Evolution of the secondary structures and compensatory mutations of the ribosomal RNAs of Drosophila melanogaster

This paper examines the effects of DNA sequence evolution on RNA secondary structures and compensatory mutations. Models of the secondary structures of Drosophila melanogaster 18S ribosomal RNA (rRNA) and of the complex between 2S, 5.8S, and 28S rRNAs have been drawn on the basis of comparative and energetic criteria. The overall AU richness of the D. melanogaster rRNAs allows the resolution of some ambiguities in the structures of both large rRNAs. Comparison of the sequence of expansion segment V2 in D. melanogaster 18S rRNA with the same region in three other Drosophila species and the tsetse fly (Glossina morsitans morsitans) allows us to distinguish between two models for the secondary structure of this region. The secondary structures of the expansion segments of D. melanogaster 28S rRNA conform to a general pattern for all eukaryotes, despite having highly divergent sequences between D. melanogaster and vertebrates. The 70 novel compensatory mutations identified in the 28S rRNA show a strong (70%) bias toward A-U base pairs, suggesting that a process of biased mutation and/or biased fixation of A and T point mutations or AT-rich slippage-generated motifs has occurred during the evolution of D. melanogaster rDNA. This process has not occurred throughout the D. melanogaster genome. The processes by which compensatory pairs of mutations are generated and spread are discussed, and a model is suggested by which a second mutation is more likely to occur in a unit with a first mutation as such a unit begins to spread through the family and concomitantly through the population. Alternatively, mechanisms of proofreading in stem-loop structures at the DNA level, or between RNA and DNA, might be involved. The apparent tolerance of noncompensatory mutations in some stems which are otherwise strongly supported by comparative criteria within D. melanogaster 28S rRNA must be borne in mind when compensatory mutations are used as a criterion in secondary-structure modeling. Noncompensatory mutation may extend to the production of unstable structures where a stem is stabilized by RNA- protein or additional RNA-RNA interactions in the mature ribosome. Of motifs suggested to be involved in rRNA processing, one (CGAAAG) is strongly overrepresented in the 28S rRNA sequence. The data are discussed both in the context of the forces involved with the evolution of multigene families and in the context of molecular coevolution in the rDNA family in particular.      

Heterologous expression of an engineered truncated form of human Lewis fucosyltransferase (Fuc-TIII) by the methylotrophic yeast Pichia pastoris

A stable GS115 Pichia pastoris recombinant strain was constructed to secrete a truncated form of the human alpha(1,3/4) fucosyltransferase (amino acids 45-361). Enzyme production resulted from a secretory pathway based on the pre-pro- alpha mating factor signal sequence of the yeast Saccharomyces cerevisiae . Following its transit through the Golgi apparatus, the enzyme accumulated in the periplasmic space before its release in the culture broth (about 30 mg/l). Cell-enclosed enzyme ( approximately 0.16%) proved to be fairly stable for many freezing and thawing cycles and could be used several times as an immobilized catalyst. Soluble enzyme (>99.8%) representing the main protein of the culture broth (10%) has been characterized by Western-blotting, substrate specificities and kinetic parameters. The two forms (cell- enclosed and soluble) of recombinant enzyme may be used for in vitro synthesis of Lewisadeterminants.      

Worldwide patterns of mitochondrial DNA differentiation in the harbor seal (Phoca vitulina)

The harbor seal (Phoca vitulina) has one of the broadest geographic distributions of any pinniped, stretching from the east Baltic, west across the Atlantic and Pacific Oceans to southern Japan. Although individuals may travel several hundred kilometers on annual feeding migrations, harbor seals are generally believed to be philopatric, returning to the same areas each year to breed. Consequently, seals from different areas are likely to be genetically differentiated, with levels of genetic divergence increasing with distance. Differentiation may also be caused by long-standing topographic barriers such as the polar sea ice. We analyzed samples of 227 harbor seals from 24 localities and defined 34 genotypes based on 435 bp of control region sequence. Phylogenetic analysis and analysis of molecular variance showed that populations in the Atlantic and Pacific Oceans and east and west coast populations of these oceans are significantly differentiated. Within these four regions, populations that are geographically farthest apart generally are the most differentiated and often do not share genotypes or differ in genotype frequency. The average corrected sequence divergence between populations in the Atlantic and Pacific Oceans is 3.28% +/- 0.38% and those among populations within each of these oceans are 0.75% +/- 0.69% and 1.19% +/- 0.65%, respectively. Our results suggest that harbor seals are regionally philopatric, on the scale of several hundred kilometers. However, genetic discontinuities may exist, even between neighboring populations such as those on the Scottish and east English coasts or the east and west Baltic. The mitochondrial data are consistent with an ancient isolation of populations in both oceans, due to the development of polar sea ice. In the Atlantic and Pacific, populations appear to have been colonized from west to east with the European populations showing the most recent common ancestry. We suggest the recent ancestry of European seal populations may reflect recolonization from Ice Age refugia after the last glaciation.      

