Figure 1.
Targeted insertion of EGFP by homologous recombination using URACm-Cm and URACm-Gs as selection markers.
(A) Schematic diagram of the domain architectures of the two selection markers is shown on the top. Alignment of amino-acid sequences surrounding the OMP-decarboxylase domain of C. merolae and G. sulphuraria is shown in the bottom. The gray bar indicates the conserved OMP-decarboxylase domain. The red squares indicate amino-acid residues that play key roles in the enzymatic activity of OMP-decarboxylase [20]. (B) Schematic diagrams of targeted gene insertion by homologous recombination. The first line indicates the introduced DNA fragment, whereas the second line indicates the genomic structure of the parental strain M4. For efficient expression of EGFP, the 5′-UTR of the CMO250C gene and the 3′-UTR of the β-tubulin gene were utilized as a promoter and a putative polyadenylation signal sequence, respectively. The third and fourth lines indicate the predicted genomic structures in which a single copy is inserted by double-crossover homologous recombination in each case. The arrowheads indicate the positions of PCR primers used. (C) PCR analysis of CC and CG strains isolated independently, along with 10D (wild-type strain) and M4 (parental strain), to confirm homologous recombination events. Primers used were F1 (No. 25), R1 (No. 26), F2 (No. 27) and R2 (No. 28) shown in Table S1. The predicted sizes of PCR products are as follows: F1/R1, 3.4 kb for CC and CG, no band for 10D and M4; F2/R2, 2.4 kb for CC and CG, no band for 10D and M4; F1/R2, 8.2 kb for CC and CG, 3.9 kb for 10D and M4.
Figure 2.
Characterization of the genome structure of CC and CG strains.
(A) Southern blotting analyses of CC strains (CC14, CC18, CC24 and CC27) and CG strains (CG1, CG2, CG3 and CG6) along with 10 D and M4. Schematic representations include the position of the probe (yellow bars) and restriction enzyme sites used in the Southern blotting analyses. The URACm-Gs gene is a chimera of C. merolae (1 to 515 bp) and G. sulphuraria (516 to 1416 bp) sequences. The hybridization probe recognizes the introduced fragments (both URACm-Gs and URACm-Cm) as well as the endogenous URA5.3 gene. The arrowhead indicates the predicted position of a fragment produced by a single-copy-insertion event. The double arrowhead indicates the predicted position of a fragment derived from the endogenous URA5.3 locus. The predicted sizes of these fragments are as follows: KpnI, 12.4 kb for CC and CG, 5.8 kb for endogenous URA5.3; EcoRV, 8.2 kb for CC and CG, 11.4 kb for endogenous URA5.3. A part of the ethidium bromide (EtBr)-stained gel is shown as a loading control. (B) Quantitative-PCR analyses of CC and CG strains, along with 10 D and M4, to estimate the copy number of CMD184C-end (b) and EGFP (c). The value of (b) and (c) in each strain was normalized against that of CMD184C-mid (a), located outside of the introduced DNA fragment, to estimate their copy number. The bars indicate the SD (n = 3). (C) PCR analysis to detect tandem insertion. The positions of PCR primers (F3 and R3) are shown in (B) and their sequences were shown in Table S1 with No. 29 and No. 30, respectively.
Figure 3.
Growth of and EGFP expression from the transformants.
(A) Growth curves of the CC and CG strains in MA2 medium in the presence or absence of uracil. (B) Immunoblotting analysis against total lysates with anti-GFP. The arrowhead indicates the position of EGFP. A CBB-stained part of the gel is shown as a loading control. (C) Fluorescent images showing EGFP signals detected in the CC and CG strains. The CG strains emitted much higher levels of EGFP signals than the CC strains. Exposure times under blue-light excitation are 0.5 sec for 10 D, M4 and CC stains, and 0.2 sec for CG strains. The lower panels show typical G1 and M phase cells (CG1), indicating that the EGFP signals distributed throughout the cytosol. Note that the chloroplasts emitted red autofluorescence. Bars, 5 µm (upper panels) and 1 µm (lower panels). (D) Flow cytometry analysis of EGFP fluorescence. The broken line indicates the mode value calculated from the 10 D data, representing the sum of background and autofluorescence signals.