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• W1974768178 abstract "The Drosophila ninaG mutant is characterized by low levels of Rh1 rhodopsin, because of the inability to transport this rhodopsin from the endoplasmic reticulum to the rhabdomere. ninaG mutants do not affect the biogenesis of the minor opsins Rh4 and Rh6. A genetic analysis placed the ninaG gene within the 86E4-86E6 chromosomal region. A sequence analysis of the 15 open reading frames within this region from the ninaGP330 mutant allele identified a stop codon in the CG6728 gene. Germ-line transformation of the CG6728 genomic region rescued the ninaG mutant phenotypes, confirming that CG6728 corresponds to the ninaG gene. The NinaG protein belongs to the glucose-methanol-choline oxidoreductase family of flavin adenine dinucleotide-binding enzymes catalyzing hydroxylation and oxidation of a variety of small organic molecules. High performance liquid chromatography analysis of retinoids was used to gain insight into the in vivo role of the NinaG oxidoreductase. The results show that when Rh1 is expressed as the major rhodopsin, ninaG flies fail to accumulate 3-hydroxyretinal. Further, in transgenic flies expressing Rh4 as the major rhodopsin, 3-hydroxyretinal is the major retinoid in ninaG+, but a different retinoid profile is observed in ninaGP330. These results indicate that the ninaG oxidoreductase acts in the biochemical pathway responsible for conversion of retinal to the rhodopsin chromophore, 3-hydroxyretinal. The Drosophila ninaG mutant is characterized by low levels of Rh1 rhodopsin, because of the inability to transport this rhodopsin from the endoplasmic reticulum to the rhabdomere. ninaG mutants do not affect the biogenesis of the minor opsins Rh4 and Rh6. A genetic analysis placed the ninaG gene within the 86E4-86E6 chromosomal region. A sequence analysis of the 15 open reading frames within this region from the ninaGP330 mutant allele identified a stop codon in the CG6728 gene. Germ-line transformation of the CG6728 genomic region rescued the ninaG mutant phenotypes, confirming that CG6728 corresponds to the ninaG gene. The NinaG protein belongs to the glucose-methanol-choline oxidoreductase family of flavin adenine dinucleotide-binding enzymes catalyzing hydroxylation and oxidation of a variety of small organic molecules. High performance liquid chromatography analysis of retinoids was used to gain insight into the in vivo role of the NinaG oxidoreductase. The results show that when Rh1 is expressed as the major rhodopsin, ninaG flies fail to accumulate 3-hydroxyretinal. Further, in transgenic flies expressing Rh4 as the major rhodopsin, 3-hydroxyretinal is the major retinoid in ninaG+, but a different retinoid profile is observed in ninaGP330. These results indicate that the ninaG oxidoreductase acts in the biochemical pathway responsible for conversion of retinal to the rhodopsin chromophore, 3-hydroxyretinal. Vitamin A provides the retinal chromophores that mediate the sensing of light quanta by visual pigments. Vitamin A is a fat-soluble vitamin that cannot be synthesized de novo by most animals. Rather vitamin A is obtained from the diet, either as preformed vitamin A such as retinol from animal sources, or as provitamin A carotenoids from plant sources. These dietary forms must be both transported to the retina and chemically modified to play a key role in visual transduction (1Blomhoff R. Green M.H. Berg T. Norum K.R. Science. 