Algorithms and software for support of gene identification experiments

MOTIVATION: Gene annotation is the final goal of gene prediction algorithms. However, these algorithms frequently make mistakes and therefore the use of gene predictions for sequence annotation is hardly possible. As a result, biologists are forced to conduct time-consuming gene identification experiments by designing appropriate PCR primers to test cDNA libraries or applying RT-PCR, exon trapping/amplification, or other techniques. This process frequently amounts to 'guessing' PCR primers on top of unreliable gene predictions and frequently leads to wasting of experimental efforts. RESULTS: The present paper proposes a simple and reliable algorithm for experimental gene identification which bypasses the unreliable gene prediction step. Studies of the performance of the algorithm on a sample of human genes indicate that an experimental protocol based on the algorithm's predictions achieves an accurate gene identification with relatively few PCR primers. Predictions of PCR primers may be used for exon amplification in preliminary mutation analysis during an attempt to identify a gene responsible for a disease. We propose a simple approach to find a short region from a genomic sequence that with high probability overlaps with some exon of the gene. The algorithm is enhanced to find one or more segments that are probably contained in the translated region of the gene and can be used as PCR primers to select appropriate clones in cDNA libraries by selective amplification. The algorithm is further extended to locate a set of PCR primers that uniformly cover all translated regions and can be used for RT-PCR and further sequencing of (unknown) mRNA.      

Intracellular localization of regulatory proteins of the German cockroach Blattella germanica densovirus

The use of western blot analysis of nuclear and cytoplasmic extracts of BgDNV densovirusinfected German cockroach, Blattella germanica, the intracellular localization of the regulatory proteins of the corresponding densovirus was investigated in cell culture. It was demonstrated that two proteins, namely NS1 and NS3, were predominantly localized in the nucleus, whereas NS2 protein was equally distributed in the nuclei and the cytoplasm. The data obtained are important for understanding the potential functions of densovirus regulatory proteins. The intracellular localization of NS3 protein was determined for the first time for any densovirus.     

Domain organization of the ORF2 C-terminal region of the German cockroach retroposon R1

Using cosmid vector, a gene library of German cockroach Blattella germanica was constructed. From this library, clones containing full-length copies of two subfamilies of R1 retroposons were selected. Retroposons R1 of German cockroach belonging to different subfamilies were shown to be different in domain organization of the ORF2 C-terminal region. For the first time, retroposons transmembrane domains were identified in the sequences of R1. It was demonstrated that two retroposon R1 subfamilies of German cockroach arose as a result of intragenomic divergence rather than via horizontal transfer of alien mobile element into cockroach genome. The differences in domain organization appeared not as a result of saltatory recombination processes, but as a consequence of gradual mutation accumulation, which led to either degeneration, or to domain formation.