Screening Of Anti-apoptotic Genes Interacting With Bax From Human Embryo Brain CDNA Library By Yeast Two-hybrid System

Apoptosis is one of the normal physiological phenomena and closely related to the growth,development and the respondence to the environmental stress in life process.The apoptosis is genetically controlled by a series of genes,including anti-apoptotic genes and pro-apoptotic genes.Up to now,some of the genes related to apoptosis have been cloned.For example,the pro-apoptotic gene Bax may induce cells to undergo apoptosis when ectopically expressed in yeast.Bcl-2 family genes play an evolutionarily conserved role in regulating the death of the cell.However,the molecular mechanism of apoptosis is still unfortunately dimness.In the present study,we identified some novel anti-apoptotic genes from a human embryo brain cDNA library by yeast-two hybrid technique,and preliminary studied their possible interactions with Bax gene.1.We successfully constructed the pJG45-PpBI-1 plasmid for studying the function of the anti-apoptotic gene PpBI-1(a plant Bax inhibitor-1 gene cloned from Phyllostachys praecox),and also constructed the pGilda-Bax plasmid for screening the anti-apoptotic genes in human embryo brain cDNA library.2.On the basis of the principle of pro-apoptotic function of Bax,26 clones that can normally grow in the selection medium has been screened using yeast-two hybrid system.The PCR tests of the total DNA of these clones with Bax's primers (Baxp1/Baxp2)showed that there were 24 clones containing the Bax sequences (possess 92.3%),suggesting the normal growth is due to the interactions of Bax with suppressors from the human embryo brain cDNA library,and not the losing of Bax。3.Among 24 clones,11 clones were selected to PCR amplification,sequencing and BLAST analysis of those cDNA library sequences.It was showed that they all contain cDNA fragments of human genomes.These cDNA fragments might suppress the function of Bax.4.Five of 11 clones were selected to determine the roles of these cDNA fragments in inhibition to apoptosis induced by Bax.The clones with the Bax plasmids but without the cDNA library plasmids were isolated through subcultures. The growth test showed that these clones contain normal growth in the selection medium,suggesting stable inhibition to apoptosis.5.We transformed these cDNA library plasmids(named B6,A10,A19 respectively)into a new yeast strain(BF264-15Dau/Yep51-Bax)and tested the growth characteristic.As a result,the clones could grow in the selection medium with Gal.On the basis of the results obtained in the present study,the roles of these cDNA fragments were preliminary analyzed.A further understanding of the association between cDNA fragments and Bax from protein level should facilitate the functions of these cDNAs.