[Different KEL gene mRNA transcripts in reticulocyte and non-reticulocyte cells].
OBJECTIVE
To investigate the difference of the transcripts between reticulocyte and non-reticulocyte cells in human blood.


METHODS
Genomic DNA, reticulocyte RNA and total RNA of K-, K+ and Kell-null(K0) were extracted, then PCR, reverse transcription-PCR(RT-PCR) and nested PCR followed by sequencing or cloning-sequencing were used to analyze the KEL gene mRNA exons 1-19 and exons 2-8. Four kinds of monoclonal antibodies were labeled to detect the expression of Kell glycoprotein on red cells or leukocytes with flow cytometry.


RESULTS
In reticulocyte, only one normal KEL transcript faithful to the genomic structure was found in all tested samples except K0 which had 4 different transcripts. Sequence analysis of exons 2-8 of total RNA confirmed the alternative KEL transcripts existed in different samples, mostly caused by abnormal splicing, among them, skipping of exon 3 and a 16 bp insertion of intron 6 at the beginning of exon 7 were the most frequent. Although only one band was observed after amplifying the exons 1-19 from total RNA, the sequencing result showed it was a mixture of different sequences. There was strong expression of Kell glycoprotein on red cells except K0, but no or low expression on leucocytes by flow cytometry.


CONCLUSIONS
Alternative transcripts of KEL gene exist in different cells, which would be responsible for different Kell glycoprotein expression patterns on different cells. This study suggested that reticulocyte RNA was more suitable than total RNA for molecular study of KEL gene transcription.
Wang
Lingling
L
Blood Group Reference Laboratory, Shanghai Blood Centre, Shanghai 200051, P.R. China.
Yang
Ying
Y
Wang
Chen
C
Zhang
Jiamin
J
Guo
Zhonghui
Z
Li
Qin
Q
Chen
Heping
H
Zhu
Ziyan
Z
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005091:Exons / Q000235:genetics; D015894:Genome, Human; D023281:Genomics; D006801:Humans; D007438:Introns / Q000235:genetics; D007626:Kell Blood-Group System / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000097:blood* / Q000235:genetics; D012156:Reticulocytes / Q000166:cytology / Q000378:metabolism*; D017422:Sequence Analysis, DNA
D007626:Kell Blood-Group System; D012333:RNA, Messenger; D004247:DNA