Cytoplasmic localization of transcripts of a complex G+C-rich crab satellite DNA.
The primary sequence and higher order structures of a G+C-rich satellite DNA of the Bermuda land crab Gecarcinus lateralis have been described previously. The repeat unit of the satellite is approximately 2.1 kb. In exploring a possible function for this satellite, we asked whether 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 conserved 3' end of the satellite DNA. During polymerase chain reaction (PCR) amplification, the probe was simultaneously either radiolabeled or biotinylated. Tissue- and stage-specific transcripts were observed when blots of poly(A)+ mRNAs recovered from polysomes isolated from crab tissues [including midgut gland (hepatopancreas), limb bud, and claw muscle] were probed with the satellite DNA fragment. The presence of satellite transcripts in polysomal mRNAs is strong evidence that the transcripts had reached the cytoplasm. To corroborate the presence of transcripts in the cytoplasm, we investigated in situ hybridization of satellite probes with RNAs in tissue sections. Biotinylated satellite DNA probes were applied to sections of midgut gland, limb bud papilla, ovary, or testis of anecdysial crabs. Retention of RNAs in tissue sections 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.
Varadaraj
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Biology Division, Oak Ridge National Laboratory, TN 37831.
Skinner
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D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D003386:Brachyura / Q000235:genetics* / Q000254:growth & development; D003593:Cytoplasm / Q000737:chemistry*; D004276:DNA, Satellite / Q000235:genetics*; D005260:Female; D018507:Gene Expression Regulation, Developmental; D008297:Male; D008969:Molecular Sequence Data; D009928:Organ Specificity; D011132:Polyribosomes / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis*; D014158:Transcription, Genetic
D004276:DNA, Satellite; D012333:RNA, Messenger