting high sequence conservation from yeast to mammals.

RNA secondary structure is a critical determinant of RNA function in ribosome assembly, pre-mRNA splicing, mRNA translation and RNA stability. The 'DEAD/H' family of putative RNA helicases may help regulate these processes by utilizing intrinsic RNA-dependent ATPase activity to catalyze conformational changes in RNA secondary structure. To investigate the repertoire of DEAD/H box proteins expressed in mammals, we used PCR techniques to clone from mouse erythroleukemia (MEL) cells three new DEAD box cDNAs with high similarity to known yeast (Saccharomyces cerevisiae) genes. mDEAD2 and mDEAD3 (mouse DEAD box proteins) are > 95% identical to mouse PL10 but exhibit differential tissue-specific expression patterns; mDEAD2 and mDEAD3 are also approx. 70% identical (at the aa level) to yeast DED1 and DBP1 proteins. Members of this DEAD box subclass contain C-terminal domains with high content of Arg, Ser, Gly and Phe, reminiscent of the RS domain in several Drosophila and mammalian splicing factors. mDEAD5 belongs to a second class related to translation initiation factors from yeast (TIF1/TIF2) and mammals (eIF-4A); this class contains a novel conserved peptide motif not found in other DEAD box proteins. Northern blotting shows that mDEAD5 is differentially expressed in testis vs. somatic tissues. Thus, mouse erythroid cells produce two highly conserved families of putative RNA helicases likely to play important roles in RNA metabolism and gene expression.

In vitro activity of sparfloxacin (AT-4140 and CI-978), a new quinolone antimicrobial agent, against Haemophilus and gram-positive cocci.

Sparfloxacin (AT-4140 and CI-978) was evaluated for activity against 194 clinical isolates of staphylococci, streptococci, Enterococcus faecalis, anaerobic Gram-positive cocci, and Haemophilus sp. The MIC of sparfloxacin for greater than 93% of the strains tested was less than or equal to 0.5 microgram/ml. Sparfloxacin demonstrated increased activity against enterococci, staphylococci, pneumococci, and anaerobic cocci when compared with ciprofloxacin.

The effects of inhibitors of glucosidase I on the formation of Sindbis virus.

We have examined the effects of deoxynojirimycin and castanospermine, compounds known to inhibit the removal of glucose from high mannose asparagine-linked oligosaccharides, on the formation of Sindbis virus. These drugs inhibited virion formation in baby hamster kidney (BHK) cells, 15B - the CHO cell line that lacks GlcNAc transferase activity, and chicken embryo fibroblasts, although our results with the latter cells were variable. We analyzed the [3H]mannose-labeled oligosaccharides from Sindbis virus infected 15B cells. Those from control cells were predominantly GlcNAc2Man5. Oligosaccharides from the treated cells were larger than the Man5 species and as expected, were partially resistant to alpha-mannosidase. The growth of Sindbis virus was inhibited to a much greater extent at 37 degrees C than at 30 degrees C in BHK cells treated with either deoxynojirimycin or castanospermine. Both of these compounds also inhibited the proteolytic cleavage of the viral glycoprotein precursor, PE2, to the virion glycoprotein, E2, but did not prevent the migration of the glycoprotein to the cell surface. These results, taken together with our earlier studies with vesicular stomatitis virus (Schlesinger et al., 1984) provide strong evidence that the removal of glucose residues during the processing of asparagine-linked oligosaccharides is critical for some proteins to achieve a functional conformation.

Pyruvate orthophosphate dikinase: intracellular site of synthesis in maize leaf cells.

Pyruvate orthophosphate dikinase is synthesized in non-green leaf cells of the maize mutant iojap. Since iojap plastids lack ribosomes, it is concluded that the site of synthesis of pyruvate orthophosphate dikinase in maize leaf cells is on ribosomes in the cytoplasm.

Glyoxylate and glutamate effects on photosynthetic carbon metabolism in isolated chloroplasts and mesophyll cells of spinach.

