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An Entry/Gateway cloning system for general expression of genes with molecular tags in Drosophila melanogaster.

Akbari OS, Oliver D, Eyer K, Pai CY - BMC Cell Biol. (2009)

Bottom Line: We have developed an efficient cloning system for expressing dosage-sensitive proteins in Drosophila melanogaster.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biology Department, University of Nevada, Reno, 1664 N. Virginia Street, M/S 314, Reno, NV 89557, USA. omar@unr.nevada.edu

ABSTRACT

Background: Tagged 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.

Results: The 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.

Conclusion: We 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.

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The pGWS entry clones are efficient in the Clonase II LR reactions. (A) The diagram of LR reaction of the pGWS.eGFP entry clone and pDEST17. Shown are features of the two parental plasmids and the recombined product encoding the eGFP protein with poly-His tag. The arrows indicate the primers for determining the bacteria colonies that contain the correctly recombined plasmid. (B) Colonies of the eGFP-His expressing bacteria are fluorescent. (C) The bacterial lysate and the His-bind-chromatography-purified eGFP-His protein were analyzed by SDS-PAGE and stained with Coomassie blue. The arrow points to the purified His-eGFP protein.
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Figure 3: The pGWS entry clones are efficient in the Clonase II LR reactions. (A) The diagram of LR reaction of the pGWS.eGFP entry clone and pDEST17. Shown are features of the two parental plasmids and the recombined product encoding the eGFP protein with poly-His tag. The arrows indicate the primers for determining the bacteria colonies that contain the correctly recombined plasmid. (B) Colonies of the eGFP-His expressing bacteria are fluorescent. (C) The bacterial lysate and the His-bind-chromatography-purified eGFP-His protein were analyzed by SDS-PAGE and stained with Coomassie blue. The arrow points to the purified His-eGFP protein.

Mentions: To test the efficiency of pGWS entry clones in the LR Clonase II™ recombination reactions, we inserted the 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 II™ reaction (figure 3A). Two clones containing the eGFP insert were analyzed by sequencing and both clones encode the 6 × His-eGFP fusion protein in the predicted reading frame. The 6 × His-GFP fusion protein was fluorescent in bacteria (figure 3B) and can be purified by a His-binding Ni2+ column (figure 3C). The result indicates that pGWS is efficient in the LR reaction. We compared the pGWS result with other LR reactions we performed with pENTR/D-TOPO (Invitrogen) entry clones, and the LR reaction efficiencies are very similar between pGWS-based and pENTR/D-TOPO-based entry clones (data not shown).


An Entry/Gateway cloning system for general expression of genes with molecular tags in Drosophila melanogaster.

Akbari OS, Oliver D, Eyer K, Pai CY - BMC Cell Biol. (2009)

The pGWS entry clones are efficient in the Clonase II LR reactions. (A) The diagram of LR reaction of the pGWS.eGFP entry clone and pDEST17. Shown are features of the two parental plasmids and the recombined product encoding the eGFP protein with poly-His tag. The arrows indicate the primers for determining the bacteria colonies that contain the correctly recombined plasmid. (B) Colonies of the eGFP-His expressing bacteria are fluorescent. (C) The bacterial lysate and the His-bind-chromatography-purified eGFP-His protein were analyzed by SDS-PAGE and stained with Coomassie blue. The arrow points to the purified His-eGFP protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654426&req=5

Figure 3: The pGWS entry clones are efficient in the Clonase II LR reactions. (A) The diagram of LR reaction of the pGWS.eGFP entry clone and pDEST17. Shown are features of the two parental plasmids and the recombined product encoding the eGFP protein with poly-His tag. The arrows indicate the primers for determining the bacteria colonies that contain the correctly recombined plasmid. (B) Colonies of the eGFP-His expressing bacteria are fluorescent. (C) The bacterial lysate and the His-bind-chromatography-purified eGFP-His protein were analyzed by SDS-PAGE and stained with Coomassie blue. The arrow points to the purified His-eGFP protein.
Mentions: To test the efficiency of pGWS entry clones in the LR Clonase II™ recombination reactions, we inserted the 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 II™ reaction (figure 3A). Two clones containing the eGFP insert were analyzed by sequencing and both clones encode the 6 × His-eGFP fusion protein in the predicted reading frame. The 6 × His-GFP fusion protein was fluorescent in bacteria (figure 3B) and can be purified by a His-binding Ni2+ column (figure 3C). The result indicates that pGWS is efficient in the LR reaction. We compared the pGWS result with other LR reactions we performed with pENTR/D-TOPO (Invitrogen) entry clones, and the LR reaction efficiencies are very similar between pGWS-based and pENTR/D-TOPO-based entry clones (data not shown).

Bottom Line: We have developed an efficient cloning system for expressing dosage-sensitive proteins in Drosophila melanogaster.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biology Department, University of Nevada, Reno, 1664 N. Virginia Street, M/S 314, Reno, NV 89557, USA. omar@unr.nevada.edu

ABSTRACT

Background: Tagged 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.

Results: The 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.

Conclusion: We 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.

Show MeSH
Related in: MedlinePlus