Caeiro F, Costa J V
Gulbenkian Institute of Science, Oeiras Codex, Portugal.
Virology. 1989 Dec;173(2):728-32. doi: 10.1016/0042-6822(89)90587-4.
A cell-free system for the study of transcription of African swine fever virus (ASFV) mRNA was developed from cytoplasmic extracts of infected cells permeabilized with lysolecithin. Extracts prepared from infected cells early and late after infection incorporated [alpha-32P]UTP into acid-insoluble material that was resistant to DNase and sensitive to RNase. The incorporation was inhibited by actinomycin D but not by alpha-amanitin. The presence of the nuclei was not required. In vitro transcription was optimal at pH 7.9 and at concentrations of 100 mM NH4Cl, 5 mM magnesium acetate, and 250 microM MnCl2. Early infected cell extracts transcribed from endogenous viral DNA a set of RNAs similar in electrophoretic migration to that observed in intact infected cells. Late infected cell extracts seemed to be unable to transcribe new RNA species besides those transcribed early after infection. The activity of the extracts could be made dependent on exogenous templates by digestion with micrococcal nuclease. RNAs transcribed after addition of native or denatured viral DNA to nuclease-treated extracts were indistinguishable from those transcribed from endogenous viral DNA. Late infected cell extracts digested with micrococcal nuclease were also active in transcribing virus-specific RNA from p2SB21, a recombinant plasmid containing the SalI B fragment of ASFV DNA.
利用溶血卵磷脂使感染细胞的细胞质提取物透化，开发了一种用于研究非洲猪瘟病毒（ASFV）mRNA转录的无细胞系统。在感染后早期和晚期从感染细胞制备的提取物将[α-32P]UTP掺入对DNase有抗性且对RNase敏感的酸不溶性物质中。掺入受到放线菌素D的抑制，但不受α-鹅膏蕈碱的抑制。不需要细胞核的存在。体外转录在pH 7.9以及100 mM NH4Cl、5 mM乙酸镁和250 μM MnCl2的浓度下最为适宜。早期感染细胞提取物从内源性病毒DNA转录出一组RNA，其电泳迁移与在完整感染细胞中观察到的相似。晚期感染细胞提取物似乎除了在感染后早期转录的那些RNA外，无法转录新的RNA种类。通过用微球菌核酸酶消化，提取物的活性可以依赖于外源模板。向经核酸酶处理的提取物中加入天然或变性病毒DNA后转录的RNA与从内源性病毒DNA转录的RNA无法区分。用微球菌核酸酶消化的晚期感染细胞提取物在从p2SB21（一种含有ASFV DNA的SalI B片段的重组质粒）转录病毒特异性RNA方面也具有活性。
