The role of TcsC in the phosphorylation of SakA.

<p>Protein extracts of resting conidia (RC)(panel A) and germlings (panel B) were analyzed by immunoblot using specific antibodies to phosphorylated SakA and as a loading control mitochondrial MnSOD. Extracts were prepared from germlings treated with 10 µg/ml fludioxonil and 1.2 M sorbitol for 2 and 20 min, respectively. A: parental strain AfS35, B: Δ<i>tcsC</i> mutant, C: complemented mutant.</p

Growth of the Δ<i>tcsC</i> mutant.

<p>Colonies of the AfS35 wild type and the Δ<i>tcsC</i> mutant grown for 72 h on AMM plates are shown in panels A/C and B/D, respectively. Magnifications of the edge of the colonies are depicted in panels C and D. Note the reduced number of extending hyphae in the mutant. Panel E: Quantification of the radial growth of AfS35 (black), Δ<i>tcsC</i> mutant (white) and complemented mutant colonies (gray) on AMM plates after 48 h and 96 h at 37°C or 48°C. Panel F: Quantification of the radial growth after 96 h of AfS35 (black), Δ<i>tcsC</i> mutant (white) and complemented mutant colonies (gray) on AMM plates supplemented with 1.4 M NaNO3 or 0.2 M ammonium tartrate at 37°C. The experiments shown in panels E and F were done in triplicate. Standard deviations are indicated. Student’s <i>t</i>-test: *p<0.005; **p<0.001.</p

Infection of immuno-compromized mice.

<p>Intranasal infection of cortisone-acetate treated mice infected with 1×10⁶ conidia of the Δ<i>tcsC</i> mutant (n = 20), the parental strain AfS35 (n = 20) and the complemented strain (n = 20). Controls received PBS only. Survival of mice is shown over time.</p

The impact of cAMP and light on the fluffy growth phenotype.

<p>Drop dilution assays were performed on AMM plates (supplemented with ammonium). The plates were supplemented or treated as indicated and incubated in incubator. When indicated plates were incubated under white light produced by an LED light source. Pictures were taken after 48 h at 37°C.</p

The Δ<i>tcsC</i> mutant is sensitive to hyperosmotic stress and resistant to fludioxonil.

<p>Drop dilution assays were performed on AMM plates (supplemented with ammonium). Panel A: control; B: 1.2 M sorbitol; C: 1 M KCl; D: 100 µg/ml congo red; E: 1 µg/ml fludioxonil. The depicted colonies were obtained after 48 h at 37°C. Top: AfS35; middle: Δ<i>tcsC</i>; bottom: complemented strain.</p

Impact of fludioxonil on <i>A. fumigatus</i> germ tubes.

<p>Conidia of the Δ<i>tcsC</i> mutant (panels A, C, E, G) and its parental strain AfS35 (panels B, D, F, H) were seeded on glass cover slips and incubated overnight in AMM at 30°C. The resulting germ tubes were treated with 1 µg/ml fludioxonil for 2 h (A, B), 4 h (C, D) and 6 h (E–H) at 37°C. A DAPI staining is shown in panels G and H. Arrows indicate lysed cells that lack intracellular nuclei and are associated with amorphous extracellular material. All bars represent 10 µm.</p

The role of TcsC in the stress-induced developmental program leading to a fluffy growth phenotype.

<p>Drop dilution assays were performed on AMM plates (supplemented with ammonium). Panel A: 1% oxygen; B: 2% oxygen; C: 2 mM farnesol; D: 200 µM farnesol; E: 2 mM farnesol; F: 100 mM MgSO₄; G: 100 mM CaCl₂; H: 100 mM MgSO₄; I: 50 mM CaCl₂; J: 500 mM CaCl₂. Side views of colonies from C and D are shown in panels E and F. The depicted colonies were photographed after 48 h at 37°C. AfS35 (top/left); Δ<i>tcsC</i> (middle); complemented strain (bottom/right).</p

Schematic model of the biological activities of <i>A. fumigatus</i> TcsC.

<p>Schematic model of the biological activities of <i>A. fumigatus</i> TcsC.</p

The Holstein Friesian Lethal Haplotype 5 (HH5) Results from a Complete Deletion of <i>TBF1M</i> and Cholesterol Deficiency (CDH) from an ERV-(LTR) Insertion into the Coding Region of <i>APOB</i>

<div><p>Background</p><p>With the availability of massive SNP data for several economically important cattle breeds, haplotype tests have been performed to identify unknown recessive disorders. A number of so-called lethal haplotypes, have been uncovered in Holstein Friesian cattle and, for at least seven of these, the causative mutations have been identified in candidate genes. However, several lethal haplotypes still remain elusive. Here we report the molecular genetic causes of lethal haplotype 5 (HH5) and cholesterol deficiency (CDH). A targeted enrichment for the known genomic regions, followed by massive parallel sequencing was used to interrogate for causative mutations in a case/control approach.</p><p>Methods</p><p>Targeted enrichment for the known genomic regions, followed by massive parallel sequencing was used in a case/control approach. PCRs for the causing mutations were developed and compared to routine imputing in 2,100 (HH5) and 3,100 (CDH) cattle.</p><p>Results</p><p>HH5 is caused by a deletion of 138kbp, spanning position 93,233kb to 93,371kb on chromosome 9 (BTA9), harboring only dimethyl-adenosine transferase 1 (<i>TFB1M</i>). The deletion breakpoints are flanked by bovine long interspersed nuclear elements Bov-B (upstream) and L1ME3 (downstream), suggesting a homologous recombination/deletion event. TFB1M di-methylates adenine residues in the hairpin loop at the 3’-end of mitochondrial 12S rRNA, being essential for synthesis and function of the small ribosomal subunit of mitochondria. Homozygous <i>TFB1M</i>^-/- mice reportedly exhibit embryonal lethality with developmental defects. A 2.8% allelic frequency was determined for the German HF population. CDH results from a 1.3kbp insertion of an endogenous retrovirus (ERV2-1-LTR_BT) into exon 5 of the <i>APOB</i> gene at BTA11:77,959kb. The insertion is flanked by 6bp target site duplications as described for insertions mediated by retroviral integrases. A premature stop codon in the open reading frame of <i>APOB</i> is generated, resulting in a truncation of the protein to a length of only <140 amino acids. Such early truncations have been shown to cause an inability of chylomicron excretion from intestinal cells, resulting in malabsorption of cholesterol. The allelic frequency of this mutation in the German HF population was 6.7%, which is substantially higher than reported so far. Compared to PCR assays inferring the genetic variants directly, the routine imputing used so far showed a diagnostic sensitivity of as low as 91% (HH5) and 88% (CDH), with a high specificity for both (≥99.7%).</p><p>Conclusion</p><p>With the availability of direct genetic tests it will now be possible to more effectively reduce the carrier frequency and ultimately eliminate the disorders from the HF populations. Beside this, the fact that repetitive genomic elements (RE) are involved in both diseases, underline the evolutionary importance of RE, which can be detrimental as here, but also advantageous over generations.</p></div

Lipid status of cattle grouped by CD mutation.

<p>Lipid status of cattle grouped by CD mutation.</p