Study on the relationship between erythroid differentiation and expression of blood group substances.

红系分化与血型物质表达关系的研究

• 批准号: 07671220
• 负责人: KAJII Eiji
• 金额: $ 1.47万
• 依托单位: Jichi Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07671220/
• 关键词: Red blood cell; Differentiation; Blood group system; Rh blood group; Duffy blood group; Splicing isoform; Gene; mRNA; Rh抗原; Duffy抗原; 遺伝子導入; Rh50糖蛋白; Rh変異型; mRNA; Rh蛋白; Duffy蛋白; スプライシング

Gene structure analyzes of the Rh blood group system revealed that both the RHD and RHCE genes consist of 10 exons and 9 introns that a 8kb-spacer region connects these two genes. By a systemic analysis of Rh-related cDNA isoforms based on reverse transcription-polymerase chain reaction, 11 and 5 truncated isoforms of the RhCE and RhD cDNAs, respectively. Studying on Rh-related gene expression during human erythroid differentiation exhibited the existence of isoforms inducible during erythroid maturation.We performed epitope analysis of Rh antigen by constructing retroviral gene coding 6 normal and chimera Rh cDNAs. The cDNAs were introduced into KU812E cells. The results indicated that the Rh epitopes were not constructed only with short polymorphic exofacial peptide loops but with other peptide fragments and other membrane components. Co-expression studies of RH50 and RHD or RHCE gene in non-erythroid cells, 293, did not show any Rh antigens and suggested that at least a second co-expressing factor was needed to express RhD or RhCE antigens on the plasma membrane. The gene analysis of a propositus with Rhnull phenotype confirmed the Rh50 glycoprotein as one of the co-expressing factors.We found a novel first exon and spliced form of Duffy mRNA that is the predominant transcript in both erythroid and postcapillary venule endothelium. We also identified two inverse GATA motifs in the 600 bp 5' flanking region. The proximal GATA was positionated downstream from the start position of endothelium and upstream from that of erythroid. Gel shift assay showed the proximal GATA gene is the target sequence of GATA-1. Deletion mutagenesis study revealed that this GATA motif represent the erythroid regulatory core region for Duffy gene.

Rh血型系统基因结构分析表明，RHD和RHCE基因均由10个外显子和9个内含子组成，其中8 kb的间隔区将这两个基因连接起来。通过逆转录-聚合酶链反应（RT-PCR）对RhCE和RhD相关cDNA异构体的系统分析，分别获得了11个和5个RhCE和RhD cDNA的截短异构体。对Rh相关基因在人红系分化过程中表达的研究表明，在红系成熟过程中存在可诱导的亚型，我们构建了编码6个正常和嵌合体Rh基因的逆转录病毒基因，对Rh抗原进行了表位分析。将cDNA导入KU 812 E细胞。结果表明，Rh抗原表位不仅由短的多态性外表面肽环构成，而且还由其他肽片段和其他膜组分构成。在非红系细胞中的RH 50和RHD或RHCE基因的共表达研究，293，没有显示任何Rh抗原，并表明至少需要第二个共表达因子来在质膜上表达RhD或RhCE抗原。对1例Rhnull先证者的基因分析证实了Rh 50糖蛋白是共表达因子之一，并发现了一个新的Duffy mRNA的第一外显子和剪接形式，它是红系和毛细血管后微静脉内皮细胞的主要转录本。我们还在600 bp的5'侧翼区鉴定了两个反向加塔基序。近端加塔位于内皮细胞起始位置的下游和红系细胞起始位置的上游。凝胶迁移实验表明，加塔基因的近端是加塔-1的靶序列。缺失突变研究表明，该加塔基序代表Duffy基因的红系调控核心区。

项目成果

Omi T,et al: "Identification of the truncated Durry mRNA in erytroid." Int J Hematology. (in press).

Omi T 等人：“红系中截短的 Durry mRNA 的鉴定”。

Tanaka M,Seno T,Shibata H,Okubo Y,Okuda H,Kajii E,and Utsumi R: "Genotyping for RhC/c and RhE/e by PCR using allele-specific oligonucleotide primers." Jpn J Legal Med. 51. 32-38 (1997)

Tanaka M、Seno T、Shibata H、Okubo Y、Okuda H、Kajii E 和 Utsumi R：“使用等位基因特异性寡核苷酸引物通过 PCR 进行 RhC/c 和 RhE/e 基因分型。”

LiJ,Iwamoto S,Sugimoto N,Okuda H,and Kajii E: "Dinucleotide repeat in the 3' flanking region provides a clue to the molecular evolution of the Duffy gene." Hum Genet. (in press).

LiJ、Iwamoto S、Sugimoto N、Okuda H 和 Kajii E：“3 侧翼区域的二核苷酸重复为 Duffy 基因的分子进化提供了线索。”

Eiji Kajii, et al: "Intricate combinatorial patterns of exon splicing generate multiple Rh-related isoforms in human erythroid cells." Human Genetics. 95. 657-665 (1995)

Eiji Kajii 等人：“外显子剪接的复杂组合模式在人类红系细胞中产生多种 Rh 相关亚型。”

Iwamoto S,et al: "Characterization of the Duffy gene promoter:Evidence for tissue specific abolishment of expression in Fy(a-b-) of black individuals." Biochem Biophys Res Commun. 222. 852-859 (1996)

Iwamoto S 等人：“Duffy 基因启动子的表征：黑人个体 Fy(a-b-) 中组织特异性表达消除的证据。”