Yan W, Lewin A, Hauswirth W
Department of Molecular Genetics and Microbiology, University of Florida, Gainesville 32610, USA.
Invest Ophthalmol Vis Sci. 1998 Dec;39(13):2529-36.
To investigate the molecular mechanism relating phenotype and genotype in the rd mouse, mRNA and pre-mRNA levels derived from the wild-type and position-347 nonsense mutant beta-phosphodiesterase (beta-PDE) genes were determined and compared with the corresponding gene copy ratios.
Total RNA and genomic DNA was isolated from the retinas of three heterozygous rd/+ mouse strains. For each, quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the ratio of wild-type and rd beta-phosphodiesterase pre-mRNA and mature mRNA. The gene copy ratio between wild-type and rd beta-PDE was also determined by quantitative PCR.
The pre-mRNA ratio of wild-type versus nonsense mutant was close to 1:1, whereas the corresponding mRNA ratio was greater than 3:1, even though the gene copy ratio was confirmed to be 1:1.
The equivalence of pre-mRNA ratio level for wild-type and nonsense mutant in the rd/+ retina indicates that both genes were transcribed at similar levels. Thus, neither the nonsense mutation at position 347 nor the intron 1 retroviral insertion also present in the rd gene seem to have affected gene transcription. In contrast, the strain-independent bias favoring wild-type mature mRNA in vivo suggests a specific degradation of mutant transcript during or after pre-mRNA splicing. This allele-specific degradation serves to decrease mutant transcript levels dramatically in all rd strains, and suggests that photoreceptor cells have the capacity to reduce the level of an mRNA containing a nonsense mutation.
研究rd小鼠表型与基因型相关的分子机制，测定源自野生型和347位无义突变β -磷酸二酯酶（β -PDE）基因的mRNA和前体mRNA水平，并与相应的基因拷贝比进行比较。
从三个杂合rd/+小鼠品系的视网膜中分离总RNA和基因组DNA。对于每个品系，使用定量逆转录聚合酶链反应（RT-PCR）来测定野生型和rdβ -磷酸二酯酶前体mRNA与成熟mRNA的比例。野生型和rdβ -PDE之间的基因拷贝比也通过定量PCR测定。
野生型与无义突变体的前体mRNA比例接近１:１，而相应的mRNA比例大于３:１，尽管基因拷贝比经确认是１:１。
rd/+视网膜中野生型和无义突变体前体mRNA比例水平相等，表明两个基因转录水平相似。因此，rd基因中存在的347位无义突变和内含子1逆转录病毒插入似乎均未影响基因转录。相反，体内野生型成熟mRNA存在不依赖品系的偏向性，提示前体mRNA剪接过程中或之后突变转录本发生特异性降解。这种等位基因特异性降解可显著降低所有rd品系中的突变转录本水平，提示光感受器细胞有能力降低含有无义突变的mRNA水平。
