Analysis Of Composition And Structure Of Genes In 349â… 12 BAC Positive Clone In MHC Classâ…¡b Region Of Chinese Merino Genome

Objective:A sequenced clone named 349112 BAC is located on Chinese Merino sheep MHC Classâ…¡b egion, which is digested to make 32P radioisotope labeled probe to screen cDNA expression library by phage in situ hybridization for getting some cDNA sequences. The cDNA expression library was constructed by Chinese Merino sheep peripheral immune organs. These screened cDNA sequeces are used to learn about the composition and structure of genes in the genomic DNA fragment of 349112 BAC. This work is a foundation for constructing sheep MHC gene map and researching on economic traits and disease esistance and susceptibility of sheep.Methods:First, to predict genes in the Chinese Merino sheep genomic DNA fragment which was inserted 349112 BAC clone by GENSCAN software. Second, simulated digestion in gel electrophoresis of the Chinese Merino sheep genomic DNA fragment was used for selecting suitable restriction enzyme by GeneQuest procedure in DNAStar software package. The digested fragments by the suitable restriction enzyme were used to prepare radioactive isotope 32P end-labeled DNA probe for phage in situ hybridization creening Chinese Merino sheep cDNA library. Then, a single positive clone were picked out from Chinese Merino sheep cDNA library and delivered them to Beijing Genomics Institute for sequencing. Finally, asing SeqMan and MegAlign procedures in DNAStar software package to splice sequences and get rid of dentical sequences, using VecScreen software to cancel the vector sequences, and using SIM4 Software to ocate these available cDNA sequences on the Chinese Merino sheep genomic DNA fragment for researching composition and structure of genes in the genomic DNA fragment.Results:1. To predict genes of the Chinese Merino sheep 150kb genomic DNA fragment which was nserted in 349112 BAC clone by GENSCAN software, the results show that the inserted genomic DNA Fragment contained 10 genes,9 among them were split genes, the rest one was not complete gene, and 2 imong the 9 genes were single exon gene. The predicted results provided the reference information about compositions and structures of genes for the follow-up experiments.2. The restriction enzyme BsaJ I was picked out by GeneQuest procedure in DNAStar software oackage to digest 349112 BAC clone into small fragment with sticky ends, and then marked the sticky ends sing radioactive isotope 32P by Klenow fragment to prepare radioactive probe for the phage in situ lybridization screening cDNA library. Through multiple times phage in situ hybridization experiments,19 positive clones were obtained. These 19 positive clones were extracted, phagemid cyclization and digested with EcoR I and Xhol I to check the size of inserted fragment, and then sequenced them. After a set of data processing, finally,17 different cDNA sequences were obtained.3. Aligning the 17 cDNA sequences with Chinese Merino sheep MHC Classâ…¡b region genomic DNA )y SIM4 software. The result of spliced alignmen analysis show that there were 10 sequences can be precisely targeted on genomic DNA.Conclusion:17 different cDNA sequences were obtained, through phage in situ hybridization screening ;DNA library.10 among 17 cDNA sequences could be precisely located on the Chinese Merino sheep 4HC Classâ…¡b region genomic DNA, indicating that the phage in situ hybridization to screen cDNA ibrary is an effective method for separation of gene express sequences.This work provides a foundation for constructing sheep MHC gene map and researching on economic raits and disease resistance and susceptibility of sheep.