T*****e
发帖数: 247 | 1 http://www.nature.com/nature/journal/vaop/ncurrent/full/nature1
标题 DNA-guided DNA interference by a prokaryotic Argonaute
摘要
RNA interference is widely distributed in eukaryotes and has a variety of
functions, including antiviral defence and gene regulation1, 2. All RNA
interference pathways use small single-stranded RNA (ssRNA) molecules that
guide proteins of the Argonaute (Ago) family to complementary ssRNA targets:
RNA-guided RNA interference1, 2. The role of prokaryotic Ago variants has
remained elusive, although bioinformatics analysis has suggested their
involvement in host defence3. Here we demonstrate that Ago of the bacterium
Thermus thermophilus (TtAgo) acts as a barrier for the uptake and
propagation of foreign DNA. In vivo, TtAgo is loaded with 5′-phosphorylated
DNA guides, 13–25 nucleotides in length, that are mostly plasmid derived
and have a strong bias for a 5′-end deoxycytidine. These small interfering
DNAs guide TtAgo to cleave complementary DNA strands. Hence, despite
structural homology to its eukaryotic counterparts, TtAgo functions in host
defence by DNA-guided DNA interference.
大意
1. 转化得到多少plasmid的效果跟这个DNA介导的DNA干扰有关
用突变掉这个Ago酶的菌株，以后max prep都可以被mini prep替换了？
2. DNA作引子可以切割DNA
有点像CRISPR啊
3. 新的细菌免疫机制 | r*****t
发帖数: 4793 | 2 有趣
确实细胞内除基因组外的DNA是个好题目
有没有，有多少，什么样什么功能，哇呀呀，很可以展开啊 | p******b
发帖数: 379 | 3 这个文章很有意思啊， 昨天我跟老板说了下想做“DNA作引子可以切割DNA”， 老板说
现在才开始做太迟了，不推荐 呵呵， | p******b
发帖数: 379 | 4 另一个有意思的问题是不知道真核细胞里面有没有这种类似的机制？ 真核里面有AGO1-
4， 说不定某个AGO 就有这种作用 | s******y
发帖数: 28562 | 5 有意思。谢谢分享！
targets:
bacterium
【在 T*****e 的大作中提到】
: http://www.nature.com/nature/journal/vaop/ncurrent/full/nature1
: 标题 DNA-guided DNA interference by a prokaryotic Argonaute
: 摘要
: RNA interference is widely distributed in eukaryotes and has a variety of
: functions, including antiviral defence and gene regulation1, 2. All RNA
: interference pathways use small single-stranded RNA (ssRNA) molecules that
: guide proteins of the Argonaute (Ago) family to complementary ssRNA targets:
: RNA-guided RNA interference1, 2. The role of prokaryotic Ago variants has
: remained elusive, although bioinformatics analysis has suggested their
: involvement in host defence3. Here we demonstrate that Ago of the bacterium
| T*****e
发帖数: 247 | 6 这个Ago的存在方式可能不大像CRISPR的Cas9酶。
据我所知，不同细菌种类的Cas9可能作用的方式略有区别，但单体原理一致。
但这个Ago在文章中有个例子，他们发现另一种细菌Rhodobacter sphaeroides的Ago用
RNA介导而非DNA。
所以是不是说Ago的版本会更丰富呢。 | K**4
发帖数: 1015 | 7 这个文章提出的DNA-guidedDNAinterference by bacterial Argonaute (or PIWI) 好
像不是很充分阿。首先他自己的实验也表明，Argonaute即结合RNA也结合DNA。
Analysis of co-purifiednucleicacidsrevealedthatTtAgo-associatedRNA (10–150
nucleotides) is preferentially 32 P-labelled in a polynucleotide kinase (PNK
) forward reaction, indicating the presence of 5' hydroxyl groups (Extended
Data Fig. 1c). By contrast, co-purified DNA has a more defined length (13–
25 nucleotides), and is preferentially labelled in a PNKexchangereaction,
indicatingphosphorylated 5'ends(Fig.2b).
至于他排除RNA的原因是：During isolation of anactive
site double mutant, TtAgoDM (TtAgo(D478A,D546A); Fig. 2a), only
RNAs co-purify (10–150 nucleotides; Extended Data Fig. 1c). This
suggests that active site residues are involved in processing and/or
binding of the ssDNA molecules.这个实际上似乎还是有点牵强的。
第三个原因，在前面的文章中，bacterial Argonaute表明可以结合 both RNA and DNA
.所以忽略RNA，现在就提出这个siDNA 有点早。
Structure of an argonaute silencing complex with a seed-containing guide DNA
and target RNA duplex
http://www.nature.com/nature/journal/v456/n7224/abs/nature07666
Nucleation, propagation and cleavage of target RNAs in Ago
silencing complexes.
http://www.nature.com/nature/journal/v461/n7265/full/nature0843
targets:
bacterium
【在 T*****e 的大作中提到】
: http://www.nature.com/nature/journal/vaop/ncurrent/full/nature1
: 标题 DNA-guided DNA interference by a prokaryotic Argonaute
: 摘要
: RNA interference is widely distributed in eukaryotes and has a variety of
: functions, including antiviral defence and gene regulation1, 2. All RNA
: interference pathways use small single-stranded RNA (ssRNA) molecules that
: guide proteins of the Argonaute (Ago) family to complementary ssRNA targets:
: RNA-guided RNA interference1, 2. The role of prokaryotic Ago variants has
: remained elusive, although bioinformatics analysis has suggested their
: involvement in host defence3. Here we demonstrate that Ago of the bacterium
| b*****s
发帖数: 169 | 8 赞！
150
PNK
Extended
【在 K**4 的大作中提到】
: 这个文章提出的DNA-guidedDNAinterference by bacterial Argonaute (or PIWI) 好
: 像不是很充分阿。首先他自己的实验也表明，Argonaute即结合RNA也结合DNA。
: Analysis of co-purifiednucleicacidsrevealedthatTtAgo-associatedRNA (10–150
: nucleotides) is preferentially 32 P-labelled in a polynucleotide kinase (PNK
: ) forward reaction, indicating the presence of 5' hydroxyl groups (Extended
: Data Fig. 1c). By contrast, co-purified DNA has a more defined length (13–
: 25 nucleotides), and is preferentially labelled in a PNKexchangereaction,
: indicatingphosphorylated 5'ends(Fig.2b).
: 至于他排除RNA的原因是：During isolation of anactive
: site double mutant, TtAgoDM (TtAgo(D478A,D546A); Fig. 2a), only
| P****d
发帖数: 564 | 9 Thanks!
150
PNK
Extended
【在 K**4 的大作中提到】
: 这个文章提出的DNA-guidedDNAinterference by bacterial Argonaute (or PIWI) 好
: 像不是很充分阿。首先他自己的实验也表明，Argonaute即结合RNA也结合DNA。
: Analysis of co-purifiednucleicacidsrevealedthatTtAgo-associatedRNA (10–150
: nucleotides) is preferentially 32 P-labelled in a polynucleotide kinase (PNK
: ) forward reaction, indicating the presence of 5' hydroxyl groups (Extended
: Data Fig. 1c). By contrast, co-purified DNA has a more defined length (13–
: 25 nucleotides), and is preferentially labelled in a PNKexchangereaction,
: indicatingphosphorylated 5'ends(Fig.2b).
: 至于他排除RNA的原因是：During isolation of anactive
: site double mutant, TtAgoDM (TtAgo(D478A,D546A); Fig. 2a), only
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