A single base mutation in the 5' splice site of intron 7 of the lck gene is responsible for the deletion of exon 7 in lck mRNA of the JCaM1 cell line.
The failure of signal transduction in the JCaM1 cell line was associated with the presence of an abnormal lck mRNA deleted of the exon 7 encoding for an inactive p56lck kinase. Our study of the lck mRNA from various T cell lines and from peripheral blood lymphocytes of healthy donors has revealed the presence of both complete and exon 7-deleted lck transcripts. Thus the exon 7-deleted lck transcript initially described in the JCaM1 mutant cell line, arises from an alternative splicing event occurring in each cells expressing the lck gene. Genomic DNA sequencing of the lck exons 6-8 portion from both the mutant JCaM1 and its parental Jurkat cell lines revealed as the only difference, the presence of a A to G mutation within the 5' splice site of intron 7 in the JCaM1 cell line DNA. To demonstrate the role of this point mutation in the lck pre-mRNA maturation, COS cells were transfected by lck minigenes from the Jurkat and JCaM1 cell lines. In COS cells transfected with minigene from the Jurkat cell line both lck transcripts (with and without exon 7) were observed whereas only the exon 7-spliced lck transcript was observed in COS cells transfected with minigene from the JCaM1 cell line. Thus the mutation is per se responsible for the deletion of exon 7 and the absence of complete lck mRNA in the JCaM1 cell line. Presence of a restriction site (HphI) in the 5' splice site of lck intron 7 from Jurkat DNA allowed to confirm the presence of the mutation on both alleles in the JCaM1 cell line.
Rouer
E
E
CJF97-03 Interactions Moléculaires Hôte-Pathogène, ICGM, Bâtiment G. Roussy, CHU Cochin-Port-Royal, Paris, France.
Brule
F
F
Benarous
R
R
D000818:Animals; D015139:Blotting, Southern; D019556:COS Cells; D002522:Chlorocebus aethiops; D004273:DNA, Neoplasm / Q000235:genetics; D005091:Exons / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007438:Introns / Q000235:genetics*; D019169:Jurkat Cells / Q000378:metabolism*; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000235:genetics* / Q000502:physiology; D009363:Neoplasm Proteins / Q000235:genetics* / Q000502:physiology; D017354:Point Mutation; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics*; D012334:RNA, Neoplasm / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics; D014162:Transfection
D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)