Molecular cloning and embryonic expression of Xenopus Six homeobox genes

Six genes are vertebrate homologues of the homeobox-containing gene sine oculis, which plays an essential role in controlling Drosophila compound eye development. Here we report the identification and expression patterns of all three subfamilies of Xenopus Six genes. Two Six2 subfamily genes (Six1, Six2) showed very similar expression patterns in cranial ganglia, otic placodes and the eyes. Non-neural expression of Six1 and Six2 was observed with mesodermal head mesenchyme, somites and their derivatives, the muscle anlagen of the embryonic trunk. In addition, Six2 expression was also found with mesenchyme associated with the developing stomach and pronephros. Expression of Six3 subfamily genes (Six3.1, Six3.2, Six6.1, and Six6.2) was restricted to the developing head, where expression was especially observed in derivatives of the forebrain (eyes, optic stalks, the hypothalamus and pituitary gland). Interestingly, expression of all Six3 subfamily members but Six6.2 was also found with the pineal gland primordium and the tegmentum. Expression of Six4 subfamily genes (Six4.1, Six4.2) was present in the developing visceral arches, placodal derivatives (otic vesicle, olfactory system), head mesenchyme and the eye. The observed dynamic expression patterns are largely conserved between lower and higher vertebrates and imply important roles of Six family genes not only in eye formation and myogenesis, but also in the development of the gut, the kidney and of placode-derived structures.     

Effect of age on sexually dimorphic selenoprotein expression in mice

Clinical data suggest that selenium (Se) supplementation decreases disease predisposition and severity and accelerates recovery in a variety of pathologies. Pre-supplementation Se levels and sex represent important determinants of these Se-dependent health effects. Accordingly, we previously reported on sexually dimorphic expression patterns of Se-dependent glutathione peroxidase 1, type I deiodinase, and selenoprotein P in young mice. In the present study we investigated whether these differences vary with age. The strong sexual dimorphic expression of hepatic type I deiodinase that was observed in young mice vanished both at the mRNA and enzyme activity level by 1 year of age. In contrast, the strong sex-specific differences in renal type I deiodinase mRNA expression were sustained with age. Accordingly, deiodinase enzymatic activities differed in male and female kidneys, largely independent of age [average of 6.8 vs. 15.7 pmol/(min mg) in males vs. females]. In parallel, hepatic Se concentrations and glutathione peroxidase activities increased in female mice compared to male littermates, establishing a new sexual dimorphism in liver. Thus, age represents another important modifier of the dynamic sex- and tissue-specific selenoprotein expression patterns. These data highlight again the unique physiological regulatory mechanisms that have evolved to control Se metabolism according to the actual needs of the organism.     

Calcium limitation in Daphnia magna

The role of ambient calcium concentrations on survival, moulting,growth and egg production was assessed in the cladoceran Daphniamagna. A threshold for survival was found in the range 0.1–0.5mg Ca l^–1, even when ionic strength of the medium waskept constant. Accumulated length and length specific dry weightwas retarded at low Ca (0.5–1.0 mg Ca l^–1) at foodconcentrations above incipient limiting level. For lower foodlevels, the effect of Ca on growth was less clear. The effectof low Ca on growth rate was most manifest during the firstdays after hatching, reflecting the higher Ca demands of theearly juveniles. Age-specific egg production was strongly reducedat Ca concentrations <10 mg Ca l^–1. This was partlyan indirect effect of reduced growth rates, but could also bean effect of higher energetic costs associated with Ca uptakein a Ca-poor medium. The high Ca demands in D.magna may notbe representative of other Daphnia species. Nevertheless, highspecific Ca content seems to be a common property of Daphniaspp. and Ca deficiency could be a major determinant of speciessuccess and community structure among crustacean zooplankton;it also puts constraints on carbon sequestration in the pelagicfood web of softwater lakes.     

Molecular cloning and expression of the ilvGEDAY genes from Salmonella typhimurium

The ilvGEDAY genes of Salmonella typhimurium were cloned in Escherichia coli K-12 by in vitro recombination techniques. A single species of recombinant plasmid, designated pDU1, was obtained by selecting for Valr Ampr transformants of strain SK1592. pDU1 was shown to contain a 14-kilobase EcoRI partial digestion product of the S. typhimurium chromosome inserted into the EcoRI site of the pVH2124 cloning vector. The ilvGEDAY genes were found to occupy a maximum length of 7.5 kilobases. Restriction endonuclease analysis of the S. typhimurium ilv gene cluster provided another demonstration of the gene order as well as established the location of ilv Y between ilvA and ilvC. The presence of a ribosomal ribonucleic acid operon on the pDU1 insert, about 3 kilobases from the 5' end of ilvG, was shown by Southern hybridization. The expression of the ilvGEDA operon from pDU1 was found to be elevated, reflecting the increased gene dosage of the multicopy plasmid. A polarity was observed with respect to ilvEDA expression which is discussed in terms of the possible translational effects of the two internal promoter sequences, one located proximal to ilvE and the other located proximal to ilvD.     

