Correction EVOLUTION Correction for “Two different domains of the luciferase gene in the heterotrophic dinoflagellate Noctiluca scintillans occur as two separate genes in photosynthetic species,” by Liyun Liu and J. Woodland Hastings, which appeared in issue 3, January 16, 2007, of Proc Natl Acad Sci USA (104:696–701; first published November 27, 2006; 10.1073/pnas.0607816103). The authors note that the data deposition footnote should instead appear as “Data deposition: The sequence reported in this paper has been deposited in the GenBank database (ac- cession no. JF838193).” www.pnas.org/cgi/doi/10.1073/pnas.1111002108 CORRECTION www.pnas.org PNAS | August 23, 2011 | vol. 108 | no. 34 | 14371 Downloaded by guest on September 25, 2021 Two different domains of the luciferase gene in the heterotrophic dinoflagellate Noctiluca scintillans occur as two separate genes in photosynthetic species Liyun Liu* and J. Woodland Hastings† Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138 Contributed by J. Woodland Hastings, September 6, 2006 This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 29, 2003. Noctiluca scintillans, a heterotrophic unarmored unicellular biolu- but cell extracts also were shown to react with dinoflagellate minescent dinoflagellate, occurs widely in the oceans, often as a luciferin to give light. This emission allowed the identification of bloom. Molecular phylogenetic analysis based on 18S ribosomal a luciferase clone whose expressed protein has a very interesting DNA sequences consistently has placed this species on the basal structure. It lacks the N-terminal Ϸ100-aa sequence present in branch of dinoflagellates. Here, we report that the structural the other seven dinoflagellate luciferases and is composed of two organization of its luciferase gene is strikingly different from that major domains. The N-terminal region codes for a protein with of the seven luminous species previously characterized, all of luciferase activity and has sequence similarity to the individual which are photosynthetic. The Noctiluca gene codes for a polypep- domains of the three-domain luciferase of Lp, whereas the tide that consists of two distinct but contiguous domains. One, C-terminal region has sequence similarity to the LBP of Lp. which is located in the N-terminal portion, is shorter than but In the course of this work, it also was discovered that both the similar in sequence to the individual domains of the three-domain LBP region identified in Noctiluca and the Lp LBP have an luciferases found in all other luminous dinoflagellates studied. The internal repeat structure with four domains. However, these other, situated in the C-terminal part, has sequence similarity to the domains are less well conserved among themselves than are the luciferin-binding protein of the luminous dinoflagellate Lingulo- three LCF domains of the seven photosynthetic dinoflagellates. dinium polyedrum, encoded there by a separate gene. Western analysis shows that the native protein has the same size (Ϸ100 Results and Discussion kDa) as the heterologously expressed polypeptide, indicating that Molecular Cloning of the Full-Length Noctiluca Luciferase Gene and it is not a polyprotein. Thus, sequences found in two proteins in the the Two-Domain Protein. Because Noctiluca luciferase in crude L. polyedrum bioluminescence system are present in a single extracts cross-reacts biochemically with dinoflagellate luciferin polypeptide in Noctiluca. to emit light, we screened a Noctiluca cDNA expression library for light emission with added Pyrocystis luciferin. From Ϸ2 ϫ 104 ͉ ͉ ͉ bioluminescence luciferin-binding protein gene fusion colonies, two were found to emit light. Subsequent sequencing ͉ gene fission domain duplication of plasmid DNA from the two showed that they were identical, both lacking 5Ј and 3Ј untranslated regions, poly(A) tail, and he bioluminescent systems of seven previously studied bi- portions of N- and C-terminal regions of the ORF. Toluminescent marine dinoflagellates have been found to be We cloned the full-length gene and the intergenic sequence by similar, but unusual in several respects, and very different from PCR amplification from genomic DNA by using primers derived any of the other groups of luminous organisms (1). In the case from the partial luciferase cDNA. A search of the National of Lingulodinium polyedrum (Lp, formerly Gonyaulax polyedra), Center for Biotechnology Information database by BLAST light is emitted as flashes from specialized organelles called revealed that the N-terminal part of the Noctiluca sequence scintillons, which are formed as outpocketings from the cyto- shares similarity only to the single domains of all other plasm into the acidic vacuole (2, 3). The scintillons contain the dinoflagellate luciferases, whereas its C-terminal part has se- three components required for light emission: dinoflagellate quence similarity only to the LBP of the