Tolerance, behaviour and oxygen consumption in the sand goby, Pomatoschistus minutus (Pallas), exposed to hypoxia

Behavioural and metabolic reactions of Pomatoschistus minutus (Pallas) exposed to various degrees of hypoxia were studied. At 15°C and 20‰ mortality was 50% at 15.2% oxygen saturation. Avoidance and oxygen saturation showed a linear inverse relationship. At levels lower than 60% saturation increased activity occurred; avoidance was significant at 30% saturation. Active, routine and standard MO₂ correlated linearly with weight at 6 and 15°C (salinity = 19‰). During hypoxia at 15°C routine MO₂ rose significantly at 60–50% and 40–30% saturation expressed either as MO₂ during longer periods at night or MO₂ at shorter intervals during the day. Standard MO₂ was unaffected by hypoxia at 15°C. Haemoglobin concentration was significantly increased when P. minutus was acclimatized to 35% saturation.     

Carbon-14 Uptake by Picoplankton and Total Phytoplankton in Eight New Zealand Lakes

Eight New Zealand lakes were surveyed for ¹⁴C uptake by phytoplankton as a function of light intensity. The results support the view that the photosynthetic picoplankton is an important contributor to primary productivity in oligotrophic lakes but is relatively unimportant in more eutrophic lakes. A comparison of carbon uptake vs. light intensity characteristics (P vs. I) of the picoplankton size class vs. that of the total phytoplankton community supports the view that the picoplankton size class may be adapted to utilization of dimmer light.     

Solid-Phase synthesis of peptide nucleic acids

Peptide nucleic acids (PNA) were synthesized by a modified Merrifield method using several improvements. Activation by O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate in combination with in situ neutralization of the resin allowed efficient coupling of all four Boc-protected PNA monomers within 30 min. HPLC analysis of the crude product obtained from a fully automated synthesis of the model PNA oligomer H-CGGACTAAGTCCATTGC-Gly-NH₂, indicated an average yield per synthetic cycle of 97.1%. N¹-benzyloxycarbonyl-N6³-methylimidazole triflate substantially outperformed acetic anhydride as a capping reagent. The resin-bound PNAs were successfully cleaved by the ‘low–high’ trifluoromethanesulphonic acid procedure.     

Horizontal Transfer of Genetic Material among Saccharomyces Yeasts

The genus Saccharomyces consists of several species divided into the sensu stricto and the sensu lato groups. The genomes of these species differ in the number and organization of nuclear chromosomes and in the size and organization of mitochondrial DNA (mtDNA). In the present experiments we examined whether these yeasts can exchange DNA and thereby create novel combinations of genetic material. Several putative haploid, heterothallic yeast strains were isolated from different Saccharomyces species. All of these strains secreted an a- or alpha-like pheromone recognized by S. cerevisiae tester strains. When interspecific crosses were performed by mass mating between these strains, hybrid zygotes were often detected. In general, the less related the two parental species were, the fewer hybrids they gave. For some crosses, viable hybrids could be obtained by selection on minimal medium and their nuclear chromosomes and mtDNA were examined. Often the frequency of viable hybrids was very low. Sometimes putative hybrids could not be propagated at all. In the case of sensu stricto yeasts, stable viable hybrids were obtained. These contained both parental sets of chromosomes but mtDNA from only one parent. In the case of sensu lato hybrids, during genetic stabilization one set of the parental chromosomes was partially or completely lost and the stable mtDNA originated from the same parent as the majority of the nuclear chromosomes. Apparently, the interspecific hybrid genome was genetically more or less stable when the genetic material originated from phylogenetically relatively closely related parents; both sets of nuclear genetic material could be transmitted and preserved in the progeny. In the case of more distantly related parents, only one parental set, and perhaps some fragments of the other one, could be found in genetically stabilized hybrid lines. The results obtained indicate that Saccharomyces yeasts have a potential to exchange genetic material. If Saccharomyces isolates could mate freely in nature, horizontal transfer of genetic material could have occurred during the evolution of modern yeast species.     

