Leyva-Baca I, Schenkel F, Martin J, Karrow N A
Centre for Genetic Improvement of Livestock, Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada, N1G 2W1.
J Dairy Sci. 2008 Jan;91(1):407-17. doi: 10.3168/jds.2007-0142.
Identification of regulatory elements in 5' regions of chemokine genes is fundamental for understanding chemokine gene expression in response to infection diseases. The CXCR1 receptor is expressed on the surface of neutrophils and interacts primarily with CXCL8 (IL-8), the most potent chemoattractant for neutrophils. The aim of this study was to characterize the 5' upstream region (2.1 kb) of the bovine CXCR1 chemokine receptor gene for polymorphism content and to identify in silico potential transcription-factor binding sites. The 5' flanking region was found by mining the NCBI GenBank (www.ncbi.nlm.nih.gov/). A DNA sequence from the whole genome shotgun sequence project with reference number AC150887.4 contained the CXCR1 5' flanking sequence. Computer analysis revealed potential binding sites for the transcription factors nuclear factor kappaB (NF-kappaB), binding factor GATA-1, barbiturate inducible element (Barbie), nuclear factor of activated T-cells, and activator protein 1. Polymorphism discovery in this region was conducted by constructing an inclusive DNA pool including 2 phenotypic extreme groups, 20 bulls with high estimated breeding values (EBV) for somatic cell score (SCS), and 20 bulls with low EBV for SCS. Independent amplicons along the 5' flanking region of bovine CXCR1 were generated for polymorphism discovery by sequencing. Three novel single nucleotide polymorphisms (SNP), CXCR1c.-344T>C, CXCR1c.-1768T>A, and CXCR1c.-1830A>G, and a previously identified SNP in the coding region, CXCR1c.777G>C, were identified. The 4 SNP were genotyped in Canadian Holstein bulls (n = 338) using tetra-primer amplification refractory mutation system (ARMS)-PCR. Average allele substitution effects were estimated to investigate associations between the 4 SNP and EBV for SCS in first, second, and third and later lactations. Multiple trait analysis revealed that the SNP CXCR1c.-1768T>A was associated with EBV for SCS in the first and second lactations and over all 3 lactations. Haplotype analysis substantiated this association with EBV for SCS in the first lactation. Given the location of SNP CXCR1c.-1768T>A and the surrounding potential binding recognition sequences for NF-kappaB, GATA-1, and Barbie transcription-factors, this SNP may be implicated in gene regulation and warrants further research.
鉴定趋化因子基因5'区域的调控元件对于理解趋化因子基因在感染性疾病反应中的表达至关重要。CXCR1受体在中性粒细胞表面表达，主要与CXCL8（白细胞介素-8）相互作用，CXCL8是对中性粒细胞最有效的趋化因子。本研究的目的是对牛CXCR1趋化因子受体基因的5'上游区域（2.1 kb）进行多态性分析，并通过计算机模拟鉴定潜在的转录因子结合位点。通过挖掘NCBI基因库（www.ncbi.nlm.nih.gov/）找到5'侧翼区域。来自全基因组鸟枪法测序项目、编号为AC150887.4的DNA序列包含CXCR1的5'侧翼序列。计算机分析揭示了转录因子核因子κB（NF-κB）、结合因子GATA-1、巴比妥酸盐诱导元件（Barbie）、活化T细胞核因子和活化蛋白1的潜在结合位点。通过构建一个包含2个表型极端组的包容性DNA池进行该区域的多态性发现，20头体细胞评分（SCS）估计育种值高的公牛和20头SCS估计育种值低的公牛。通过测序生成沿牛CXCR1 5'侧翼区域的独立扩增子用于多态性发现。鉴定出3个新的单核苷酸多态性（SNP），即CXCR1c.-344T>C、CXCR1c.-1768T>A和CXCR1c.-1830A>G，以及编码区一个先前鉴定的SNP，CXCR1c.777G>C。使用四引物扩增不应性突变系统（ARMS）-PCR对338头加拿大荷斯坦公牛中的这4个SNP进行基因分型。估计平均等位基因替代效应以研究这4个SNP与第一、第二、第三及以后泌乳期SCS的估计育种值之间的关联。多性状分析表明，SNP CXCR1c.-1768T>A与第一和第二泌乳期以及所有3个泌乳期的SCS估计育种值相关。单倍型分析证实了该SNP与第一泌乳期SCS的估计育种值之间的这种关联。鉴于SNP CXCR1c.-1768T>A的位置以及周围NF-κB、GATA-1和Barbie转录因子的潜在结合识别序列，该SNP可能与基因调控有关，值得进一步研究。
