Evolutionary analysis of the multigene pregnancy-specific β1-Glycoprotein family: Separation of historical and nonhistorical signals

The pregnancy-specific ₁-glycoproteins (PSG) form a large family of closely related proteins. Using newly developed methods of sequence analysis, in combination with protein modeling, we provide a framework for investigating the evolution and biological function of genes like the PSG. Evolutionary trees, based on C-terminal sequence, group PSG genes in a manner consistent with their genomic organization. Trees constructed using the N-terminal domain sequences are unreliable as an indicator of phylogeny because of non-neutral processes of sequence change. During duplication of the PSG genes, evolutionary pressures have resulted in a gradient of constrained change across each gene. The N-terminal domains show a nonrandom pattern of amino acid substitutions clustered in the immunoglobulin complementarity-determining region (CDR)-like regions, which appear to be important in the function of the protein.     

A T7 promoter-specific, inducible protein expression system forBacillus subtilis

The adaptation and application of theEscherichia coli T7 RNA polymerase system for regulated and promoter-specific gene expression inBacillus subtilis is reported. The expression cassette used inBacillus subtilis was tightly regulated and T7 RNA polymerase (T7 RNAP) appeared 30 min after induction. The efficiency of T7 promoter-specific gene expression inB. subtilis was studied using one secretory and two cytosolic proteins of heterologous origin. The accumulation ofE. coli -galactosidase, as well as a 1,4--glucosidase fromThermoanaerobacter brockii inB. subtilis after T7 RNAP induction was strongly enhanced by rifampicin inhibition of host RNAP activity. The-amylase ofThermoactinomyces vulgaris, a secretory protein, was found to accumulate in the culture supernatant up to levels of about 70 mg/l 10–20 h after T7 RNAP induction, but was also deposited in cellular fractions. The addition of rifampicin inhibited-amylase secretion, but unexpectedly, after a short period, also prevented its further (intra)cellular accumulation     

Combination of solid phase extraction and a microalgal test system based on pulse-amplitude modulated fluorescence to detect photosystem 11 herbicides up to 0.05 μeq l−1

This note reports a microalgal test system which usesin-vivo chlorophyll a-fluorescence in combination with a solid phase extraction procedure to monitor photosystem II-herbicides in natural waters up to 0.05 g L^–1.     

Application of 3-Dimensional Homology Modeling of Cytochrome P450 2B1 for Interpretation of Site-Directed Mutagenesis Results

Abstract Three-dimensional structures of cytochrome P450 2B1 were modeled based on the crystallographic structure of P450(cam). The effect of the alignment, loop choice, and minimization with or without water was assessed. Although final models were similar in overall structure, the identity of active site residues depended upon the alignment. An example is Phe-206, which may or may not form part of the active site. The choice of the loop conformation had a lesser effect, while including water in the final minimization step was essential for preserving the shape and size of the active site. The best model (model 2) was in good agreement with the data from site-directed mutagenesis studies, and correctly predicted the effect of substitutions at 9 out of 10 amino acid positions. Thus, residues important for P450 2B1 activity, such as Ile- 114, Phe-206, Ile-290, Thr-302, Val-363, and Gly-478, constitute part of the active site and are able to interact with the substrate androstenedione through hydrophobic interactions. On the other hand, Ser-303, Ser-360 and Lys-473 are far from the active site and/or cannot interact with the substrate, in agreement with experimental data. The model indicates other residues likely to be important for enzyme function, such as Tyr- 111, Leu-209, Ile-477, and Ile- 480, which can be tested experimentally. The substrate may assume numerous binding orientations consistent with observed patterns of hydroxylation at C(5) and C(6). The replacement in the model of certain amino acid residues to mimic residue substitutions from site-directed mutagenesis studies and docking of the substrate into the modified active site allowed a plausible explanation for alterations in regio- and stereospecificities of some mutants of P450 2B1, such as Gly-478 → Ala or Val-363 Ala.     

Brood size and the cost of provisioning nestlings: interpreting Lack's hypothesis

A recent model of parental provisioning (the tradeoff model) suggests that the maximum delivery rate of food to nestlings represents a tradeoff between parental residual reproductive value and nestling survival. In contrast, Lack's hypothesis suggests that maximum provisioning rate determines brood size and therefore delivery rates are limited by shortages of food or foraging time, not by tradeoffs of parental investment. Several authors have examined the shape of the per-nestling feeding curves to test the tradeoff model against Lack's hypothesis. We show that Lack's hypothesis can produce per-nestling feeding curves consistent with the tradeoff model. Therefore, the shape of the per-nestling feeding curve cannot be used to distinguish between the models.