Gene sequence and expression of an analog of proliferating cell nuclear antigen (PCNA) in the alga Tetraselmis chui and detection of the encoded protein with anti-rat PCNA monoclonal antibody.
To identify a phytoplankton cell cycle marker detected by a monoclonal antibody against mammalian proliferating cell nuclear antigen (PCNA) (S. Lin, J. Chang, and E. J. Carpenter, J. Phycol. 30:449-456, 1994), a PCNA gene fragment was isolated by reverse transcription-PCR from the marine unicellular alga Tetraselmis chui Butcher (Prasinophyceae). The gene fragment was 616 bp in length and contained an open reading frame of 205 amino acids. The deduced amino acid sequence showed 80 and 88% similarity to human and rice PCNA, respectively. Southern hybridization indicated that the isolated gene fragment was part of the T. chui genome, with up to three copies in each haploid nucleus. Northern hybridization was used to detect a PCNA mRNA with a size of 1.2 kb from an exponentially growing algal culture. The T. chui gene fragment has been cloned into an expression vector, and a fusion protein was subsequently generated. Anti-rat PCNA simultaneously recognized the PCNA fusion protein and a single 33-kDa band in T.chui total protein extract. Our results indicate that T. chui PCNA is highly similar to its mammalian counterpart and that anti-rat PCNA is a good tool for detecting phytoplankton PCNA in general.
Cheng
L C
LC
Institute of Marine Biology, Taiwan Ocean University, Keelung, Republic of China.
Hwang
S P
SP
Chang
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002453:Cell Cycle / Q000235:genetics; D000460:Chlorophyta / Q000166:cytology / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D005796:Genes; D005819:Genetic Markers; D006801:Humans; D008969:Molecular Sequence Data; D010839:Phytoplankton / Q000166:cytology / Q000235:genetics; D016133:Polymerase Chain Reaction; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics* / Q000276:immunology; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D005819:Genetic Markers; D018809:Proliferating Cell Nuclear Antigen