Combinatory microarray and SuperSAGE analyses identify pairing-dependently transcribed genes in Schistosoma mansoni males, including Follistatin

Background: Schistosomiasis is a disease of world-wide importance and is caused by parasitic flatworms of the genus Schistosoma. These parasites exhibit a unique reproduction biology as the female’s sexual maturation depends on a constant pairing-contact to the male. Pairing leads to gonad differentiation in the female, and even gene expression of some gonad-associated genes is controlled by pairing. In contrast, no morphological changes have been observed in males, although first data indicated an effect of pairing also on gene transcription in males. Methodology/Principal Findings: To investigate the influence of pairing on males, we performed a combinatory approach applying SuperSAGE and microarray hybridization, generating the most comprehensive data-set on differential transcription available to date. Of 6,326 sense transcripts detected by both analyses, 29 were significantly differentially transcribed. Besides mutual confirmation, the two methods complemented each other as shown by data comparison and real-time PCR, which revealed a number of genes with consistent regulation across all methods. One of the candidate genes, follistatin of S. mansoni (SmFst) was characterized in more detail by in situ hybridization and yeast two-hybrid (Y2H) interaction analyses with potential binding partners. Conclusions/Significance: Beyond confirming previously hypothesized differences in metabolic processes between pairingexperienced (EM) and pairing-unexperienced males (UM), our data indicate that neuronal processes are involved in malefemale interaction but also TGFb-signaling. One candidate revealing significant down-regulation in EM was the TGFbpathway controlling molecule follistatin (SmFst). First functional analyses demonstrated SmFst interaction with the S. mansoni TGFb-receptor agonists inhibin/activin (SmInAct) and bone morphogenic protein (SmBMP), and all molecules colocalized in the testes. This indicates a yet unknown role of the TGFb-pathway for schistosome biology leading to male competence and a possible influence of pairing on the male gonad

The Diamond STING server

Diamond STING is a new version of the STING suite of programs for a comprehensive analysis of a relationship between protein sequence, structure, function and stability. We have added a number of new functionalities by both providing more structure parameters to the STING Database and by improving/expanding the interface for enhanced data handling. The integration among the STING components has also been improved. A new key feature is the ability of the STING server to handle local files containing protein structures (either modeled or not yet deposited to the Protein Data Bank) so that they can be used by the principal STING components: (Java)Protein Dossier ((J)PD) and STING Report. The current capabilities of the new STING version and a couple of biologically relevant applications are described here. We have provided an example where Diamond STING identifies the active site amino acids and folding essential amino acids (both previously determined by experiments) by filtering out all but those residues by selecting the numerical values/ranges for a set of corresponding parameters. This is the fundamental step toward a more interesting endeavor—the prediction of such residues. Diamond STING is freely accessible at and

The Genome of the Natural Genetic Engineer Agrobacterium tumefaciens C58

ro- teobacterium of the family Rhizobiaceae and a member of the diverse Agrobacterium genus. A ubiquitous soil organism and etiological agent of the plant disease crown gall (1), A. tumefaciens infects more than 90 families of dicotyledonous plants, resulting in major agronomic losses (2, 3). The gall results from the transfer, integration, and expression of a discrete set of genes (T-DNA) located on the tumor-inducing (Ti) plasmid. Expression of these genes leads to biosynthesis of plant growth hormones as well as a bacterial nutrient source called opines (4 ). The processing and transfer of the T-DNA is mediated by the Ti plasmid virulence (vir) genes, and several virulence determinants initially characterized in A. tumefaciens have been found in plant symbionts and animal pathogens (5--7 ). The genes within the T-DNA can be replaced by any DNA sequence, making A. tumefaciens an ideal vehicle for gene transfer and an essential tool for plant research and transgenic crop production

Selected genes significantly differentially transcribed according to both analyses.

<p>Of all transcripts detected in the SuperSAGE and microarray analyses, 29 were differentially transcribed according to both methods. Of these, 21 with functional annotations are presented here, excluding those, which were annotated as ’hypothetical protein’. Follistatin (Smp_123300) is highlighted (see also text). Genes, gene annotations, log₂(EM/UM) ratios of microarray analyses (M- log₂) and SuperSAGE (S-log₂) as well as exon/intron presence of the SuperSAGE tags (S-tag) are given.</p

Venn diagram of SuperSAGE and microarray data analyses.

<p>For final analyses we selected 9344 and 7494 genes for which sense transcripts were detected by SuperSAGE and microarray, respectively. The upper diagram (detected) presents numbers of genes, whose transcripts were identified by either one (light grey, left: SuperSAGE, 3018; dark grey, right: Microarray, 1168) or both methods (intersection; grey, middle part, 6326). The lower diagram (significantly differentially) presents genes, whose transcripts were significantly differentially (Super SAGE, p<1⁻¹⁰; Microarray, q<0.01; log₂ratios <−0.585 or >0.585) regulated between EM and UM according to at least one analysis (outside the intersection: light grey, left: SuperSAGE, 47; dark grey, right: microarray, 110; within the intersection: grey, left: 144 found as differentially transcribed according to SuperSAGE only; grey, right: 365 found as differentially transcribed according to microarray data analysis only; light orange, left, 33: found as significantly regulated by both analyses but differentially transcribed only according to SuperSAGE; light orange, right, 22: found as significantly regulated by both analyses but differentially transcribed according to microarray data analysis only). Finally, a number of 29 transcripts (dark orange, middle) were found representing genes being differentially transcribed according to the analyses of the data sets obtained by both methods.</p

SmFst <i>in situ</i>-hybridization.

<p><i>In situ</i>-hybridization experiments with sense and antisense probes resulted in variable SmFst signals, but always in the reproductive organs of couples and UM. A: UM, B: EM, C: EF, D: EF. t: testicular lobes, o: ovary, p: parenchyma, v: vitellarium, vs: ventral sucker. Scale bar: 50 µM.</p

Exemplary results from real-time PCR analyses.

<p>Represented results are sorted according to technical categories. Log₂ratios (EM/UM) are given for all biological replicas tested in either microarray, SuperSAGE or real-time PCR. grey: microarray; black: SuperSAGE; light gray: real-time PCR. Stars in graphs indicate significance in the according analysis. nd: no transcripts for this gene detected in this analysis; no: this gene is not represented by an oligo on the microarray; i: detected transcripts were aligned to a predicted intron. To ease comparison between graphs all y-axis intervals were set to 0.5.</p

SmBMP <i>in situ</i>-hybridization.

<p>Both probes detected transcripts in the reproductive organs of couples and UM. Sense and antisense transcripts were detected. The pictures are representative examples for the detection of SmBMP transcripts, independent of probes and strand. A: EM, B: UM, C: EF, D: EF. t: testicular lobes, g: gut, o: ovary, ot: ootype, p: parenchyma, v: vitellarium, vs: ventral sucker. Scale bar: 50 µM.</p

Selection of transcripts detected by microarray only.

<p>Shown are 10 of 110 genes, which were detected only in the microarrays and found to be significantly and differentially transcribed. Smp_numbers and annotations other than ‘hypothetical protein’ existed for these 10 genes only. Genes, gene annotations, log₂(EM/UM) ratios, q-values and presence or absence (yes/no) of <i>Nla</i>III restriction enzyme recognition sites are indicated.</p