Maxwell I H, Maxwell F, Hahn W E
Nucleic Acids Res. 1980 Dec 11;8(23):5875-94. doi: 10.1093/nar/8.23.5875.
cDNA of modal size approximately 1600 nucleotides, transcribed from mouse brain polyadenylated mRNA, was annealed with excess of high molecular weight (approximately kb) genomic DNA. The S1 nuclease method was then applied to determine possible sequence discontinuity between the cDNA and genomic DNA. A substantial reduction in the average size of the annealed cDNA was observed following S1 nuclease treatment. Large single copy genomic DNA, annealed with excess high molecular weight DNA, and cDNA, hybridized with its template mRNA, were resistant to cleavage by S1 nuclease. We interpret these results to indicate a high frequency of discontinuous coding sequences in the genomic DNA that annealed with the cDNA. The same result was obtained using fractionated cDNA, enriched in transcripts of relatively infrequent or abundant mRNA species. The result obtained with the infrequent sequence class cDNA indicates that tens of thousands of split genes exist in the mouse genome. Extensive cleavage of the cDNA by S1 nuclease was also observed after hybridization with greater than 30S nuclear RNA, indicating that intervening sequences are generally transcribed.
从小鼠脑多聚腺苷酸化mRNA转录而来的、大小约为1600个核苷酸的cDNA，与过量的高分子量（约kb）基因组DNA进行退火。然后应用S1核酸酶法来确定cDNA与基因组DNA之间可能存在的序列间断性。在S1核酸酶处理后，观察到退火cDNA的平均大小显著减小。与过量高分子量DNA退火的大的单拷贝基因组DNA，以及与其模板mRNA杂交的cDNA，对S1核酸酶的切割具有抗性。我们将这些结果解释为表明与cDNA退火的基因组DNA中存在高频的间断编码序列。使用富集了相对稀少或丰富mRNA种类转录本的分级分离cDNA也得到了相同的结果。用稀少序列类cDNA获得的结果表明，小鼠基因组中存在数以万计的断裂基因。与大于30S核RNA杂交后，也观察到S1核酸酶对cDNA的广泛切割，表明间隔序列通常会被转录。