1990; 250: 399-404Crossref PubMed Scopus (522) Google Scholar, 2Yeum K.J. Russell R.M. Annu. Rev. Nutr. 2002; 22: 483-504Crossref PubMed Scopus (441) Google Scholar). Drosophila nina mutations have provided some insights into processing of vitamin A for use in vision. Eight Drosophila nina genes are known (3Pak W.L. Investig. Ophthalmol. Vis. Sci. 1995; 36: 2340-2357PubMed Google Scholar). These nina mutants, named for the “neither inactivation nor afterpotential” electroretinogram defect, all possess low rhodopsin levels responsible for this phenotype. Vitamin A-deprived flies also show the same electroretinogram defects and low rhodopsin levels (4Harris W.A. Ready D.F. Lipson E.D. Hudspeth A.J. Stark W.S. Nature. 1977; 266: 648-650Crossref PubMed Scopus (93) Google Scholar, 5Ozaki K. Nagatani H. Ozaki M. Tokunaga F. Neuron. 1993; 10: 1113-1119Abstract Full Text PDF PubMed Scopus (71) Google Scholar), establishing the importance of vitamin A availability in the production of rhodopsin. The ninaB and ninaD mutants are also known to lower rhodopsin levels by disrupting vitamin A availability. The ninaD gene encodes a scavenger receptor (6Kiefer C. Sumser E. Wernet M.F. Von Lintig J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10581-10586Crossref PubMed Scopus (205) Google Scholar) required for efficient use of carotenoids, such as β-carotene, from the diet. The ninaB encodes an oxygenase enzyme (7von Lintig J. Dreher A. Kiefer C. Wernet M.F. Vogt K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1130-1135PubMed Google Scholar) responsible for cleaving β-carotene to generate retinal. Both of these gene products act outside of the retina to carry out their roles in vitamin A metabolism (8Gu G. Yang J. Mitchell K.A. O'Tousa J.E. J. Biol. Chem. 2004; 279: 18608-18613Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Five different retinoids are known to serve as visual pigment chromophores within the animal kingdom. These are retinal, (3,4)-didehydroretinal, (3R)-3-hydroxyretinal, (3S)-3-hydroxyretinal, and (3R)-4-hydroxyretinal. Although the 3R enantiomer of 3-hydroxyretinal is found among many insect orders, the 3S enantiomer is found only among the Cyclorrhapha suborder of Diptera (9Seki T. Vogt T.F. Comp. Biochem. Physiol. B. 1998; 119: 53-64Crossref Scopus (43) Google Scholar) that includes Drosophila (10Seki T. Isono K. Ito M. Katsuta Y. Eur. J. Biochem. 1994; 226: 691-696Crossref PubMed Scopus (19) Google Scholar). The biochemical pathways responsible for production of this retinoid and the other modified chromophores from retinal or other vitamin A precursors have not been characterized. Here we describe the molecular characterization and phenotypic analysis of ninaG. The ninaG gene encodes a member of the glucose-methanol-choline (GMC) 1The abbreviations used are: GMC, glucose-methanol-choline; PDA, prolonged depolarizing afterpotential; HPLC, high performance liquid chromatography; MS, mass spectroscopy; GFP, green fluorescent protein.1The abbreviations used are: GMC, glucose-methanol-choline; PDA, prolonged depolarizing afterpotential; HPLC, high performance liquid chromatography; MS, mass spectroscopy; GFP, green fluorescent protein. family of oxidoreductases (11Lamark T. Kaasen I. Eshoo M.W. Falkenberg P. McDougall J. Strom A.R. Mol. Microbiol. 1991; 5: 1049-1064Crossref PubMed Scopus (210) Google Scholar). The biochemical substrates of most of these enzymes are not known, but some of the enzymes are dehydrogenases and oxidases carrying out redox reactions on a wide variety of organic metabolites. We show that NinaG is required for the biogenesis of the Drosophila rhodopsin chromophore, (3S)-3-hydroxyretinal. This study provides the first molecular description of an enzyme active in modification of retinal to an alternative visual pigment chromophore. ninaG Mutant Analysis—The ethylmethane sulfonate-induced ninaGP330 strain was obtained from W. L. Pak, Purdue University (3Pak W.L. Investig. Ophthalmol. Vis. Sci. 1995; 36: 2340-2357PubMed Google Scholar). ninaG1 was identified following