Nakagomi O, Oyamada H, Nakagomi T
Department of Laboratory Medicine, Akita University School of Medicine, Japan.
Mol Cell Probes. 1989 Sep;3(3):263-71. doi: 10.1016/0890-8508(89)90007-8.
A recently developed alkaline northern blot hybridization assay (Li, J. K. K., Parker, B. & Kowalik, T., Analytical Biochemistry 163, 210-18, 1987) was used to assess the genetic relatedness of the fourth gene of human rotavirus strains recovered from children with diarrhea and from asymptomatic neonates. Genomic double stranded (ds) RNAs of the rotavirus strains were separated by polyacrylamide gel electrophoresis and were blotted to nylon membranes (Gene Screen Plus or Zeta Probe membranes). The blotted RNAs were then probed with 32P-labelled single-stranded (ss) RNA probes prepared by in vitro transcription from single-shelled particles of the different strains. When analysed under a condition of high stringency (52 degrees C, 2.5 x SSC, 50% formamide) that allowed up to 21% of nucleotide mismatch, a high degree of the fourth gene homology was observed among strains recovered from asymptomatic neonates (asymptomatic rotaviruses) or among strains recovered from infants and children with diarrhea (symptomatic rotaviruses), while the homology of the fourth gene between the asymptomatic and symptomatic strains was considerably lower. It is of particular interest that the fourth gene of the AU-1 and AU228 strains recovered from children with diarrhea failed to hybridize to the corresponding gene of either asymptomatic or symptomatic rotavirus strains but showed a high degree of homology with the fourth gene of a feline rotavirus recovered from an apparently healthy cat. These data indicate that a new group of the fourth gene is present among symptomatic rotaviruses and that the fourth gene of this group is genetically related to the corresponding gene of a feline rotavirus.
最近开发的碱性Northern印迹杂交分析方法（Li, J. K. K., Parker, B. & Kowalik, T., 《分析生物化学》163, 210 - 18, 1987）被用于评估从腹泻儿童和无症状新生儿中分离出的人类轮状病毒株第四基因的遗传相关性。轮状病毒株的基因组双链（ds）RNA通过聚丙烯酰胺凝胶电泳分离，并印迹到尼龙膜（Gene Screen Plus或Zeta Probe膜）上。然后，用通过体外转录从不同毒株的单壳颗粒制备的32P标记单链（ss）RNA探针探测印迹的RNA。在允许高达21%核苷酸错配的高严谨条件（52℃，2.5×SSC，50%甲酰胺）下进行分析时，在从无症状新生儿中分离出的毒株（无症状轮状病毒）之间或从腹泻婴幼儿中分离出的毒株（有症状轮状病毒）之间观察到第四基因的高度同源性，而无症状毒株和有症状毒株之间第四基因的同源性则低得多。特别有趣的是，从腹泻儿童中分离出的AU - 1和AU228毒株的第四基因未能与无症状或有症状轮状病毒株的相应基因杂交，但与从一只看似健康的猫中分离出的猫轮状病毒的第四基因显示出高度同源性。这些数据表明，在有症状轮状病毒中存在一组新的第四基因，并且该组的第四基因与猫轮状病毒的相应基因在遗传上相关。
