Tamara Maes

The dTph1 transposable element family of Petunia hybrida line W138 consists of between 100 and 200 members. A strategy that allows simultaneous detection of individual elements is described. Sequences flanking dTph1 elements are amplified by means of a ligation-mediated PCR. The resulting fragments are locus-specific and can be analysed by polyacrylamide gel electrophoresis. One of the applications of Transposon Display is the isolation of dTph1-tagged genes. Fragments that co-segregate with a mutant phenotype can be extracted from the gel and reamplified, providing access to tagged genes, as demonstrated in a reconstruction experiment. Data on the molecular identification of a phenotypic mutant, isolated in a random tagging experiment is also presented. Upon sequencing, the obtained candidate fragment was found to be identical to part of the previously identified Fbp1 gene.

The nitrogen-fixing endophyte Azoarcus sp. strain BH72 infects roots of Kallar grass and rice inter- and intra-cellularly and can spread systemically into shoots without causing symptoms of plant disease. Although cellulose or its breakdown products do not support growth, this strain expresses an endoglucanase, which might be involved in infection. Sequence analysis of eglA places the secreted 34-kDa protein into the glycosyl hydrolases family 5, with highest relatedness (40% identity) to endoglucanases of the phytopathogenic bacteria Xanthomonas campestris and Ralstonia solanacearum. Transcriptional regulation studied by eglA:: gusA fusion was not significantly affected by cellulose or its breakdown products or by microaerobiosis. Strongest induction (threefold) was obtained in bacteria grown in close vicinity to rice roots. Visible sites of expression were the emergence points of lateral roots and root tips, which are the primary regions of ingress into the root. To study the role in endophytic colonization, eglA was inactivated by transposon mutagenesis. Systemic spreading of the eglA mutant and of a pilAB mutant into the rice shoot could no longer be detected by polymerase chain reaction. Microscopic inspection of infection revealed that the intracellular colonization of root epidermis cells was significantly reduced in the eglA- mutant BHE6 compared with the wild type and partially restored in the complementation mutant BHRE2 expressing eglA. This provides evidence that Azoarcus sp. endoglucanase is an important determinant for successful endophytic colonization of rice roots, suggesting an active bacterial colonization process.

Endometrial cancer (EC) is the most frequent of the invasive tumors of the female genital tract. Although usually detected in its initial stages, a 20% of the patients present with advanced disease. To date, no characterized molecular marker has been validated for the diagnosis of EC. In addition, new methods for prognosis and classification of EC are needed to combat this deadly disease. We thus aimed to identify new molecular markers of EC and to evaluate their validity on endometrial aspirates. Gene expression screening on 52 carcinoma samples and series of real-time quantitative PCR validation on 19 paired carcinomas and normal tissue samples and on 50 carcinoma and noncarcinoma uterine aspirates were performed to identify and validate potential biomarkers of EC. Candidate markers were further confirmed at the protein level by immunohistochemistry and Western blot. We identified ACAA1, AP1M2, CGN, DDR1, EPS8L2, FASTKD1, GMIP, IKBKE, P2RX4, P4HB, PHKG2, PPFIBP2, PPP1R16A, RASSF7, RNF183, SIRT6, TJP3, EFEMP2, SOCS2 and DCN as differentially expressed in ECs. Furthermore, the differential expression of these biomarkers in primary endometrial tumors is correlated to their expression level in corresponding uterine fluid samples. Finally, these biomarkers significantly identified EC with area under the receiver-operating-characteristic values ranging from 0.74 to 0.95 in uterine aspirates. Interestingly, analogous values were found among initial stages. We present the discovery of molecular biomarkers of EC and describe their utility in uterine aspirates. These findings represent the basis for the development of a highly sensitive and specific minimally invasive method for screening ECs.