生物工程专业英语翻译,帮忙翻译一下吧.拜托啦、2.8. RNase H assayIn a 10 Al volume, 1 nM a-32P-labeled uidA RNAtranscript, 50 nM oligodesoxynucleotide, 40 mM Tris–HCl pH 7.5, 4 mM MgCl2, 1 mM DTT and 5 unitsRNase H were mixed and incubated at 37 8C for 1 min.The reaction was sto - 唯久问答

生物工程专业英语翻译,帮忙翻译一下吧.拜托啦、2.8. RNase H assayIn a 10 Al volume, 1 nM a-32P-labeled uidA RNAtranscript, 50 nM oligodesoxynucleotide, 40 mM Tris–HCl pH 7.5, 4 mM MgCl2, 1 mM DTT and 5 unitsRNase H were mixed and incubated at 37 8C for 1 min.The reaction was sto

2019-03-30

生物工程专业英语翻译,帮忙翻译一下吧.拜托啦、
2.8. RNase H assay
In a 10 Al volume, 1 nM a-32P-labeled uidA RNA
transcript, 50 nM oligodesoxynucleotide, 40 mM Tris–
HCl pH 7.5, 4 mM MgCl2, 1 mM DTT and 5 units
RNase H were mixed and incubated at 37 8C for 1 min.
The reaction was stopped by addition of 5 Al stopping
buffer. Reaction products were separated by polyacryl-
amide gel electrophoresis (5%) and gels were exposed
to an X-ray film after drying on Whatman filter paper.
Reaction products for each oligodesoxynucleotide were
determined twice in separate assays.
3. Results
3.1. In vitro analyses of ribozymes targeting the uidA
mRNA
The secondary structure of the uidA mRNA (1811 bp)
was analysed in silico using the program RNAdraw
(Matzura and Wennborg, 1996) and searched for sin-
gle-stranded regions containing a GUC sequence, which
follows the NHH rule and represents an efficient ribo-
zyme cleavage site (Ohkawa et al., 1995; Kore et al.,
1998). Seven such sites were identified within the uidA
mRNA. Four target sites are located in the 5V-region (nt
10, 67, 219, 297), two target sites are located in the
middle region (nt 476, 762) and one site within the 3V-
region of the substrate mRNA (nt 1332; data not shown).
Ribozymes targeting these cleavage sites (rz4–rz10)
were developed (Table 1), transcribed in vitro and ex-
amined qualitatively for cleavage activity of in vitro
transcribed uidA mRNAs. Three uidA substrate lengths
were tested, namely a short version buidAshortQ (nt 1–
217), a medium size version buidAmedQ (nt 1–1090) and
a full-length version buidAlongQ (nt 1–1811). We chose
three different substrate lengths as RNA transcripts
often show reduced stability with increased transcript
length.
Hendry and McCall (1996) reported that a relatively
short arm I (5 bp) together with a longer arm III (10 bp)
improves the cleavage activity of hammerhead ribo-
zymes. To analyse the influence of binding arm length
on cleavage activity, we constructed the ribozymes rz4
and rz5 in two versions, i.e. a symmetric version with
10 binding nucleotides on both arms I and III (rz4sym
and rz5sym), and an asymmetric version with five
binding nucleotides in arm I and 10 binding nucleotides
in arm III (rz4asym and rz5asym). Fig. 3 shows exem-
plarily the result of an in vitro ribozyme assay with
uidAshort as substrate and the symmetric and asym-
metric versions of ribozymes rz4 and rz5. All four

