Cyclin B1 Destruction Box-Mediated Protein Instability: The Enhanced Sensitivity of Fluorescent-Protein-Based Reporter Gene System

Abstract

The periodic expression and destruction of several cyclins are the most important steps for the exact regulation of cell cycle. Cyclins are degraded by the ubiquitin-proteasome system during cell cycle. Besides, a short sequence near the N-terminal of cyclin B called the destruction box (D-box; CDB) is also required. Fluorescent-protein-based reporter gene system is insensitive to analysis because of the overly stable fluorescent proteins. Therefore, in this study, we use human CDB fused with both enhanced green fluorescent protein (EGFP) at C-terminus and red fluorescent protein (RFP, DsRed) at N-terminus in the transfected human melanoma cells to examine the effects of CDB on different fluorescent proteins. Our results indicated that CDB-fused fluorescent protein can be used to examine the slight gene regulations in the reporter gene system and have the potential to be the system for screening of functional compounds in the future.

Oncogenic Role of Tumor Necrosis Factor α-Induced Protein 8 (TNFAIP8)

“…Interestingly, 80% of the TIPE3 D-Box consensus sequence is similar to the hTNFAIP8 and mTNFAIP8, and a significant variation in TIPE1 and TIPE2 D-Box consensuses with hTNFAIP8 was observed (Figure 1B). The D-Box has been reported in cell cycle-related proteins like p21 [51], cyclin B1 [52], cyclin A2 [53], Nek2A [54] and STK31 [55] (Figure 2C). The D-Box consensus facilitates the degradation of cell cycle-related proteins by anaphase-promoting complex or cyclosome (APC/C) during cell cycle progression [56].…”

Cell-by-cell dissection of phloem development links a maturation gradient to cell specialization

“…References (39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53) are cited only in the supplementary material…”

Establishment of a melanogenesis regulation assay system using a fluorescent protein reporter combined with the promoters for the melanogenesis-related genes in human melanoma cells

“…The measurements were set to excitation/emission of 488/507 nm for EGFP. The measured intensity was expressed as relative fluorescent intensity [18].…”

“…PCR was performed using 1.5 mM MgCl2, 0.2 mM dNTP, 2.5 units of Taq DNA polymerase and 0.1 M each of the primers ( Table 1). The amplified products were separated in 2% agarose gel in Tris-borate-EDTA (TBE) buffer and stained with ethidium bromide [18].…”

“…Fluorescent proteins are frequently used in reporter gene systems because of their simplicity, rapidity and clarity [17][18][19]. In this study, we established a melanogenesis regulation assay using a fluorescent protein reporter combined with the promoters for the MITF, Tyr and Dct genes in MeWo human melanoma cells.…”