QUANTITATIVE GENETIC ANALYSIS OF MORPHOLOGICAL VARIATION IN AN ANTARCTIC DIATOM GROWN AT TWO LIGHT INTENSITIES1

Ten clonal isolates of Thalassiosira tumida (Janisch) Hasle were grown in duplicate semi-continuous batch cultures at 116 and 11.6 μE·m⁻²·s⁻¹ acclimated cells were harvested during exponential growth and cleaned for examination by light microscopy (LM) and scanning electron microscopy (SEM). Number of strutted processes surrounding the central annulus (SP) and average number of satellite pores per process (AVSAT) were counted using SEM on 20 valves from each culture grown in high light, for a total of 400 valves examined; number of marginal labiate processes (LP) and overall diameter (DIAM) were measured using LM on 20 valves from each culture grown in both high and low light for a total of 800 valves examined. Univariate analysis of variance showed that bottle effects resulting from microenvironmental differences between replicates were a small but significant source of variation in DIAM, LP, and SP but not AVSAT. Significant differences among clones were observed for all characters. Decreased irradiance resulted in a significant decrease in valve diameter but no significant effect on LP; no light × clone interaction was observed. Significant covariance between characters among clones was also observed; since valve diameter is known to decrease during asexual growth, the correlation coefficients for SP, AVSAT, and LP with DIAM were used to correct the data for this source of nongenetic differences between clones. Analysis of the size-corrected data showed that the proportion of total phenotypic variance in SP, LP, and AVSAT caused by genetic differences among clones was 0.14, 0.14, and 0.30, respectively. This indicates that the majority of total phenotypic variance was due to environmental or developmental causes, but that sufficient genetic variability exists to support rapid phenotypic evolution in SP, LP, and AVSAT under continued directional selection. Finally, the results of the genetic analysis revealed a high (0.82) genetic correlation between SP and LP.     

Cloning and expression of cellulase and xylanase genes in Lactobacillus plantarum

Summary Eleven cellulase genes from Gram-positive bacteria were cloned in a Lactobacillus plantarum silage inoculum. Eight of these genes were expressed as active enzymes from their original promotors and translation signals. Where tested, the enzymes produced by transformed L.plantarum had the same temperature and pH optimum as enzymes produced in the original host, or in transformed Escherichia coli. Using chloramphenicol acetyltransferase as a cell-internal marker enzyme, it could be demonstrated that at least endoglucanase D from Clostridium thermocellum was actively secreted by transformed L. plantarum. In growing L. plantarum cultures, most of the enzymes were irreversibly inactivated when the pH decreased below 4.5. If the transformed strains were to be applied as an inoculum in silage, this pH inactivation might be useful in preventing overdigestion of the crop fibre. Offprint requests to: F. Michiels     

Endocytosis in mouse blastocysts: Characterization and quantification of the fluid phase component

Fluid phase endocytosis in mouse blastocysts was characterized using the fluid phase marker, ³H-dextran, which did not bind to the membrane. This nonsaturable uptake occurred via an energy-requiring process, with only 20% accountable by diffusion as indicated by analysis at 4°C. Insulin stimulated uptake of ³H-dextran by 30% (P<0.05) over the first hr. The rate of uptake then decreased in both control and insulin-treated blastocysts. However, by 2 hr, insulin-treated blastocysts contained 38% more ³H-dextran (38%; P<0.01) than control blastocysts. Incubation of blastocysts in protein-free medium increased ³H-dextran uptake to a rate equivalent to 12% of the blastocyst volume/min (1,500 ± 240 pliter/hr), compared to 4.5% and 1.5% of the blastocyst volume/min for uptake in the presence of 0.1 g BSA/I and 10 g BSA/I, respectively. Confocal microscopic studies of fluorescently labelled dextran uptake in blastocysts, cultured in the absence of BSA, showed an increase in weak fluorescence labelling in the trophectoderm cells of blastocysts, compared to blastocysts cultured in the presence of BSA. There was no diffusion of fluorescence label into the blastocoel cavity. This is consistent with fluid being endocytosed, possibly by a large number of small pinocytic vesicles. Thus fluid-phase endocytosis in blastocysts is stimulated by insulin, increasing the delivery of nutrient-containing fluid into blastocysts. In the absence of protein, embryos also increase fluid uptake, possibly in an attempt to maintain the rate of supply of protein nutrient to trophectoderm cells. An analysis of the rate of protein delivery in both adsorbed and dissolved phases is presented, which reveals the potential for significant contributions of both phases of endocytosis to blastocyst metabolism in vivo. © 1995 Wiley-Liss, Inc.     

Direct Complementation ofChlamydomonasMutants with Amplified YAC DNA

We previously described the construction and characterization of aChlamydomonasgenomic library in yeast artificial chromosomes (YACs). Here we describe the isolation and genetic mapping of YACs at the FLA10 locus on theunichromosome as well as isolation of a YAC spanning the PF14 locus on chromosome VI. Genetic mapping of YAC end clones by RFLP analyses in interspecific crosses reveals that YACs with a physical size of 150 kb commonly span genetic intervals defined by one or two recombination events in crosses of approximately 20 tetrads. This promises to make chromosomal walking inChlamydomonasa relatively efficient enterprise. We also describe our development of a method for direct complementation of mutant genes by transformation with amplified wildtype YAC DNA. The use of positional cloning using YACs and this direct functional assay for the presence of a gene in a YAC represent powerful molecular genetic tools enabling the cloning of most anyChlamydomonasgene.     

