| Nuclear dot-associated SP100 protein is a member of Promyelocytic leukaemia nuclear bodies(PML-NBs). SP100 plays an important role in making PML-NBs stable, preventing the virus, transcriptional regulation, cell apotheosis and other life activities. The study on human and mice shows that SP100 gene exist complex alternative splicing ways. So far, there haven’t been any report on porcine SP100 gene. Thus this study was designed to clone the c DNA and splice variants of porcine SP100 gene, analyze its subcellular localization. The minigene technology was used to analysis its alternative splicing. The main results we got are as follows:(1) The porcine SP100 gene and seven splice variants of it were cloned(Gen Bank accession Nos. KC538899、KC540633-5、KF017543-4、KP735962). The full length of V1 is 1498 bp with 66 bp 5’ UTR, 1161 bp CDS and 271 bp 3’UTR, expected to coding a polypeptide chain of 386 aa. V2, 3, 5-7 are formed by V1 occurs exon skipping, V4 is formed by V1 occurs exon repeat; on the corresponding polypeptide chains, V3, 4, 6 and 7 occur internal deletion, V2 and 5 occur frameshift mutations, which lead to premature terminate of translation. The polypeptide chain length of V2-7 are 66, 336, 442, 144, 272 and 308 aa respectively.(2) Subcellular localization analysis shows that V1, V3, V4, V6 and V7 are all located on cell nucleus, V2 and V5 are uniformly distributed throughout the whole cell, which shows that frameshift mutations lead to NLS loss. Subcellular localization analysis on truncation mutants of splice variant V1 shows that, V1(221-386aa)、V1(301-386aa) are located on cell nucleus, V1(1-138aa)、V1(344-386aa) are located on both cell nucleus and cytoplasm. Combined with the sequence composition situation of VI, V3, V4, V6 and V7, we make sure that NLS located on 301-343 aa of V1. Based on bioinformatics analysis, change section 334 basic amino acid(Arg, R)by site-directed mutagenesis, which determined the affect on subcellular localization of V1 when polar amino acid changes. Found that when it changes to a neutral zmino acid(Trp, W), V1 still located on cell nucleus, when it changes to a acidic amino acids(Glu, E), V1 is located on cell cytoplasm.(3) Sequence analysis reveals that, exon 2, 8 and 9 have splice site mutation, are altrenatively spliced exons, build minigene vector for this area. By the way of minigene analysis, three new splicing mutation(NV1-3)were found in this area. In order to verify the minigene result in vivo, we design specific primer to amplify NV2, amplify the endogenous transcription SP100 gene, successfully obtained a c DNA sequence include full-length coding region in NV2 spliced(named V8), which proved minigene techniques can be used to analysis the splicing varation situation in life.(4) Alternative splice V8 is formed by V1 occurs exon skipping of exon 3-8, the length ofamplified fragment is 822 bp, include a 5’UTR of 66 bp, CDS of 198 bp and a 3’UTR of 558 bp. Exon 8 loss result in the early appearance of termination code, expected encoding a polypeptide chain of 65 aa. The sequence has been submitted to the Gen Bank database under accession No. KP939097. |
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