Glutathione Depletion Due to Copper-Induced Phytochelatin Synthesis Causes Oxidative Stress in Silene cucubalus

The relation between loss of glutathione due to metal-induced phytochelatin synthesis and oxidative stress was studied in the roots of copper-sensitive and tolerant Silene cucubalus (L.) Wib., resistant to 1 and 40 micromolar Cu, respectively. The amount of nonprotein sulfhydryl compounds other than glutathione was taken as a measure of phytochelatins. At a supply of 20 micromolar Cu, which is toxic for sensitive plants only, phytochelatin synthesis and loss of total glutathione were observed only in sensitive plants within 6 h of exposure. When the plants were exposed to a range of copper concentrations for 3 d, a marked production of phytochelatins in sensitive plants was already observed at 0.5 micromolar Cu, whereas the production in tolerant plants was negligible at 40 micromolar or lower. The highest production in tolerant plants was only 40% of that in sensitive plants. In both varieties, the synthesis of phytochelatins was coupled to a loss of glutathione. Copper at toxic concentrations caused oxidative stress, as was evidenced by both the accumulation of lipid peroxidation products and a shift in the glutathione redox couple to a more oxidized state. Depletion of glutathione by pretreatment with buthionine sulfoximine significantly increased the oxidative damage by copper. At a comparably low glutathione level, cadmium had no effect on either lipid peroxidation or the glutathione redox couple in buthionine sulfoximine-treated plants. These results indicate that copper may specifically cause oxidative stress by depletion of the antioxidant glutathione due to phytochelatin synthesis. We conclude that copper tolerance in S. cucubalus does not depend on the production of phytochelatins but is related to the plant's ability to prevent glutathione depletion resulting from copper-induced phytochelatin production, e.g. by restricting its copper uptake.     

Defective biliary secretion of bile acid 3-O-glucuronides in rats with hereditary conjugated hyperbilirubinemia

Biliary secretion of bile acid glucuronides was studied in control rats and in rats with a congenital defect in hepatobiliary transport of organic anions (GY rats). In control animals, hepatobiliary transport of [3H]lithocholic acid 3-O-glucuronide and [3H]cholic acid 3-O-glucuronide was efficient (greater than 95% in 1 h) and comparable to that of [14C]taurocholic acid. Secretion of both glucuronides was impaired in GY rats (24% and 71% at 1 h), whereas that of taurocholate was similar to control values. However, recovery of the glucuronides in bile was nearly complete within 24 h; virtually no radioactivity was found in urine. In control rats, biliary secretion of lithocholic acid 3-O-glucuronide, but not that of cholic acid 3-O-glucuronide or taurocholate, could be delayed by simultaneous infusion of dibromosulphthalein. In mutant rats, dibromosulphthalein infusion was also able to inhibit secretion of cholic acid 3-O-glucuronide. [3H]Hydroxyetianic acid, a C20 short-chain bile acid, was secreted by control rats as a mixture of 20% carboxyl-linked and 80% hydroxyl-linked (3-O-)glucuronide; secretion was very efficient (99% in 1 h). In GY rats, secretion was drastically impaired (16% at 1 h and 74% over a 24-h period). Initially, the mutant secreted more carboxyl- than hydroxyl-linked glucuronide, but the ratio reached that of control animals after 24 h. The rates of formation of both types of hydroxyetianic acid glucuronide by hepatic microsomes from mutant rats were similar or even slightly higher than those of control microsomes. These findings indicate that bile acid 3-O-glucuronides, but probably not carboxyl-linked glucuronides, are secreted into bile by a transport system shared with organic anions such as conjugated bilirubin and dibromosulphthalein, but different from that for amino acid-conjugated bile acids.     

Increase of fungitoxicity of mercuric chloride by methionine,ethionine and S-methylcysteine

The fungitoxicity of mercuric chloride to Aspergillus niger was increased in the presence of d-, l-, dl-methionine, dl-ethionine, dl-S-methylcysteine or sodium methylmercaptide. The same effect was observed with methionine for two other fungi investigated: Cladosporium cucumerinum and Scopulariopsis brevicaulis. It is suggested that this effect can be ascribed to the formation of CH₃SHg⁺ or (CH₃S)₂Hg, or the corresponding ethyl compounds. CH₃SHgCl and (CH₃S)₂Hg were synthetically prepared and proved indeed far more fungitoxic than HgCl₂. The hypothesis was further substantiated by the observation that A. niger rapidly converts dl-methionine into CH₃SH, which undoubtedly reacts with Hg²⁺ to give the above mentioned methylthiomercury compounds.