Apoptosis methods, genes and proteins

A cell and genotype technology, applied in the field of genes and proteins, which can solve problems such as inability to die, lack of "small" cells, and inability to respire functional mitochondria

Inactive Publication Date: 2009-05-20
MORVUS TECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0039] Greenhalf et al. (1996) demonstrated that although Bax induction always prevents yeast cell growth under any circumstances, it fails to cause death in "petite" cells which cannot because of lack of functional mitochondria breathe

Method used

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  • Apoptosis methods, genes and proteins
  • Apoptosis methods, genes and proteins
  • Apoptosis methods, genes and proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0564] Example 1: Generation of DNA constructs

[0565] Generation of construct DNA allowing expression of pro-apoptotic Bax

[0566] Constructs that induce expression of Bax from different chromosomal loci in yeast

[0567] Yeast integration vectors pRS305 (with S. cerevisiae LEU2 gene as selectable marker), pRS402 (with S. cerevisiae ADE2 gene as selectable marker) and pRS404 (with S. Saccharomyces cerevisiae TRP1 gene).

[0568] The XhoI-SacIGAL1p-MS fragment, which contains the Saccharomyces cerevisiae GAL1 gene promoter fragment and the SUC2 gene terminator fragment (S) preceding the c-myc tag fragment (M), was isolated from construct pBGAL1MS (a pBlueScript KS(+) derivative; figure 1 ; Figure 61 GAL1MS sequence shown in SEQ ID NO: 1), and ligated to XhoI-SacI digested pRS305, pRS402 and pRS404 vectors, so as to integrate at LEU2, ADE2, TRP1 and HIS3 chromosomal loci. Four resulting plasmids yIPLEUG1 ( figure 2 ), yIPADEG1( image 3 ), yIPTRPG1 ( Figure 4 ) a...

Embodiment 2

[0582] Example 2: Optimization of a Yeast Screening System for Selection of New Anti-apoptosis Genes from Human Hippocampus (YAS)

[0583] Yeast growth conditions

[0584] YPD (1% Bacto yeast extract, 2% Bacto peptone and 2% glucose) is a rich medium. Synthetic minimal medium (SC) consisted of 0.67% yeast nitrogen source (Difco) and 2% glucose (SD) or 2% galactose (SG). Appropriate growth supplements (adenine, histidine, lysine, leucine, uracil or tryptophan) were added to SD or SG for growth of a particular strain depending on the plasmid carried by the strain. Save the supplement as a stock solution and add to an appropriate volume of medium. The concentrations and volumes of stock solutions given below are required to prepare 1 liter of medium. The final concentration of adenine, uracil, tryptophan and histidine is 20 mg / l, lysine and tyrosine are 30 mg / l, and leucine is 100 mg / l.

[0585] Establishment of Yeast Anti-apoptosis Screening System

[0586] In order to ...

Embodiment 3

[0616] Example 3: Screening of anti-apoptotic genes in human hippocampus

[0617] Construction and Amplification of Human Hippocampal cDNA Library

[0618] A human hippocampal cDNA library was routinely synthesized using BioCat GmbH (Heidelberg, Germany). The cDNA library was made from whole human adult normal brain hippocampus mRNA. First-strand cDNA synthesis was performed using oligo(dT)-NotI primer and M-MLV-RNase H-reverse transcriptase. Second-strand synthesis was performed using random linker primers and Klenow Exo-DNA polymerase. For directional cloning, sticky-ended cDNAs were first digested with Not I followed by size fractionation on a 1.3% agarose gel. After elution of the cDNA, the cDNA larger than 0.6 kb was ligated into NotI and BsaBI digested yeast expression vector pSYE224 (Figure 39). The plasmid cDNA library was obtained as a glycerol stock. In order to preserve all the genetic information of this library, a method using semi-solid amplification was e...

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Abstract

A W303a Saccharomyces cerevisiae yeast cell which contains a polynucleotide that encodes a functional Bax polypeptide under the control of a galactose- inducible promoter that is integrated at the LEU2 chromosomal locus. A kit of parts comprising the yeast cells and a yeast plasmid vector suitable for transforming a cDNA library into the yeast cells. Use of the yeast cell for screening a cDNA library for a polynucleotide that is or encodes an inhibitor of B ax-mediated apoptosis. Genes and polypeptides that inhibit Bax-mediated apoptosis and which were identified from a human hippocampus cDNA library screened in the yeast cells. A method of combating Bax-mediated apoptosis in a cell using an inhibitor of Bax-mediated apoptosis which was identified from a human hippocampus cDNA library screened in the yeast cells. A method of promoting Bax-mediated apoptosis in a cell using an inhibitor or antagonist of the anti-apoptotic polypeptides identified from a human hippocampus cDNA library screened in the yeast cells.

Description

technical field [0001] The present invention relates to the identification of genes and proteins that regulate apoptosis. In particular, the present invention relates to yeast strains useful in methods for identifying modulators of apoptosis, screening methods using said yeast strains, and genes and proteins identified thereby. The invention also relates to medical uses of the identified genes and proteins. Background technique [0002] Abnormal expression of apoptosis regulatory proteins is often the cause of various diseases such as cancer, rheumatoid arthritis, neurodegeneration and cardiovascular diseases. Numerous lines of evidence strongly suggest that apoptosis contributes to the death of neuronal cells in various neurodegenerations. Such as in stroke, spinal cord trauma (spinal cord trauma), head injury (head injury), spinal muscular atrophy (spinal muscular atrophy, SMA), amyotrophic lateral sclerosis (amyotrophic lateral sclerosis, ALS), Alzheimer's Apoptosis pl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/81
CPCA61K38/52A61K38/51C07K14/4747A61K38/177C12Y502/01008A61K38/00A61K38/1716A61K38/46A61K38/1709G01N2510/00A61K38/45A61K38/44A61P3/10A61P9/00A61P9/10A61P19/02A61P25/00A61P25/14A61P25/16A61P25/28A61P27/02A61P27/06A61P27/12A61P29/00A61P35/00A61P43/00
Inventor 巴巴托什·乔杜里
Owner MORVUS TECH LTD
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