Multiple alpha- and beta-tubulin genes in Chlamydomonas and regulation of tubulin mRNA levels after deflagellation

We constructed and characterized recombinant cDNA clones containing alpha- and beta-tubulin DNA sequences. The inserted DNA was determined to code for alpha- and beta-tubulin by positive hybridization-selection. The selected mRNA was translated in vitro, and the translation products were shown to be alpha- or beta-tubulin by comigration with flagellar alpha- and beta-tubulin on one- and two-dimensional gels and by immunoprecipitation with antibodies specific for alpha- and beta-tubulin. Hybridization of the cloned tubulin probes with Chlamydomonas DNA indicated that there are at least two genes each for alpha- and beta-tubulin in this organism. No evidence of cross-hybridization between alpha- and beta-tubulin DNA sequences was found. Because previous experiments had shown that tubulin synthesis was stimulated in response to flagellar amputation, the tubulin clones were used to analyze the levels of tubulin sequences in RNA from cells before and after deflagellation. Hybridization of the tubulin cDNA probes with total or polyadenylated RNA indicated that tubulin sequences in RNA increased within 8 min following deflagellation, reached maximal levels by 50 min and began to decrease by 80 min after deflagellation. One hybridization band was detected with use of the beta-tubulin probe, but RNA in two size classes hybridized to the alpha-tubulin probe.     

Effects of prostaglandins E2 and F2α on lecithin biosynthesis by cultured lung cells

Transformed cells from human lung carcinoma (Line A549), resembling type II pneumocytes, were cultured in monolayer at 37°C and incubated for five hours with ³H-choline and ¹⁴C-palmitate in the presence of various concentrations of prostaglandins (PGs) E₂ and F_2α. In the control (no PG) the level of % palmitate incorporation was 13.5 × as high as that of choline, after taking isotope dilution into account. Between the concentrations studied, 0.1 and 10 μM, both prostaglandins stimulated markedly the incorporation of both precursors, though choline up to 3 × better than palmitate. This was indicated by a change in the palmitate/choline incorporation ratio from 13.5 to as low as 4.2. At the lowest PG concentration, 0.1 μM, PGE₂ was much more effective than PGF_2α in stimulating the incorporation of both precursors.     

Using RAD‐seq to develop sex‐linked markers in a haplodiplontic alga

For many taxa, including isomorphic haplodiplontic macroalgae, determining sex and ploidy is challenging, thereby limiting the scope of some population demographic and genetic studies. Here, we used double‐digest restriction site‐associated DNA sequencing (ddRAD‐seq) to identify sex‐linked molecular markers in the widespread red alga Agarophyton vermiculophyllum. In the ddRAD‐seq library, we included 10 female gametophytes, 10 male gametophytes, and 16 tetrasporophytes from one native and one non‐native site (N = 40 gametophytes and N = 32 tetrasporophytes total). We identified seven putatively female‐linked and 19 putatively male‐linked sequences. Four female‐ and eight male‐linked markers amplified in all three life cycle stages. Using one female‐ and one male‐linked marker that were sex‐specific, we developed a duplex PCR and tested the efficacy of this assay on a subset of thalli sampled at two sites in the non‐native range. We confirmed ploidy based on the visual observation of reproductive structures and previous microsatellite genotyping at 10 polymorphic loci. For 32 vegetative thalli, we were able to assign sex and confirm ploidy in these previously genotyped thalli. These markers will be integral to ongoing studies of A. vermiculophyllum invasion. We discuss the utility of RAD‐seq over other approaches previously used, such as RAPDs (random amplified polymorphic DNA), for future work designing sex‐linked markers in other haplodiplontic macroalgae for which genomes are lacking.     

Stream Water Chemistry in Mixed-Conifer Headwater Basins: Role of Water Sources,Seasonality, Watershed Characteristics,and Disturbances

Ecosystems - Understanding the transport of dissolved organic carbon (DOC) and nitrogen (N) as water flows through headwater basins is important for predicting downstream water quality. With...     

In Silco Mapping of ESTs from the Turkey (Meleagris Gallopavo)

Sequence similarity was used to predict the position of expressed sequence tags (ESTs) in the genome of the turkey (Meleagris gallopavo). Turkey EST sequences were compared with the draft assembly of the chicken whole-genome sequence and the chicken EST database by BLASTN. Among the 877 ESTs examined, 788 had significant matches in the chicken genome sequence. Position of orthologous sequences in the chicken genome and the predicted position of the EST loci in the turkey genome are presented. Genetic assignments suggest a high level of accuracy for the COMPASS predictions.