Comparison of the zebrafish (Danio rerio) Id2 with that of other organisms shows evolutionary conservation of this protein. A: The predicted amino acid sequences of the zebrafish id2 gene (accession no. DQ186992) aligned with those of rainbow trout, Xenopus, chicken, mouse, and human. Yellow-boxed letters indicate conserved amino acids. Blue outlined text highlights the conserved cyclinE/A-CDK2 phosphorylation site SPVR. Two helix regions indicated by red outline and loop region highlighted by green outline, together make up the helix-loop-helix region. B: Phylogenetic analysis (Clustal Method) shows that zebrafish Id2 is correctly placed in the second subclass under the Id family. Refer to the Experimental Procedures section for the GenBank accession numbers of the other Id genes. C: Reverse transcriptase polymerase chain reaction (RT-PCR) detects the continuous presence of transcripts of id2 during early development. RT-PCR was performed using id2-specific primers. The embryonic stages are indicated on top of each lane. id2 is present at high levels before mid-blastula transition (MBT). To confirm the early maternal expression, bands were cut out from the agarose gel and sequenced. EF1α was used as a positive control. RT-negative controls were carried out at the same time and no band was detected (data not shown).
|
