CFTR illegitimate transcription in lymphoid cells: quantification and applications to the investigation of pathological transcripts.
Since the isolation of the cystic fibrosis transmembrane conductance regulator gene (CFTR) and the characterization of the main mutation (delta F508) in 1989, a large number of rare mutations has been found. Full screening of the CFTR gene is difficult because it is split into 27 exons covering 250 kb of genomic DNA. This gene is essentially expressed in the lung and intestinal tract, neither of which are easily accessible for routine investigations. The recent description of a faint transcription of highly tissue-specific genes in any cell, a phenomenon known as illegitimate transcription, would facilitate the research of mutations and the characterization of truncated m-RNA caused by splicing mutations. Using the polymerase chain reaction on cDNA (cDNA-PCR), we detected transcripts of the CFTR gene in lymphocytes and lymphoblast cells at a very low level (about 300 times less than in lung or intestine). This strategy allowed us to obtain a sufficient amount of cDNA-PCR product compatible with further molecular analyses. We have, therefore, analyzed a cDNA fragment overlapping exons 10 and 11 by polyacrylamide gel electrophoresis and direct sequencing, and detected the delta F508 mutation at this level. Our protocol can be generalized to the investigation of the total 4.5-kb CFTR coding sequence.
CNRS UA 1147, INSERM U 129, Paris, France.
N
N
Fonknechten
J
J
Chelly
J
J
Lepercq
A
A
Kahn
J C
JC
Kaplan
A
A
Kitzis
J C
JC
Chomel
D000818:Animals; D001483:Base Sequence; D003550:Cystic Fibrosis / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D005091:Exons; D006801:Humans; D008099:Liver / Q000201:enzymology; D008214:Lymphocytes / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D009928:Organ Specificity; D016133:Polymerase Chain Reaction / Q000379:methods; D011770:Pyruvate Kinase / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D015183:Restriction Mapping; D014158:Transcription, Genetic
C505032:CFTR protein, human; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA; D011770:Pyruvate Kinase