A schematic map of the UP6 locus on chromosome 6. The map shows a 35.7 kb genomic DNA region that harbors the UP6 and eight genes located upstream of it. Each arrow represents a gene. The name of the gene is given on top of the arrow. The black numbers on the top of arrows denote sizes of genes (bp) while black numbers below denote distances in between genes (bp). 

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... linearized CHLI1-pDBle was used to complement chli1-1 . Transformed cells were plated onto fresh TAP plates containing 15 μg/ml zeocin (Z) and placed in the dark at 25°C. Single colonies were picked and transferred onto fresh TAP+Z plates using a numbered grid template for screening of potential chli1-1 rescued transformants. Screening of chli1-1 rescued transformants was done by visual inspection of green coloration and monitoring growth of light adapted complement strain cells either on TAP in the dark or in the dim light or HS plates under medium light (300 μmol photons m -2 s -1 ). Chlamydomonas cells from different strains grown in TAP in the dark were harvested, washed twice with fresh medium and resus- pended in TEN buffer (10 mM Tris-HCl, 10 mM EDTA and 150 mM NaCl; pH 8). Protein concentrations of samples were determined by the method of Lowry et al. (1951) 21 with bovine serum albumin as standard. Gel lanes were either loaded with an equal amount of Chl (4 μg Chl) or with 40 μg of protein. Resuspended cell suspen- sion was mixed in a 1:1 ratio with the sample solubilization buffer SDS-urea buffer (150 mM Tris-HCl, pH 6.8; 7% w/v SDS; 10% w/v glycerol; 2 M urea, bromophenol blue and 10% β -mercaptoethanol) and were incubated at room temperature for about thirty minutes, with intermittent vortexing. The sample solubilization buffer was prepared according to the protocol of Smith et al . (1990) 22 using reagents from Fisher (Pittsburgh, PA). After incubation, the solubi- lized protein samples were vortexed and spun at a maximum speed of 20,000 g in a microcentrifuge for five minutes at 4°C. The solu- ble fraction was loaded on a “any kD  Mini-PROTEAN ® TGX  Precast Gel” (BioRad, Hercules, CA) and SDS-PAGE analysis was performed according to Laemmli (1970) 23 using a Page Ruler pre- stained or unstained protein ladder (Fermentas, Glen Burnie, Maryland) at a constant current of 80 V for 2 hours. Gels were stained with colloi- dal Coomassie Gel code blue stain reagent (Thermo Fisher Scientific, Rockford, IL) for protein visualization. Electrophoretic transfer of the SDS-PAGE resolved proteins onto an Immobilon P–PVDF membrane (Millipore, Billerica, MA) was carried out for 2 hours at a constant current of 400 mA in the transfer buffer (25 mM Tris, 192 mM glycine and 20% methanol). The CHLI1 polyclonal antibody was raised in rabbit against the full length Arabidopsis thaliana CHLI1 mature protein that lacks the predicted transit peptide 24 . This antibody is a gift from Dr. Robert Larkin (Michigan State University). CHLI1 primary antibodies were diluted to a ratio of 1:2,000 before being used as a primary probe. The secondary antibodies used for Western blotting were conjugated to horseradish peroxidase (Pierce protein research product, Thermo Fisher Scientific, Rockford, IL) and diluted to a ratio of 1:20,000 with the antibody buffer. Western blots were developed by using the Supersignal West Pico chemiluminescent substrate kit (Pierce protein research product, Thermo Fisher Scientific, Rockford, IL). Cell density (number of cells per ml of the culture) was calculated by counting the cells using a Neubauer ultraplane hemacytometer (Hausser Scientific, Horsham, PA). Pigments from intact cells were extracted in 80% acetone and cell debris was removed by centrifu- gation at 10,000 g for 5 minutes. The absorbance of the supernatant was measured with a Beckman Coulter DU 730 Life science UV/Vis spectrophotometer (Brea, CA). Chl a and b concentrations were determined by Arnon (1949) 25 equations, with corrections as described by Melis et al. (1987) 26 . Mutant 5A7 was generated by random insertional mutagenesis of the Chlamydomonas reinhardtii wild type strain 4A+ (137c genetic background). 5A7 lacks detectable chlorophyll, appears yellowish- brown in color and grows only under heterotrophic conditions in the dark or in the dim light in the presence of acetate in the growth media (Figure 2). It is incapable of photosynthesis and is sensitive to light intensities higher than 20 μmol photons m -2 s -1 (Figure 2A). The linearized plasmid pBC1 was used to generate 5A7 (Figure 1). To find the insertion of the APHVIII end of the plasmid in 5A7 , a modified TAIL (Thermal Asymmetric InterLaced) PCR method was used. Figure 3A shows the position of the vector specific TAIL PCR primers and also shows the arbitrary position of the random non- degenerate primer. A 850 bp DNA product from TAIL2 PCR was purified from the agarose gel (Figure 3B, Table 1). This purified DNA product was used for PCR using internal primers specific to the 3 ́UTR (UnTranslated Region) of the APHVIII gene. The PCR results confirmed that the 850 bp DNA product contains the 3 ́UTR of the APHVIII gene (Figure 3C). Sequencing of the 850 