Isolation of cDNA and genomic DNA clones encoding a calmodulin-binding protein related to a family of ATPases involved in cell division and vesicle fusion

Teerapong Buaboocha, Birong Liao, Raymond E. Zielinski

Research output: Contribution to journalArticle

Abstract

Calmodulin (CAM), a primary Ca2+ receptor in all eukaryotic cells, is a multifunctional protein that functions by interacting with and modulating the activities of a wide variety of target proteins. Identifying and characterizing these CaM-binding target proteins is essential to define the pathways by which Ca2+-regulated signals are transduced. An Arabidopsis thaliana L. flower cDNA expression library constructed in λZAPII was screened for CaM-binding proteins with 35S-labeled CaM. A partial cDNA whose encoded protein shares a high level of similarity with yeast CDC48p was isolated. A genomic clone was isolated using the partial length cDNA clone as a probe, and its nucleotide sequence was determined. The genomic DNA sequence was used to design oligonucleotide primers for polymerase-chainreaction (PCR) experiments that facilitated cloning and reconstructing a full-length, 3.4-kb cDNA clone. The cDNA encodes a 111-kDa CaM-interacting protein (CIP111) containing motifs characteristic of a diverse family of ATPases, including proteins involved in cell cycle regulation, protein degradation, and vesicle-mediated protein transport. A truncated fusion protein encoded by the carboxy-terminal region of CIP111 was produced in Escherichia coli and shown to bind CaM in a Ca2+-dependent manner by protein gel blot and affinity chromatography binding assays. Reverse-transcription PCR analyses demonstrated that CIP111 mRNA is expressed in all organs examined including flowers, siliques, floral stalks, leaves, and roots. DNA blot hybridization analyses indicate that a single-copy gene in Arabidopsis is likely to encode CIP111.

Original languageEnglish (US)
Pages (from-to)774-781
Number of pages8
JournalPlanta
Volume212
Issue number5-6
DOIs
StatePublished - May 8 2001

Fingerprint

Calmodulin-Binding Proteins
calmodulin
Cell Division
adenosinetriphosphatase
Adenosine Triphosphatases
cell division
binding proteins
Complementary DNA
Clone Cells
clones
genomics
DNA
Proteins
proteins
Arabidopsis
calcium
Carrier Proteins
Cell Cycle Proteins
DNA Primers
flowers

Keywords

  • ATPase Calcium
  • Arabidopsis (ATPase, Ca)
  • Protein-protein interaction
  • Signal transduction

ASJC Scopus subject areas

  • Genetics
  • Plant Science

Cite this

Isolation of cDNA and genomic DNA clones encoding a calmodulin-binding protein related to a family of ATPases involved in cell division and vesicle fusion. / Buaboocha, Teerapong; Liao, Birong; Zielinski, Raymond E.

In: Planta, Vol. 212, No. 5-6, 08.05.2001, p. 774-781.

Research output: Contribution to journalArticle

Buaboocha, Teerapong ; Liao, Birong ; Zielinski, Raymond E. / Isolation of cDNA and genomic DNA clones encoding a calmodulin-binding protein related to a family of ATPases involved in cell division and vesicle fusion. In: Planta. 2001 ; Vol. 212, No. 5-6. pp. 774-781.
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