Molecular characterization of a ribonucleic acid transcript that is highly up-regulated at the time of ovulation in the brook trout (Salvelinus fontinalis) ovary.
Using stage-specific subtractive cDNA cloning, a 0.8-kilobase (kb) cDNA (TOP-1; trout ovulatory protein-1) was previously obtained that hybridized only with ovarian RNA and was highly up-regulated at the time of ovulation. In the present study, a 1.4-kb cDNA (TOP-2; trout ovulatory protein-2) was obtained after rescreening a brook trout ovulatory cDNA library using TOP-1 as a probe. The levels of ovarian transcripts hybridizing on Northern blots probed with TOP-2 begin to increase after the breakdown of the germinal vesicle in the oocyte and appear to peak approximately 24 h postovulation. Antibodies produced to a recombinant maltose binding protein-TOP-2 fusion protein recognize 3-4 protein bands (53, 39, 29, and 24 kDA) on Western blots of ovarian tissue and/fluid. Levels of the two largest proteins increase immediately after ovulation and decrease significantly by 4-8 days postovulation. TOP-2 has an open reading frame of 262 amino acids. The presumed amino acid sequence of TOP-2 is rich in cysteine and is arranged in 5 polypeptide domains consisting of two types, one of which is homologous to the domains of TOP-1. Both domain types also share homology with a group of mammalian leukocytic protease inhibitors called antileukoproteinases.
Garczynski
M A
MA
Department of Biological Sciences, University of Notre Dame, Indiana 46656, USA.
Goetz
F W
FW
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D001826:Body Fluids / Q000378:metabolism; D003001:Cloning, Molecular; D005260:Female; D015698:Genomic Library; D007074:Immunoglobulin G / Q000096:biosynthesis; D008969:Molecular Sequence Data; D010053:Ovary / Q000378:metabolism; D010060:Ovulation / Q000502:physiology*; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D012313:RNA / Q000096:biosynthesis* / Q000235:genetics*; D014158:Transcription, Genetic; D014337:Trout / Q000502:physiology*; D015854:Up-Regulation / Q000502:physiology*
D007074:Immunoglobulin G; D011506:Proteins; C108860:trout ovulatory protein 1; C108861:trout ovulatory protein 2; D012313:RNA