Biochemical and Biophysical Research Communications
Molecular cloning and characterization of rat LC3A and LC3B—Two novel markers of autophagosome
Section snippets
Materials and methods
Cloning of LC3A and LC3B cDNAs. The amino acid sequences of human LC3A (GenBank Accession No.: AF276658), LC3B (GenBank Accession No.: AF087871) and LC3C (GenBank Accession No.: AF276659) were used to search the rat expressed sequence tag (EST) database at GenBank (http://www.ncbi.nlm.nih.gov). Homologous ESTs were obtained and assembled into three EST contigs and checked manually. Two pairs of primers (named LC3A/B/-A and B; see Table 1) were designed based on the contig sequences and used for
Identification and isolation of LC3A and LC3B
Mann and Hammarback [5] purified the bovine brain LC3 by molecular sieve chromatography from salt-extracted MAPs, and the sequence of the first 20 amino acids of gel-purified bovine LC3 was determined by Edman degradation. They obtained two amino acid sequences, and they used the major sequence PSDRPFKQRRSFADDVKEVQ to search the GenBank database and cloned the rat LC3 gene, but the sequence similarity is significantly low [5]. We previously identified three human LC3 family members (LC3A, LC3B,
Conclusion
In present work, we cloned LC3A and LC3B, two novel variants of rat LC3. The expression patterns of LC3, LC3A, and LC3B were different in 12 normal rat adult tissues. LC3A and LC3B undergo a characteristic C-terminal cleavage and are associated with autophagic membranes. The conserved Gly120 residues of LC3A and LC3B are essential for their post-translational modification and localization to autophagic membranes. Present data also suggested that LC3A and LC3B could be used as two novel
Acknowledgments
This work was supported by the National 973 Program and 863 High Technology Program of China, as well as the National Natural Science Foundation of China.
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