de Wett W, Sippola M, Tromp G, Prockop D, Chu M L, Ramirez F
J Biol Chem. 1986 Mar 15;261(8):3857-62.
R-loop mapping of DNA:RNA hybrids formed between mutant pro-alpha 2(I) mRNAs and appropriate human pro-alpha 2(I) genomic clones was employed to define the location of mutations which result in the synthesis of shortened pro-alpha 2(I) chains in skin fibroblasts from two variants of osteogenesis imperfecta. Hybridization of the genomic clone NJ-9 with pro-alpha 2(I) mRNA from a patient with a mild atypical form of the disease resulted in the identification of mutant pro-alpha 2(I) mRNA lacking the sequences which correspond to exon 11 of the pro-alpha 2(I) collagen gene. Exon 11, a 54-base pair exon, encodes amino acids 73 to 90 of the alpha 2(I) chain. Also, electron microscopy of R-loop structures formed between the genomic clone NJ-1 and mRNA from a variant with a perinatal lethal form of osteogenesis imperfecta visualized pro-alpha 2(I) mRNAs which did not hybridize to the sequences of exon 28, a 54-base pair exon coding for amino acids 448 to 465 of the alpha 2(I) chain. Moreover, nuclease S1 mapping of the variant's mutant pro-alpha 2(I) mRNA, employing the human pro-alpha 2(I) cDNA clone Hf-15, confirmed the location of the mismatch to the sequences corresponding to exon 28. Although the data do not determine the exact nature of the mutations, they illustrate the use of R-loop mapping as an alternative approach to S1 mapping analysis for the detection and localization of collagen mRNA deletions.
RNA杂交体的R环图谱分析：利用突变型前α2（I）mRNA与合适的人源前α2（I）基因组克隆形成的DNA:RNA杂交体，来确定导致成骨不全两种变体的皮肤成纤维细胞中合成缩短的前α2（I）链的突变位置。将基因组克隆NJ - 9与一名患有轻度非典型疾病患者的前α2（I）mRNA杂交，结果鉴定出突变型前α2（I）mRNA缺失了与前α2（I）胶原基因第11外显子相对应的序列。第11外显子是一个54个碱基对的外显子，编码α2（I）链的第73至90个氨基酸。此外，对基因组克隆NJ - 1与围产期致死型成骨不全变体的mRNA形成的R环结构进行电子显微镜观察，发现了未与第28外显子序列杂交的前α2（I）mRNA，第28外显子是一个54个碱基对的外显子，编码α2（I）链的第448至465个氨基酸。此外，利用人源前α2（I）cDNA克隆Hf - 15对该变体的突变型前α2（I）mRNA进行核酸酶S1图谱分析，证实了错配位置与第28外显子相对应的序列。虽然这些数据并未确定突变的确切性质，但它们说明了R环图谱分析可作为S1图谱分析的替代方法，用于检测和定位胶原mRNA缺失。
