Aleksandra Comino

Bacillus licheniformis bacitracin-resistance ABC transporter: relationship to mammalian multidrug resistance.

The nucleotide sequence of the Bacillus licheniformis bacitracin‐resistance locus was determined. The presence of three open reading frames, bcrA, bcrB and bcrC, was revealed. The BcrA protein shares a high degree of homology with the hydrophilic ATP‐binding components of the ABC family of transport proteins. The bcrB and bcrC genes were found to encode hydro‐phobic proteins, which may function as membrane components of the permease. Apart from Bacillus subtilis, these genes also confer resistance upon the Gram‐negative Escherichia coli. The presumed function of the Bcr transporter is to remove the bacitracin molecule from its membrane target. In addition to the homology of the nucleotide‐binding sites, BcrA protein and mammalian multidrug transporter or P‐glycoprotein share collateral detergent sensitivity of resistant cells and possibly the mode of Bcr transport activity within the membrane. The advantage of the resistance phenotype of the Bcr transporter was used to construct deletions within the nucleotide‐binding protein to determine the Importance of various regions in transport.

Assessing pathogenicity of MLH1 variants by co-expression of human MLH1 and PMS2 genes in yeast

BackgroundLoss of DNA mismatch repair (MMR) in humans, mainly due to mutations in the hMLH1 gene, is linked to hereditary nonpolyposis colorectal cancer (HNPCC). Because not all MLH1 alterations result in loss of MMR function, accurate characterization of variants and their classification in terms of their effect on MMR function is essential for reliable genetic testing and effective treatment. To date, in vivo assays for functional characterization of MLH1 mutations performed in various model systems have used episomal expression of the modified MMR genes. We describe here a novel approach to determine accurately the functional significance of hMLH1 mutations in vivo, based on co-expression of human MLH1 and PMS2 in yeast cells.MethodsYeast MLH1 and PMS1 genes, whose protein products form the MutLα complex, were replaced by human orthologs directly on yeast chromosomes by homologous recombination, and the resulting MMR activity was tested.ResultsThe yeast strain co-expressing hMLH1 and hPMS2 exhibited the same mutation rate as the wild-type. Eight cancer-related MLH1 variants were introduced, using the same approach, into the prepared yeast model, and their effect on MMR function was determined. Five variants (A92P, S93G, I219V, K618R and K618T) were classified as non-pathogenic, whereas variants T117M, Y646C and R659Q were characterized as pathogenic.ConclusionResults of our in vivo yeast-based approach correlate well with clinical data in five out of seven hMLH1 variants and the described model was thus shown to be useful for functional characterization of MLH1 variants in cancer patients found throughout the entire coding region of the gene.

Interaction trap experiment with CDC6.

CDC6 is an essential gene of yeast Saccharomyces cerevisiae. Although DNA sequence of the gene is available for a long time, biochemical function of Cdcó protein in the cell cycle remains unclear. Using the interaction trap experiment we were looking for proteins interacting specifically with Cdc6. Four gene products interacting with Cdc6 were detected. By sequence analysis we found that ECM11 codes for the protein involved in the cell wall synthesis, YNL201 codes for the protein of unknown function, probably involved in the carbon metabolism, YOR279 codes for protein of completely unknown function with no significant similarity with any known protein, and the interaction with Tyl retrotransposition element was also found. The strongest interaction with Cdc6 bait measured as β-galactosidase activity was observed with ECM11 and YNL201; YOR279 interacts slightly weaker. The weakest β-galactosidase activity was obtained by Ty1A element. The strongest suppression of cdc6-1 mutation was observed by Ty1A element, the slight one with ECM11 and YNL201 but no suppression of thermosensitive mutation was detected for YOR279.

Ecm11 is located in the cell nucleus throughout mitosis

Abstract It was found previously that Ecm11 interacts with Cdc6 in a two-hybrid screen. To verify that these two gene products can actually encounter in the living cell, ECM11 was expressed as a fusion with GFP. The fusion protein was located in the cell nucleus throughout all stages of mitosis. The protein was detected in discrete clusters in the nucleus. Clusters may be due to Ecm11 protein polymerisation and association with discrete structures in the nucleus. Two Ecm11 molecules interact in the two-hybrid assay, suggesting the presence of functional dimmers. Ecm11 does not promote transcription of β-galactosidase reporter gene, when fused with LexA DNA binding domain and fetch to the promoter of the reporter gene. According to our observation, we suppose that Ecm11 is a nuclear protein involved in the upholding chromatin structure.