Mammalian a-Polymerase: Cloning of Partial Complementary DNA and Immunobinding of Catalytic Subunit in Crude Homogenate Protein Blots

D. N. SenGupta, P. Kumar, B. Z. Zmudzka, C. Parrott, S. H. Wilson, S. Coughlin, J. K. Vishwanatha, F. A. Robey

Research output: Contribution to journalArticle

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Abstract

A new polyclonal antibody against the a-polymerase catalytic polypeptide was prepared by using homogeneous HeLa cell a-polymerase. The antibody neutralized a-polymerase activity and was strong and specific for the a-polymerase catalytic polypeptide (Afr 183000) in Western blot analysis of crude extracts of HeLa cells. The antibody was used to screen a cDNA library of newborn rat brain poly(A+) RNA in Agtil. A positive phage was identified and plaque purified. This phage, designated Apolal.2, also was found to be positive with an antibody against Drosophila a-polymerase. The insert in Apolal.2 (1183 base pairs) contained a poly(A) sequence at the 3’ terminus and a short in-phase open reading frame at the 5’ terminus. A synthetic oligopeptide (eight amino acids) corresponding to the open reading frame was used to raise antiserum in rabbits. Antibody affinity purified from this serum was found to be immunoreactive against purified a-polymerase by enzyme-linked immunosorbent assay and was capable of immunopreci-pitating a-polymerase. This indicated the Apolal.2 insert encoded an a-polymerase epitope and suggested that the cDNA corresponded to an a-polymerase mRNA. This was confirmed in hybrid selection experiments using pUC9 containing the cDNA insert and poly(AH-) RNA from newborn rat brain; the insert hybridized to mRNA capable of encoding a-polymerase catalytic polypeptides. Northern blot analysis of rat brain poly(AH-) RNA revealed that this mRNA is ~5.4 kilobases.

Original languageEnglish
Pages (from-to)956-963
Number of pages8
JournalBiochemistry
Volume26
Issue number3
DOIs
StatePublished - 1 Jan 1987

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Cloning
Organism Cloning
Catalytic Domain
Complementary DNA
Messenger RNA
Antibodies
Rats
Brain
HeLa Cells
Bacteriophages
Peptides
Open Reading Frames
Proteins
RNA
Oligopeptides
Poly A
Antibody Affinity
Complex Mixtures
Immunosorbents
Gene Library

Cite this

SenGupta, D. N., Kumar, P., Zmudzka, B. Z., Parrott, C., Wilson, S. H., Coughlin, S., ... Robey, F. A. (1987). Mammalian a-Polymerase: Cloning of Partial Complementary DNA and Immunobinding of Catalytic Subunit in Crude Homogenate Protein Blots. Biochemistry, 26(3), 956-963. https://doi.org/10.1021/bi00377a041
SenGupta, D. N. ; Kumar, P. ; Zmudzka, B. Z. ; Parrott, C. ; Wilson, S. H. ; Coughlin, S. ; Vishwanatha, J. K. ; Robey, F. A. / Mammalian a-Polymerase : Cloning of Partial Complementary DNA and Immunobinding of Catalytic Subunit in Crude Homogenate Protein Blots. In: Biochemistry. 1987 ; Vol. 26, No. 3. pp. 956-963.
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Mammalian a-Polymerase : Cloning of Partial Complementary DNA and Immunobinding of Catalytic Subunit in Crude Homogenate Protein Blots. / SenGupta, D. N.; Kumar, P.; Zmudzka, B. Z.; Parrott, C.; Wilson, S. H.; Coughlin, S.; Vishwanatha, J. K.; Robey, F. A.

In: Biochemistry, Vol. 26, No. 3, 01.01.1987, p. 956-963.

Research output: Contribution to journalArticle

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T2 - Cloning of Partial Complementary DNA and Immunobinding of Catalytic Subunit in Crude Homogenate Protein Blots

AU - SenGupta, D. N.

AU - Kumar, P.

AU - Zmudzka, B. Z.

AU - Parrott, C.

AU - Wilson, S. H.

AU - Coughlin, S.

AU - Vishwanatha, J. K.

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N2 - A new polyclonal antibody against the a-polymerase catalytic polypeptide was prepared by using homogeneous HeLa cell a-polymerase. The antibody neutralized a-polymerase activity and was strong and specific for the a-polymerase catalytic polypeptide (Afr 183000) in Western blot analysis of crude extracts of HeLa cells. The antibody was used to screen a cDNA library of newborn rat brain poly(A+) RNA in Agtil. A positive phage was identified and plaque purified. This phage, designated Apolal.2, also was found to be positive with an antibody against Drosophila a-polymerase. The insert in Apolal.2 (1183 base pairs) contained a poly(A) sequence at the 3’ terminus and a short in-phase open reading frame at the 5’ terminus. A synthetic oligopeptide (eight amino acids) corresponding to the open reading frame was used to raise antiserum in rabbits. Antibody affinity purified from this serum was found to be immunoreactive against purified a-polymerase by enzyme-linked immunosorbent assay and was capable of immunopreci-pitating a-polymerase. This indicated the Apolal.2 insert encoded an a-polymerase epitope and suggested that the cDNA corresponded to an a-polymerase mRNA. This was confirmed in hybrid selection experiments using pUC9 containing the cDNA insert and poly(AH-) RNA from newborn rat brain; the insert hybridized to mRNA capable of encoding a-polymerase catalytic polypeptides. Northern blot analysis of rat brain poly(AH-) RNA revealed that this mRNA is ~5.4 kilobases.

AB - A new polyclonal antibody against the a-polymerase catalytic polypeptide was prepared by using homogeneous HeLa cell a-polymerase. The antibody neutralized a-polymerase activity and was strong and specific for the a-polymerase catalytic polypeptide (Afr 183000) in Western blot analysis of crude extracts of HeLa cells. The antibody was used to screen a cDNA library of newborn rat brain poly(A+) RNA in Agtil. A positive phage was identified and plaque purified. This phage, designated Apolal.2, also was found to be positive with an antibody against Drosophila a-polymerase. The insert in Apolal.2 (1183 base pairs) contained a poly(A) sequence at the 3’ terminus and a short in-phase open reading frame at the 5’ terminus. A synthetic oligopeptide (eight amino acids) corresponding to the open reading frame was used to raise antiserum in rabbits. Antibody affinity purified from this serum was found to be immunoreactive against purified a-polymerase by enzyme-linked immunosorbent assay and was capable of immunopreci-pitating a-polymerase. This indicated the Apolal.2 insert encoded an a-polymerase epitope and suggested that the cDNA corresponded to an a-polymerase mRNA. This was confirmed in hybrid selection experiments using pUC9 containing the cDNA insert and poly(AH-) RNA from newborn rat brain; the insert hybridized to mRNA capable of encoding a-polymerase catalytic polypeptides. Northern blot analysis of rat brain poly(AH-) RNA revealed that this mRNA is ~5.4 kilobases.

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