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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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Expression of GFP- or mRFP-tagged CP190 in transgenic flies. (A-B) A third instar larva expressing eGFP-tagged CP190 (A), or mRFP-tagged CP190 (B). (C-D) Fluorescent signals of CP190eGFP in a salivary gland (C), and in the nucleus of a salivary gland cell (D). (E-F) Fluorescent signals of CP190mRFP in a salivary gland (E), and in the nucleus of a salivary gland cell (F). (G-I) The distribution of CP190mRFP (G) and Mod67.2 (H) proteins at the tip of X chromosome. The polytene chromosome was prepared from a y2 3rd instar larva. The white arrows point to the y locus where contains a copy of the gypsy insulator. The red arrow point to the location that contain only CP190 protein but do not contain Mod67.2 protein.
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Figure 5: Expression of GFP- or mRFP-tagged CP190 in transgenic flies. (A-B) A third instar larva expressing eGFP-tagged CP190 (A), or mRFP-tagged CP190 (B). (C-D) Fluorescent signals of CP190eGFP in a salivary gland (C), and in the nucleus of a salivary gland cell (D). (E-F) Fluorescent signals of CP190mRFP in a salivary gland (E), and in the nucleus of a salivary gland cell (F). (G-I) The distribution of CP190mRFP (G) and Mod67.2 (H) proteins at the tip of X chromosome. The polytene chromosome was prepared from a y2 3rd instar larva. The white arrows point to the y locus where contains a copy of the gypsy insulator. The red arrow point to the location that contain only CP190 protein but do not contain Mod67.2 protein.

Mentions: To circumvent the over-expression problems we experienced with the Gal4/UAS system, we used our P-DEST vectors (pUWG and pUWR). As described above, these vectors contain the Ubi-63E promoter for driving the eGFP- or mRFP-tagged fusion proteins. We obtained multiple transgenic lines from each P-element, including two mRFP lines and eight eGFP lines. In all transgenic lines, we detected eGFP or mRFP-tagged CP190 proteins. The fluorescent signals of CP190eGFP or CP190mRFP were observed in many tissues in all developmental stages, including embryos, larvae (figure 5), and adults. We next determined if the tagged CP190 proteins behave similarly to the wildtype CP190, which has been shown to be present on polytene chromosomes as many bands [3,9]. Both eGFP- and mRFP-tagged CP190 were detected, via their fluorescent signals, as many bands on polytene chromosomes in the nuclei of living salivary gland cells (figure 5C–5F). The bands of tagged CP190 were also detected on squashed polytene chromosome samples (figure 5G–5I). The CP190mRFP fusion protein co-localizes with Mod(mdg4)67.2, another protein in the gypsy complex, at the gypsy insertion site at the yellow (y) locus on the y2 chromosome (figure 5G and 5I white arrows). This suggests that the tagged CP190 protein is recruited to the gypsy insulator complex. In addition to the bands containing gypsy insulator proteins, CP190mRFP also localizes to other Mod67.2-independent sites (figure 5G and 5I red arrows), the same as wildtype CP190 as reported previously [3]. The banding pattern of CP190mRFP proteins on polytene chromosomes was indistinguishable from that of the wildtype CP190 protein. The tagged-CP190 transgenes also rescued the lethality of the homozygous CP190 mutation (data not shown) and rescued the defective gypsy-dependent y2 and ct6 phenotypes of the homozygous CP190 mutation (explained in detail in a separate manuscript in preparation). All evidence indicates that the tagged CP190 proteins function similarly, if not exactly the same, as the wildtype CP190. Since the Ubi-63 promoter activity may be stronger with heat shock, we treated the larvae carrying the CP190mRFP transgene with one dose of heat treatment at 37°C for 20 mins and monitored the CP190mRFP signal from 2 hours post-treatment until 24 hours post-treatment. We detected only slightly elevated expression of CP190mRFP after 3 hours and no significant changes afterward, judging by the slightly increased fluorescent signal. The heat-treated larvae were viable and developed into normal flies (data not shown). This result indicates that the lethality of hs-Gal4 > UAS-CP190mRFP larvae after heat treatment described in the above section was not due to the activity of CP190mRFP during or after heat treatment but was likely due to over-expression induced by the Gal4/UAS expression system.


