RT-PCR cloning of Rab3 isoforms expressed in peritoneal mast cells.
Rab proteins are ras-like low molecular mass GTP-binding proteins, which are postulated to act as specific regulators of membrane trafficking in exocytosis and endocytosis. We have previously shown that synthetic peptides, corresponding to the effector domain of Rab3 proteins, stimulate a complete exocytotic response in mast cells. We have used a PCR-cloning strategy to investigate the presence of mRNA encoding Rab3 in mast cells. RNA based PCR was then performed on mast cell RNA using degenerate oligonucleotide primers based on two conserved sequences among Rab3 proteins. However, no PCR products were obtained, even for proteins known to be expressed in high copy numbers in mast cells (beta-actin and Fc receptor). We have found that the problem resides in the presence of mast cell secretory granule derived heparin, that copurifies with the RNA; heparin has been shown to inhibit the activity of reverse transcriptase and Taq polymerase in PCR. After treating the RNA (obtained from about 500 mast cells) with heparinase, several PCR products of varying size were obtained using primers specific for Rab3 proteins. These products were cloned and sequenced. We have found clones containing sequences that had a 100% homology at the deduced amino acid level to a portion of Rab3B and Rab3D (amino acids 16 to 83).
Oberhauser
A F
AF
Department of Physiology and Biophysics, Mayo Clinic, Rochester, MN 55905.
Balan
V
V
Fernandez-Badilla
C L
CL
Fernandez
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D015870:Gene Expression; D019763:Heparin Lyase; D017737:Macrophages, Peritoneal / Q000378:metabolism; D008407:Mast Cells / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010529:Peritoneal Cavity / Q000166:cytology*; D016133:Polymerase Chain Reaction; D011133:Polysaccharide-Lyases; D012333:RNA, Messenger / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase; D051381:Rats; D017421:Sequence Analysis; D017385:Sequence Homology; D020692:rab3 GTP-Binding Proteins
D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase; D019204:GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D011133:Polysaccharide-Lyases; D019763:Heparin Lyase