Persson A C, Stet R J, Pilström L
Department of Cell and Molecular Biology, Immunology Programme, Uppsala University, Box 596, S-751 24 Uppsala, Sweden.
Immunogenetics. 1999 Oct;50(1-2):49-59. doi: 10.1007/s002510050685.
Degenerate polymerase chain reaction (PCR) primers based on conserved residues from alignments of species with already characterized major histocompatibility complex (MHC)-encoded sequences were used in the search for class I and beta(2)-microglobulin (b(2)m) genes in Atlantic cod (Gadus morhua L. ). After PCR amplification and subsequent sequencing a putative class I sequence was identified, from which a probe was designed and used to screen a spleen cDNA library from one single individual. The full-length clone obtained was sequenced and shown to be a classical Mhc class I-encoded sequence. It revealed the characteristic alpha1-, alpha2-, and alpha3-domains and transmembrane and cytoplasmic region, with several conserved amino acids. A PCR amplification from the alpha2-domain to the CY-region was performed on the same library, using a proof-reading enzyme. At least 11 unique additional sequences were isolated. Moreover, sequencing of the additional cDNA clones resulted in a total of 17 different Mhc class I sequences in this individual. A Southern hybridization of DNA from four different individuals using an alpha3-specific probe confirmed this large number of genes. Interestingly, based on differences mainly in their transmembrane region, the sequences obtained could be divided into two distinct groups. Within the groups no support could be obtained for any further subdivision. Southern experiments using an alpha1-specific probe gave almost the same restriction fragment length polymorphism with a high number of hybridizing bands, suggesting a low divergence in this part of the gene. Sequencing of PCR clones obtained with a proof-reading enzyme confirmed this at the nucleotide level. PCR amplification to isolate and characterize the b(2)m gene resulted in a sequence which was used to screen a thymus cDNA library. Two different alleles were obtained and these showed the characteristic features of known teleostean beta(2)m sequences. A Southern hybridization with genomic DNA from four different individuals suggested the presence of one b(2)m locus in Atlantic cod.
基于已鉴定的主要组织相容性复合体（MHC）编码序列的物种比对中的保守残基，设计了简并聚合酶链反应（PCR）引物，用于在大西洋鳕鱼（Gadus morhua L.）中寻找I类基因和β2-微球蛋白（b2m）基因。经过PCR扩增和后续测序，鉴定出一个推定的I类序列，并据此设计了探针，用于筛选来自单个个体的脾脏cDNA文库。获得的全长克隆经测序显示为经典的Mhc I类编码序列。它揭示了特征性的α1、α2和α3结构域以及跨膜和胞质区域，还有几个保守氨基酸。使用校对酶对同一文库进行从α2结构域到CY区域的PCR扩增。至少分离出11个独特的额外序列。此外，对额外的cDNA克隆进行测序后，该个体中总共得到了17种不同的Mhc I类序列。使用α3特异性探针对来自四个不同个体的DNA进行Southern杂交，证实了存在大量此类基因。有趣的是，基于主要在其跨膜区域的差异，获得的序列可分为两个不同的组。在这些组内，无法支持进一步细分。使用α1特异性探针的Southern实验给出了几乎相同的限制性片段长度多态性，有大量杂交带，表明该基因这部分的差异较小。用校对酶获得的PCR克隆测序在核苷酸水平上证实了这一点。用于分离和鉴定b2m基因的PCR扩增得到了一个序列，该序列用于筛选胸腺cDNA文库。获得了两个不同的等位基因，它们显示出已知硬骨鱼类β2m序列的特征。用来自四个不同个体的基因组DNA进行Southern杂交表明，大西洋鳕鱼中存在一个b2m基因座。
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