Abstract
BackgroundTagged fusion proteins are priceless tools for monitoring the activities of biomolecules in living cells. However, over-expression of fusion proteins sometimes leads to the unwanted lethality or developmental defects. Therefore, vectors that can express tagged proteins at physiological levels are desirable tools for studying dosage-sensitive proteins. We developed a set of Entry/Gateway® vectors for expressing fluorescent fusion proteins in Drosophila melanogaster. The vectors were used to generate fluorescent CP190 which is a component of the gypsy chromatin insulator. We used the fluorescent CP190 to study the dynamic movement of related chromatin insulators in living cells.ResultsThe Entry/Gateway® system is a timesaving technique for quickly generating expression constructs of tagged fusion proteins. We described in this study an Entry/Gateway® based system, which includes six P-element destination vectors (P-DEST) for expressing tagged proteins (eGFP, mRFP, or myc) in Drosophila melanogaster and a TA-based cloning vector for generating entry clones from unstable DNA sequences. We used the P-DEST vectors to express fluorecent CP190 at tolerable levels. Expression of CP190 using the UAS/Gal4 system, instead, led to either lethality or underdeveloped tissues. The expressed eGFP- or mRFP-tagged CP190 proteins are fully functional and rescued the lethality of the homozygous CP190 mutation. We visualized a wide range of CP190 distribution patterns in living cell nuclei, from thousands of tiny particles to less than ten giant ones, which likely reflects diverse organization of higher-order chromatin structures. We also visualized the fusion of multiple smaller insulator bodies into larger aggregates in living cells, which is likely reflective of the dynamic activities of reorganization of chromatin in living nuclei.ConclusionWe have developed an efficient cloning system for expressing dosage-sensitive proteins in Drosophila melanogaster. This system successfully expresses functional fluorescent CP190 fusion proteins. The fluorescent CP190 proteins exist in insulator bodies of various numbers and sizes among cells from multiple living tissues. Furthermore, live imaging of the movements of these fluorescent-tagged proteins suggests that the assembly and disassembly of insulator bodies are normal activities in living cells and may be directed for regulating transcription.
Highlights
Tagged fusion proteins are priceless tools for monitoring the activities of biomolecules in living cells
Important features of pGWS include: (i) a restriction enzyme site (CCCGGG) that becomes bluntended after the SmaI digestion; (ii) attL1 and attL2 sequences flanking the SmaI site for LR recombination; (iii) Gen as the selectable marker; (iv) an insert in the first frame in pGWS will be in frame, after recombination, to the tags in all the P-element destination vectors (P-DEST) vectors described below and in frame with the tags of the commerciallyavailable destination vectors from Invitrogen
To test the efficiency of pGWS entry clones in the LR Clonase IITM recombination reactions, we inserted the enhanced green fluorescent protein (eGFP) sequence into pGWS and obtained the entry clone pGWS.eGFP, which was subsequently recombined with the pDEST17 Gateway® vector (Invitrogen) in an LR Clonase IITM reaction
Summary
Tagged fusion proteins are priceless tools for monitoring the activities of biomolecules in living cells. The "Entry/Gateway®" technology is a recently-developed plasmid construction strategy for rapidly cloning one DNA sequence into multiple destination plasmids. This technology greatly reduces the labor-intensive and timeconsuming procedures of classical plasmid construction. It is useful to create multiple plasmids for expressing various tagged versions of a specific protein or for expressing the protein under various promoters To use this technology, first a donor plasmid containing the DNA of interest is created, known as an "entry" clone. Due to the precision of the recombinase reaction, the desired sequence is inserted at the designated position of the destination vector This makes the technology useful in many applications, for example generating epitope-tagged fusion proteins in which controlling the reading frame is critical
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