Thermal avoidance and preference in Daphnia magna

1. Water fleas (Daphnia magna) bred at 23°C were non-responsive to temperatures between 13 and 25°C.

2. At the lower (11°C) and upper limits (30°C) their klinokinetic avoidance behaviour showed a larger intraindividual than interindividual variation.

3. Thermal sensitivity for avoidance responses in D. magna was about 1.5°C.

4. For D. magna bred for one parthenogenetic generation at 14°C heat avoidance temperature was about 8°C lower, and cold avoidance temperature was about 1°C higher than in D. magna from 23°C.

5. In group experiments the animals showed some preference for the acclimation temperature.

6. Cold induced stenothermy and warm induced eurythermy in D. magna were related to the mode of reproduction.

Intersex and male development in Daphnia magna

Sex induction is environmentally stimulated in Daphnia and involves a cue-dependent response for sex determination. Somatic growth was shown to be similar in males and females during juvenile instars, but divergent due to a reduction in male somatic growth, at about the time that females produce ovaries. At this time, males appeared morphologically adult with respect to secondary sex characteristics. Intersex was rare and is unlikely to be important in natural populations. Intersex could be induced in both sexes, and observed more frequently after longer exposure to high temperature, or in the second generation following a temperature change. This indicates an impact on the ability of mothers to determine the sex of the offspring. It may be possible to use intersex characteristics for manipulative investigation of sex-determination mechanisms in Daphnia. Although sex-determination is initiated before birth, intersex occurrence suggests that development of male characters requires an additional process, probably involving hormone activity, during juvenile development and maturation.     

Reproducibility of short-term and reproduction toxicity experiments with Daphnia magna and comparison of the sensitivity of Daphnia magna with Daphnia pulex and Daphnia cucullata in short-term experiments

Short-term toxicity tests with Daphnia magna are reasonably reproducible. The sensitivity of Daphnia pulex, Daphnia cucullata and Daphnia magna proved to be about the same in short-term tests with 15 different chemical compounds.Reproduction toxicity experiments with Daphnia magna can easily be carried out within three weeks. At least duplicate experiments are necessary to arrive at an approximate no-effect level.     

Optimization of mRNA design for protein expression in the crustacean Daphnia magna

The water flea Daphnia is a new model organism for ecological, evolutionary, and toxicological genomics. Detailed functional analysis of genes newly discovered through genomic approaches often requires overexpression of the identified protein. In the present study, we report the microinjection of in vitro-synthesized RNAs into the eggs as a method for overexpressing ubiquitous proteins in Daphnia magna. We injected a 1.3-kb mRNA that coded for the red fluorescent protein (DsRed2) flanked by UTRs from the ubiquitously expressed elongation factor 1α-1 (EF1α-1) into D. magna embryos. DsRed2 fluorescence in the embryos was measured 24 h after microinjection. Unexpectedly, the reporter RNA containing the 522-bp full-length EF1α-1 3′ UTR failed to induce fluorescence. To assess reporter expression, the length of the 3′ UTR that potentially contained negative regulatory elements of protein expression, including AU-rich regions and Musashi binding elements, was serially reduced from the 3′ end. Assessing all injected RNA alternatives, mRNA containing the first 60 bp of the 3′ UTR gave rise to the highest fluorescence, 14 times the Daphnia auto-fluorescence. In contrast, mRNA lacking the entire 3′ UTR hardly induced any change in fluorescence intensity. This is the first evaluation of UTRs of mRNAs delivered into Daphnia embryos by microinjection for overexpressing proteins. The mRNA with truncated 3′ UTRs of Daphnia EF1α-1 will be useful not only for gain-of-function analyses but also for labeling proteins and organelles with fluorescent proteins in Daphnia.     

Prenatal sex ratios and expression of sexually dimorphic traits in three snake species

Variation in intrauterine exposure to hormones associated with variation in the sex of litter mates has well-established and far-reaching effects on sexual development in some mammals. Research on this phenomenon in reptiles is scant, but suggests that lizards may follow the mammalian model whereas snakes may be affected differently. We examined sex-specific expression of four sexually dimorphic traits (tail length, head length, ventral scale count, swimming speed) in three species of snakes (Nerodia sipedon, Thamnophis sirtalis, T. sauritus) relative to litter sex ratios. We found little evidence that traits in either sex were masculinized or feminized in response to variation in litter sex ratio. The one significant result appeared best explained as a statistical artifact attributable to a single litter. Our results indicate that snakes are different from the one lizard studied to date. Unlike previous suggestions that prenatal hormonal mechanisms operate differently in snakes and lizards, however, the difference appears to be that development of sexually dimorphic traits in lizards is affected by litter sex ratios whereas in snakes it is not.     