an ethylmethane sulfonate screen done in the O'Tousa laboratory and ninaG2 was identified within an existing laboratory stock. Deficiency chromosomes were obtained from the Drosophila Stock Center, Bloomington, Indiana and W. Engels, University of Wisconsin. Drosophila strains were maintained at room temperature and usually reared on standard cornmeal-molasses medium. Pseudopupil and protein blots assessment of rhodopsin content was carried out as described in Hsu et al. (12Hsu C.D. Adams S.M. O'Tousa J.E. Vision Res. 2002; 42: 507-516Crossref PubMed Scopus (7) Google Scholar). Electroretinogram and electron microscopic analyses were performed as described previously (13Washburn T. O'Tousa J.E. Genetics. 1992; 130: 585-595Crossref PubMed Google Scholar), in both cases using 2-day-old flies maintained in 12-h light/12-h dark light cycle. Rhodopsin cellular location was visualized using transgenic flies carrying pRh1:Rh1-GFP, constructed using the Rh1-GFP coding sequence described by Pichaud and Desplan (14Pichaud F. Desplan C. Development. 2001; 128: 815-826PubMed Google Scholar) and the Rh1 promoter region. 2K. Hibbard and J. E. O'Tousa, unpublished data. Ommatidia of newly eclosed Drosophila carrying this element in appropriate genetic backgrounds were prepared by modification of described protocols (15Hardie R.C. Voss D. Pongs O. Laughlin S.B. Neuron. 1991; 6: 477-486Abstract Full Text PDF PubMed Scopus (66) Google Scholar, 16Hardie R.C. J. Neurosci. 1991; 11: 3079-3095Crossref PubMed Google Scholar). The dispersed ommatidia were immersed in a drop of chilled phosphate-buffered saline, pH 7.4, on a glass slide. To-Pro-3 iodide (Molecular Probes) was added to visualize nuclei, anti-fade reagent (Molecular Probes) was added in equal volume, and the GFP fluorescence was then imaged with Bio-Rad MRC 1024 Scanning Confocal microscope. Vitamin A-free medium was prepared as described by Nichols and Pak (17Nichols R. Pak W.L. Drosoph. Inf. Serv. 1985; 61: 195Google Scholar). To generate flies deficient in vitamin A, a life cycle (from egg to adult) was completed on this medium. Replenishment with all-trans-retinal (Sigma) was done by placing adult flies on a supplemented medium for 3 days prepared by adding 10 μl of a 10 mm solution/ml of vitamin A-free medium. For Rh1 RNA analysis, adult flies were flash-frozen in liquid nitrogen and vortexed to separate the heads from the bodies. Total RNA was isolated from different fly strains using the RNeasy mini kit (Qiagen). 15 μg of total RNA was subjected to 1.2% formaldehyde-agarose gel electrophoresis. 18 and 28 S ribosomal RNA standards were visualized on the same gel with ethidium bromide staining to evaluate RNA integrity and as loading controls. The gel was blotted onto Hybond-XL nylon transfer membrane (Amersham Biosciences) using the manufacturer's instructions, probed with Rh1 cDNA, random-labeled with [α-32P]dCTP using the Megaprime DNA labeling system (Amersham Biosciences) and autoradiographed to visualize Rh1 RNA levels. FRT/FLP (flippase recombinase/flippase recombinase target) genetic mosaics were constructed and analyzed as described previously (8Gu G. Yang J. Mitchell K.A. O'Tousa J.E. J. Biol. Chem. 2004; 279: 18608-18613Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). ninaG Molecular Analysis—For sequence analysis of the 15 genes in the ninaG region, 1 μl of genomic DNA, prepared using the single fly DNA prep protocol (18Gloor G.B. Preston C.R. Johnson-Schlitz D.M. Nassif N.A. Phillis R.W. Benz W.K. Robertson H.M. Engels W.R. Genetics. 