The Location Of Web2 With Ddc2 In S Phase Checkpoint Pathway Of S. Cerevisiae

PrefaceThe event of cell cycles takes place in the order of timing and space,which ensures that cell division and chromosome segreation are completed with high fidelity. In principle, the order of cell cycle events could be accomplished by requiring the completion of previous event. Checkpoint respond to DNA damage and DNA replication block by arresting the cell cycle to provide time for repair and by inducing transcription of genes that facilitate repair. Until the damage was repaired ,the cell cycle processed into division cycle. Checkpoint loss results in genomic instability and has been implicated in the evolution of normal cells to cancer cells.Previous experiments have suggested that the role of web2 in S checkpoint regulation and web2 gene acts on upstream of rad53 in S checkpoint signal pathway. There are several genes function upstream to rad53 such as pol2,mec1,tell and ddc2. ddc2 gene of S. cerevisiae encodes an essential protein kinase that is required for activition of replication - sensitive and DNA damage - sensitive checkpoint arrest and induction of transcription responses as rad53 gene. So we elected ddc2 gene as a marker to make sure the position of web2 in S checkpoint regulation mechanism and the interaction of web2 and other genes.MethodsTotal DNA were extracted from S. cerevisiae Ymw genome. Using the genome DNA as template, PCR produced a 2244bp fragment of ddc2 by using P1 and P2 which are designed restrict sites BamHI,EcoRI and stop coden TAA.The 2244bp fragment was recovered by double digestion, and was cloned into pMD18 - T vector . The gene of ddc2 was proofed by the DNA sequencing. pMD18 -T -ddc2d was double digested by BamHI and EcoRI ,the product of ddc2 was recovered and linked into pRS316 which is prepared by double digestion by BamHI and EcoRI. The novel vector pRS316 - ddc2 was transformed into web2 mutant strain. Transformants were chosen that were ura - but ura + when grown on SG - ura plates. HU sensitivity rescue test and RT - PCR was performed.ResultsIn the present study, by using PCR, we got the ddc2 gene and inserted it into the expression vector pRS316 to form the the expression vector pRS316 - ddc2 successfully. The ddc2 gene in pRS316 - ddc2 is under the control of GAL promoter, when the transformants grew in galactose, the ddc2 gene could express. ddc2 rescue test indicate web2 colonies growing YPG that were treated with HU after 1 hr are far less than those without HU. And the longer the time of HU be present ,the less the colonies grow. The coloines of web2 mutant with ddc2 plas-mid is higher than web2 mutant in the presence if HU,approaching the level of wild type. There also existed the same ddc2 rescue in ddc2 mutant. But the ddc2 plasmid couldn ' t rescue the mutant ' s survival in glucose. The pRS316 a-lone can ' t rescue web2 mutant in both galactose and glucose. These dates indicated ddc2 gene could relieve web2 mutant ' s HU sensitivity. The temperature control showed web2(ddc2) could grow at the permissive (30t ) and non-permissive (31X.) temperature sensitivity. ddc2 mRNA in yeast cells treated with HU was detected by RT - PCR. Consistent with wild - type, web2 ( ddc2 ) showed high level of ddc2 mRNA. The mRNA level didn ' t raise in web2 mutant and web2 ( pRS316 ) strain.DiscussionTaken together,the overexpression of ddc2 in respond to HU indicate that...