Objective
The long term goal of this project is the physical and functional mapping of the human chromosome 5 critical region q23-32. m is goal falls within the larger scope of the original proposal P930070, which is the identification, isolation and characterization of important genes that reside on human chromosome 5 and play an important role on regulating the growth of the cells and exhibiting as well the additional property of suppression. The main aim of our laboratory is the physical mapping by the construction of YAC and cosmid contigs covering the critical region 5q31-33. The specific phases of the project are the following: 1. Analysis of the primary structure of the region 5q31-33, the design of STS derived from this region. 2. Screening with the help of STSs selected from the YAC library of human DNA. 3. The selection of cosmid clones completely overlapping the YAC clones selected by the STSs.
4. The building of one contig or several ones from the selected cosmid clones overlapping the region of interest. During the first phase STSs will be designed for all the markers, preferrably human-specific, since eventual construction of YAC and cosmid contigs will be carried in DNA from mouse x human somatic cell hybrid carrying the fragments of human chromosome 5. In the second phase isolated DNA from all the 164 pools of a YAC library of human DNA will be screened with the help of STSs using PCR.
The detection of 3-6 positive YAC clones overlapping up to 1 million base pairs could be expected in the case of each marker taking account of fivefold coverage of the human genome by the YAC library. Preliminary screening of using marker D5S119 has resulted in 3 positive pools. In the third phase we will proceed to the construction of YAC contigs using pulsed field gel electrophoresis (PFGE). The degree of overlapping will be determined by restriction fingerprints analysis.
Furthermore, using a combination of several methodological approaches (cross-hybridaziation of terminal probes, restriction fingerprint analysis and a 'walk on chromosome' technique) we will construct several cosmid contigs connected by YAC clones between each other. The mutual arrangement of these contigs could be defined by the method of blotthybridization with the products of YAC clone DNA, taking into account their mutual. The above approaches will yield a concrete physical map of the area 5q31-33. Additionally we will proceed to the functional mapping of the 5q23-33 region by the isolation and characterization of the region-specific cDNA clones, followed by the preparation of monospecific antisera against the cDNA expression products. These studies are expected to complement the ongoing research within the framework of the original proposal and will provide novel information regarding the physical and funtional mapping of the critical region 5q23-32, which contains important genes, including tumor suppressor genes.
Call for proposal
Data not availableFunding Scheme
CSC - Cost-sharing contractsCoordinator
71110 Heraklion
Greece