Functional genomics and proteomics analysis of cDNA and encoded proteins

Sammanfattning: With the identification of all genes in the human genomewithin reach, there is a natural shift in focus from geneidentification to gene characterization. Functionalcharacterization of the 60,000 - 100,000 genes expected to befound in the human genome will prove an immense challenge forthe scientific community. The success of such a huge task isnot possible to achieve with today's technologies but dependson new innovative developments including improvements andextensions of existing strategies and the creation ofcompletely new approaches. Improvements will be needed forlaboratory methods but also for bioinformatics tools, which arebecoming crucial to the success of various projects indifferent areas of molecular biology.The determination of spatiotemporal localization of proteinson a cellular and sub-cellular level at different cellularconditions, e.g. stage in cell cycle and phenotype, is animportant step in the elucidation of gene function, since itwould provide important clues to function, potentially revealinteracting proteins, and suggest further, valuable additionalexperiments. This thesis deals with the development of systems,aimed at large-scale localization of cDNA-encoded proteins.Significant efforts have thus been devoted to the developmentof robustE. coliexpression systems, suitable for high-throughputproduction and affinity purification of cDNA encoded proteins.Such proteins have been used to generate antibodies, capable ofrecognizing the native proteins, corresponding to the cDNAs.Results from characterization of two genes (TSG118 and TEKT1)by antibodies generated by these approaches are describedbelow.The Tsg118 protein was found to be mainly expressed inproliferating somatic cells and male germ cells.Immunolocalization studies showed a temporally regulatedlocalization of Tsg118 to the dense fibrillar component of thenucleolus. This nulceolar localization detected during meioticphase in germ cells and interphase in somatic cells were inmitotic somatic cells replaced by localization to the surfaceof the condensed chromosomes.The first mammalian (murine) tektin gene TEKT1 (originallydenoted MT14) was further described. Tektins are filamentousproteins found in cilia, flagella, and centrioles. Thelocalization of TEKT1 expression was studied in mouse embryo byin situhybridization. Expression of TEKT1 was found inadult brain, choroid plexus, the forming retina, and olfactoryreceptor neurons showing a striking correlation with knownpresence of either motile or primary cilia.The TEKT1 gene expression was also characterized byimmunolocalization in mouse testis. The Tekt1 protein was foundto be localized to the centrosome of round haploid germ cellsand displayed a diffuse localization to the caudal end ofelongated haploid germ cells. In earlier and later stages ofsperm development the localization of Tekt1 could not beestablished. This result indicated a temporal and spatialcorrelation of localization with the origin of sperm taildevelopment suggesting a possible role of Tekt1 in theformation of sperm flagella.The fragmented nature of cDNA libraries result in a problemof retrieving the upstream coding sequence of genes. Apolymerase chain reaction (PCR)-assisted biotin-capture methodwas devised that was performed directly on poly(A)+ RNA togenerate a full-coding sequence of a gene with only partiallyknown sequence, TSG118, and for which a full-length clone ofthe gene was not found in existing cDNA libraries.The last part of this thesis describes a software for thevisualization of global gene expression profiles. The systemprovides an automated classification of gene expressiongenerated by expressed sequence tag (EST)-sequencing, and wouldconstitute a valuable tool in global transcript analysis, whichis an important part in present functional genomicsapproaches.Key words:affinity purification, bioinformatics, cDNA,E. coliexpression, functional genomics, genecharacterization, immunolocalization, mouse testis, proteomics,rabbit antibodies, spermatogenesis, tektin, transcriptionanalysis, virtual chip.