dinoflagellate Lp. luciferase (LCF) (4), its luciferin substrate, and a luciferin- Various combinations of PCR amplification from genomic DNA binding protein (LBP) (5). ruled out the possibility that the chimeric feature of this lucif- Ϸ Lp luciferase (Mr, 137 kDa) has an internal triplication, thus erase gene might have resulted from cloning artifacts. three homologous domains within a single molecule, each of As is known for luciferase genes in the other dinoflagellates which, when isolated as a separate peptide, is catalytically active Ϸ (10), the Noctiluca gene also occurs as tandem copies (Fig. 1). in vitro (6). There is also an N-terminal sequence of 100 aa of However, the intergenic nucleotide sequence (GenBank acces- unknown function. LBP is a smaller protein (Mr, Ϸ75 kDa), whose N-terminal Ϸ100 aa has a sequence identity of Ϸ50% with the corresponding luciferase region (7). The activities of both Author contributions: L.L. and J.W.H. designed research; L.L. performed research; L.L. and proteins are strongly regulated by pH (8). J.W.H. analyzed data; and L.L. and J.W.H. wrote the paper. In six other photosynthetic dinoflagellates, the structures of The authors declare no conflict of interest. the luciferases were found to be essentially the same as in Lp, and Abbreviations: LBP, luciferin binding protein; LCF, luciferase; Lp, Lingulodinium poly- all were found to occur in multiple and tandem copies, albeit edrum; Ns, Noctiluca scintillans; Pl, Pyrocystis lunula; Pr, Protoceratium reticulatum. with very different intergenic sequences (9, 10). In some of these Data deposition: The sequence reported in this paper has been deposited in the GenBank species, a luciferin-binding protein has not yet been found, but database (accession no. 828400). in vitro, all react to emit light with the luciferin of Pyrocystis See accompanying Profile on page 693. lunula (Pl), which was established to be an open tetrapyrrole (11) *Present address: Department of Biology, California State University, Northridge, and will be called dinoflagellate luciferin. CA 91330. Based on 18S ribosomal DNA, the heterotrophic unarmored †To whom correspondence should be addressed. E-mail: [email protected]. species Noctiluca scintillans (Ns) is phylogenetically distant (12), © 2006 by The National Academy of Sciences of the USA 696–701 ͉ PNAS ͉ January 16, 2007 ͉ vol. 104 ͉ no. 3 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0607816103 INAUGURAL ARTICLE Fig. 1. A schematic representation showing the genomic structure of Ns lcf genes and the domain organization of their predicted proteins in comparison with the second domain of Lp LCF and Lp LBP. At least two copies of the Ns lcf genes are tandemly arranged in a head-to-tail fashion, each consisting of 4,360 bp. The precise boundary between the intergenic region and 5Ј UTR was not precisely mapped, and together they account for 1,518 bp. The 3Ј UTR of the Ns lcf is only 97 bp long, therefore, much shorter than the 3Ј UTRs of lcf genes of seven photosynthetic dinoflagellates, which range from 194 to 432 bp. The largest ORF of Ns lcf predicts a protein of 914 aa with two major domains, an N-terminal luciferase-like (LCF-like) domain and a C-terminal luciferin-binding protein-like (LBP-like) domain. The LCF-like domain shares a 56% sequence identity with but is shorter at the N terminus by 60 aa residues than the individual domainsof Lp LCF. The LBP-like domain of 591 aa residues is 41% identical with the comparable region of Lp LBP. No significant similarity was found between the most N-terminal sequence of 29 aa residues of the Ns LCF and any sequences in the database. sion no. 828400) has no similarity to those of any of the other erases, based on the protein sequences (Fig. 2), is consistent dinoflagellate tandem genes reported, which themselves are with this conclusion. different from each other. Previous studies revealed that the amount of bias in codon As also shown in Fig. 1, the full-length Noctiluca protein usage varies in the central regions of the domains of the possesses a short N-terminal sequence of 29 aa residues followed luciferases of the seven photosynthetic dinoflagellates (9, 10). by two domains, referred to as the luciferase-like and the The effective numbers of codons is a measure of codon biases LBP-like domains, which are linked by a sequence of 11 aa (higher value, lower bias). This value is lowest for Lp luciferase Ϸ Ϸ residues. No significant similarity was found between the 29-aa ( 33) and highest for Pl luciferase ( 50); the corresponding N-terminal Noctiluca sequence and the Ϸ100-aa N-terminal value for the Noctiluca luciferase-like domain (55) is even higher. sequence of any of the seven other dinoflagellate luciferases, or Guanosine and cytosine account for only 40% at the third of Lp LBP, or any other sequence in the GenBank. position of its codons, a value that is far lower than that for the seven other luciferases (64–87%).