1993; 135: 81-95Crossref PubMed Google Scholar), was used in PCR with Platinum Taq DNA High Fidelity proofreading polymerase (Invitrogen) and unique oligonucleotide primers designed to amplify a 1-kb segment from the region. The PCR products were cloned into pCR4 TOPO cloning vector (Invitrogen). Plasmids containing the correct inserts were bidirectionally sequenced using T3 and T7 sequencing primers and Big Dye Terminator v.3 on ABI Prism® 3700 automated sequencer (Applied Biosystems). For transgenic rescue of ninaG, a 3.8-kb KpnI-StuI fragment from BAC013PO8 containing 1.1 kb of the ninaG (CG6728) promoter region, the entire open reading frame, and 0.7 kb of the 3′-untranslated region was cloned into the P element vector pCaSpeR4 (19Thummel C.S. Boulet A.M. Lipshitz H.D. Gene (Amst.). 1988; 74: 445-456Crossref PubMed Scopus (472) Google Scholar) and transformed into the ninaGP330 mutant using standard procedures (20Spradling A.C. Roberts D.B. Drosophila a Practical Approach. IRL Press Limited, Oxford, England1986: 175-196Google Scholar). Retinal Extraction and HPLC/MS Analysis—Methodology for retinal extraction was developed from the published protocol of Suzuki et al. (21Suzuki T. Fujita Y. Noda Y. Miyata S. Vision Res. 1986; 26: 425-429Crossref PubMed Scopus (46) Google Scholar). 700-800 Drosophila were flash frozen in liquid nitrogen, and the heads were separated from bodies by vigorous shaking, homogenized in 100 μl of 6 m formaldehyde, 0.1 m phosphate buffer (pH 7.5), and incubated at 30 °C for 2 min. 0.5 ml of dichloromethane was then added, the homogenate was vortexed, and further incubated at 30 °C for 10 min. 1 ml of n-hexane was added, mixed, and spun at 3,000 rpm for 5 min. The upper organic layer was collected, the aqueous phase was extracted again with dichloromethane and n-hexane, and the pooled organic layers were evaporated to dryness under vacuum. Samples were resuspended in 50 μl of the initial HPLC mobile phase, and then filtered through a 0.22 μm PTFE filter (Nalgene) prior to HPLC analysis. A Waters 2690 HPLC system equipped with a 996 photodiode array detector and interfaced with a Micromass Quattro LC triple quadrupole mass spectrometer (Waters) was used for liquid chromatography/UV-visible/MS experiments. Absorbance spectra were monitored from 200 to 800 nm, and mass spectra were acquired over the mass range 150-800 at a scan rate of 2 s. A 10-μl sample was injected onto a 4.6 × 20-mm, 3-μm particle size Atlantis dC18 guard column and a 4.6 × 150-mm, 3-μm particle size Atlantis dC18 analytical column (Waters). The mobile phase gradient consisted of 80:20 (v/v) acetonitrile:water for 3 min, a 12-min linear gradient to 100% acetonitrile, and 5 min of 100% acetonitrile delivered at a flow rate of 1 ml/min. The HPLC eluent was split 50:50 prior to injection into the electrospray ion source of the mass spectrometer, and formic acid (0.1%) was added to all mobile phase solvents as a means of increasing ionization efficiency for MS. The ninaG Mutant Phenotype—A collection of vision-defective mutants called nina, for “neither inactivation nor afterpotential” were identified on the basis of their electroretinogram phenotype (22Stephenson R.S. O'Tousa J. Scavarda N.J. Randall L.L. Pak W.L. Symp. Soc. Exp. Biol. 1983; 36: 477-501PubMed Google Scholar). In wild type, a bright blue stimulus induces a depolarized state that persists after termination of the stimulus (Fig. 1A, top trace). This is referred to as the prolonged depolarizing afterpotential (PDA). The lack of the PDA response in nina mutants, such as shown for ninaGP330 in Fig. 1A, bottom trace, is because of low levels of Rh1 rhodopsin within the R1-6 photoreceptors (3Pak W.L. Investig. Ophthalmol. Vis. Sci. 1995; 36: 2340-2357PubMed Google Scholar, 23O'Tousa J.E. Baehr W. Martin R.L. Hirsh J. Pak W.L. Applebury M.L. Cell. 1985; 40: 839-850Abstract Full Text PDF PubMed Scopus (376) Google Scholar). The ninaG mutant possesses very low levels of Rh1 rhodopsin