Generation of the Brucella melitensis ORFeome version 1.1.

The bacteria of the Brucella genus are responsible for a worldwide zoonosis called brucellosis. They belong to the alpha-proteobacteria group, as many other bacteria that live in close association with a eukaryotic host. Importantly, the Brucellae are mainly intracellular pathogens, and the molecular mechanisms of their virulence are still poorly understood. Using the complete genome sequence of Brucella melitensis, we generated a database of protein-coding open reading frames (ORFs) and constructed an ORFeome library of 3091 Gateway Entry clones, each containing a defined ORF. This first version of the Brucella ORFeome (v1.1) provides the coding sequences in a user-friendly format amenable to high-throughput functional genomic and proteomic experiments, as the ORFs are conveniently transferable from the Entry clones to various Expression vectors by recombinational cloning. The cloning of the Brucella ORFeome v1.1 should help to provide a better understanding of the molecular mechanisms of virulence, including the identification of bacterial protein-protein interactions, but also interactions between bacterial effectors and their host's targets

Quantitative evaluation of the expression of MAGE genes in tumors by limiting dilution of cDNA libraries.

The MAGE-A genes are expressed in tumor cells but not in healthy tissues, except in male germ line cells and in placenta. They encode tumor-specific antigens recognized by autologous cytolytic T lymphocytes (CTLs). On the basis of semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assays, 6 of the 12 members of the MAGE-A family, including MAGE-A1, were previously reported to have a high level of expression in tumors, whereas 5 other members, including MAGE-A10, were expressed at a much lower level, deemed to be insufficient for CTL recognition. However, analysis with antibodies has shown that some melanoma cell lines contain equivalent amounts of MAGE-A1 and MAGE-A10 proteins. This discrepancy appeared to be due to the low efficacy of the primers that had been used for the previous MAGE-A10 RT-PCR assays. This led us to develop a method that is independent of the efficacy of the PCR primers to evaluate MAGE-A gene expression. cDNA libraries from tumor cell lines were introduced into bacteria, of which 200 pools of about 500 bacteria were maintained in microcultures. The frequencies of the MAGE-A cDNA clones in each library were evaluated by performing PCR assays on each of these pools. The abundance of MAGE-A10 cDNAs was found to be similar to that of MAGE-A1 in 3 of the libraries that were analyzed, including 2 with high expression (1/6,400), confirming that MAGE-A10 is expressed at a high level. MAGE-A2, A3, A4, A6 and A12 cDNAs were also confirmed often to be present at a frequency of more than 1/10,000, a level of expression that should suffice for recognition of antigenic peptides encoded by these genes by cytolytic T cells. The remaining MAGE genes are either not expressed in tumors or are expressed at a very low level, with the exception of MAGE-A8 and 11, which show high expression in a very small number of tumors. This method also allowed us to isolate 5 MAGE-A cDNAs that we had not obtained previously, enabling us to delineate the exons in the sequences of genes MAGE-A5, A8, A9, A10 and A11

Generation of the Brucella melitensis ORFeome version 1.1.

The bacteria of the Brucella genus are responsible for a worldwide zoonosis called brucellosis. They belong to the alpha-proteobacteria group, as many other bacteria that live in close association with a eukaryotic host. Importantly, the Brucellae are mainly intracellular pathogens, and the molecular mechanisms of their virulence are still poorly understood. Using the complete genome sequence of Brucella melitensis, we generated a database of protein-coding open reading frames (ORFs) and constructed an ORFeome library of 3091 Gateway Entry clones, each containing a defined ORF. This first version of the Brucella ORFeome (v1.1) provides the coding sequences in a user-friendly format amenable to high-throughput functional genomic and proteomic experiments, as the ORFs are conveniently transferable from the Entry clones to various Expression vectors by recombinational cloning. The cloning of the Brucella ORFeome v1.1 should help to provide a better understanding of the molecular mechanisms of virulence, including the identification of bacterial protein-protein interactions, but also interactions between bacterial effectors and their host's targets