Structure-function analysis of Escherichia coli translation initiation factor IF3: tyrosine 107 and lysine 110 are required for ribosome binding.

Translation initiation factor IF3 is required for peptide chain initiation in Escherichia coli. IF3 binds directly to 30S ribosomal subunits ensuring a constant supply of free 30S subunits for initiation complex formation, participates in the kinetic selection of the correct initiator region of mRNA, and destabilizes initiation complexes containing noninitiator tRNAs. The roles that tyrosine 107 and lysine 110 play in IF3 function were examined by site-directed mutagenesis. Tyrosine 107 was changed to either phenylalanine (Y107F) or leucine (Y107L), and lysine 110 was converted to either arginine (K110R) or leucine (K110L). These single amino acid changes resulted in a reduced affinity of IF3 for 30S subunits. Association equilibrium constants (M-1) for 30S subunit binding were as follows: wild-type, 7.8 x 10(7); Y107F, 4.1 x 10(7); Y107L, 1 x 10(7); K110R, 5.1 x 10(6); K110L, < 1 x 10(2). The mutant IF3s were similarly impaired in their abilities to specifically select initiation complexes containing tRNA(fMet). Toeprint analysis indicated that 5-fold more Y107L or K110R protein was required for proper initiator tRNA selection. K110L protein was unable to mediate this selection even at concentrations up to 10-fold higher than wild type. The results indicate that tyrosine 107 and lysine 110 are critical components of the ribosome binding domain of IF3 and, furthermore, that dissociation of complexes containing noninitiator tRNAs requires prior binding of IF3 to the ribosomes.     

Structural organization and polymorphism of the alpha satellite DNA sequences of chromosomes 13 and 21 as revealed by pulse field gel electrophoresis

Summary More than 30 unrelated individuals were analysed by pulse field gel electrophoresis for the alphoid centromeric sequences of chromosomes 13 and 21. These individuals had DNA patterns all different from each other and were most probably heterozygous at both loci. When several nuclear families were analysed in this manner, segregation was shown to be Mendelian, and no recombination event was detected over the 150 meioses scored in this study. Alphoid DNA sequences, therefore, constitute highly polymorphic centromeric markers, which can be used in linkage analysis for loci close to the centromeres of chromosomes 13 and 21.     

Construction and characterization of a partial library of yeast artificial chromosomes from human chromosome 21

We report a protocol for cloning large DNA fragments in yeast artificial chromosomes (YAC). A partial library has been constructed from a somatic hybrid containing chromosome 21 as the single source of human DNA. About 4.0 Mb of human DNA was recovered in 17 YAC clones. Three clones were analyzed by in situ hybridization and mapped on chromosome 21. One clone hybridized with the chromosome 21 centromeric region and may provide new insight both on the molecular structure of centromere and on the localization of Alzheimer disease gene.     

New pyrrole-based histone deacetylase inhibitors: binding mode, enzyme- and cell-based investigations

Aroyl-pyrrolyl-hydroxy-amides (APHAs) are a class of synthetic HDAC inhibitors described by us since 2001. Through structure-based drug design, two isomers of the APHA lead compound 1, the 3-(2-benzoyl-1-methyl-1H-pyrrol-4-yl)-N-hydroxy-2-propenamide 2 and the 3-(2-benzoyl-1-methyl-1H-pyrrol-5-yl)-N-hydroxy-2-propenamide 3 (iso-APHAs) were designed, synthesized and tested in murine leukemia cells as antiproliferative and cytodifferentiating agents. To improve their HDAC activity and selectivity, chemical modifications at the benzoyl moieties were investigated and evaluated using three maize histone deacetylases: HD2, HD1-B (class I human HDAC homologue), and HD1-A (class II human HDAC homologue). Docking experiments on HD1-A and HD1-B homology models revealed that the different compounds selectivity profiles could be addressed to different binding modes as observed for the reference compound SAHA. Smaller hydrophobic cap groups improved class II HDAC selectivity through the interaction with HD1-A Asn89-Ser90-Ile91, while bulkier aromatic substituents increased class I HDAC selectivity. Taking into account the whole enzyme data and the functional test results, the described iso-APHAs showed a behaviour of class I/IIb HDACi, with 4b and 4i preferentially inhibiting class IIb and class I HDACs, respectively. When tested in the human leukaemia U937 cell line, 4i showed altered cell cycle (S phase arrest), joined to high (51%) apoptosis induction and significant (21%) differentiation activity.     

High taxonomic level fingerprint of the human intestinal microbiota by Ligase Detection Reaction - Universal Array approach

Background  

Affecting the core functional microbiome, peculiar high level taxonomic unbalances of the human intestinal microbiota have been recently associated with specific diseases, such as obesity, inflammatory bowel diseases, and intestinal inflammation.