when assayed in protein blots (Fig. 1B, upper panel). The requirement for ninaG in production of other rhodopsins was tested by protein blots against the R7 opsin Rh4 and the R8 opsin Rh6. Fig. 1B, middle panels, show that Rh4 and Rh6 opsin production does not require ninaG activity. Similar results were seen for two other ninaG alleles. Thus, ninaG function is only required for efficient biogenesis of a subset of Drosophila visual pigments. To investigate whether maturation of other transmembrane proteins is affected in ninaG mutants, Trp and RdgB protein levels were examined. Trp is a calcium channel that co-localizes with rhodopsin in the rhabdomeres (24Niemeyer B.A. Suzuki E. Scott K. Jalink K. Zuker C.S. Cell. 1996; 85: 651-659Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). RdgB is a membrane protein that is localized to the photoreceptor subrhabdomeric cisternae (25Vihtelic T.S. Goebl M. Milligan S. O'Tousa J.E. Hyde D.R. J. Cell Biol. 1993; 122: 1013-1022Crossref PubMed Scopus (160) Google Scholar). Neither Trp nor RdgB protein levels are affected in ninaG mutants (Fig. 1B, lower panels). Northern analysis was used to determine whether ninaG was required for expression of Rh1 mRNA. Similar levels of ninaE transcript of 1.5 kb representing the Rh1 opsin mRNA was detectable by Northern analysis in wild type, ninaGP330, ninaG1, and ninaG2 heads (Fig. 1C). Heads from the eya (eyes absent) mutant, lacking the compound eyes, were used as the negative control. These results show that ninaG mutants do not affect Rh1 mRNA levels, establishing that ninaG acts in translational or posttranslational control of rhodopsin production. Defective Rh1 Transport in ninaG Mutants—To investigate the effect of the ninaG mutation on the localization of Rh1 rhodopsin in the photoreceptor cells, we created flies containing rhodopsin-GFP (Rh1-GFP) transgene driven by the Drosophila Rh1 promoter. The location of Rh1-GFP was visualized in whole mounts of isolated ommatidia, the unit cluster of the R1-8 photoreceptor cells. In the ninaG+ background, the Rh1-GFP localizes predominantly to the rhabdomeres of R1-6 cells (Fig. 2A, labeled R). In ninaG mutants, Rh1-GFP localized exclusively to the cytoplasmic compartments (Fig. 2B, labeled C), failing to decorate the rhabdomeres (R). The redistribution of Rh1-GFP seen in ninaG mutants is similar to that observed in vitamin A-deprived flies (data not shown). At the ultrastructural level, ninaGP330 (Fig. 2D) shows a reduction in the size of the R1-6 rhabdomeres (Fig. 2, C and D, one rhabdomere is labeled R) when compared with wild type (Fig. 2C). We also observed proliferation of the endoplasmic reticulum in the cell bodies (Fig. 2D, ER), and similar results were observed in ninaG2 (data not shown). The ninaGP330 mutant shows these defects at all ages examined, with no other significant changes, including retinal degeneration, observed up to 20 days of age (data not shown). The Retina Is the Site of ninaG Activity—To test whether NinaG activity is required within photoreceptors, we created genetic mosaics in which only the eye tissue was mutant for ninaG. Fig. 3 shows the rhodopsin content in ninaG mosaics and an appropriate set of control genotypes. In the ninaG mosaic (Fig. 3, far right), the retinal tissue is mutant for ninaG, but the rest of the animal is ninaG+. These mosaic flies show low levels of rhodopsin, showing that NinaG activity is required within photoreceptors or other cell types of the retina for normal rhodopsin expression. Identification of the ninaG Gene—We determined the cytological position of ninaG as a first step toward identifying and characterizing the ninaG gene. Flies heterozygous for ninaG and each of a set of deficiencies spanning much of the right arm