Fluorescence In Situ Hybridization Technology To Build A Sketch Of A Physical Map Of The Genome Of Schistosoma Japonicum

[Objective] To construct the genome physical maps of Schistosoma japonicum using fluorescence in situ hybridization technique to confirm the localications of BAC clones on the chromosomes of S. japonicum. To obtain more useful imformation and to find the genes localized on the sex chromosomes for obtaining the clues for potential vaccine and drug targets, providing the basis for fine sequencing and improving the suitation of vaccine and drug of S. japonicum.[Method] One hundred BAC clones were randomly selected from the library of BAC clones of S. japonicum, and their DNA were extracted by Large-Construct Kit (QIAGEN company) for the FISH. The slides of metaphase chromosomes of S. japonicum were prepared using infected snails collected from Gui Chi in An Hui province. With the C-band technique,8 pairs of chromosomes of S. japonicum were recognized and the relative length and arm ratio of each chromosome were calculated based on the measurement of 20 division phases of the metaphase chromosomes. As the probes,100 BAC clone DNAs were labelled with either biotin or digoxin, or the both (double FISH) and hybridized with the metaphase chromosomes, then were reacted with FITC-avidin or rhodamine -anti- digoxin which was as the antibody. The signals of the localication of BAC clones were observed after the chromosomes counterstained by DAPI. The gene prediction and function annotations were carried out for the 9 BAC clones which were completely sequences.[Results] The DNAs of S. japonicum were extracted from the 100 BAC clones randomly selected for the FISH. Through prediction, the size of the 100 BAC clones randomly selected was found to be 75kb-140kb. A total of 300 slides of metaphase chromosomes of S. japonicum were prepared using the infected snails. The karyotype formula of chromosomes of S. japonicum was found to be 2n=6m+6st+ 4sm(ZZ) and 2n=5m+6st+5sm (ZW). Eight pairs of chromosomes were recognized by C-band, and the ideograph of C-band of the chromosomes was established. By the FISH technique,97 BAC clones were localized resperctively on seven pairs of autosomal and one pair of sex chromosomes of the parasite. The signals of 97 BAC clones were observed on the schistosome chromosomes 1 (22 clones),2(14 clones), 3(14 clones),4(9 clones),5(3 clones),6(3 clones),7(3 clones), Z and W (38 clones). By using the double FISH, localization was made for 20 BAC clones in 9 supercontigs, and it was found that not all BAC clones from one single supercontig were localized on the same region of the same chromosome. The gene prediction and function annotations were carried out for the 9 BAC clones selevted, of which the full length had been sequenced. In each BAC clone,12-25 proteins sequences were identified with the size larger than 90 amino acid residues.[Conclusion] Using the FISH and double FISH technique, the genome physical maps of S. japonicum was constructed for the first time. The S. japonicum has 7 pairs of autosomal and one pair of sex chromosomes. According to the size, the 8 pairs of chromosomes may be divided into 3 groups, i.e. chromosome 1 and sex chromosomes were in the group 1, chromosome 2,3 and 4 were in the group 2, and chromosome 5,6 and 7 were in the group 3. From the preliminary study, the measurement of arm ratio showed the karyotype formula of chromosomes of S. japonicum was 2n= 6m+6st+4sm (ZZ) and 2n= 5m+6st+5sm (ZW), which means the sex chromosome Z should categorized as the m (median region), and the W as the sm (submedian region), different from the sex chromosomes of S.mansoni. A total of 97 BAC clones randomly selected from the genome library were localized resperctively on 8 pairs of chromosomes of S. japonicum for physical mapping. On the sex chromosomes,38 BAC clones were localized and their sequecnces will be useful for finding of targets for new drugs and vaccines of S. japonicum. Interestingly, the fact that not all the BAC clones from one single supercontig were localized on the same region of the same chromosome by double FISH may imply the assembling of the working draft of 5. japonicum fenome remained to be improved. Through the gene prediction and function annotations, about 12-25 proteins sequences in each BAC clone from the 9 BAC clones with full length of the sequences were identified and may be informative for further mining of genes of interests.