Amino acid substitution analysis of E. coli thymidylate synthase: The study of a highly conserved region at the N-terminus

Amino acid substitution analysis within a highly conserved region of Escherichia coli thymidylate synthase (TS), using suppression of amber mutations by tRNA suppressors, has yielded a bank of 124 new mutationally altered TS proteins. These mutant proteins have been used to study the structure-function relationship of the Escherichia coli TS protein at the N-terminus corresponding to residues 20 through 35. This region contains a block of amino acids whose sequence has been well conserved among other known TS proteins from various organisms. Positions 20 through 25 contain a surface loop structure and positions 26 through 35 encompass a β-strand. We find that residues surrounding a β-bulge structure within the β-strand are particularly sensitive to amino acid substitution, suggesting that this structure is maintained by a highly ordered packing arrangement. Three residues in the surface loop that are present at the base of the substrate binding pocket are also sensitive to amino acid substitution. The remainder of the conserved sites, including those at the dimer interface, are tolerant to most, if not all, of the substitutions tested. © 1992 Wiley-Liss, Inc.     

Testicular expression of PC4 in the rat: molecular diversity of a novel germ cell-specific Kex2/subtilisin-like proprotein convertase.

The rat cDNA sequence of PC4 (rPC4), representing a new member of the Kex2/subtilisin-like proprotein convertases, demonstrated the presence of at least three rPC4 mRNAs resulting in the production of rPC4-A (654 amino acids), rPC4-B (619 amino acids), and rPC4-C (609 amino acids) with different C-terminal sequences. Analogous to rat PC4, three cDNAs were also found for the mouse PC4. The observed molecular diversity of PC4 mRNA possibly results from the differential splicing and/or exon skipping of the parent gene. PC4 mRNA, with a major form at 2.8 kilobases, was highly abundant in the rat testis but could not be detected by Northern analysis in any other tissues including the central nervous system and peripheral tissues. Testicular cell separation studies combined with Northern analysis indicate the high expression levels of PC4 in germ cells but not in Leydig, Sertoli, or peritubular cells. In situ hybridization histochemistry confirms the site of PC4 gene expression as the pachytene spermatocytes and the round spermatids but not in the elongating spermatids. We also demonstrate the colocalization of PC4 with proenkephalin in testicular germ cells by in situ hybridization. A study of the ontogeny of PC4 indicated that PC4 mRNA was first expressed postnatally between days 19 and 22, coinciding with the first stages of spermiogenesis. The stage-specific expression of PC4 in testis indicates its potential role in the developmental maturation of germ cells and that this convertase may play a specific physiological function in reproduction.     

Paleobiology of a Neoproterozoic tidal flat/lagoonal complex: the Draken Conglomerate Formation, Spitsbergen

Carbonates and rare shales of the ca 700-800 Ma old Draken Conglomerate Formation, northeastern Spitsbergen, preserve a record of environmental variation within a Neoproterozoic tidal flat/lagoon complex. Forty-two microfossil taxa have been recognized in Draken rocks, and of these, 39 can be characterized in terms of their paleoenvironmental distributions along a gradient from the supratidal zone to permanently submerged lagoons. Supratidal to subtidal trends include: increasing microbenthic diversity, increasing abundance and diversity of included allochthonous (presumably planktonic) elements, decreasing sheath thickness of mat-building organisms (with significant taphonomic consequences), and an increasing sediment/fossil ratio in fossiliferous rocks. Five principal and several minor biofacies can be distinguished. The paleoecological resolution obtainable in the Draken Conglomerate Formation rivals that achieved for most Phanerozoic fossil deposits. It documents the complexity and diversity of Proterozoic coastal ecosystems and indicates that both environment and taphonomy need to be taken into explicit consideration in attempts to understand evolutionary trends in early fossil record. Three species, Coniunctiophycus majorinum, Myxococcoides distola, and M. chlorelloidea, are described as new; Siphonophycus robustum, Siphonophycus septatum, and Gorgonisphaeridium maximum are proposed as new combinations.     

Biochemical ecology of the forest tent caterpillar: responses to dietary protein and phenolic glycosides

Summary Interactions between quaking aspen (Populus tremuloides) and the forest tent caterpillar (Malacosoma disstria) are likely to be influenced by leaf protein and phenolic glycoside levels, and insect detoxication activity. We investigated the direct and interactive effects of dietary protein and phenolic glycosides on larval performance and midgut enzyme activity of forest tent caterpillars. We conducted bioassays with six artificial diets, using both first and fourth stadium larvae. Four of the diets comprised a 2×2 factorial design-two levels of protein, each with and without phenolic glycosides. Additionally, we assayed high protein diets containing S,S,S-tributylphosphorotrithioate (DEF, an esterase inhibitor) and DEF plus phenolic glycosides. Enzyme solutions were prepared from midguts of sixth instars and assayed for -glucosidase, esterase and glutathione transferase activities. First instar mortality and development times were higher for larvae on diets low in protein or containing phenolic glycosides. Effects of phenolic glycosides were especially pronounced at low protein levels and when administered with DEF. Fourth instar development times were prolonged, and growth rates reduced, in response to consumption of low protein diets. Effects of phenolic glycosides on growth were less pronounced, although the effect for larvae on the low protein diet was nearly significant. Activity of each of the enzyme systems was reduced in larvae reared on low protein diets, and esterase activity was induced in larvae fed phenolic glycosides. Our results suggest that larval performance may be strongly affected by levels of protein and phenolic glycosides commonly occurring in aspen foliage, and that these factors may play a role in differential defoliation of aspen by forest tent caterpillars.     

The effect of established plants on recruitment in the annual forb Sinapis arvensis

Summary The germination response of Sinapis arvensis to the presence of established plants was investigated in a greenhouse experiment. Established conspecific and heterospecific plants were found to inhibit germination and reduce the probability of recruitment of those seeds that germinate. Established plants have no effect on seed mortality in the soil. Using a simple recruitment model, it is demonstrated that the combination of variance in germination time coupled with the interaction between buried seeds and established plants can generate density dependence. The implications of these results for community processes, such as succession, are discussed.     

Seasonal carbon isotope discrimination in a grassland community

Summary Grassland communities of arid western North America are often characterized by a seasonal increase in ambient temperature and evaporative demand and a corresponding decline in soil moisture availability. As the environment changes, particular species could respond differently, which should be reflected in a number of physiological processes. Carbon isotope discrimination varies during photosynthetic activity as a function of both stomatal aperture and the biochemistry of the fixation process, and provides an integrated measure of plant response to seasonal changes in the environment. We measured the seasonal course of carbon isotope discrimination in 42 grassland species to evaluate changes in gas exchange processes in response to these varying environmental factors. The seasonal courses were then used to identify community-wide patterns associated with life form, with phenology and with differences between grasses and forbs. Significant differences were detected in the following comparisons: (1) Carbon isotope discrimination decreased throughout the growing season; (2) perennial species discriminated less than annual species; (3) grasses discriminated less than forbs; and (4) early flowering species discriminated more than the later flowering ones. These comparisons suggested that (1) species active only during the initial, less stressful months of the growing season used water less efficiently, and (2) that physiological responses increasing the ratio of carbon fixed to water lost were common in these grassland species, and were correlated with the increase in evaporative demand and the decrease in soil moisture.