of the third chromosome were examined for rhodopsin content using the presence/absence of the deep dark pseudopupil (26Franceschini N. Snyder A.W. Menzel R. Photoreceptor Optics. Springer-Verlag, New York, NY1975: 98-125Crossref Google Scholar). The presence of this structure in white-eyed flies is indicative of high rhodopsin levels. Df(3R)M-Kx1/ninaG and Df(3R)T-32/ninaG and Df(3R)T-61/ninaG flies showed no dark deep pseudopupil suggestive of low rhodopsin levels. Reduced rhodopsin levels in these three genotypes were confirmed by Western analysis (data not shown). This analysis placed the map location of ninaG within region 86E3-87A9 on the right arm of the third chromosome. To further refine this map position, P-element-induced deletions within the 86E region (27Kusano K. Johnson-Schlitz D.M. Engels W.R. Science. 2001; 291: 2600-2602Crossref PubMed Scopus (97) Google Scholar) were tested for complementation with ninaG mutants. Df(3R)thoR1, Df(3R)pros235, and Df(3R)pros640, but not Df(3R)T7, failed to complement the low rhodopsin phenotype of ninaG (Fig. 1B, upper panel). These results placed ninaG into a small region bracketed proximally by the pros gene and distally by the tho gene. P-element alleles of pros (P[PZ]pros10419) and tho (P[PZ] tho1) complemented ninaG. Therefore, ninaG is likely to lie in the region between pros and tho. This is a genomic region of 79,639 bp containing 15 annotated genes. To identify the gene responsible for the ninaG mutant, we sequenced the coding regions of the 15 identified genes within the 86E5-86E8-E11 cytological region from ninaGP330. 36 different PCRs generated products of ∼1-kb length to examine all genes. The coding sequences within these PCR products were compared with the Drosophila genome sequence (28Adams M.D. Celniker S.E. Holt R.A. Evans C.A. Gocayne J.D. Amanatides P.G. Scherer S.E. Li P.W. Hoskins R.A. Galle R.F. George R.A. Lewis S.E. Richards S. Ashburner M. Henderson S.N. Sutton G.G. Wortman J.R. Yandell M.D. Zhang Q. Chen L.X. Brandon R.C. Rogers Y.H. Blazej R.G. Champe M. Pfeiffer B.D. Wan K.H. Doyle C. Baxter E.G. Helt G. Nelson C.R. Gabor G.L. Abril J.F. Agbayani A. An H.J. Andrews-Pfannkoch C. Baldwin D. Ballew R.M. Basu A. Baxendale J. Bayraktaroglu L. Beasley E.M. Beeson K.Y. Benos P.V. Berman B.P. Bhandari D. Bolshakov S. Borkova D. Botchan M.R. Bouck J. Brokstein P. Brottier P. Burtis K.C. Busam D.A. Butler H. Cadieu E. Center A. Chandra I. Cherry J.M. Cawley S. Dahlke C. Davenport L.B. Davies P. de Pablos B. Delcher A. Deng Z. Mays A.D. Dew I. Dietz S.M. Dodson K. Doup L.E. Downes M. Dugan-Rocha S. Dunkov B.C. Dunn P. Durbin K.J. Evangelista C.C. Ferraz C. Ferriera S. Fleischmann W. Fosler C. Gabrielian A.E. Garg N.S. Gelbart W.M. Glasser K. Glodek A. Gong F. Gorrell J.H. Gu Z. Guan P. Harris M. Harris N.L. Harvey D. Heiman T.J. Hernandez J.R. Houck J. Hostin D. Houston K.A. Howland T.J. Wei M.H. Ibegwam C. Jalali M. Kalush F. Karpen G.H. Ke Z. Kennison J.A. Ketchum K.A. Kimmel B.E. Kodira C.D. Kraft C. Kravitz S. Kulp D. Lai Z. Lasko P. Lei Y. Levitsky A.A. Li J. Li Z. Liang Y. Lin X. Liu X. Mattei B. McIntosh T.C. McLeod M.P. McPherson D. Merkulov G. Milshina N.V. Mobarry C. Morris J. Moshrefi A. Mount S.M. Moy M. Murphy B. Murphy L. Muzny D.M. Nelson D.L. Nelson D.R. Nelson K.A. Nixon K. Nusskern D.R. Pacleb J.M. Palazzolo M. Pittman G.S. Pan S. Pollard J. Puri V. Reese M.G. Reinert K. Remington K. Saunders R.D. Scheeler F. Shen H. Shue B.C. Siden-Kiamos I. Simpson M. Skupski M.P. Smith T. Spier E. Spradling A.C. Stapleton M. Strong R. Sun E. Svirskas R. Tector C. Turner R. Venter E. Wang A.H. Wang X. Wang Z.Y. Wassarman D.A. Weinstock G.M. Weissenbach J. Williams S.M. Woodage T. Worley K.C. Wu D. Yang S. Yao Q.A. Ye J. Yeh R.F. Zaveri J.S. Zhan M. Zhang G. Zhao Q. Zheng L. Zheng X.H. Zhong F.N. Zhong W. Zhou X. Zhu S. Zhu X. Smith H.O. Gibbs R.A. Myers E.W. Rubin G.M. Venter J.C. Science. 