Parameters interacting with mannose selection employed for the production of transgenic sugar beet

The mannose selection system employs the phosphomannose isomerase (PMI) gene as selectable gene and mannose, converted to mannose-6-phosphate by endogenous hexokinase, as selective agent. The transgenic PMI-expressing cells have acquired the ability to convert mannose-6-phosphate to fructose-6-phosphate, while the non-transgenic cells accumulate mannose-6-phosphate with a concomitant consumption of the intracellular pools of phosphate and ATP. Thus, certain steps of mannose selection depend on the cells’ own metabolism which may be affected by a number of factors, some of which are studied here using Agrobacterium tumefaciens-mediated gene transfer to sugar beet cotyledonary explants. Four frequently employed saccharides (sucrose, glucose, fructose, and maltose) were tested at various concentrations and were found to interact strongly with the phytotoxic effect of mannose, glucose being able to counteract nearly 100% of an almost complete mannose-induced growth inhibition. Sucrose, maltose, and fructose also alleviated significantly the mannose-induced growth inhibition, but were 4-, 5-, and 7-fold less potent than glucose, respectively (calculated as hexose equivalents). The transformation frequencies were also dependent on the nature and concentration of the added carbohydrates, but in this respect sucrose resulted in the highest transformation frequencies, about 1.0%, while glucose and fructose gave significantly lower frequencies. The selection efficiencies were highest in the presence of maltose where no non-transgenic escapes were found over a range of concentrations. The effect of the light intensity was also investigated and the transformation frequencies were positively correlated to light intensity, although the relative impact of light on growth in the presence of mannose appeared not to be dependent on the mannose concentration. Additional phosphate in the selection media had a strong positive effect on the transformation frequencies, suggesting phosphate limitation during selection. The mannose selection system was found to be relatively genotype-independent, provided a slight optimization of the mannose concentrations during selection. Analysis of F₁-offspring showed that all studied primary transformants resulted in PMI-expressing plantlets and that the segregational patterns were in accordance with expectations in at least 50% of the transformants, confirming the stable and active inheritance of the PMI-gene.     

Multiple α-Glucoside Transporter Genes in Brewer’s Yeast

Maltose and maltotriose are the two most abundant fermentable sugars in brewer’s wort, and the rate of uptake of these sugars by brewer’s yeast can have a major impact on fermentation performance. In spite of this, no information is currently available on the genetics of maltose and maltotriose uptake in brewing strains of yeast. In this work, we studied 30 brewing strains of yeast (5 ale strains and 25 lager strains) with the aim of examining the alleles of maltose and maltotriose transporter genes contained by them. To do this, we hybridized gene probes to chromosome blots. Studies performed with laboratory strains have shown that maltose utilization is conferred by any one of five unlinked but highly homologous MAL loci (MAL1 to MAL4 and MAL6). Gene 1 at each locus encodes a maltose transporter. All of the strains of brewer’s yeast examined except two were found to contain MAL11 and MAL31 sequences, and only one of these strains lacked MAL41. MAL21 was not present in the five ale strains and 12 of the lager strains. MAL61 was not found in any of the yeast strains. In three of the lager strains, there was evidence that MAL transporter gene sequences occurred on chromosomes other than those known to carry MAL loci. Sequences corresponding to the AGT1 gene, which encodes a transporter of several α-glucosides, including maltose and maltotriose, were detected in all but one of the yeast strains. Homologues of AGT1 were identified in three of the lager strains, and two of these homologues were mapped, one to chromosome II and the other to chromosome XI. AGT1 appears to be a member of a family of closely related genes, which may have arisen in brewer’s yeast in response to selective pressure.     

