Zhang H, Robison B, Thorgaard G H, Ristow S S
Washington State University Department of Animal Sciences, Pullman, WA 99164-6351, USA.
Biochim Biophys Acta. 2000 Nov 15;1494(1-2):14-22. doi: 10.1016/s0167-4781(00)00198-6.
Utilizing a splenic cDNA library and rapid amplification of cDNA 5' ends (5'-RACE), a C-type lectin gene was cloned from a homozygous cloned rainbow trout. The 1176 bp cDNA contains a 714 bp open reading frame from which a 238-amino-acid (aa) (27 kDa) protein was deduced. It was confirmed that this protein belongs to the C-type animal lectins, and is a type II membrane receptor. The predicted protein from this sequence contains a 48 aa cytoplasmic domain, a 20 aa transmembrane domain (TM), a 46 aa stalk region and a 124 aa carbohydrate-recognition domain (CRD). The stalk region contains a leucine-zipper, and an N-glycosylation site was also found in the CRD. Sequence alignment and phylogenetic analysis of the CRD indicate that the protein has similarity with human dendritic cell immunoreceptor (DCIR), gp120 binding C-type lectin (gp120BCL) and mammalian hepatic lectins. The N-terminus (aa 4-183) has similarity with NKG2, a group of C-type lectin receptors important in human natural killer cell function. The genomic DNA (gDNA) containing this gene was amplified and sequenced. The 4569 bp gDNA contains five exons and four introns. The first three exons encode the cytoplasmic domain, the TM and stalk region, respectively. Unlike the other type II C-type lectin receptors in which the CRD was encoded by three exons, the CRD of this lectin was encoded by two exons. A transposon Tc1-like fragment was found in intron III. Intron IV is composed of a simple repeat. Tissue-specific expression of the gene was studied by RT-PCR, and it was mainly expressed in spleen and peripheral blood leukocyte (PBL). Using AluI to digest the fragment containing exon I, intron I and exon II, an RFLP was produced between the sequences of this gene in two cloned fish, OSU 142 and Arlee (AR). Seventy-one doubled haploids (DH) of OSU X AR were screened, and the gene was mapped to linkage group XIV on the published map (Young et al., Genetics 148 (1998) 839).
利用脾脏cDNA文库和cDNA 5′末端快速扩增技术（5′-RACE），从纯合克隆虹鳟鱼中克隆出一个C型凝集素基因。该1176 bp的cDNA包含一个714 bp的开放阅读框，由此推导的蛋白质为238个氨基酸（aa）（27 kDa）。已证实该蛋白质属于C型动物凝集素，是一种II型膜受体。该序列预测的蛋白质包含一个48 aa的胞质结构域、一个20 aa的跨膜结构域（TM）、一个46 aa的柄区和一个124 aa的碳水化合物识别结构域（CRD）。柄区含有一个亮氨酸拉链，在CRD中也发现了一个N-糖基化位点。CRD的序列比对和系统发育分析表明，该蛋白质与人类树突状细胞免疫受体（DCIR）、gp120结合C型凝集素（gp120BCL）和哺乳动物肝脏凝集素具有相似性。N端（aa 4 - 183）与NKG2相似，NKG2是一组在人类自然杀伤细胞功能中起重要作用的C型凝集素受体。扩增并测序了包含该基因的基因组DNA（gDNA）。4569 bp的gDNA包含5个外显子和4个内含子。前三个外显子分别编码胞质结构域、TM和柄区。与其他CRD由三个外显子编码的II型C型凝集素受体不同，该凝集素的CRD由两个外显子编码。在内含子III中发现了一个类似转座子Tc1的片段。内含子IV由一个简单重复序列组成。通过RT-PCR研究了该基因的组织特异性表达，其主要在脾脏和外周血白细胞（PBL）中表达。用AluI消化包含外显子I、内含子I和外显子II的片段，在两个克隆鱼OSU 142和阿利（AR）中该基因序列之间产生了一个限制性片段长度多态性（RFLP）。筛选了71个OSU X AR的双单倍体（DH），并将该基因定位到已发表图谱（Young等人，《遗传学》148（1998）839）上的连锁群XIV。