2000; 287: 2185-2195Crossref PubMed Scopus (4744) Google Scholar, 29Celniker S.E. Wheeler D.A. Kronmiller B. Carlson J.W. Halpern A. Patel S. Adams M. Champe M. Dugan S.P. Frise E. Hodgson A. George R.A. Hoskins R.A. Laverty T. Muzny D.M. Nelson C.R. Pacleb J.M. Park S. Pfeiffer B.D. Richards S. Sodergren E.J. Svirskas R. Tabor P.E. Wan K. Stapleton M. Sutton G.G. Venter C. Weinstock G. Scherer S.E. Myers E.W. Gibbs R.A. Rubin G.M. Genome Biology. 2002; (http://genomebiology.com/2002/3/12/research/0079)Google Scholar). The gene designated CG6728 showed a notable alteration in the ninaGP330 mutant allele, a C to T change at nucleotide 241. This resulted in a nonsense codon, altering a CAG (Gln) codon to a TAG (STOP) codon. No other mutations resulting in nonsense mutations were identified, although three other genes contained single nucleotide changes resulting in amino acid substitutions. As a result of the sequence analysis, CG6728 emerged as the best candidate for the ninaG gene. Fig. 4A shows a diagram of the gene structure, identifying the site of the mutation found in ninaGP330. We used a 3.8-kb genomic KpnI-StuI fragment that contained this open reading frame and 1.1 kb of upstream sequences to construct transgenic flies. Fig. 4B shows that this CG6728 genomic fragment rescues the low rhodopsin phenotype of ninaGP330 mutants. Further, electroretinogram analysis showed that the CG6728 transgene restores the PDA response in ninaG mutants (Fig. 4C). These results establish that CG6728 is the ninaG gene. NinaG Is a Member of GMC Oxidoreductase Enzyme Family—Comparison of the NinaG protein sequence with GenBank™ protein databases indicated that NinaG is a member of the GMC oxidoreductase family. This is a diverse family of flavin adenine dinucleotide flavoproteins. There are 15 identified Drosophila genes in this family. Only one, glucose dehydrogenase (30Schiff N.M. Feng Y. Quine J.A. Krasney P.A. Cavener D.R. Mol. Biol. Evol. 1992; 9: 1029-1049PubMed Google Scholar), codes for a characterized enzyme. Fig. 5A shows the neighbor-joining gene tree (31Saitou N. Nei M. Mol. Biol. Evol. 1987; 4: 406-425PubMed Google Scholar) of the Drosophila GMC oxidoreductase family members and characterized GMC oxidoreductase enzymes from other organisms. The analysis shows high divergence within this family in general and the NinaG protein in particular. Such results preclude any inference of substrate specificity or enzymatic action from this type of analysis. Sequence analysis showed that the amino-terminal region of NinaG contains a signal recognition sequence (Fig. 5B). This amino-terminal domain is predicted to contain a signal peptide in NinaG protein with cleavage between residues 26 and 27 (32von Heijne G. Nucleic Acids Res. 1986; 14: 4683-4690Crossref PubMed Scopus (3685) Google Scholar). The other members of the family lack any predicted signal sequence. Other features of the NinaG protein structure are as expected for the GMC oxidoreductase family, including the ADP-binding domain described as the βαβ fold (33Wierenga R.K. Drenth J. Schulz G.E. J. Mol. Biol. 1983; 167: 725-739Crossref PubMed Scopus (178) Google Scholar) within the amino-terminal region. NinaG Acts in Chromophore Production—The identification of NinaG as a member of the GMC oxidoreductase family suggests that the NinaG is an enzyme responsible for a redox reaction on small organic molecules. Given that the Rh1 accumulation in the endoplasmic reticulum observed in ninaG mutants is similar to other genotypes, and vitamin A-free diets deficient in rhodopsin chromophore availability, we hypothesized that the