Multiple polyadenylation sites in a Drosophila tropomyosin gene are used to generate functional mRNAs.

The gene encoding muscle tropomyosin I in Drosophila is alternatively spliced in embryonic and thoracic muscle to generate two sizes classes of RNAs. By Northern blot analysis, the embryonic RNA class shows a broad RNA band of hybridization of 1.3 kb and a more sharply defined, less abundant RNA band at 1.6 kb. The thoracic class of RNAs, on the other hand, consists of a broad hybridization band at 1.7 kb and a more sharply defined band at 1.9 kb. Each size class of RNA encodes a different tropomyosin isoform. The two classes of alternatively spliced RNAs utilize the same 3' terminal exon of the gene. The DNA sequence of this exon reveals a cluster of several polyadenylation signals (AAUAAA) or polyadenylation-like signals. We show here by S1 nuclease protection analysis that at least five and possibly seven of these polyadenylation or polyadenylation-like sequences are associated with in vivo embryonic and thoracic mRNA cleavage processing sites. Six of these S1 sites are clustered within 119 bp and a seventh is located 255 bp downstream. At least one of the polyadenylation-like signal sequences appears to be an unusual AACAAA sequence. In addition we also show that these mRNAs function in vitro to synthesize muscle tropomyosins.     

Effects of temperature and moisture during incubation on carcass composition of hatchling snapping turtles (Chelydra serpentina)

Summary Flexible-shelled eggs of common snapping turtles (Chelydra serpentina) were incubated on each of two substrates (vermiculite, sand) at each of three temperatures (26.0°C, 28.5°C, 31.0°C) and three moisture regimes (wet, intermediate, dry). Embryos developing in cool, wet environments mobilized the largest amounts of protein from their yolk and attained the largest size before hatching, whereas turtles developing in warm, dry environments mobilized the smallest quantities of protein and were the smallest in body size at hatching. Embryos on wet substrates mobilized more lipid from their yolk than did embryos on dry media, but ambient temperature had no demonstrable influence on patterns of lipid mobilization. The total reserve of neutral lipid available in residual yolk plus carcass to sustain neonates in the interval prior to the beginning of feeding was largest in hatchlings from dry environments and smallest in animals from wet environments, but was unaffected by temperature during incubation. Hydration of tissues in hatchlings was higher when incubation was in cool, moist conditions than when incubation was in warm, dry settings, thereby indicating that some of the effects of moisture and temperature on mobilization of nutrients by embryos may be mediated by differences in intracellular water. Patterns of response to temperature and moisture recorded for turtles emerging from eggs on sand were similar to those recorded for hatchlings on vermiculite, so no important conclusion would have been affected by incubating eggs on one medium instead of the other.     

Experiments on resting site selection by nocturnal moths

When different species of moths are presented with a choice between black and white resting backgrounds, there is a strong correlation between the colour selected and the reflectance of the forewings. Under more natural conditions, light-coloured moths usually rest on fresh vegetation whilst dark-winged species select tree bark or rest upon the ground, and different defensive strategies appear to have been adopted by species in these two latter situations. Studies on the mechanism of background selection, and on background selection in polymorphic species, are reviewed.     

Photoreactions of cytochrome b-559 and cyclic electron flow in Photosystem II of intact chloroplasts

The high potential cytochrome b-559 of intact spinach chloroplasts was photooxidized by red light with a high quantum efficiency and by far-red light with a very low quantum efficiency, when electron flow from water to Photosystem II was inhibited by a carbonyl cyanide phenylhydrazone (FCCP or CCCP). Dithiothreitol, which reacts with FCCP or CCCP, reversed the photooxidation of cytochrome b-559 and restored the capability of the chloroplasts to photoreduce CO₂ showing that the FCCP/CCCP effects were reversible. The quantum efficiency of cytochrome b-559 photooxidation by red or far-red light in the presence of FCCP was increased by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone which blocks oxidation of reduced plastoquinone by Photosystem I. When the inhibition of water oxidation by FCCP or CCCP was decreased by increased light intensities, previously photooxidized cytochrome b-559 was reduced. Red light was much more effective in photoreducing oxidized high potential cytochrome b-559 than far-red light. The red/far-red antagonism in the redox state of cytochrome b-559 is a consequence of the different sensitivity of the cytochrome to red and far-red light and does not indicate that the cytochrome is in the main path of electrons from water to NADP. Rather, cytochrome b-559 acts as a carrier of electrons in a cyclic path around Photosystem II. The redox state of the cytochrome was shifted to the oxidized side when electron transport from water became rate-limiting, while oxidation of water and reduction of plastoquinone resulted in its shifting to the reduced side.     

The stacking of chloroplast thylakoids: Quantitative analysis of the balance of forces between thylakoid membranes of chloroplasts,and the role of divalent cations

An analysis is made of the van der Waals dispersion attractive forces and electrostatic repulsive forces between the grana thylakoid membranes of chloroplasts. These forces are determined for negatively charged surfaces with a pK_a value of 4.7 for a bulk pH of 7.0 with a range of mono- and divalent cation concentrations and intermembrane spacing in the range 10 to 80 Å. For equilibrium under dark conditions, it is concluded that either there is extensive electrostatic binding of divalent cations (Mg²⁺) to the negatively charged membrane groups (phospholipid, sulfolipid, and protein carboxyl), or a redistribution of these groups between stacked and unstacked regions must be invoked.     

