Biomedical subjects

P Stoilov

Publications and source records attributed to P Stoilov.

3 recordsLinked to original sources

PubMed2000

An alternative-exon database and its statistical analysis.

We compiled a comprehensive database of alternative exons from the literature and analyzed them statistically. Most alternative exons are cassette exons and are expressed in more than two tissues. Of all exons whose expression was reported to be specific for a certain tissue, the majority were expressed in the brain. Whereas the length of constitutive exons follows a normal distribution, the distribution of alternative exons is skewed toward smaller ones. Furthermore, alternative-exon splice sites deviate more from the consensus: their 3' splice sites are characterized by a higher purine content in the polypyrimidine stretch, and their 5' splice sites deviate from the consensus sequence mostly at the +4 and +5 positions. Furthermore, for exons expressed in a single tissue, adenosine is more frequently used at the -3 position of the 3' splice site. In addition to the known AC-rich and purine-rich exonic sequence elements, sequence comparison using a Gibbs algorithm identified several motifs in exons surrounded by weak splice sites and in tissue-specific exons. Together, these data indicate a combinatorial effect of weak splice sites, atypical nucleotide usage at certain positions, and functional enhancers as an important contribution to alternative-exon regulation.

Algorithms↗

PubMed1999

The in vivo minigene approach to analyze tissue-specific splicing.

The exact mechanisms leading to alternative splice site selection are still poorly understood. However, recently cotransfection studies in eukaryotic cells were successfully used to decipher contributions of RNA elements (cis-factors), their interacting protein components (trans-factors) or the cell type to alternative pre-mRNA splicing. Splice factors often work in a concentration dependent manner, resulting in a gradual change of alternative splicing patterns of a minigene when the amount of a trans-acting protein is increased by cotransfections. Here, we give a detailed description of this technique that allows analysis of large gene fragments (up to 10-12 kb) under in vivo condition. Furthermore, we provide a summary of 44 genes currently investigated to demonstrate the general feasibility of this technique.

Alternative Splicing↗

PubMed1997

Repair pattern in the beta-globin gene cluster of human fibroblasts after ultraviolet irradiation.

We have developed a novel technique to determine repair of structurally different DNA lesions. It was used to address the question of whether DNA repair in the absence of transcription occurs in a uniformly random manner or with preferences for certain regions. Human fibroblasts were exposed to ultraviolet light (3-10 J/m2) and treated with 7.5 mM hydroxyurea to inhibit replicative DNA synthesis. During the first hours after irradiation cells were treated with 5-bromodeoxyuridine to label the regions undergoing repair, with the presumption that the regions that have been more efficiently repaired would incorporate more of the nucleoside. A 155-kb DNA sequence containing the entire human beta-globin domain was reconstructed using sequences deposited in the EMBL gene bank. Twelve uniformly long single-copy RNA probes spanning the beta-globin cluster were synthesised in vitro and immobilized on microtiter plates. They were hybridized with DNA from the irradiated cells. The amount of 5-bromodeoxyuridine, incorporated as a result of repair in the DNA fractions hybridized to the different RNA probes, was determined immunochemically using antibody to this nucleoside. By this technique we registered increased repair efficiency in the zone of the permanent scaffold attachment region at the 5'-end of the beta-globin domain during the first hours after ultraviolet irradiation. This result was confirmed and by the more conventional T4 endonuclease V technique detecting the removal of cyclobutane pyrimidine dimers.

Bromodeoxyuridine↗