Generation of Translin and Trax GFP-, CFP- and YFP-Fusions

SUPPLEMENTAL DATA

Generation of Translin and Trax GFP-, CFP- and YFP-fusions:

To create P{w+, translin::GFP} transgenic animals, a 1.9 kb HindIII/NdeI fragment containing the entire translin genomic region was cloned into the pSL1180 vector (GE Healthcare) and then subcloned with HindIII/XbaI into the pBS(KS+) (Stratagene). After removing the translin 5’ HindIII/SacI fragment, a BglII site was introduced right before the termination codon of translin by site directed mutagenesis. The BamHI fragment from the pRSET clone containing GFP S65T (Heim et al. 1994) was inserted into this unique BglII site and a clone with the correct orientation was selected. The 3’ part of translin containing the GFP ORF was cut out with SacI/XbaI and ligated into the above mentioned SacI/XbaI cut pSL1180 plasmid containing the translin genomic region. The HindIII/XbaI fragment containing the translin genomic region with the GFP insertion was then subcloned in the pLitmus28 vector (New England Biolabs) and the BamHI/XbaI fragment of the resulting fusion gene was inserted into the BamHI/XbaI sites of a modified pCaSpeR (Pirrotta 1988) transformation vector. The fusion gene was finally introduced into flies by P-element mediated transformation.

To create P{w+, translin::eCFP} transgenic animals, a BamHI fragment from a modified pECFP (Clontech) containing a second BamHI-site created by linker-ligation into the NotI site, was inserted into the unique BglII site of the abovementioned pBS(KS+) containing the 3’ part of the translin gene. Similarly to the GFP fusion gene construct, the CFP fusion gene sequence was completed with the 3’ part of the translin gene and then subcloned via several steps (using pSL1180 and pLitmus28 plasmids) into the BamHI/XbaI sites of the modified pCaSpeR transformation vector. To obtain P{w+,translin+} transgenic animals, the translin genomic region was amplified by PCR from a BAC clone. A ClaI/NdeI fragment of this PCR product was then ligated into the ClaI/NdeI sites of the pLitmus28 vector containing the translin CFP fusion construct, thereby replacing the CFP sequences. The construct was further subcloned into pCaSpeR by using the BamHI/XbaI sites.

To obtain P{w+, trax::eYFP} transgenic flies, a 2.1 kb HindIII/SphI fragment containing the entire trax region was cloned into pLitmus39 (New England Biolabs) and a slightly larger HindIII/SalI fragment was then moved into pBS KS(+). This allowed us to use the adjacent BamHI and KpnI sites to clone the gene into the pCaSpeR transformation vector. To insert the eYFP fragment into the trax gene, the trax 5’ HindIII/StuI fragment was removed from the abovementioned pLitmus39 vector and a BglII site was introduced at the 3’ end of the trax ORF by site directed mutagenesis. Into this unique BglII site, YFP was inserted as a BamHI fragment of a modified, additional restriction sites containing pEYFP (Clontech). This 3’ part of the trax gene containing an in-frame fusion of eYFP was then ligated into the SphI and StuI sites of the above mentioned trax gene in the pCaSpeR, thereby replacing the original trax 3’ part.

Literature cited:

HEIM, R., D. PRASHER and R. TSIEN, 1994 Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci. USA 91: 12501-12504.

PIRROTTA, V., 1988 Vectors for P-mediated transformation in Drosophila, pp. 437-456 in Vectors, a survey of molecular cloning vectors and their uses, edited by R. L. RODRIGUEZ, AND DENHARDT, D.T. Butterworths.