bp TAIL2 PCR product revealed that the APHVIII end of the plasmid has been inserted in the fourth exon of a hypothetical gene which we have named as UP6 (Figure 4). UP6 (Cre06.g306500) is located on chromosome 6. Figure 5 shows a schematic map of the UP6 locus with its eight neighboring genes UP4 (Cre06.g306100), UP3 (Cre06.g306150), UP1 (Cre06.g306250), UP2 (Cre06.g306200) CHLI1 (Cre06. g306300), FDX3 (Cre06.g306350), AMT (g7098) and UP5 (Cre06. g306450). It is to be noted that we have named all of these genes arbitrarily for our study except for the CHLI1 and FDX3 genes, which were annotated in the Chlamydomonas genome database. Readers are requested to identify these unknown genes by the gene locus number (Cre or g number) in the Phytozome database. PCR analyses with the genomic DNA of 4A+ and 5A7 were performed using primers specific to four neighboring genes upstream of the CHLI1 (including UP6 ) and four neighboring genes downstream of the CHLI1 locus (Table 2 and Table 3; Figure 6A and 6B). PCR analyses revealed that all eight genes neighboring the CHLI1 locus were deleted or displaced from their native location (Figure 6A and 6B). UP5 primers gave nonspecific multiple products in 5A7 (Figure 6B). The first two exons of UP6 are present in the 5A7 genome as the UP6 primers spanning the first and the second exon, gave similar genomic DNA PCR product of the expected size as in the 4A+ lane (Figure 6B). Reverse transcription (RT-PCR) analyses using the same UP6 primers on 5A7 and 4A+ cDNA did not yield a PCR product in 5A7 unlike that in 4A+ (Figure 6C; Table 3). This shows that the insertion of the plasmid in the fourth exon of UP6 in 5A7 has hampered the transcription of the UP6 gene. Taken together the data shows that at least a 35,715 bp genomic region has been deleted and or/displaced when the plasmid got inserted in the 5A7 genome. Except for the CHLI1 gene, the functions of the remaining eight genes (including UP6 ) are not known. We do not yet know the exact location of the pUC origin (pUC ori) end of the plasmid (Figure 1) in the 5A7 genome. As CHLI plays a role in Chl biosynthesis, we checked for the presence/absence of the CHLI1 and CHLI2 in 5A7 . RT-PCR results show that CHLI1 transcript is absent and CHLI2 transcript is present in 5A7 (Figure 7A, Table 4). Figure 7B shows the presence of the CHLI2 gene in 5A7 . We will be referring to strain 5A7 as chli1-1 from here onward. As our chli1-1 lacks Chl and CHLI1 is involved in Chl biosynthesis, we cloned the CHLI1 cDNA in the pDBle vector to transform chli1-1 (Figure 8, Table 4). CHLI1 expression is driven by the constitutive PsaD promoter in the CHLI1-pDBle construct (Figure 8). pDBle has two Ble genes that confer resistance to the antibiotic zeocin. Figure 9 shows growth phenotypes of two chli1-1 rescued transformants ( chli1-7 and chli1-8 ); chli1-1 and 4A+. chli1-1 rescued transformants are able to synthesize Chl, are not light sensitive and are capable of photosynthesis (Figure 9). As the chli1-1 rescued transformants harbor the Ble gene (from the pDBle vector) and APHVIII gene (derived from the parental strain chli1-1 ), they can grow both on zeocin and paromomycin media plates unlike chli1-1 and 4A+ (Figure 9). Chl analyses show that both chli1-1 rescued transformants are about 33–46% Chl deficient. chli1-1 rescued transformants have a similar Chl a / b ratio as that of the wild type (Table 5, Data File below). Figure 10A and 10B show a schematic figure of the native Chlamydomonas CHLI1 gene and the trans CHLI1 gene used for complementation, respectively. PCR analyses using the genomic DNA show that the chli1-1 rescued transformants have the trans CHLI1 gene (Figure 10C and 10D). In Figure 10D the genomic DNA PCR product sizes in the two chli1-1 rescued transformant lanes are smaller than that in the 4A+ lane as we have cloned the CHLI1 cDNA for complementation. The Chlamydomonas CHLI1 protein has about 71% sequence identity to the Arabidopsis CHLI1 protein. Figure 11A shows a stained protein gel. The two chli1-1 rescued transformants and the 4A+ were loaded on an equal Chl basis in each lane in the protein gel (Figure 11A). As chli1-1 lacks Chl, the maximum amount of protein (40 μg) that can be loaded in a mini protein gel, was used (Figure 11A). Light harvesting complex proteins (LHCs) can barely be detected in the chli1-1 mutant (Figure 11A). Western analyses of the two chli1-1 rescued transformants with a CHLI1 antibody show that the CHLI1 protein is absent in the chli1-1 mutant but present in the chli1-1 rescued transformants (Figure 11B). Western analyses also show that the Arabidopsis CHLI1 antibody detects both the CHLI1 (40 kDa) and CHLI2 (42 kDa) protein in Chlamydomonas as the Chlamydomonas CHLI2 has about 62% sequence identity to the Arabidopsis CHLI1 (Figure 11B). In the wild type the CHLI2 protein amount is much lower than that of CHLI1. As the chli1-1 rescued transformants are Chl deficient compared to the wild type, the two rescued transformant lanes show higher amount of protein loadings (Figure 11A). Although more protein was loaded in the chli1-1 lane in the protein gel compared to that in the 4A+ and the chli1-1 rescued ...