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)

Expression of GFP- or mRFP-tagged CP190 in transgenic flies. (A-B) A third instar larva expressing eGFP-tagged CP190 (A), or mRFP-tagged CP190 (B). (C-D) Fluorescent signals of CP190eGFP in a salivary gland (C), and in the nucleus of a salivary gland cell (D). (E-F) Fluorescent signals of CP190mRFP in a salivary gland (E), and in the nucleus of a salivary gland cell (F). (G-I) The distribution of CP190mRFP (G) and Mod67.2 (H) proteins at the tip of X chromosome. The polytene chromosome was prepared from a y2 3rd instar larva. The white arrows point to the y locus where contains a copy of the gypsy insulator. The red arrow point to the location that contain only CP190 protein but do not contain Mod67.2 protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Expression of GFP- or mRFP-tagged CP190 in transgenic flies. (A-B) A third instar larva expressing eGFP-tagged CP190 (A), or mRFP-tagged CP190 (B). (C-D) Fluorescent signals of CP190eGFP in a salivary gland (C), and in the nucleus of a salivary gland cell (D). (E-F) Fluorescent signals of CP190mRFP in a salivary gland (E), and in the nucleus of a salivary gland cell (F). (G-I) The distribution of CP190mRFP (G) and Mod67.2 (H) proteins at the tip of X chromosome. The polytene chromosome was prepared from a y2 3rd instar larva. The white arrows point to the y locus where contains a copy of the gypsy insulator. The red arrow point to the location that contain only CP190 protein but do not contain Mod67.2 protein.
Mentions: To circumvent the over-expression problems we experienced with the Gal4/UAS system, we used our P-DEST vectors (pUWG and pUWR). As described above, these vectors contain the Ubi-63E promoter for driving the eGFP- or mRFP-tagged fusion proteins. We obtained multiple transgenic lines from each P-element, including two mRFP lines and eight eGFP lines. In all transgenic lines, we detected eGFP or mRFP-tagged CP190 proteins. The fluorescent signals of CP190eGFP or CP190mRFP were observed in many tissues in all developmental stages, including embryos, larvae (figure 5), and adults. We next determined if the tagged CP190 proteins behave similarly to the wildtype CP190, which has been shown to be present on polytene chromosomes as many bands [3,9]. Both eGFP- and mRFP-tagged CP190 were detected, via their fluorescent signals, as many bands on polytene chromosomes in the nuclei of living salivary gland cells (figure 5C–5F). The bands of tagged CP190 were also detected on squashed polytene chromosome samples (figure 5G–5I). The CP190mRFP fusion protein co-localizes with Mod(mdg4)67.2, another protein in the gypsy complex, at the gypsy insertion site at the yellow (y) locus on the y2 chromosome (figure 5G and 5I white arrows). This suggests that the tagged CP190 protein is recruited to the gypsy insulator complex. In addition to the bands containing gypsy insulator proteins, CP190mRFP also localizes to other Mod67.2-independent sites (figure 5G and 5I red arrows), the same as wildtype CP190 as reported previously [3]. The banding pattern of CP190mRFP proteins on polytene chromosomes was indistinguishable from that of the wildtype CP190 protein. The tagged-CP190 transgenes also rescued the lethality of the homozygous CP190 mutation (data not shown) and rescued the defective gypsy-dependent y2 and ct6 phenotypes of the homozygous CP190 mutation (explained in detail in a separate manuscript in preparation). All evidence indicates that the tagged CP190 proteins function similarly, if not exactly the same, as the wildtype CP190. Since the Ubi-63 promoter activity may be stronger with heat shock, we treated the larvae carrying the CP190mRFP transgene with one dose of heat treatment at 37°C for 20 mins and monitored the CP190mRFP signal from 2 hours post-treatment until 24 hours post-treatment. We detected only slightly elevated expression of CP190mRFP after 3 hours and no significant changes afterward, judging by the slightly increased fluorescent signal. The heat-treated larvae were viable and developed into normal flies (data not shown). This result indicates that the lethality of hs-Gal4 > UAS-CP190mRFP larvae after heat treatment described in the above section was not due to the activity of CP190mRFP during or after heat treatment but was likely due to over-expression induced by the Gal4/UAS expression system.

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