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DNA (deoxyribonucleic acid) phosphorothioation modifier gene cluster

A technology for phosphorylation and modification of genes, which is applied in the field of DNA phosphorylation modification of gene clusters, and can solve problems such as no discovery.

Active Publication Date: 2015-11-25
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Surprisingly, no common dnd gene cluster

Method used

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  • DNA (deoxyribonucleic acid) phosphorothioation modifier gene cluster
  • DNA (deoxyribonucleic acid) phosphorothioation modifier gene cluster
  • DNA (deoxyribonucleic acid) phosphorothioation modifier gene cluster

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: Preliminary inference of CpsC phosphorylation gene cluster

[0055] Download the genome file of marine halophilic Vibrio FF75 from the NCBI website, and upload it to the Rapidannotationusingsubsystemtechnology website for genome annotation. After the analysis is complete, use thioredoxin (thioredoxin, PAPS reductase) and cysteinedesulfurase (cysteine ​​desulfurase) as keywords to search for all about 4,500 genes. The obtained candidate genes were compared upstream and downstream one by one, and a gene cluster composed of three genes with a length of about 6 kb was found, and the third gene was PAPS reductase. At the same time, it was found that there is a gene encoding cysteine ​​desulfurase at about 7 kb upstream of the gene cluster. Other genes in and around this region do not belong to any sulfur metabolism system, so it is inferred that this region is very likely to be a CpsC phosphorylation-modified gene cluster encoding DNA. These 4 genes are named as v...

Embodiment 2

[0056] Example 2: Proof of gene knockout vpt1, vpt2, vpt3 and vpt4 Vibrio halophilus Vibriocyclitrophicus Essential genes for phosphorylation of FF75DNA

[0057] According to the genome sequence of marine halophilus Vibrio FF75, the primers corresponding to the four genes were designed. Using the extracted total DNA of Vibrio FF75 as a template, PCR on the left and right arms of each gene, the second PCR synthesis of left and right arm conjoined, the conjoined are about 1.5kb. The left and right arms of the TA clone were joined together for sequencing. After the result is correct, use the SpeI and SacI double enzyme digestion system to process the recombinant T vector to obtain the left and right arm conjoined fragments; use the same endonuclease combination to process the suicide vector pJC4 plasmid to obtain a double-nicked linear plasmid band. Those are connected by T4 ligase.

[0058] Convert the connected product into E.coli WM6024 (the bacterium is DAP auxotrophic), spr...

Embodiment 3

[0061] Example 3: Marine halophilic Vibrio Vibriocyclitrophicus Boundary determination of FF75 DNA phosphorylation modified gene cluster

[0062] The mutant strains XXL-4, XXL-2, XXL-5, XXL-7 in Example 2 (knockout respectively vpt1 , vpt2 , vpt3 , vpt4 ) Loss of DNA phosphorylation modification, so according to the gene knockout method in Example 2, the upstream and downstream were knocked out to obtain mutant XXL-1 (knockout 1585) and mutant XXL-3 (knockout Except for 1587-1590), XXL-6 (knockout 1594), tested by LC-MS / MS, experiments show that mutants XXL-1, XXL-3 and XXL-6 still have DNA phosphorylation modification (see respectively under Figure 5 , 6 , 7), therefore, Vibrio Vibriocyclitrophicus The upstream boundary of the FF75 DNA phosphorylation modification gene cluster was determined between 1585 and vpt4 Between, the downstream boundary is determined at vpt3 And 1594, vpt4 with vpt1 There are 1587-1590 between. When knocked out in Example 2 vpt1 , vpt2 , ...

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Abstract

The invention discloses a DNA (deoxyribonucleic acid) phosphorothioation modifier gene cluster. DNA phosphorothioation modifiers controlled by the gene cluster are single-chain modifiers, selective sequence sites are CpsC, the integral phosphorothioation modifier gene cluster comprises four genes including an encoding cysteine desulfurase gene and a 3' phosphoadenosine-5' phosphosulfate (PAPS) reductase gene, and encoding proteins of the two other genes are members of ABC transport protein families and DUF4007 super-families respectively. The DNA phosphorothioation modifier gene cluster has the advantages that the genes and the proteins of the genes can be used in the bio-pharmaceutical science fields of heterogeneous-source host DNA modification, epigenetics, cell-free phosphorothioation modification system construction, oligonucleotide medicine modification and the like.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a gene cluster modified by phosphorothioation of DNA. Background technique [0002] It is well known that DNA is the carrier of genetic information, and various chemical modifications on DNA can cause changes in genetic information. Studies have found that there are many types of chemical modifications on DNA, which can be generally divided into two categories: one occurs on the bases of DNA, and the other occurs on the backbone of DNA. The first type of modification exists widely in organisms, and people have studied it thoroughly. The most famous one is the DNA base methylation modification found in bacteria in 1965. On the one hand, this modification can be used by organisms to mark their own DNA to distinguish foreign DNA, thereby limiting its invasion and ensuring the stability of biological inheritance; The development has played a milestone contributio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/54C12N15/53C12N15/10C12N15/70C12N15/76C12N15/81C12N15/82
Inventor 王连荣陈实熊啸林邓子新黎寒
Owner WUHAN UNIV
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