CYTOPLASMIC LOCALIZATION OF TRANSCRIPTS OF A COMPLEX G-RICH CRAB SATELLITE DNA(C)

The primary sequence and higher order structures of a G-C-rich satelli te DNA of the Bermuda land crab Gecarcinus lateralis have been describ ed previously. The repeat unit of the satellite is approximately 2.1 k b. In exploring a possible function for this satellite, we asked wheth er it is transcribed. As a probe for transcripts, we used a segment of DNA amplified from a 368 bp EcoRI fragment from the very highly conse rved 3' end of the satellite DNA. During polymerase chain reaction (PC R) amplification, the probe was simultaneously either radiolabeled or biotinylated. Tissue- and stage specific transcripts were observed whe n blots of poly(A)+ mRNAs recovered from polysomes isolated from crab tissues [including midgut gland (hepatopancreas), limb bud, and claw m uscle] were probed with the satellite DNA fragment. The presence of sa tellite transcripts in polysomal mRNAs is strong evidence that the tra nscripts had reached the cytoplasm. To corroborate the presence of tra nscripts in the cytoplasm, we investigated in situ hybridization of sa tellite probes with RNAs in tissue sections. Biotinylated satellite DN A probes were applied to sections of midgut gland, limb bud papilla, o vary, or testis of anecdysial crabs. Retention of RNAs in tissue secti ons was improved by UV-irradiation prior to hybridization. Transcripts were abundant in the cytoplasm of all tissues except testis. Sections of crab midgut gland treated with RNase A prior to hybridization and sections of mouse pancreatic tumor served as controls; neither showed any signals with the probe.