Revision as of 15:18, 26 October 2020

clb2 promoter (during G2 phase)

Purpose

Works for the cell synchronization device, B-type cyclin involved in cell cycle progression; activates Cdc28p to promote the transition from G2 to M phase;

Principle

It is a promoter which promotes transciption in G2 of the yeast cell cycle .If you want to express your proteins in G2 of the yeast cell cycle ,you can add the cln2 sequence before your gene sequence ,then put them in a yeast plasmid .After transformation ,you can get the protein you want from the metabolic product of the Bacterial you use for transformation .
Sequence and Features

BNU-China 2020 - Contribution

We know from many documents that the Clb2 promoter can periodically regulate the transcription of foreign proteins. At the same time, there is a UAS sequence available for regulation on the promoter sequence. And it is feasible to couple the promoter to GFP. This can be used as a way to verify the expression of the promoter. In the research by Trcek, Tatjana et al. 2011，replaced the coding sequence in clb2 with DOA1 and applied the original regulatory part of clb2, and the result of the cycle change in mRNA level is shown in the figure. The transcription effect of this promoter in G2 phase was verified.

Figure1. DOA1 mRNA expressed from the CLB2 promoter and the integration cassette used. Colors denote gene origins: ACT1, blue; DOA1 pale blue; CLB2 violet.The transcription effect of CLB2 promoter varies with different stages of the cell cycle(Trcek, Tatjana. et al, 2011)

In another research(Maher M, 1995), they sequenced the region of Clb2 promoter. The major start site at +1 is indicated by an arrow, and other start sites are indicated by asterisks. Other features shown are the ATG translation initiator codon at position 362, putative TATA boxes at positions -19 and -113 (underlined), and four sequences which represent possible Mcm1 binding sites.

Figure2. Sequence and features of the CLB2 promoter. The major start site at 11 is indicated by an arrow. The ATG translation initiator codon is at position 362. Putative TATA boxes is at positions -19 and -113 (underlined). Four sequences which represent possible Mcm1 binding sites is in boldface type and underlined.

Then, they constructed a series of deletions and assayed promoter function. This analysis defined a region from positions -362 to -131 as being important for CLB2. Multiple elements in this region contribute to the overall control of CLB2 transcription, suggesting that the region from position-362 to -131 promoter fragment can be used as a UAS for cell cycle regulation.

Some researchers indicated that GFP, a polypeptide of similar size as Myc12, has no appreciable effect on Clb2 activity. Clb2-GFP has been applied for earlier localization studies, implying that GFP properly folds and adopts a defined 3D structure.

Figure 3. Ability of UAS deletions to support cell cycle-regulated transcription. UAS sequences mapped by deletion analysis as for panel A were inserted upstream of a ubiYlacZ reporter gene. Cells were synchronized with a-factor, and ubiYlacZ, CLB2, H2A, and Prt1 transcript levels were assessed by Northern analysis in a synchronous population of cells.

Reference

[1]Trcek, Tatjana et al. “Single-molecule mRNA decay measurements reveal promoter- regulated mRNA stability in yeast.” Cell vol. 147,7 (2011): 1484-97. doi:10.1016/j.cell.2011.11.051 [2]Maher M, Cong F, Kindelberger D, Nasmyth K, Dalton S. Cell cycle-regulated transcription of the CLB2 gene is dependent on Mcm1 and a ternary complex factor. Mol Cell Biol. 1995;15(6):3129-3137. doi:10.1128/mcb.15.6.3129 [3]Hood JK, Hwang WW, Silver PA. The Saccharomyces cerevisiae cyclin Clb2p is targeted to multiple subcellular locations by cis- and trans-acting determinants. J Cell Sci. 2001 Feb;114(Pt 3):589-97. [4]Kuczera T, Bayram Ö, Sari F, Braus GH, Irniger S. Dissection of mitotic functions of the yeast cyclin Clb2. Cell Cycle. 2010 Jul 1;9(13):2611-9. doi: 10.4161/cc.9.13.12082. [5]Veis, J., Klug, H., Koranda, M., & Ammerer, G. (2007). Activation of the G2/M-specific gene CLB2 requires multiple cell cycle signals. Molecular and cellular biology, 27(23), 8364–8373. https://doi.org/10.1128/MCB.01253-07