Agarose slab-gel electrophoresis equipment.

Simple slab-gel molds which utilize the electrophoresis apparatus described by F. W. Studier (J. Mol. Biol.79, 237 (1973)) have been designed for pouring and running agarose slab-gels. Analytical gels in which many samples are run simultaneously facilitate the assay of many enzymes which lead to physical changes in DNA, whereas the preparative gels allow the separation of large quantities (1–20 mg) of DNA fragments.     

Expression of Telomerase and Telomere Length Are Unaffected by either Age or Limb Regeneration in Danio rerio

Background

The zebrafish is an increasingly popular model for studying many aspects of biology. Recently, ztert, the zebrafish homolog of the mammalian telomerase gene has been cloned and sequenced. In contrast to humans, it has been shown that the zebrafish maintains telomerase activity for much of its adult life and has remarkable regenerative capacity. To date, there has been no longitudinal study to assess whether this retention of telomerase activity equates to the retention of chromosome telomere length through adulthood.

Methodology/Principal Findings

We have systematically analyzed individual organs of zebrafish with regard to both telomere length and telomerase activity at various time points in its adult life. Heart, gills, kidney, spleen, liver, and intestine were evaluated at 3 months, 6 months, 9 months, and 2 years of age by Southern blot analysis. We found that telomeres do not appreciably shorten throughout the lifespan of the zebrafish in any organ. In addition, there was little difference in telomere lengths between organs. Even when cells were under the highest pressure to divide after fin-clipping experiments, telomere length was unaffected. All aged (2 year old) tissues examined also expressed active amounts of telomerase activity as assessed by TRAP assay.

Conclusions/Significance

In contrast to several other species including humans, the retention of lifelong telomerase and telomeres, as we have reported here, would be necessary in the zebrafish to maintain its tremendous regenerative capacity. The ongoing study of the zebrafish''s ability to maintain telomerase activity may be helpful in unraveling the complexity involved in the maintenance (or lack thereof) of telomeres in other species such the mouse or human.     

Tori lines mitigate seabird bycatch in a pelagic longline fishery

Reviews in Fish Biology and Fisheries - Albatross bycatch has been increasing over the past decade in the US tuna longline fishery of the central North Pacific. A controlled field...     

Constitutive and transport-related endocytotic pathways in turtle bladder epithelium

Summary Proton secretion in the urinary bladder of the fresh-water turtle is mediated by a proton pump located in the apical membrane of a population of cells characteristically rich in carbonic anhydrase. Earlier studies have demonstrated that these cells exhibit apical-membrane endocytotic and exocytotic processes which are thought to be involved in the regulation of the rate of proton transport via alterations in the number of pumps within the apical membrane. In this study, we sought to characterize these processes using two different methods. Analysis of transepithelial impedance yielded estimates of membrane capacitance which could be related to membrane area, thereby allowing one to monitor net changes in apical-membrane area resulting from changes in the net rates of endo-and exocytosis. Uptake of the fluid-phase marker FITC-dextran provided a measure of net extracellular volume uptake which was related to net rates of endocytosis. Our major conclusions are summarized as follows. The bladder cells exhibit a high baseline rate of endocytosis which appears to be a constitutive process similar to pinocytosis. This process is completely inhibited when ambient temperature is reduced to 15°C. In addition, serosal application of 0.5mm acetazolamide causes a transient increase in the rate of endocytosis, concomitant with a decrease in the rate of transport. Reduction of ambient temperature to 15°C reduces the rate of acetazolamide-induced endocytosis, but does not abolish it. Addition of 1mm serosal azide not only prevents the acetazolamide-induced increase in endocytosis, but also prevents the decrease in transport caused by acetazolamide. Azide has no effect on the baseline rate of endocytosis, nor does it prevent inhibition of carbonic anhydrase by acetazolamide. The specificity of azide, coupled with the different temperature sensitivities, demonstrate that the constitutive and transport-dependent endocytotic pathways are distinct processes. The observation that azide prevents both the acetazolamide-induced increase in endocytosis and the decrease in transport strongly supports the notion that endocytosis of proton-pump-containing membrane is requisite for the inhibition of transport by acetazolamide. Finally, the results also demonstrate that acetazolamide does not inhibit proton secretion simply by inhibiting carbonic anhydrase.     

Isolation and identification of eight microsatellite loci in the Cherokee darter (Etheostoma scotti) and their variability in other members of the genera Etheostoma, Ammocrypta, and Percina

The Cherokee darter Etheostoma scotti is a federally threatened fish endemic to the Etowah River system of northwest Georgia. In order to analyse the population structure and genetic diversity of this fish, eight tetranucleotide microsatellite genetic markers were developed. The marker set was applied to 13 additional darter species to test cross-species amplification and polymorphism. Successful amplification was obtained for all eight loci in each of the 13 other species of darters, with between seven and eight polymorphic loci per species.     

Mapping one of the 2 genes controlling lemon ray flower color in sunflower (Helianthus annuus L.)

In an F2 population of 120 plants derived from a cross between 2 breeding lines with yellow ray flowers, we observed 111 plants with yellow-colored and 9 plants with lemon-colored ray flowers. The segregation pattern fits a 15:1 (chi2(15:1) = 0.32, P > 0.5) ratio, suggesting that the lemon ray flower color is conditioned by 2 independent recessive genes that had been contributed individually by each of the parents. We sampled 111 plants from the 3 F(2:3) families displaying a 3 to 1 segregating ratio for genotyping with molecular markers. One of the genes, Yf(1), was mapped onto linkage group 11 of the public sunflower map. A targeted region amplified polymorphism marker (B26P17Trap13-68) had a genetic distance of 1.5 cM to Yf(1), and one simple sequence repeat marker (ORS733) and one expressed sequence tag (EST)-based marker (HT167) previously mapped to linkage group 11 were linked to Yf(1) with distances of 9.9 and 2.3 cM, respectively.     

CD4 cell surface downregulation in HIV-1 Nef transgenic mice is a consequence of intracellular sequestration.

The Nef gene product is a regulatory protein of HIV whose biological function is poorly understood. Nef has been thought to have a negative effect on viral replication in vitro but has been shown in studies with SIV to be necessary in the establishment of viraemia in vivo. In vitro studies in various human cell lines have shown that Nef downregulates the expression of cell surface CD4 and thus could have effects on the immune response. We have generated four transgenic mouse lines, with constructs containing two different Nef alleles under the control of CD2 regulatory elements to examine the interaction of Nef with the host immune system in vivo. In adult transgenic mice we have found marked downregulation in the level of CD4 on the surface of double positive thymocytes and a decrease in the number of CD4+ T cells in the thymus. Functional analyses have revealed a decrease in the total activation of transgenic thymocytes by anti-CD3 epsilon antibody. By specific intracellular staining of T cells in such mice we have found CD4 colocalizing with a Golgi-specific marker. These results strongly suggest a Nef mediated effect on developing CD4 thymocytes resulting from interference of Nef in the intracellular trafficking or post-translational modification of CD4.     

Cation loss during accumulation of heavy metal cations by Saccharomyces cerevisiae

Summary Cu²⁺ accumulation byS. cerevisiae resulted in rapid release of 70% of cellular K⁺, followed by a slower release of approximately 60% of cellular Mg²⁺, but little loss of Ca²⁺. Co²⁺ was accumulated in smaller quantities and caused a smaller loss of physiological cations than either Cu²⁺ or Cd²⁺. Mg²⁺ release during copper accumulation was maximal at pH 6. Mg²⁺ release during Cu²⁺ accumulation increased with temperature and salinity of the suspension.