Purification of sea urchin ribosomal RNA genes with a single-strand specific nuclease.
Ribosomal RNA genes (rDNA) from Lytechinus variegatus were isolated by selective heat denaturation of main band DNA followed by single-strand specific nuclease (S1) treatment to remove the single-stranded DNA. After S1 nuclease treatment the partially purified fraction contained 10% rDNA, representing a 50-fold purification. Preparative CsCl centrifugation of this fraction resulted in highly purified rDNA with an average molecular weight of 1.9 - 10(7) and no single-strand breaks. High molecular weight sea urchin DNA was refractory to selective heat denaturation. DNA with an average molecular weight of greater than or equal to 2.9 - 10(7) was only 60-80% denatured after heating 13 degrees C above the Tm, whereas, DNA with an average molecular weight of less than or equal to 1.9 - 10(7) was 98% denatured. This phenomenon appears not to be due to time, buffer, or pH, but is dependent on size.
D000818:Animals; D004247:DNA / Q000302:isolation & purification*; D004277:DNA, Single-Stranded / Q000302:isolation & purification*; D005796:Genes; D008970:Molecular Weight; D009691:Nucleic Acid Denaturation; D012335:RNA, Ribosomal / Q000096:biosynthesis*; D012260:Ribonucleases; D012617:Sea Urchins / Q000032:analysis / Q000378:metabolism*; D013696:Temperature; D014158:Transcription, Genetic
D004277:DNA, Single-Stranded; D012335:RNA, Ribosomal; D004247:DNA; D012260:Ribonucleases