Abstract
The circadian expressed luciferin-binding protein (LBP) gene from the marine bioluminescent alga Gonyaulax polyedra represents the first dinoflagellate gene that has been cloned and sequenced at both cDNA and genomic levels. Starting with a fragment from the 3'-end of the LBP cDNA that was found by immunoscreening of a cDNA library, genomic clones were obtained by the inverse polymerase chain reaction technique. Full-length cDNA clones were selected by screening a cDNA library by plaque hybridizations and by polymerase chain reaction amplifications. The LBP sequence has a 2004-nucleotide open reading frame coding for a protein of 668 amino acids (approximately 75 kDa). The reading frame and identity of the clone were confirmed by the sequence of an octapeptide obtained from a purified fragment of CNBr-treated LBP. A variant LBP cDNA was found to differ in sequence by approximately 11% at the DNA level. The untranslated regions of the mRNA are 111 nucleotides (5'-untranslated region) and 158 nucleotides (3'-untranslated region) long, respectively. The LBP gene contains no introns and exhibits certain features not typical for a eukaryotic gene. Its promoter does not include the typical TATA box within approximately 50 nucleotides upstream of the transcription start site, and the usual poly(A+) signal (AAUAAA) is not present on the end of the LBP mRNA. The copy number of the gene is very high (approximately 1000 copies/cell). However, the universal genetic code and conserved positions relevant for the translational apparatus are maintained.
Highlights
Molecular Cloning and Genomic Organization of a Gene for Luciferinbinding Protein from the Dinoflagellate Gonyaulax polyedru”
Box within -50 nucleotides upstream of the transcrip- Since the protein is synthesized i n vivo for only a short time tion start site, and thuesual poly(A+)signal (AAUAAA) in the early night, it was concluded that thetranslation of the is not present on the endof the LBP mRNA
The enzymes AatII, HindIII, EcoRII, EagI, SalI, XbaI, MluI, BssHII, BstEII,and Sac1 all restricted the DNA poorly or not a t all. This could bedue to the absence or paucity of the requisite sites and/orto thepresence of the modified nucleotide 5'-hydroxymethyluracilin place of thymine residues (Rae, 1976)
Summary
Molecular Cloning and Genomic Organization of a Gene for Luciferinbinding Protein from the Dinoflagellate Gonyaulax polyedru”. Such controlis widespread in biological systems (LBP) gene from the marinebioluminescent alga Gon- from microorganisms to humans; the sleep/wake cycle and yaulaxpolyedra representsthefirstdinoflagellate activity in animals, photosynthesis in plants, and nitrogen sga3cne’r-ndeeeengntdehinnaoofgtmhtohiacfeslbeaLevBeecnlDPs.NccSDlAotNanrleAitdbiantrhngaadrytws,ewiqtgahueasenfnoofcmrueanidgcdamctbelbyonontitmehsfmrcoDwumNenroAet-heHftoixatasettmiionpngosirneatlcaycla.o,nno1tb9roa9cl1bt)ye,ratihsaiiassrcpelhooaclktloempxeaermciohpdalniecisstomifmp(iErnogdcmeosfusrneedspssr,uo1bd9ju8ecc8-t; obtained by theinverse polymerase chainreaction tion in both plants and animals LBP is rapidly synthesized during the early night phase and can reach -1% of the total cellular protein levels. Several hours later, it is preferentially degraded by an unknown mechanism. Box within -50 nucleotides upstream of the transcrip- Since the protein is synthesized i n vivo for only a short time tion start site, and thuesual poly(A+)signal (AAUAAA) in the early night, it was concluded that thetranslation of the is not present on the endof the LBP mRNA. The universal genetic code and conserved positions relevant for the translational apparatusare maintained
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