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RNA interferases and methods of use thereof

A technology of use and enzyme polypeptide, applied in the direction of DNA preparation, recombinant DNA technology, biochemical equipment and methods, etc.

Inactive Publication Date: 2014-07-23
UNIV OF MEDICINE & DENTISTRY OF NEW JERSEY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no evidence that Kid(PemK) inhibits the replication of chromosomal DNA

Method used

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  • RNA interferases and methods of use thereof
  • RNA interferases and methods of use thereof
  • RNA interferases and methods of use thereof

Examples

Experimental program
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Effect test

Embodiment I

[0285] As described here, treatment with toluene permeabilized E. coli cells, and these cells were used to prove that MazF inhibited translation, but did not inhibit RNA synthesis or DNA replication. Moreover, it was shown that MazF specifically cleaves mRNA between the A and C residues of the ACA sequence in a ribosome-independent manner. Therefore, the present invention proves that MazF interferes with the function of mRNA by cutting the mRNA at a specific site. Therefore, the inventors of the present invention discovered that MazF is a new type of endoribonuclease, and named it "mRNA interfering enzyme" here.

[0286] Materials and Method

[0287] Strains and plasmids. E. coli BL21 (DE3), BW25113 (Datsenko and Wanner, Proc Natl Acad Sci US A97, 6640-5 (2000)) and MRE600 (Swaney et al., Antimicrob Agents Chemother 42, 3251-5 (1998)) were used. The plasmid pET-21cc-MazEF was constructed from the plasmid pET-21cc (Novagen), which was modified to express MazE and MazF (His) under...

Embodiment II

[0326] Importantly, before the discovery of the present invention, the cellular target of MazF has not been identified. As shown here, MazF acts as a highly sequence-specific endoribonuclease, which cuts cellular mRNA at the ACA site. This activity may achieve partial or complete inhibition of protein synthesis in cells. Standard calculations are performed based on the same principle that any one of the four nucleotides is incorporated into each of the three nucleotide positions in the ACA sequence. Based on this standard calculation, the ACA sequence appears in The predicted frequency in RNA transcripts is 1 / 64. It should be understood that some RNA transcripts contain lower or higher frequencies of ACA sequences compared to predicted frequencies. Therefore, the sensitivity of a particular RNA transcript or a family of related RNA transcripts to cleavage by MazF endoribonuclease depends on the frequency of the ACA sequence or MazF target sequence in the transcript. Moreover...

Embodiment III

[0328] As described above, in E. coli, programmed cell death is considered to be mediated through the "addiction component" system, each of which consists of a pair of co-expressed genes, which encode a stable toxin And unstable antitoxin. Their expression is automatically regulated by the toxin / antitoxin complex or only by the antitoxin. When co-expression is inhibited, the antitoxin is rapidly degraded by proteases, allowing the toxin to act on its target. In E. coli, extrachromosomal elements are the main genetic system of bacterial programmed cell death. The most studied extrachromosomal addictive component is the phd-doc on phage P1 (Lehnherr et al. (1993) J Mol Biol 233, 414-428; Lehnherr and Yarmolinsky (1995) Proc Natl Acad Sci USA 92, 3274-3277; Magnuson and Yarmolinsky (1998) J Bacteriol180, 6342-6351; Gazit and Sauer (1999) J Biol Chem274, 16813-16818; Gazit and Sauer (1999) J Biol Chem274, 2652-2657), ccdA-ccdB on the F factor (Tam and Kline (1989) J Bacteriol171...

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Abstract

The present invention is directed to the discovery of a novel family of enzymes designated herein as mRNA interferases that exhibit endoribonuclease activity. The novel finding of the present inventors, therefore, presents new applications for which mRNA interferase nucleic and amino acid sequences, and compositions thereof may be used to advantage. The invention also encompasses screening methods to identify compounds / agents capable of modulating mRNA interferase activity and methods for using such compounds / agents. Also provided is a kit comprising mRNA interferase nucleic and / or amino acid sequences, mRNA interferase activity compatible buffers, and instruction materials.

Description

[0001] This application is a divisional application for an invention patent application with an application date of June 14, 2004, an application number of 200480023092.X, and an invention title of "RNA interfering enzyme and its method of use". Invention field [0002] The present invention relates to the field of molecular biology, especially the discovery of new enzyme activities. In particular, the present invention relates to the identification of a new protein family named here as mRNA interferase (interferase). Exemplary members of the family described here include MazF and PemK, as well as their homologs and orthologs. More particularly, the present invention relates to the characterization of the biochemical properties of MazF and PemK polypeptides as endoribonuclease or mRNA interferase. The invention also includes the analysis of related proteins, which function to inhibit the activity of mRNA interfering enzymes. In particular, the characterization of MazE protein f...

Claims

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Application Information

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IPC IPC(8): C12N15/70A61K48/00A61K38/46A61P31/04A61P29/00A61P37/02A61P17/00A61P17/06A61P37/08A61P11/06A61P9/10A61P41/00A61P43/00C12N15/10C12NC12Q1/42
CPCC12P19/34C12N15/70C12Q1/42G01N2333/922G01N2800/26C12N9/22A61P11/06A61P17/00A61P17/06A61P29/00A61P31/04A61P35/00A61P37/02A61P37/06A61P37/08A61P41/00A61P43/00A61P9/00A61P9/10A61K38/465C12N15/635C12Q1/44
Inventor M·伊诺耶J·张Y·L·张
Owner UNIV OF MEDICINE & DENTISTRY OF NEW JERSEY
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