NinaG enzyme acts in the production of 3-hydroxyretinal, the Rh1 chromophore. To investigate this possibility, HPLC was used to profile the retinal extracts from ninaG+ and ninaG mutant heads. A similar number of heads were used for preparation of these extracts, and the results reported here were reproduced in independently prepared samples. Fig. 6 displays representative chromatograms of these extracts at two wavelengths, 324 and 374 nm. These chromatograms have been scaled to the same y axis to allow direct comparison of the samples. Significant differences were observed among three peaks eluting at 3.5, 3.8, and 4.1 min, whereas the remaining chromatographic profiles of all samples were similar. To prove that all observed components are because of retinoid species, wild type flies were reared on a vitamin A-free diet. Extracts from such flies showed no chromatographic peaks in the region of interest at the wavelengths listed above. Furthermore, feeding retinal back to these adult flies restored the chromatographic profile observed for wild type flies reared on normal medium (data not shown). Fig. 6A shows that wild type flies possess a major retinoid species with a retention time of 4.1 min, with a secondary species indicated by the peak at 3.8 min. The ninaG flies extract yielded significantly smaller peaks at 4.1 and 3.8 min, indicating that they possess lower levels of both of these species. The λmax of both the 4.1- and 3.8-min peaks was 374 nm, and the electrospray mass spectrum (data not shown) of each peak showed an ion at m/z 301.5 for [M+H]+ of 3-hydroxyretinal. These peaks were assigned as 11-cis-3-hydroxyretinal (4.1 min) and all-trans-3-hydroxyretinal (3.8 min), in accordance with earlier work showing these are the predominant retinoids extracted from Drosophila heads (10Seki T. Isono K. Ito M. Katsuta Y. Eur. J. Biochem. 1994; 226: 691-696Crossref PubMed Scopus (19) Google Scholar). Extracts from wild type flies raised in the dark and provided with all-trans-retinal as the only retinoid source yielded a large increase in the 3.8-min peak at the expense of the 4.1-min peak (data not shown). This result is in agreement with the isomer assignments of the chromatographic peaks, because flies require light to convert 3-hydroxyretinal from the all-trans to the 11-cis isomer (34Isono K. Tanimura T. Oda Y. Tsukahara Y. J. Gen. Physiol. 1988; 92: 587-600Crossref PubMed Scopus (41) Google Scholar). Fig. 6B shows the chromatograms at 324 nm for wild type and ninaG extracts. The predominant retinoid elutes at 3.5 min and has a λmax of 324 nm. Interestingly, the electrospray mass spectrum of the corresponding chromatographic peak showed an ion at m/z 285.5 (data not shown), which is the expected m/z value for [M+H]+ of retinal. The HPLC/UV-visible/MS results of the all-trans-retinal standard yielded a chromatographic peak at 13.2 min with a λmax of 374 nm and m/z 285.5 (data not shown). Although the presence of an ion at m/z 285.5 suggests that the 3.5-min peak is an isomer of retinal, the major change in λmax (324 versus 374 nm) and retention time (3.5 versus 13.2 min) relative to the all-trans-retinal standard does not support this assignment. If the majority of the 3-hydroxyretinal extracted from wild type is complexed to Rh1 rhodopsin, the absence of 3-hydroxyretinal in the ninaG mutant background might be a secondary effect of ninaG action reducing Rh1 rhodopsin protein production. To discount this possibility, the profile of retinoids in flies expressing Rh4 in place of Rh1 (refe" @default.
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• W1974768178 title "The Drosophila ninaG Oxidoreductase Acts in Visual Pigment Chromophore Production" @default.
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