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2.8. 核糖核酸酶H鉴定
在10ml反应体系中,包含1纳摩尔的磷32标记的uidA基因的转录RNA,50纳摩尔的寡脱氧核苷酸,40毫摩尔pH值7.5的Tris – HCl缓冲液,4毫摩尔氯化镁,1毫摩尔DTT和5units（酶活单位）的核酸酶H,混合均匀后,于37摄氏度条件下孵育1分钟.加入5ml的终止缓冲液终止该反应.反应产物用聚丙烯酰胺凝胶电泳（5％）分离,将凝胶置于沃特曼滤纸上通过X射线检测.每个寡脱氧核苷酸反应产物分别检测两次.
3. 结果
3.1. uidA信使RNA核酶的体外分析
采用RNAdraw（Matzura和Wennborg,1996年）对硅片中uidA信使RNA（1811 bp）的二级结构进行了分析,根据NHH原则,单链中包含GUC序列表示其包含一个有效的核酶切割位点（Ohkawa等,1995; Kore的等,1998）.在uidA信使RNA中共鉴定获得了七个核酶切割位点.其中四个位于5V区域（nt10,67,219,297）,两个位于中间区域（nt476,762）,一个位于3V 区域（nt1332;数据未显示）.核酶作用于这些位点（rz4 - rz10,详见表1）进行转录并定性地检验其转录uidA信使RNA的活性.实验检测到三个不同长度的uidA底物,即一个短的buidAshortQ（nt1-217）,一个中等大小的buidAmedQ（nt1-1090）和全长的buidAlongQ（nt1-1811）.实验对三个不同的长度的底物进行RNA转录表明,随着长度的增加稳定逐渐降低.亨德利和麦考尔（1996）研究表明,同时增加一个较短臂I（5 BP）和一个较长臂III（10 bp）可提高了钝化的核酶的切割活性.为了分析粘性臂长度对切割活性的影响,实验分别构建了核酶rz4和rz5,即在臂I和臂III均加入10个粘性核苷酸的对称型（rz4sym和rz5sym）,以及分别在臂I增加5个核苷酸和在臂III增加10个核苷酸的非对称型（rz4asym和rz5asym）.图. 3显示了以短链uidA为底物的核酶及对称型和非对称型核酶rz4和rz5的检测结果.所有四个 2.8. 核糖核酸酶H鉴定
在10ml反应体系中,包含1纳摩尔的磷32标记的uidA基因的转录RNA,50纳摩尔的寡脱氧核苷酸,40毫摩尔pH值7.5的Tris – HCl缓冲液,4毫摩尔氯化镁,1毫摩尔DTT和5units（酶活单位）的核酸酶H,混合均匀后,于37摄氏度条件下孵育1分钟.加入5ml的终止缓冲液终止该反应.反应产物用聚丙烯酰胺凝胶电泳（5％）分离,将凝胶置于沃特曼滤纸上通过X射线检测.每个寡脱氧核苷酸反应产物分别检测两次.
3. 结果
3.1. uidA信使RNA核酶的体外分析
采用RNAdraw（Matzura和Wennborg,1996年）对硅片中uidA信使RNA（1811 bp）的二级结构进行了分析,根据NHH原则,单链中包含GUC序列表示其包含一个有效的核酶切割位点（Ohkawa等,1995; Kore的等,1998）.在uidA信使RNA中共鉴定获得了七个核酶切割位点.其中四个位于5V区域（nt10,67,219,297）,两个位于中间区域（nt476,762）,一个位于3V 区域（nt1332;数据未显示）.核酶作用于这些位点（rz4 - rz10,详见表1）进行转录并定性地检验其转录uidA信使RNA的活性.实验检测到三个不同长度的uidA底物,即一个短的buidAshortQ（nt1-217）,一个中等大小的buidAmedQ（nt1-1090）和全长的buidAlongQ（nt1-1811）.实验对三个不同的长度的底物进行RNA转录表明,随着长度的增加稳定逐渐降低.亨德利和麦考尔（1996）研究表明,同时增加一个较短臂I（5 BP）和一个较长臂III（10 bp）可提高了钝化的核酶的切割活性.为了分析粘性臂长度对切割活性的影响,实验分别构建了核酶rz4和rz5,即在臂I和臂III均加入10个粘性核苷酸的对称型（rz4sym和rz5sym）,以及分别在臂I增加5个核苷酸和在臂III增加10个核苷酸的非对称型（rz4asym和rz5asym）.图. 3显示了以短链uidA为底物的核酶及对称型和非对称型核酶rz4和rz5的检测结果.所有四个

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