RT-PCR cloning of Rab3 isoforms expressed in peritoneal mast cells

Andres Oberhauser, Vijayan Balan, Carmen L. Fernandez-Badilla, Julio M. Fernandez

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

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-eloning 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 (β-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).

Original languageEnglish (US)
Pages (from-to)171-174
Number of pages4
JournalFEBS Letters
Volume339
Issue number1-2
DOIs
StatePublished - Feb 14 1994
Externally publishedYes

Fingerprint

rab3 GTP-Binding Proteins
Cloning
Mast Cells
Organism Cloning
Protein Isoforms
RNA
Polymerase Chain Reaction
Heparin
Heparin Lyase
Taq Polymerase
Amino Acids
ras Proteins
DNA Primers
Fc Receptors
RNA-Directed DNA Polymerase
Molecular mass
GTP-Binding Proteins
Actins
Conserved Sequence
Exocytosis

Keywords

  • Heparin inhibition
  • PCR cloning
  • Peritoneal mast cell
  • Rab3 protein
  • RT-PCR

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

RT-PCR cloning of Rab3 isoforms expressed in peritoneal mast cells. / Oberhauser, Andres; Balan, Vijayan; Fernandez-Badilla, Carmen L.; Fernandez, Julio M.

In: FEBS Letters, Vol. 339, No. 1-2, 14.02.1994, p. 171-174.

Research output: Contribution to journalArticle

Oberhauser, A, Balan, V, Fernandez-Badilla, CL & Fernandez, JM 1994, 'RT-PCR cloning of Rab3 isoforms expressed in peritoneal mast cells', FEBS Letters, vol. 339, no. 1-2, pp. 171-174. https://doi.org/10.1016/0014-5793(94)80409-5
Oberhauser, Andres ; Balan, Vijayan ; Fernandez-Badilla, Carmen L. ; Fernandez, Julio M. / RT-PCR cloning of Rab3 isoforms expressed in peritoneal mast cells. In: FEBS Letters. 1994 ; Vol. 339, No. 1-2. pp. 171-174.
@article{d6784ea9496141e0b240e09ab2d51fb7,
title = "RT-PCR cloning of Rab3 isoforms expressed in peritoneal mast cells",
abstract = "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-eloning 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 (β-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).",
keywords = "Heparin inhibition, PCR cloning, Peritoneal mast cell, Rab3 protein, RT-PCR",
author = "Andres Oberhauser and Vijayan Balan and Fernandez-Badilla, {Carmen L.} and Fernandez, {Julio M.}",
year = "1994",
month = "2",
day = "14",
doi = "10.1016/0014-5793(94)80409-5",
language = "English (US)",
volume = "339",
pages = "171--174",
journal = "FEBS Letters",
issn = "0014-5793",
publisher = "Elsevier",
number = "1-2",

}

TY - JOUR

T1 - RT-PCR cloning of Rab3 isoforms expressed in peritoneal mast cells

AU - Oberhauser, Andres

AU - Balan, Vijayan

AU - Fernandez-Badilla, Carmen L.

AU - Fernandez, Julio M.

PY - 1994/2/14

Y1 - 1994/2/14

N2 - 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-eloning 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 (β-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).

AB - 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-eloning 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 (β-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).

KW - Heparin inhibition

KW - PCR cloning

KW - Peritoneal mast cell

KW - Rab3 protein

KW - RT-PCR

UR - http://www.scopus.com/inward/record.url?scp=0028220267&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028220267&partnerID=8YFLogxK

U2 - 10.1016/0014-5793(94)80409-5

DO - 10.1016/0014-5793(94)80409-5

M3 - Article

C2 - 7508866

AN - SCOPUS:0028220267

VL - 339

SP - 171

EP - 174

JO - FEBS Letters

JF - FEBS Letters

SN - 0014-5793

IS - 1-2

ER -