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Mitochondrion RNA polymerase subsidiary transcription factor as well as polymerase-transcription factor compound and application thereof

An RNA polymerase and transcription factor technology, applied in the field of novel polymerase-transcription factor complexes, which can solve problems such as mitochondrial DNA deletion

Inactive Publication Date: 2011-07-20
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, Mtf1-knockout budding yeast showed smaller cell morphology, loss of mitochondrial DNA, and inability to grow on non-fermentative carbon source medium

Method used

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  • Mitochondrion RNA polymerase subsidiary transcription factor as well as polymerase-transcription factor compound and application thereof
  • Mitochondrion RNA polymerase subsidiary transcription factor as well as polymerase-transcription factor compound and application thereof
  • Mitochondrion RNA polymerase subsidiary transcription factor as well as polymerase-transcription factor compound and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1: Cloning of novel mitochondrial RNA polymerase Rpo41 and accessory transcription factor Mtf1 from fission yeast

[0077] According to the amino acid sequence of the homologous protein in budding yeast, Blast comparison was carried out on Genedb, the website of the Sanger Research Center in the United Kingdom. The results showed that there was a highly conserved homologous mitochondrial RNA polymerase in fission yeast, and the system name was SPAC26H5.12 , and a highly conserved mitochondrial transcription factor, the system name SPAC1002.08c. The amino acid sequence comparison results between Mtf1 homologous proteins in fission yeast and budding yeast can be found in figure 1 , by comparison, the applicant found that fission yeast Mtf1 has a conserved N-terminal domain and a conserved C-terminal domain, see figure 2 .

[0078] In the following embodiments of the present invention, unless otherwise specified, SPAC26H5.12 is referred to as Rpo41 for short, an...

Embodiment 2

[0079] Example 2: Sublocalization of fission yeast Mtf1 in cells

[0080] In order to study the function of fission yeast Mtf1 in cells, the present invention uses the ultracentrifugation method to extract fission yeast mitochondria, nucleus and cytoplasmic components respectively, and uses the Western-blot method to detect the sublocalization of Mtf1 in cells, such as Figure 4 As shown, Mtf1 is localized in both the nucleus and mitochondria. This result suggests that Mtf1 not only functions in the mitochondria of fission yeast, but also has the function of transcribing genes in the nucleus.

Embodiment 3

[0081] Example 3: Fission yeast Mtf1 knockout mutants have defects in cell cycle regulation and cell morphology

[0082] In order to explore its cell function, the present invention knocks out the Mtf1 gene of fission yeast. The knockout is carried out in diploid cells, and the spores produced by mating are observed under a microscope after germination. Cells lacking Mtf1 exhibited a similar morphology, ie, the knockout strain failed to form colonies and could only divide a few times on the plate. image 3 showed that cells lacking Mtf1 were morphologically longer and larger than wild-type cells, and became egg- or bottle-shaped. DAPI staining showed that Mtf1-deficient cells had no apparent nuclear defects. Staining with fluorescent white Calcofluor (Calcofluor fluorescent stain) showed that cells lacking Mtf1 had a higher proportion of cell compartments, some of which contained multiple cell compartments. This result suggests that Mtf1 may have the function of regulating c...

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PUM

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Abstract

The invention belongs to the technical field of biology and relates to a mitochondrion RNA polymerase subsidiary transcription factor Mtf1 and a polymerase-transcription factor compound. The amino acid sequence of the mitochondrion RNA polymerase subsidiary transcription factor Mtfl provided by the invention comprises 1-366 sites in a sequence table SEQ ID NO:3. RNA polymerase protein and Mtf1 can act to each other to form the polymerase-transcription factor compound. The Mtf1 can be used for transcribing RNA, relates to regulation on a cell cycle, and can be used as a drug target for screening antitumor drugs. The invention provides a set of accurate and effective transcription tool for the gene transcription in cell nucleus and also provides a new approach for the study of gene functions. Meanwhile, the Mtf1 relates to the regulation on the cell cycle, a gene knockout strain of the Mtf1 cannot form cloning, and the Mtf1can be used as a target for screening antitumor drugs.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a novel polymerase-transcription factor complex. More specifically, the present invention relates to the gene cloning, protein preparation, function research and application in the field of biotechnology of a mitochondrial RNA polymerase Rpo41-transcription factor Mtf1 complex. Background technique [0002] Transcription of DNA into RNA in cells is one of the most important processes in life. Among them, the transcription complex composed of DNA-dependent RNA polymerase and accessory transcription factors is the core device to complete the above process [References 1, 2, 3, 4]. Therefore, the research on the transcription complex has always been a hot spot in the scientific community. It is now generally accepted that three classes of RNA polymerases (I, II, and III) responsible for transcription of nuclear genes are encoded in eukaryotes [Ref. 5]. Among them, the most important tran...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/39C12N15/31C12N9/00C12P21/02C12Q1/68C12Q1/02
Inventor 阿拉斯太尔·马歇尔王哲陈东戎孙文夏蒋恒义张静
Owner FUDAN UNIV