Genetic diversity of the genus Malus and implications for linkage mapping with SNPs

Knowledge about the sequence-based genetic diversity of a crop species is important in order to develop highly informative genotyping assays, which will eventually positively impact breeding practice. Diversity data were obtained from two pools of 185 and 75 accessions each, representing most of the species belonging to the genus Malus, by re-sequencing 27 gene-specific amplicons and by screening 237 Malus × domestica SNPs using the multiplex genotyping technology SNPlex™. Nucleotide diversity and insertion/deletion rates in M. × domestica were estimated as π = 0.0037 and 1/333 bp, respectively. The SNP frequency was estimated as 0.0194 (1 SNP/52 bp) while within a single apple cultivar an average of one SNP in every 455 bp was found. We also investigated transferability (T _SNP) of the heterozygous state of SNPs across the species M. × domestica and the genus Malus. Raw re-sequencing showed that 12–15% of M. × domestica SNPs are transferable to a second M. × domestica cultivar, however T _SNP rose to ∼41% with SNPs selected for high minor allele frequency. T _SNP of chosen SNPs averaged ∼27% in the two M. × domestica-related species, Malus sieversii and Malus sylvestris, but was much lower in more distantly related species. On the basis of T _SNP, simulations, and empirical results, we calculated that a close-design, multiplexed genotyping array with at least 2,000 SNPs is required for building a highly saturated linkage maps within any M. × domestica cross. The same array would gradually lose informativeness in increasingly phylogenetically distant Malus species.     

Avoidance of protein oxidation correlates with the desiccation and radiation resistance of hot and cold desert strains of the cyanobacterium Chroococcidiopsis

To investigate the relationship between desiccation and the extent of protein oxidation in desert strains of Chroococcidiopsis a selection of 10 isolates from hot and cold deserts and the terrestrial cyanobacterium Chroococcidiopsis thermalis sp. PCC 7203 were exposed to desiccation (air-drying) and analyzed for survival. Strain CCMEE 029 from the Negev desert and the aquatic cyanobacterium Synechocystis sp. PCC 6803 were further investigated for protein oxidation after desiccation (drying over silica gel), treatment with H₂O₂ up to 1 M and exposure to γ-rays up to 25 kGy. Then a selection of desert strains of Chroococcidiopsis with different survival rates after prolonged desiccation, as well as Synechocystis sp. PCC 6803 and Chroococcidiopsis thermalis sp. PCC 7203, were analyzed for protein oxidation after treatment with 10 and 100 mM of H₂O₂. Results suggest that in the investigated strains a tight correlation occurs between desiccation and radiation tolerance and avoidance of protein oxidation.

     

Functional ultrasound imaging of the brain

We present functional ultrasound (fUS), a method for imaging transient changes in blood volume in the whole brain at better spatiotemporal resolution than with other functional brain imaging modalities. fUS uses plane-wave illumination at high frame rate and can measure blood volumes in smaller vessels than previous ultrasound methods. fUS identifies regions of brain activation and was used to image whisker-evoked cortical and thalamic responses and the propagation of epileptiform seizures in the rat brain.     

Synthesis of 6-thio pseudo glycolipids and their orientation on a gold slide studied by IRRAS

We have synthesized four 6-thio pseudo glycolipid analogues and assessed how two of them self-assembled on a gold surface. These structures were designed as candidate tethers molecules to anchor bilayer lipid membranes on gold. 6-Deoxy-6-thiogalactose was chosen to anchor the macromolecule to the gold and define an aqueous zone at the gold surface. A long alkane chain (C-12 or C-18) linked to the anomeric position of the sugar residue was chosen to anchor a bilayer lipid membrane. The linkage between the carbohydrate and the hydrophobic chains is either a glycosidic bond or a 1,4-disubstituted triazole formed by copper(I)-catalysed alkyne-azide cycloaddition (CuAAC) of the propargyl glycoside with azido-dodecane and azido-octadecane. We are expecting that the hydrocarbon chains will orient themselves perpendicular to the gold surface and be incorporated into the first leaflet of the bilayer membrane. We have studied self assembled monolayers of the C-12 aglycone analogues on gold using infrared reflection absorption spectroscopy (IRRAS). We compared the results given by the IRRAS experiments to the IR spectra recorded by attenuated total reflection (ATR) spectroscopy on films of the randomly oriented analogues. Our results demonstrate that the C-12 analogues did bind to gold and did orient themselves perpendicular to the gold slide.     

Circadian rhythm-dependent alterations of gene expression in Drosophila brain lacking fragile X mental retardation protein

Fragile X syndrome is caused by the loss of the FMR1 gene product, fragile X mental retardation protein (FMRP). The loss of FMRP leads to altered circadian rhythm behaviors in both mouse and Drosophila; however, the molecular mechanism behind this phenomenon remains elusive. Here we performed a series of gene expression analyses, including of both mRNAs and microRNAs (miRNAs), and identified a number of mRNAs and miRNAs (miRNA-1 and miRNA-281) with circadian rhythm-dependent altered expression in dfmr1 mutant flies. Identification of these RNAs lays the foundation for future investigations of the molecular pathway(s) underlying the altered circadian rhythms associated with loss of dFmr1.     

Escherichia coli “TatExpress” strains export several g/L human growth hormone to the periplasm by the Tat pathway

Escherichia coli is a heavily used platform for the production of biotherapeutic and other high-value proteins, and a favored strategy is to export the protein of interest to the periplasm to simplify downstream processing and facilitate disulfide bond formation. The Sec pathway is the standard means of transporting the target protein but it is unable to transport complex or rapidly folding proteins because the Sec system can only transport proteins in an unfolded state. The Tat system also operates to transport proteins to the periplasm, and it has significant potential as an alternative means of recombinant protein production because it transports fully folded proteins. Here, we have tested the Tat system's full potential for the production of biotherapeutics for the first time using fed-batch fermentation. We expressed human growth hormone (hGH) with a Tat signal peptide in E. coli W3110 “TatExpress” strains that contain elevated levels of the Tat apparatus. This construct contained four amino acids from TorA at the hGH N-terminus as well as the initiation methionine from hGH, which is removed in vivo. We show that the protein is efficiently exported to the periplasm during extended fed-batch fermentation, to the extent that it is by far the most abundant protein in the periplasm. The protein was shown to be homogeneous, disulfide bonded, and active. The bioassay showed that the yields of purified periplasmic hGH are 5.4 g/L culture whereas an enzyme-linked immunosorbent assay gave a figure of 2.39 g/L. Separate analysis of a TorA signal peptide linked to hGH construct lacking any additional amino acids likewise showed efficient export to the periplasm, although yields were approximately two-fold lower.