Brown W R, Kacskovics I, Amendt B A, Blackmore N B, Rothschild M, Shinde R, Butler J E
Department of Microbiology, University of Iowa, Iowa City 52242, USA.
J Immunol. 1995 Apr 15;154(8):3836-42.
Recently published genomic and cDNA sequences for porcine IgA suggested that the splice acceptor site in the C alpha 1-C alpha 2 intron was an AA rather than an AG dinucleotide. This possibility was tested in an in vitro HeLa cell splicing system using an RNA substrate corresponding to the genomic DNA with the putative AA splice site. Data indicated that splicing occurred at a cryptic AG site 12 nucleotides into the C alpha 2 domain rather than at the AA site. The possibility that swine B cells could use either site was tested by preparing the cDNAs from 13 different samples representing nine animals and amplifying the segment from the first C alpha 1 nucleotide to nucleotide 532 in C alpha 2 (genomic DNA numbering system). Analysis on a 6% polyacrylamide sequencing gel revealed two polynucleotide products in most samples that differed by the expected 12 nucleotides, suggesting that swine could use both splice sites. Sequence analysis confirmed that the shorter form was spliced at the downstream site and the larger form at the apparent upstream AA site. However, when the genomic DNA from an animal expressing only the longer polynucleotide was cloned and sequenced, the upstream splice acceptor site was AG not AA. Thus the data suggested that porcine IgA occurred in two allelic forms, designated IgAa and IgAb, which differ by an apparent G to A mutation in the last nucleotide of intron 1 resulting in a short-hinged (two amino acids, IgAb) variant, in which the downstream cryptic splice site is used, as well as a "normal-hinged" (six amino acids, IgAa) variant. Evidence that IgAa and IgAb are allelic was confirmed by genotypic analyses of progeny from matings of IgAa/IgAb heterozygotes. Evidence that both transcripts are functional was confirmed by showing that serum IgA levels were similar in animals homozygous for each variant.
最近公布的猪IgA基因组和cDNA序列表明，Cα1 - Cα2内含子中的剪接受体位点是AA而不是AG二核苷酸。利用与具有假定AA剪接位点的基因组DNA相对应的RNA底物，在体外HeLa细胞剪接系统中对这种可能性进行了测试。数据表明，剪接发生在Cα2结构域内12个核苷酸处的一个隐蔽AG位点，而不是AA位点。通过从代表9只动物的13个不同样本中制备cDNA，并扩增从第一个Cα1核苷酸到Cα2中第532个核苷酸的片段（基因组DNA编号系统），来测试猪B细胞是否可以使用这两个位点。在6%聚丙烯酰胺测序凝胶上的分析显示，大多数样本中有两种多核苷酸产物，它们相差预期的12个核苷酸，这表明猪可以使用这两个剪接位点。序列分析证实，较短的形式在下游位点剪接，较长的形式在明显的上游AA位点剪接。然而，当克隆并测序来自只表达较长多核苷酸的动物的基因组DNA时，上游剪接受体位点是AG而不是AA。因此，数据表明猪IgA以两种等位基因形式存在，命名为IgAa和IgAb，它们在第1内含子最后一个核苷酸处存在一个明显的G到A突变，导致一个短铰链（两个氨基酸，IgAb）变体，该变体使用下游隐蔽剪接位点，以及一个“正常铰链”（六个氨基酸，IgAa）变体。IgAa和IgAb是等位基因的证据通过对IgAa/IgAb杂合子交配后代的基因型分析得到证实。两种转录本都具有功能的证据通过显示每个变体纯合子动物的血清IgA水平相似得到证实。