Aggregation-dependent turnover of flagellar adhesion molecules in chlamydomonas gametes

Previous studies on flagellar adhesion in chlamydomonas (Snell, W. and S. Roseman. 1979. J. Biol. Chem. 254:10820-10829.) have shown that as gametes adhere to flagella isolated from gametes of the opposite mating type, the adhsiveness of the added flagella but not of the gametes is lost. The studies reported here show that the addition of protein synthesis inhibitors (cycloheximide [CH] or anisomycin) to the medium of such cell- flagella mixtures causes the cells to lose their adhesiveness. This loss, however, occurs only after the cells have interacted with 4-8 flagella/cell and does not occur if the cells are kept in CH (7 h) without aggregating. The availability of an impotent (imp) mating type plus (MT(+)) mutant (provided by U.W. Goodenough), which adheres but is unable to undergo the fusion that normally follows adhesion, made it possible to determine whether a similar loss of adhesiveness occurs in mixtures of matting type minus (mt(-)) and imp mt(+) gametes. In the absence of inhibitor, mt(-) and imp mt(+) gametes adhered to each other (without fusing) for several hours; however, in the presence of CH or anisomycin, the gametes began to de-adhere 35 min after mixing, and, by 90 min, 100 percent of the cells were single again. This effect was reversible, and the rapid turnover of cells were single again. This effect was reversible, and the rapid turnover of molecules involved in adhesion occurred only during adhesion inasmuch as gametes pretreated for 4 h with CH were able to aggregate in CH for the same length of time as nonpretreated cells aggregated in CH. By the addition of CH at various times after the mt(-) and imp mt(+) gametes were mixed, measurements were made of the “pool size” of the molecules involved in adhesion. The pool reached a minimum after 25 min of aggregation, rapidly increased for the next 25 min, and then leveled off at the premixing level. These results suggest that flagellar adhesion in chlamydomonas causes modification of surface molecules (receptors, ligands), which brings about their inactivation and stimulates their replacement.     

Effects of pH and Oxygen on Photosynthetic Reactions of Intact Chloroplasts

Oxygen inhibition of photosynthesis was studied with intact spinach (Spinacia oleracea L.) chloroplasts which exhibited very high rates of photosynthetic CO₂ reduction and were insensitive to additions of photosynthetic intermediates when CO₂ was available at saturating concentrations. Photosynthetic rates were measured polarographically as O₂ evolution, and the extent of the reduction of substrate was estimated from the amount of O₂ evolved. With CO₂ as substrate, inhibition of photosynthesis by O₂ was dependent on pH. At pH values above 8, rates of O₂ evolution were strongly inhibited by O₂ and only a fraction of the added bicarbonate was reduced before O₂ evolution ceased. The extent of O₂ evolution declined with increasing O₂ concentration and decreasing initial bicarbonate concentration. At pH 7.2, the initial photosynthetic rate was inhibited about 30% at high O₂ levels, but the extent of O₂ evolution was unaffected and most of the added bicarbonate was reduced. Photosynthetic O₂ evolution with 3-phosphoglycerate as substrate was similarly dependent on pH and O₂ concentration. In contrast, there was little effect of O₂ and pH on oxaloacetate-dependent oxygen evolution. Acid-base shift experiments with osmotically shocked chloroplasts showed that ATP formation was not affected by O₂. The results are discussed in terms of a balance between photosynthetic O₂ evolution and O₂ consumption by the ribulose diphosphate oxygenase reaction.     

Characterisation of recombinant human fatty aldehyde dehydrogenase: implications for Sjögren-Larsson syndrome

Fatty aldehyde dehydrogenase (FALDH) is an NAD+-dependent oxidoreductase involved in the metabolism of fatty alcohols. Enzyme activity has been implicated in the pathology of diabetes and cancer. Mutations in the human gene inactivate the enzyme and cause accumulation of fatty alcohols in Sj?gren-Larsson syndrome, a neurological disorder resulting in physical and mental handicaps. Microsomal FALDH was expressed in E. coli and purified. Using an in vitro activity assay an optimum pH of approximately 9.5 and temperature of approximately 35 degrees C were determined. Medium- and long-chain fatty aldehydes were converted to the corresponding acids and kinetic parameters determined. The enzyme showed high activity with heptanal, tetradecanal, hexadecanal and octadecanal with lower activities for the other tested substrates. The enzyme was also able to convert some fatty alcohol substrates to their corresponding aldehydes and acids, at 25-30% the rate of aldehyde oxidation. A structural model of FALDH has been constructed, and catalytically important residues have been proposed to be involved in alcohol and aldehyde oxidation: Gln-120, Glu-207, Cys-241, Phe-333, Tyr-410 and His-411. These results place FALDH in a central role in the fatty alcohol/acid interconversion cycle, and provide a direct link between enzyme inactivation and disease pathology caused by accumulation of alcohols.