SEASONAL VARIATION IN SEXUAL SELECTION IN THE MOTTLED SCULPIN

Seasonal variation in sexual and natural selection in male mottled sculpins (Cottus bairdi) can be evaluated by calculating selection differentials, which measure the magnitude of phenotypic change resulting from selection, and by calculating indices of the opportunity for selection, which indicate the potential for phenotypic selection in a given interval. Selection differentials are high at the beginning of the breeding season and decline throughout the breeding season. The magnitude and direction of selection differentials depend on when spawning occurs and are independent of the size or age of the females that spawn. Annual selection differentials due to differences in mating success (female choice) are nearly constant between years. Annual selection differentials associated with hatching success are variable. Opportunities for selection (I = fitness variance/[mean fitness]²) show clear seasonal patterns. They are highest at the beginning and at the end of the spawning season. However, this variation is dependent on the mean used to calculate I, and hence variation in I values does not indicate a significant change in the variance of male fitness.     

The STE2 gene product is the ligand-binding component of the alpha-factor receptor of Saccharomyces cerevisiae

The STE2 gene of Saccharomyces cerevisiae encodes a 431-residue protein containing seven hydrophobic segments that is thought to be an essential component of the cell-surface receptor for alpha-factor in MATa haploids. Methods were devised to prepare membrane fractions from MATa cells that retained high levels of alpha-factor binding activity, consistent with the view that the alpha-factor receptor resides in the plasma membrane. To demonstrate that the membrane constituent responsible for alpha-factor binding was the STE2 polypeptide, specific antibodies were generated and used to identify STE2-related polypeptides by radiolabeling, immunoprecipitation, and polyacrylamide gel electrophoresis. Under conditions of complete solubilization, the major form of the STE2 gene product detected was a glycoprotein with an apparent molecular weight of 49,000. Affinity labeling of yeast membrane preparations by chemical cross-linking to 35S-alpha-factor indicated that a molecule of 49,000 molecular weight was the major alpha-factor-binding species. This alpha-factor-binding species was shown to be the product of the STE2 gene in three ways. First, MATa haploids carrying the STE2 gene on a multicopy plasmid overproduced alpha-factor binding activity about 15-fold. Second, MATa cells completely lacking a STE2 gene showed only nonspecific binding of alpha-factor (equivalent to the level displayed by MAT alpha haploids) and possessed no species that could be cross-linked to 35S-alpha-factor. Third, MATa cells expressing a truncated but functional STE2 gene (in which the COOH-terminal 135-hydrophilic residues were deleted) produced a protein detected by cross-linking to 35S-alpha-factor of apparent molecular weight 33,000, close to the size expected for the predicted abbreviated STE2 polypeptide. These findings demonstrate unequivocally that the STE2 gene product is the membrane component responsible for the ligand recognition function of the yeast alpha-factor receptor.     

Fatty Acids in the Lipids of Marine and Terrestrial Nitrifying Bacteria

Fatty acids in the lipids of 19 marine and terrestrial nitrifying bacteria have been analyzed. Ammonia-oxidizing bacteria have a very simple acid composition; palmitic and palmitoleic acid account for 96 to 100% of the total acids. The fatty acids of nitrite-oxidizing bacteria cover a wider range, from C(14) to C(19), but from two to four acids still account for more than 80% of the total acids. Branched iso- and anteiso-acids are present in traces only in 2 of the 19 bacteria. The chemical and morphological similarity between blue-green algae and these bacteria is discussed.     

Structural and functional homology between the 29 kD rat liver nucleoprotein and the high mobility group 1 protein

A 29 kD soluble rat liver nucleoprotein (p29) has increased binding affinity for the hormone responsive element (RE) of the rat haptoglobin (Hp) gene during the acute-phase reaction. In this work the possibility of its structural and functional homology to the high mobility group 1 (HMG1) nonhistone protein constituent of chromatin was examined. The results of two-dimensional gel electrophoresis, Southwestern and Western immunoblot analyses, showed that p29 and HMG1 are homologous protein species. On the basis of in vitro and in vivo phosphorylation/dephosphorylation experiments, we discuss the modulatory role of phosphate groups in view of the structure and function of p29.     

