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NONCODDNA Résumé de rapport

Project ID: 42248
Financé au titre de: FP6-MOBILITY
Pays: Croatia

Final Activity Report Summary - NONCODDNA (Structural and functional analysis of noncoding heterochromatic DNA in insect Tribolium castaneum)

The overall aim of the project was to improve knowledge and develop an experimental skill in the field of eukaryotic genome organisation and functions, in particular of heterochromatic and centromeric regions composed of repetitive, noncoding DNA. This part of the genome plays a role in genome evolution and a numerous regulatory signals important for the temporal and developmental control of gene expression reside within it. The project predicted introduction of bioinformatics tools necessary for the survey of the whole genomes with the particular emphasis on repetitive non-coding DNA. The model organism used in the studies was beetle Tribolium castaneum, an insect of economic importance whose genome has been sequenced recently, as well as few other coleopteran insects.

Following bioinformatics approach, the repetitive sequences were planned to be further experimentally characterised concerning their expression and function. These analyses were supposed to include transfer of modern molecular biological methods and techniques such as chromatin immunoprecipitation (ChIP) and gene silencing using RNAi, as well as biochemical methods for RNA-protein interaction analysis. The introduction of new methods and technologies necessary for the project realisation was planned to be achieved through recruitment of external researchers and sending of staff members from Ru?er Bo?kovi? Institute to partner institutions. The aim of the project was also to increase cooperation between researchers involved in the fields of insect genomics. The genome assemblies of Tribolium castaneum available from the ftp site enabled comprehensive analysis of repetitive sequences, in particular satellite DNAs.

In addition, genomic clones containing elements of Tribolium castaneum satellite DNA in close vicinity to genes were identified and characterised and their distribution on chromosomes was analysed. By direct genomic sequencing structure and evolution of Tribolium castaneum satellite DNA TCAST was analysed, two major satellite subfamilies identified and compared with the clones present in genome bank. RLM-RACE method was introduced for the characterisation of satellite DNA transcripts as well as method of RNA interference (RNAi) and chromatin immunoprecipitation for the analysis of their function. Using these methods transcription start sites of satellite transcripts as well as polyadenylation sites were determined and functional significance of transcripts analysed.

The obtained results indicate the role of transcripts in establishment of heterochromatin and in kinetochore formation. Based on sequences of approximately 100 orthologous genes of T. castaneum proteins in completely sequenced genomes of Endopterygota phylogenetic trees were constructed and compared to general phylogeny of Endopterygota. Those genes that gave acceptable trees were selected, and conserved PCR-primers for amplification of nuclear protein-coding genes that could be used as phylogenetic markers in deep phylogeny of Coleoptera were constructed and used for phylogenetic studies.


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