Pegasus, a small terminal inverted repeat transposable element found in the white gene of Anopheles gambiae

Pegasus, a novel transposable element, was discovered as a length polymorphism in the white gene of Anopheles gambiae. Sequence analysis revealed that this 535 bp element was flanked by 8 bp target site duplications and 8 bp perfect terminal inverted repeats similar to those found in many members of the Tcl family. Its small size and lack of long open reading frames preclude protein coding capacity. Southern analysis and in situ hybridization to polytene chromosomes demonstrated that Pegasus occurs in approximately 30 copies in the genomes of An. gambiae and its sibling species and is homogenous in structure but polymorphic in chromosomal location. Characterization of five additional elements by sequencing revealed nucleotide identities of 95% to 99%. Of 30 Pegasus-containing phage clones examined by PCR, only one contained an element exceeding 535 bp in length, due to the insertion of another transposable element-like sequence. Thus, the majority, if not all, extant Pegasus elements may be defective copies of a complete element whose contemporary existence in An. gambiae is uncertain. No Pegasus-hybridizing sequences were detected in nine other anophelines and three culicines examined, suggesting a very limited taxonomic distribution.     

Identification of novel high-frequency DNA methylation changes in breast cancer

Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis. The inherent thermodynamic stability of cytosine methylation and the apparent high specificity of the alterations for disease may accelerate the development of powerful molecular diagnostics for cancer. We report a genome-wide analysis of DNA methylation alterations in breast cancer. The approach efficiently identified a large collection of novel differentially DNA methylated loci (approximately 200), a subset of which was independently validated across a panel of over 230 clinical samples. The differential cytosine methylation events were independent of patient age, tumor stage, estrogen receptor status or family history of breast cancer. The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively. Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date. The discovery of over 50 novel DNA methylation-based biomarkers of breast cancer may provide new routes for development of DNA methylation-based diagnostics and prognostics, as well as reveal epigenetically regulated mechanism involved in breast tumorigenesis.     

MMP-13 selective alpha-sulfone hydroxamates: identification of selective P1' amides

Continuing our interest in designing compounds preferentially potent and selective for MMP-13, we report on a series of hydroxamic acids with a flexible amide P1′ substituents. We identify an amide which spares both MMP-1 and -14, and shows >500 fold selectivity for MMP-13 versus MMP-2 and -8.     

MMP-13 selective α-sulfone hydroxamates: a survey of P1' heterocyclic amide isosteres

Seeking compounds preferentially potent and selective for MMP-13, we reported in the preceding Letter on a series of hydroxamic acids with a flexible benzamide tail groups.^1a Here, we replace the amide moiety with non-hydrolyzable heterocycles in an effort to improve half-life. We identify a hydroxamate tetrazole 4e that spares MMP-1 and -14, shows >400-fold selectivity versus MMP-8 and >600-fold selectivity versus MMP-2, and has a 4.8 h half-life in rats. X-ray data (1.9 Å) for tetrazole 4c is presented.     

Sorghum genome sequencing by methylation filtration

Sorghum bicolor is a close relative of maize and is a staple crop in Africa and much of the developing world because of its superior tolerance of arid growth conditions. We have generated sequence from the hypomethylated portion of the sorghum genome by applying methylation filtration (MF) technology. The evidence suggests that 96% of the genes have been sequence tagged, with an average coverage of 65% across their length. Remarkably, this level of gene discovery was accomplished after generating a raw coverage of less than 300 megabases of the 735-megabase genome. MF preferentially captures exons and introns, promoters, microRNAs, and simple sequence repeats, and minimizes interspersed repeats, thus providing a robust view of the functional parts of the genome. The sorghum MF sequence set is beneficial to research on sorghum and is also a powerful resource for comparative genomics among the grasses and across the entire plant kingdom. Thousands of hypothetical gene predictions in rice and Arabidopsis are supported by the sorghum dataset, and genomic similarities highlight evolutionarily conserved regions that will lead to a better understanding of rice and Arabidopsis.     

Role of a surface tryptophan in defining the structure, stability, and DNA binding of the hyperthermophile protein Sac7d

Sac7d is a small, chromatin protein from Sulfolobus acidocaldarius which induces a sharp kink in DNA with intercalation of valine and methionine side chains. The crystal structure of the protein-DNA complex indicates that a surface tryptophan (W24) plays a key role in DNA binding by hydrogen bonding to the DNA at the kink site. We show here that substitution of the solvent-exposed tryptophan with alanine (W24A) led to a significant loss in not only DNA binding affinity but also protein stability. The W24A substitution proved to be one of the most destabilizing surface substitutions in Sac7d. A global linkage analysis of the pH and salt dependence of stability indicated that the protein stability surface (DeltaG vs temperature, pH, and salt concentration) was lowered overall by 2 kcal/mol (from 0 to 100 degrees C, pH 0 to 7, and 0 to 0.3 M KCl). The lower free energy of unfolding could not be attributed to significant structural perturbations of surface electrostatic interactions. Residual dipolar coupling of partially aligned protein and the NMR solution structure of W24A confirmed that the surface substitution resulted in no significant change in structure. Stabilization of this hyperthermophile protein and its DNA complex by a surface cluster of hydrophobic residues involving W24 and the two intercalating side chains is discussed.