Analysis of human extrachromosomal DNA elements originating from different β-satellite subfamilies

By screening total human DNA with probes derived from the small polydisperse circular (spc) DNA fraction of cultured human cells, we identified three clones that carry long stretches of -satellite DNA. Further experiments have shown that the three sequences belong to at least two different -satellite subfamilies, which are characterized by different higher order subunits. Members of one of these subfamilies are located in the cytological satellites of all acrocentric chromosomes, whereas members of another are located on the short arms of the acrocentrics on both sides of the stalk regions and also in the centromeric regions of chromosomes 1 and 9. This is the first time that -satellite sequences obtained from the spcDNA of human cells have been assigned to -satellite subfamilies that are organized as long arrays of tandemly arranged higher order monomers. This indicates that -satellite sequences can be excised from their chromosomal loci via intrastrand-recombination processes.This paper is dedicated to Prof. Dr. Ulrich Wolf on his 60th birthday, January 1993     

A competition smFRET assay to study ligand-induced conformational changes of the dengue virus protease

Ligand binding to proteins often is accompanied by conformational transitions. Here, we describe a competition assay based on single molecule Förster resonance energy transfer (smFRET) to investigate the ligand-induced conformational changes of the dengue virus (DENV) NS2B-NS3 protease, which can adopt at least two different conformations. First, a competitive ligand was used to stabilize the closed conformation of the protease. Subsequent addition of the allosteric inhibitor reduced the fraction of the closed conformation and simultaneously increased the fraction of the open conformation, demonstrating that the allosteric inhibitor stabilizes the open conformation. In addition, the proportions of open and closed conformations at different concentrations of the allosteric inhibitor were used to determine its binding affinity to the protease. The K_D value observed is in accordance with the IC₅₀ determined in the fluorometric assay. Our novel approach appears to be a valuable tool to study conformational transitions of other proteases and enzymes.     

Yeast glycogenin (Glg2p) produced in Escherichia coli is simultaneously glucosylated at two vicinal tyrosine residues but results in a reduced bacterial glycogen accumulation.

Saccharomyces cerevisiae possesses two glycogenin isoforms (designated as Glg1p and Glg2p) that both contain a conserved tyrosine residue, Tyr232. However, Glg2p possesses an additional tyrosine residue, Tyr230 and therefore two potential autoglucosylation sites. Glucosylation of Glg2p was studied using both matrix-assisted laser desorption ionization and electrospray quadrupole time of flight mass spectrometry. Glg2p, carrying a C-terminal (His6) tag, was produced in Escherichia coli and purified. By tryptic digestion and reversed phase chromatography a peptide (residues 219-246 of the complete Glg2p sequence) was isolated that contained 4-25 glucosyl residues. Following incubation of Glg2p with UDPglucose, more than 36 glucosyl residues were covalently bound to this peptide. Using a combination of cyanogen bromide cleavage of the protein backbone, enzymatic hydrolysis of glycosidic bonds and reversed phase chromatography, mono- and diglucosylated peptides having the sequence PNYGYQSSPAM were generated. MS/MS spectra revealed that glucosyl residues were attached to both Tyr232 and Tyr230 within the same peptide. The formation of the highly glucosylated eukaryotic Glg2p did not favour the bacterial glycogen accumulation. Under various experimental conditions Glg2p-producing cells accumulated approximately 30% less glycogen than a control transformed with a Glg2p lacking plasmid. The size distribution of the glycogen and extractable activities of several glycogen-related enzymes were essentially unchanged. As revealed by high performance anion exchange chromatography, the intracellular maltooligosaccharide pattern of the bacterial cells expressing the functional eukaryotic transgene was significantly altered. Thus, the eukaryotic glycogenin appears to be incompatible with the bacterial initiation of glycogen biosynthesis.