Preparation of a human DOPA decarboxylase cDNA probe by PCR and its assignment to chromosome 7.
The reverse transcription of mRNA of a human pheochromocytoma using an oligonucleotide complementary to rat DOPA decarboxylase (DDC) sequence as a primer, gave rise to a single strand cDNA. This resultant cDNA permitted by PCR amplification the preparation and cloning in PUC 19 of a human DDC probe of 747 base pairs. The choice of primer was dictated by the presence of tryptophan codons in the DOPA decarboxylase sequence which were conserved in different species throughout evolution. The presence of mismatches on the primers was not an obstacle to a specific amplification and the probe sequence was found identical to the human DDC cDNA sequence. This probe, labelled by nick translation detected on Southern blot of human-rodent hybrids, bands on human DNA after EcoRI, BamHI or HindIII digestion. The results with EcoRI were explicit and drove to the conclusion that DDC is located on chromosome 7. Five bands were obtained on human DNA digested with EcoRI, indicating that either numerous introns could interrupt the coding sequence of DDC gene or duplicated sequences could be present in chromosome 7.
Bruneau
G
G
Laboratoire de Biochimie Cellulaire, Collège de France, Paris.
Gross
M S
MS
Krieger
M
M
Bernheim
A
A
Thibault
J
J
Nguyen
V C
VC
D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D002874:Chromosome Mapping; D002897:Chromosomes, Human, Pair 7; D003001:Cloning, Molecular; D015342:DNA Probes; D004277:DNA, Single-Stranded / Q000096:biosynthesis; D004296:Dopa Decarboxylase / Q000235:genetics*; D006801:Humans; D006822:Hybrid Cells / Q000502:physiology; D008969:Molecular Sequence Data; D011061:Poly A / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger
D015342:DNA Probes; D004277:DNA, Single-Stranded; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D004296:Dopa Decarboxylase