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

从扩增产物中分离到两个Rh多肽基因，暂定名为RHPI和RhPII。两个克隆都有一个由1251个核苷酸组成的开放阅读框。与已发表的序列相比，RHPI克隆显示为单核苷酸替换，没有氨基酸替换。另一方面，RhPII基因克隆在开放阅读框上有41个核苷酸替换，导致31个氨基酸替换。通过对在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个截短的RHPI和RhPII cDNAs。这些异构体似乎是由6个RHPI和3个RhPII外显子的组合剪接而产生的。我们的结果表明，Rh相关多肽的异构体在C末端不同。因此，Rh相关基因的多个RNA剪接途径在两个Rh相关基因中操作，甚至在人类红系细胞的单个细胞谱系中也是如此。利用RT-PCR和Southern杂交分析纯化的人白细胞群体中Rh基因在mRNA上的表达。扩增产物在CD19、TCR-1、CD14、CD13、CD71、CD19、-TCR-1、CD19、CD14、CD19、TCR等细胞中扩增出单一条带，但在CD13、CD71、CD19、CD19、CD71、CD19、CD19、CD71、CD19、CD14、CD19等细胞中扩增出单一条带。另一方面，在所有这些细胞中，3‘区的PCR产物如预期的那样显示出多条额外的条带先于该条带迁移，这在每个细胞中表现出明显不同的条带集合。额外的条带似乎由RHPI-和RhPII-cDNA剪接异构体组成。这些结果表明，Rh基因的表达并不局限于人类红系。此外，不同的转录起始点可能优先用于Rh基因的表达。

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.