Application Of The MC1 Satellite DNA On Swine 13/17 Robertsonian Translocation

Tandem repeat sequences were distributed widely in the genome of eukaryotes. At the level of full genomes , both the abundance and distribution characteristics of repeats types, such as dinucleotide repeats and trinucleitide repeats et cetera were varied in different organisms , and the variedness also occured in different repeat classes, and across inter-chromosomes, and even between coding and noncoding regions. All of these differences indicated that the genesis and evolution of tandem repeat sequences were complex , as was typical of centromeric. In order to address these problems, the study of tandem repeat sequences self-evolution, the biology function, and their application researches should be pursued.The centromeric MC1 repeated sequences were cloned and analyzed in swine. To clarify the centromeric repeated sequences characterization of 13/17 Robertsonian tr- anslocation chromosome, metaphase fluorescence in situ hybridization assays were performed with centromeric MC1 repeated sequence monomer probe. A domestic pig cosmid library was constructed and positive clones were screened and analyzed. The chromosomal translocation method was founded by metaphase fluorescence in situ hybridization assays, which made a foundation for further research on the 13/17 Robertsonian translocation in swine.MC1 satellite DNA primers were designed according to the published centrome- ric repeated sequences in domestic pig . Expected DNA fragment were amplified by PCR. The target fragments were ligated to pucm-T vector and transformed into DH5α of Escherichia coli . Integrated DNA from recombinant plasmid was identified by PCR and then sequenced. As a result, five sequences were founded including two sequences about 263 bp with 90.50% sequence identity and three sequences about 596 bp, 598 bp and 602 bp with 90.81% sequence identity . These sequences were compared with that of Sus scrofa published in GenBank by BLAST , and high homology was found .The caryotype of normal swine was 2n=38, but the heterozygote of 13/17 Rober- tsonian translocation swine was 2n=37. A new submetacentric 13/17 Robertsonian translocation chromosome was comprised of the 13 and 17 acrocentric chromosomes. The swine centromeric heterochromatin was characterised by two distinct satellite DNA families designed MC1 and AC2. The MC1 family was comprised of the metacentric chromosomes (1-12) as well as X chromosome, and their centromeres had been shown to be composed of divergent 340 bp monomer units. The AC2 family was comprised of 14 bp monomer units consisting of the acrocentric chromosomes (13-18). Mitosis metaphase chromosome preparation of the homozygotes and heterozygotes of 13/17 Robertsonian translocation individual were made by peripheral blood lymphocyte. Metaphase chromosome fluorescence in situ hybridization assays were performed with centromeric MC1 repeated sequences as probe, which labeled by digoxin. The centromeric region of meta-/submetacentric chromosomes were hybridized, but without hybridization signal on 13/17 Robertsonian translocation chromosome.It was confirmed that the centromeric region satellite DNA type of 13/17 Robertsonian translocation chromosome was not contained meta-/submetacentric MC1 satellite DNA.Genomic DNA prepared for library construction was digested by proteinase K and extracted by phenol. The DNA was randomly sheared and end-repaired by T4 DNA Polymerase and T4 Polynucleotide Kinase. 25-40 kb fragment was recovered by 1% agarose gel electrophoresis and ligated with pCC1FOS Vector. Recombinant molecules were packaged in vitro with the Phage Packaging Extract, then transformed into E.coli EPI300. A fosmid genomic library of domestic pig was successfully constructed with the titer of 8.50×105 cfu/mL in which the insert fragment size was about 25 kb. Positive clones were achieved by in stitu hybridization with MC1satellite DNA as probe. Three positive clones was identified by PCR and then sequenced. High homology of 99.17% was found by aligment among three sequences. It was indicated that the library was of high quality which can be used to screen new satellite DNA in swine.