Addition of millimolar sodium glyoxylate to spinach (Spinacia oleracea) chloroplasts was inhibitory to photosynthetic incorporation of (14)CO(2) under conditions of both low (0.2 millimolar or air levels) and high (9 millimolar) CO(2) concentrations. Incorporation of (14)C into most metabolites decreased. Labeling of 6-P-gluconate and fructose-1,6-bis-P increased. This suggested that glyoxylate inhibited photosynthetic carbon metabolism indirectly by decreasing the reducing potential of chloroplasts through reduction of glyoxylate to glycolate. This hypothesis was supported by measuring the reduction of [(14)C]glyoxylate by chloroplasts. Incubation of isolated mesophyll cells with glyoxylate had no effect on net photosynthetic CO(2) uptake, but increased labeling was observed in 6-P-gluconate, a key indicator of decreased reducing potential. The possibility that glyoxylate was affecting photosynthetic metabolism by decreasing chloroplast pH cannot be excluded. Increased (14)C-labeling of ribulose-1,5-bis-P and decreased 3-P-glyceric acid and glycolate labeling upon addition of glyoxylate to chloroplasts suggested that ribulose-bis-P carboxylase and oxygenase might be inhibited either indirectly or directly by glyoxylate. Glyoxylate addition decreased (14)CO(2) labeling into glycolate and glycine by isolated mesophyll cells but had no effect on net (14)CO(2) fixation. Glutamate had little effect on net photosynthetic metabolism in chloroplast preparations but did increase (14)CO(2) incorporation by 15% in isolated mesophyll cells under air levels of CO(2).

Effects of glycine hydroxamate, carbon dioxide, and oxygen on photorespiratory carbon and nitrogen metabolism in spinach mesophyll cells.

The effects of added glycine hydroxamate on the photosynthetic incorporation of (14)CO(2) into metabolites by isolated mesophyll cells of spinach (Spinacia oleracea L.) was investigated under conditions favorable to photorespiratory (PR) metabolism (0.04% CO(2) and 20% O(2)) and under conditions leading to nonphotorespiratory (NPR) metabolism (0.2% CO(2) and 2.7% O(2)). Glycine hydroxamate (GH) is a competitive inhibitor of the photorespiratory conversion of glycine to serine, CO(2) and NH(4) (+). During PR fixation, addition of the inhibitor increased glycine and decreased glutamine labeling. In contrast, labeling of glycine decreased under NPR conditions. This suggests that when the rate of glycolate synthesis is slow, the primary route of glycine synthesis is through serine rather than from glycolate. GH addition increased serine labeling under PR conditions but not under NPR conditions. This increase in serine labeling at a time when glycine to serine conversion is partially blocked by the inhibitor may be due to serine accumulation via the "reverse" flow of photorespiration from 3-P-glycerate to hydroxypyruvate when glycine levels are high. GH increased glyoxylate and decreased glycolate labeling. These observations are discussed with respect to possible glyoxylate feedback inhibition of photorespiration.

Amino Acid Synthesis in Photosynthesizing Spinach Cells : EFFECTS OF AMMONIA ON POOL SIZES AND RATES OF LABELING FROM CO(2).

Isolated cells from leaves of Spinacia oleracea have been maintained in a state capable of high rates of photosynthetic CO(2) fixation for more than 60 hours. The incorporation of (14)CO(2) under saturating CO(2) conditions into carbohydrates, carboxylic acids, and amino acids, and the effect of ammonia on this incorporation have been studied. Total incorporation, specific radioactivity, and pool size have been determined as a function of time for most of the protein amino acids and for gamma-aminobutyric acid. The measurements of specific radio-activities and of the approaches to (14)C "saturation" of some amino acids indicate the presence and relative sizes of metabolically active and passive pools of these amino acids.Added ammonia decreased carbon fixation into carbohydrates and increased fixation into carboxylic acids and amino acids. Different amino acids were, however, affected in different and highly specific ways. Ammonia caused large stimulatory effects in incorporation of (14)C into glutamine (a factor of 21), aspartate, asparagine, valine, alanine, arginine, and histidine. No effect or slight decreases were seen in glycine, serine, phenylalanine, and tyrosine labeling. In the case of glutamate, (14)C labeling decreased, but specific radioactivity increased. The production of labeled gamma-aminobutyric acid was virtually stopped by ammonia.The results indicate that added ammonia stimulates the reactions mediated by pyruvate kinase and phosphoenolpyruvate carboxylase, as seen with other plant systems. The data on the effects of added ammonia on total labeling, pool sizes, and specific radioactivities of several amino acids provides a number of indications about the intracellular sites of principal synthesis from carbon skeletons of these amino acids and the selective nature of effects of increased intracellular ammonia concentration on such synthesis.