A trait‐based approach for predicting species responses to environmental change from sparse data: how well might terrestrial mammals track climate change?

Estimating population spread rates across multiple species is vital for projecting biodiversity responses to climate change. A major challenge is to parameterise spread models for many species. We introduce an approach that addresses this challenge, coupling a trait‐based analysis with spatial population modelling to project spread rates for 15 000 virtual mammals with life histories that reflect those seen in the real world. Covariances among life‐history traits are estimated from an extensive terrestrial mammal data set using Bayesian inference. We elucidate the relative roles of different life‐history traits in driving modelled spread rates, demonstrating that any one alone will be a poor predictor. We also estimate that around 30% of mammal species have potential spread rates slower than the global mean velocity of climate change. This novel trait‐space‐demographic modelling approach has broad applicability for tackling many key ecological questions for which we have the models but are hindered by data availability.     

Simulation of forest tree species’ bud burst dates for different climate scenarios: chilling requirements and photo-period may limit bud burst advancement

This study investigates whether the assumed increase of winter and spring temperatures is depicted by phenological models in correspondingly earlier bud burst (BB) dates. Some studies assume that rising temperatures lead to an earlier BB, but even later BB has been detected. The phenological model PIM (promoter-inhibitor-model) fitted to the extensive phenological database of the German Weather Service was driven by several climate scenarios. This model accounts for the complicated mechanistic interactions between chilling requirements, temperature and photo-period. It predicts BB with a r² between 0.41 and 0.62 and a RMSE of around 1 week, depending on species. Parameter sensitivities depict species dependent interactions between growth and chilling requirements as well as photo-period. A mean trend to earlier BB was revealed for the period 2002– 2100, varying between ?0.05 and ?0.11 days per year, depending on species. These trends are lower than for the period 1951– 2009. Within climate scenario period, trends are decreasing for beech and chestnut, stagnating for birch and increasing for oak. Results suggest that not fulfilled chilling requirements accompanied by an increasing dependency on photo-period potentially limit future BB advancement. The combination of a powerful phenological model, a large scale phenological database and several climate scenarios, offers new insights into the mechanistic comprehension of spring phenology.     

Isolation and Functional Characterisation of a fads2 in Rainbow Trout (Oncorhynchus mykiss) with Δ5 Desaturase Activity

Rainbow trout, Oncorhynchus mykiss, are intensively cultured globally. Understanding their requirement for long-chain polyunsaturated fatty acids (LC-PUFA) and the biochemistry of the enzymes and biosynthetic pathways required for fatty acid synthesis is important and highly relevant in current aquaculture. Most gnathostome vertebrates have two fatty acid desaturase (fads) genes with known functions in LC-PUFA biosynthesis and termed fads1 and fads2. However, teleost fish have exclusively fads2 genes. In rainbow trout, a fads2 cDNA had been previously cloned and found to encode an enzyme with Δ6 desaturase activity. In the present study, a second fads2 cDNA was cloned from the liver of rainbow trout and termed fads2b. The full-length mRNA contained 1578 nucleotides with an open reading frame of 1365 nucleotides that encoded a 454 amino acid protein with a predicted molecular weight of 52.48 kDa. The predicted Fads2b protein had the characteristic traits of the microsomal Fads family, including an N-terminal cytochrome b5 domain containing the heme-binding motif (HPPG), histidine boxes (HDXGH, HFQHH and QIEHH) and three transmembrane regions. The fads2b was expressed predominantly in the brain, liver, intestine and pyloric caeca. Expression of the fasd2b in yeast generated a protein that was found to specifically convert eicosatetraenoic acid (20:4n-3) to eicosapentaenoic acid (20:5n-3), and therefore functioned as a Δ5 desaturase. Therefore, rainbow trout have two fads2 genes that encode proteins with Δ5 and Δ6 desaturase activities, respectively, which enable this species to perform all the desaturation steps required for the biosynthesis of LC-PUFA from C₁₈ precursors.     

Specific Laboratory Test for Diagnosis of Multiple Sclerosis

Lymphocytes from patients with multiple sclerosis are much more susceptible to the inhibitory activity of linoleic acid (0·08 mg/ml) when tested for sensitization to thyroid by the macrophage electrophoretic mobility test (91% inhibition) than are those from normal subjects (57% inhibition). Cells from patients with a variety of other neurological diseases give 47% inhibition with linoleic acid. These differences are specific for multiple sclerosis and can be used as an in-vitro diagnostic test for the disease. Nearly 43% of clinically normal near relatives of patients with multiple sclerosis show an “anomalous” figure of about 77%; in the remainder the figure is the same as in the general population (57%). An anomalous result is compatible with lifelong freedom from M.S. Possibly a congenital anomalous handling of unsaturated fatty acids is a constant feature of the disease.     

Intercoil connections of the kidney of the brine shrimp,Artemia salina

Summary Maxillary glands (kidneys) of adult brine shrimp were examined by light and electron microscopy. Within a single maxillary gland there are numerous places where adjacent coils of the efferent tubule are linked by structures designated intercoil connections. At the site of such a connection the basal laminae of the two coils are continuous, and one or more cytoplasmic processes originating from a cell of one coil form a bridge to the adjacent coil. The connecting processes are branched and interdigitate complexly with complementary processes from cells of the neighboring coil. Speculations are made about the functional significance of intercoil connections in the maxillary gland of Artemia.