Use of Conserved Randomly Amplified Polymorphic DNA (RAPD) Fragments and RAPD Pattern for Characterization of Lactobacillus fermentum in Ghanaian Fermented Maize Dough

The present work describes the use of randomly amplified polymorphic DNA (RAPD) for the characterization of 172 dominant Lactobacillus isolates from present and previous studies of Ghanaian maize fermentation. Heterofermentative lactobacilli dominate the fermentation flora, since approximately 85% of the isolates belong to this group. Cluster analysis of the RAPD profiles obtained showed the presence of two main clusters. Cluster 1 included Lactobacillus fermentum, whereas cluster 2 comprised the remaining Lactobacillus spp. The two distinct clusters emerged at the similarity level of <50%. All isolates in cluster 1 showed similarity in their RAPD profile to the reference strains of L. fermentum included in the study. These isolates, yielding two distinct bands of approximately 695 and 773 bp with the primers used, were divided into four subclusters, indicating that several strains are involved in the fermentation and remain dominant throughout the process. The two distinct RAPD fragments were cloned, sequenced, and used as probes in Southern hybridization experiments. With one exception, Lactobacillus reuteri LMG 13045, the probes hybridized only to fragments of different sizes in EcoRI-digested chromosomal DNA of L. fermentum strains, thus indicating the specificity of the probes and variation within the L. fermentum isolates.     

Capacity of Bembidion lampros and Tachyporus hypnorum to survive periods of starvation in early spring

The capacity of the polyphagous predators Bembidion lampros Herbst (Coleoptera; Carabidae) and Tachyporus hypnorum F. (Coleoptera; Staphylinidae) to survive starvation in early spring was investigated. The two species are of similar size and live in the same habitat. They have similar life cycles; where adults emerge in autumn, hibernate during winter and disperse during spring into arable fields where they reproduce and then die. Both species showed an ability to survive long periods of starvation after hibernation. However, B. lampros could live without food several weeks longer than T. hypnorum.Fat content was measured to estimate the energy reserves of beetles in the field. The fat content of B. lampros was at a constant level of 23% both when fed ad libitum in the laboratory and when collected in the field during spring. The fat content of B. lampros was reduced to a level of 7% when they died by starvation. Fat content of T. hypnorum was more variable both when fed ad libitum in the laboratory (17–33%), when collected in the field during the spring (20—26%) and when they died by starvation (10–20%).Neither the mean longevity of starved beetles nor the fat content of specimens collected in the field indicated that the beetles suffered high mortality due to a food shortage in early spring before movement into arable fields.     

Free clathrin triskelions are required for the stability of clathrin-associated adaptor protein (AP-2) coated pit nucleation sites.

In this study image correlation spectroscopy was used to demonstrate the presence of two populations of clathrin in situ, on intact cells. In the periphery of the cell approximately 35% of the clathrin triskelions are free within the cytosol while approximately 65% are in large aggregates, presumably coated pits. Although endocytosis is inhibited at low temperature, free clathrin triskelions are still present and small AP-2 aggregates (of approximately 20 proteins), or coated pit nucleation sites, are still observed. Following hypertonic treatment, or cytoplasmic acidification, free clathrin triskelions within the cytosol are depleted and all of the clathrin becomes associated with the membrane. Under these conditions coated pit associated AP-2 remains while the smaller AP-2 aggregates, or coated pit nucleation sites, dissociate. This indicates that the stabilization of AP-2 coated pit nucleation sites requires the presence of free clathrin triskelions within the cytosol. Furthermore, this indicates that free clathrin is required for the early stages of coated pit formation and presumably the continuation of the clathrin-mediated endocytic process. We also provide indirect evidence that AP-2 binding to the membrane in coated pit nucleation sites may be regulated in part by binding to internalization-competent membrane receptors.     

A head-activator binding protein is present in hydra in a soluble and a membrane-anchored form.

The neuropeptide head activator plays an important role for proliferation and determination of stem cells in hydra. By affinity chromatography a 200 kDa head-activator binding protein, HAB, was isolated from the multiheaded mutant of Chlorohydra viridissima. Partial amino acid sequences were used to clone the HAB cDNA which coded for a receptor with a unique alignment of extracellular modules, a transmembrane domain, and a short carboxy-terminal cytoplasmic tail. A mammalian HAB homologue with identical alignment of these modules is expressed early in brain development. Specific antibodies revealed the presence of HAB in hydra as a transmembrane receptor, but also as secreted protein, both capable of binding head activator. Secretion of HAB during regeneration and expression in regions of high determination potential hint at a role for HAB in regulating the concentration and range of action of head activator.