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Sequence analysis of Rh polypeptide cDNAs and its application to forensic practice

Rh多肽cDNA序列分析及其在法医学实践中的应用

基本信息

批准号：
05807039
负责人：
KAJII Eiji
金额：
$ 1.22万
依托单位：
Jichi Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05807039/
关键词：
Rh blood group Polypeptide Gene Cloning Splicing Isoform Rhシステム Rhポリペプチド多型赤血球白血球 Rh式血液型

项目摘要

Two Rh polypeptide cDNA have been isolated from the PCR products and tentatively designated RhPI cDNA and RhPII cDNA.Both cDNA clones have an open reading frame composed of 1251 nucleotides. The RhPI cDNA clone shows a single nucleotide substitution with no amino acid substitution compared with the published sequence. The RhPII cDNA clone, on the other hand, differs from the above by 41 nucleotide substitutions with the open reading frame, resulting in 31 amino acid substitutions.By a systemic analysis of Rh-related mRNA isoforms expressed in reticulocytes, eleven and five truncated isoforms of the RhPI and RhPII cDNAs, respectively, were identified. These isoforms appear to be generated by combinatorial splicing of six RhPI and three RhPII exons. Our results suggest that the Rh-related polypeptide isoforms differing at the C terminus. Multiple RNA splicing pathway are thus operative in the two Rh-related genes even within a single cell lineage of human erythroid cells.The expression of the Rh gene at the mRNA was analyzed in purified populations of human leukocytes by using RT-PCR followed by Southern blot analysis. The PCR products of the 5^1-terminal region showed a single band as expected in the CD19^+TCR-1^- and CD14^+ cells but doubtfully in the CD13^+CD71^- and CD19^-TCR-1^+ cells. On the other hand, in all these cells, the PCR products of the 3' region exhibited multiple additional bands migrating ahead of the band as expected, which showed distinctly different sets of bands in each cell. The additional bands appeared to consist of RhPI-and RhPII-cDNA splicing isoforms. These results indicated that the expression of the Rh gene is not restricted to human erythroid lineage. Additionally, it was suggested that different transcription initiation sites might be utilized preferentially for Rh gene expression.

从PCR产物中分离到两个Rh多肽cDNA，分别命名为RhPI cDNA和RhPII cDNA，这两个cDNA克隆的开放阅读框均为1251个核苷酸。RhPI cDNA克隆与已发表的序列相比显示单核苷酸取代，没有氨基酸取代。另一方面，RhPII cDNA克隆与上述不同之处在于开放阅读框架的41个核苷酸替换，导致31个氨基酸替换。通过对网织红细胞中表达的Rh相关mRNA亚型的系统分析，分别鉴定了11个和5个RhPI和RhPII cDNA的截短亚型。这些亚型似乎是由6个RhPI和3个RhPII外显子的组合剪接产生的。我们的研究结果表明，Rh相关的多肽异构体不同的C端。本研究采用RT-PCR和Southern杂交技术分析了Rh基因在人白细胞中的表达情况。5^1-末端区域的PCR产物显示出单一条带，正如在CD 19 ^+TCR-1^-和CD 14 ^+细胞中所预期的那样，但在CD 13 ^+ CD 71 ^-和CD 19 ^-TCR-1^+细胞中则不确定。另一方面，在所有这些细胞中，3'区的PCR产物显示出多个额外的条带，如预期的那样迁移到条带之前，这在每个细胞中显示出明显不同的条带组。额外的条带似乎由RhPI-和RhPII-cDNA剪接同种型组成。这些结果表明Rh基因的表达不限于人红系。此外，研究表明Rh基因表达可能优先利用不同的转录起始位点。