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Examines the lacA gene encoding B-galactosidase in Bacillus subtilis. Identification of the lacA gene; Information on the experiments conducted.

Presents a study in which flow dialysis and isothermal microcalorimetry was combined to study the binding of Ca2+ and Mg2+ to SynCaM, a synthetic hybrid of mammalian and plant CaM able to activate all the CaM-dependent enzymes (37). Indication that affinity constants and enthalpy and entropy changes of Ca2+ and Mg2+ interactions with SynCaM were determined; Materials and methods used in the study; Results of the study.

Studies the integrated mapping and sequencing of a 115 kb DNA fragment from Bacillus subtilis. Rationale of the shotgun approach; Random library construction and sequencing of the clones; Ordering of scaffolds by PCR; Identification of putative coding regions and transcriptional units of fragment S1; Protein sequence analysis.

Background: Pharmacological high-throughput screening (HTS) represents a powerful strategy for drug discovery in genetic diseases, particularly when the full spectrum of pathological dysfunctions remains unclear, such as in Friedreich ataxia (FRDA). FRDA, the most common recessive ataxia, results from a generalized deficiency of mitochondrial and cytosolic iron-sulfur cluster (ISC) proteins activity, due to a partial loss of frataxin function, a mitochondrial protein proposed to function as an iron-chaperone for ISC biosynthesis. In the absence of measurable catalytic function for frataxin, a cell-based assay is required for HTS assay.Methods: Using a targeted ribozyme strategy in murine fibroblasts, we have developed a cellular model with strongly reduced levels of frataxin. We have used this model to screen the Prestwick Chemical Library, a collection of one thousand off-patent drugs, for potential molecules for FRDA.Results: The frataxin deficient cell lines exhibit a proliferation defect, associated with an ISC enzyme deficit. Using the growth defect as end-point criteria, we screened the Prestwick Chemical Library. However no molecule presented a significant and reproducible effect on the proliferation rate of frataxin deficient cells. Moreover over numerous passages, the antisense ribozyme fibroblast cell lines revealed an increase in frataxin residual level associated with the normalization of ISC enzyme activities. However, the ribozyme cell lines and FRDA patient cells presented an increase in Mthfd2 transcript, a mitochondrial enzyme that was previously shown to be upregulated at very early stages of the pathogenesis in the cardiac mouse model.Conclusion: Although no active hit has been identified, the present study demonstrates the feasibility of using a cell-based approach to HTS for FRDA. Furthermore, it highlights the difficulty in the development of a stable frataxin-deficient cell model, an essential condition for productive HTS in the future. [ABSTRACT FROM AUTHOR]