Isolation and characterization of DMRT1 and its putative regulatory region in the protogynous wrasse, Halichoeres tenuispinis.
A full-length cDNA of doublesex and mab-3-related transcription factor 1 gene (DMRT1) from wrasse testis was isolated by cDNA library screening. Wrasse DMRT1 was 3116 bp in size and contained the DM domain, with a zinc finger DNA-binding motif, and the male-specific motif. Northern blot analysis identified a 3.2-kb transcript approximately equal in size to the DMRT1 nucleotide sequence detected in the testis, but not in the ovary, confirming that this sequence is male-specific in protogynous wrasse. Southern blot analysis suggested that the wrasse genome contains two copies of the DMRT1 gene. The ORF consisted of five exons and four introns with conserved donor-acceptor splice sites at all exon-intron junctions. The 5'-flanking region of the wrasse DMRT1 gene was isolated by DNA walking, and putative regulatory sites were identified by searching data bases. The 5'-flanking region was divided into 9 elements, then 17 DMRT-luciferase chimeric plasmids were constructed. By transient transfection into Cos-1 and TM4 cells, distal element I which contains GATA-binding sites and proximal element B containing the sex-determining region on Y chromosome gene (SRY) binding site were revealed to have an important role in transcriptional regulation of the wrasse DMRT1 when an enhancer sequence was provided.
Jeong
Hyung-Bok
HB
Department of Biology, Jeju National University, 66 Jejudaehakno, Jeju-si, Jeju Special Self-Governing Province, Republic of Korea.
Park
Ji-Gweon
JG
Park
Young-Ju
YJ
Takemura
Akihiro
A
Hur
Sung-Pyo
SP
Lee
Young-Don
YD
Kim
Se-Jae
SJ
D024506:5' Flanking Region; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D004247:DNA / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005091:Exons; D015723:Gene Library; D007438:Introns; D049410:Luciferases, Renilla / Q000235:genetics / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010473:Perciformes / Q000235:genetics*; D010802:Phylogeny; D010957:Plasmids; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D012045:Regulatory Sequences, Nucleic Acid; D014157:Transcription Factors / Q000235:genetics* / Q000302:isolation & purification*; D014162:Transfection; D016335:Zinc Fingers
C110547:DMRT1 protein; D018076:DNA, Complementary; D014157:Transcription Factors; D004247:DNA; D049410:Luciferases, Renilla