Evolution of heat shock protein expression in a natural population of Daphnia magna

Populations often face changes in environmental conditions in a relatively short timescale, which may lead to microevolution of traits to cope with these changing selective pressures. Here, we demonstrate microevolution of a physiological trait in a natural population of the water flea Daphnia magna. Levels of the stress protein Hsp60 showed genetic variation, indicating in situ evolutionary potential, and the levels increased through time. The observed microevolutionary increase did not fit the historically documented changes in fish predation pressure in this pond, but it paralleled an increase in the load of infective stages of epibionts through time. In line with this, the locally most abundant epibiont caused an induction of Hsp60. Because stress proteins show evolutionary potential and protect organisms against a wide array of environmental factors, microevolution of stress proteins in natural populations is likely to be common.     

Molecular cloning and expression of flagellar radial spoke and dynein genes of Chlamydomonas

Several flagellar dynein ATPase and radial spokehead genes have been isolated from a Chlamydomonas genomic expression library in lambda gt11. The library was probed with polyclonal and monoclonal antibodies raised against purified flagellar polypeptides, and recombinant phage giving positive signals were cloned. In vitro translation of mRNAs hybrid-selected by the cloned sequences from whole cell RNA provided confirmation of identity for three of the four clones. Evidence supporting the identification of the fourth, which encodes a dynein heavy chain, was provided by antibody selection; the fusion protein produced by this clone selected heavy chain-specific antibodies from a complex polyclonal antiserum recognizing many dynein determinants. One of the radial spoke sequences isolated here is of particular interest because it encodes the wild-type allele of a locus which was defined previously by temperature-sensitive paralyzed flagella mutation pf-26ts (Huang, B., G. Piperno, Z. Ramanis, and D. J. L. Luck, 1981, J. Cell Biol., 88:80-88). The cloned sequence was used to hybrid-select mRNA from mutant pf-26ts cells, and when translated in vitro, the selected mRNA produced a mutant spokehead polypeptide with an altered electrophoretic mobility. This confirms that the pf-26ts mutation alters the primary structure of a radial spokehead polypeptide. To quantify spokehead and dynein mRNAs during flagellar regeneration, all of the cloned sequences were used as hybridization probes in RNA dot experiments. Levels increased rapidly and coordinately after deflagellation, peaked 3-10-fold above nondeflagellated controls, and then returned to control values within 2 h. This accumulation pattern was similar to that of flagellar alpha-tubulin mRNA.     

Preferential feeding behaviour of Daphnia magna

Preferential feeding behaviour by Daphnia magna was shown when Daphnia were fed on a mixture of ¹⁴C-labelled algae (Chlorella vulgaris or Scenedesmus quadricauda) and ³H-labelled bacteria (Escherichia coli). Daphnia consumption of bacteria was equal or higher in the presence of algae. On the other hand, in the presence of bacteria, algal consumption decreased by 40–70% compared to algal consumption in the absence of bacteria. ¹⁴C radioactive uptake was in good agreement with the chlorophyll content and demonstrates the preferential feeding behaviour of Daphnia.     

Macromolecular isoforms of Daphnia magna haemoglobin

The haemoglobin (Hb) of Daphnia magna acclimated to different oxygen conditions was sampled, and in its natively assembled state it was separated by chromatofocusing. The Hb isoforms were analysed for their subunit composition under denaturating conditions by two-dimensional gel electrophoresis. The Hb system is suggested to consist of three predominant Hb aggregates, which are characterised by a specific subunit composition and synthesised in response to different ambient oxygen conditions. In normoxia, a dominant Hb aggregate (DmHbI) with a pI of 4.4-4.6 was composed of subunits B, C, E, F and G. In severe hypoxia, a different dominant Hb isoform (DmHbIII) with a pI of 5.7-5.9 was composed of subunits A, B, C, D, E and F. Further analyses in moderate hypoxia provided evidence for a third Hb isoform (DmHbII) composed of subunits B, C, D, E and F. Sequence alignment and homology modelling of the tertiary structure of the D. magna Hb domains 1 and 2 revealed functionally relevant substitutions of amino acid residues at positions B10, E7 and E11, which determine the functional properties of D. magna haemoglobin in terms of haem contact, oxygen binding and affinity. Both domains are predicted to possess the common haemoglobin fold, but helices C and D are not properly formed, and helix G is interrupted by a short coil.