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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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Structure of the pGWS and P-destination vectors. (A-B) The detailed structure of P-DEST vectors with N-terminal tags (A), and with C-terminal tags (B). The sequences and the reading frame from the epitope tags are shown below the map. The attR1 and attR2 sequences for Clonase II® recombination are shaded. (C) The structure and sequences of the pGWS. The circular pGWS has a unique SmaI site which is a restriction enzyme that can cut pGWS into a linear DNA with two blunted ends. The 3'-protruding ends after Taq T-tailing for TA cloning are shown in brackets. The reading frame after LR recombination with P-DEST vectors is indicated.
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Figure 2: Structure of the pGWS and P-destination vectors. (A-B) The detailed structure of P-DEST vectors with N-terminal tags (A), and with C-terminal tags (B). The sequences and the reading frame from the epitope tags are shown below the map. The attR1 and attR2 sequences for Clonase II® recombination are shaded. (C) The structure and sequences of the pGWS. The circular pGWS has a unique SmaI site which is a restriction enzyme that can cut pGWS into a linear DNA with two blunted ends. The 3'-protruding ends after Taq T-tailing for TA cloning are shown in brackets. The reading frame after LR recombination with P-DEST vectors is indicated.

Mentions: We have created a vector for generating entry clones named pGWS that uses Gentamicin (Gen) as the selectable marker. The pGWS is particularly useful for the cloning of unstable DNA sequences using the SURE® strain of E. coli (Stratagene) which is Kanamycin (Kan) resistant. The pGWS is designed for a TA-based or blunt-end-based cloning method. Important features of pGWS include: (i) a restriction enzyme site (CCCGGG) that becomes blunt-ended 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-DEST vectors described below (figure 2) and also in frame with the tags of the commercially-available destination vectors from Invitrogen. To use pGWS in TA-based cloning, pGWS is linearized by SmaI to become blunt-ended, followed by a Taq polymerase incubation to add a "T" overhang on the 3' ends.


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)

Structure of the pGWS and P-destination vectors. (A-B) The detailed structure of P-DEST vectors with N-terminal tags (A), and with C-terminal tags (B). The sequences and the reading frame from the epitope tags are shown below the map. The attR1 and attR2 sequences for Clonase II® recombination are shaded. (C) The structure and sequences of the pGWS. The circular pGWS has a unique SmaI site which is a restriction enzyme that can cut pGWS into a linear DNA with two blunted ends. The 3'-protruding ends after Taq T-tailing for TA cloning are shown in brackets. The reading frame after LR recombination with P-DEST vectors is indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Structure of the pGWS and P-destination vectors. (A-B) The detailed structure of P-DEST vectors with N-terminal tags (A), and with C-terminal tags (B). The sequences and the reading frame from the epitope tags are shown below the map. The attR1 and attR2 sequences for Clonase II® recombination are shaded. (C) The structure and sequences of the pGWS. The circular pGWS has a unique SmaI site which is a restriction enzyme that can cut pGWS into a linear DNA with two blunted ends. The 3'-protruding ends after Taq T-tailing for TA cloning are shown in brackets. The reading frame after LR recombination with P-DEST vectors is indicated.
Mentions: We have created a vector for generating entry clones named pGWS that uses Gentamicin (Gen) as the selectable marker. The pGWS is particularly useful for the cloning of unstable DNA sequences using the SURE® strain of E. coli (Stratagene) which is Kanamycin (Kan) resistant. The pGWS is designed for a TA-based or blunt-end-based cloning method. Important features of pGWS include: (i) a restriction enzyme site (CCCGGG) that becomes blunt-ended 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-DEST vectors described below (figure 2) and also in frame with the tags of the commercially-available destination vectors from Invitrogen. To use pGWS in TA-based cloning, pGWS is linearized by SmaI to become blunt-ended, followed by a Taq polymerase incubation to add a "T" overhang on the 3' ends.

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