To identify avian FAR sequences, amino acid similarity search was carried out with the "basic local alignment search tool" (BLASTP 2.2.25, NCBI) [40
] using the following queries: human, HsFAR1 [NCBI: NP_115604]; human, HsFAR2 [NCBI: NP_060569.3]; mouse, MmFAR1 [NCBI: NP_080419.2]; mouse, MmFAR2 [NCBI: NP_848912.1] and jojoba, ScFAR [NCBI: AAD38039.1] (for further accession numbers see additional file 1
). Protein property analyses were conducted with "TMHMM" [42
] and "NCBI Conserved Domain Search" [45
]. For the illustration of conserved domains and transmembrane regions "PROSITE MyDomains" on the ExPASy server was used. Amino acid sequence alignments were carried out with ClustalX2.1 [48
] and GeneDoc [49
]. Phylogenetic analysis of FAR amino acid sequences was conducted with MEGA5 [50
], using the Neighbor-Joining method with 1000 bootstrap replicates. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site.
Tissues of domestic chicken and domestic goose were obtained from Putenfarm Peter Ritte in Wegberg-Rickelrath, tissues of barn owl were obtained from the Institute for Biology II, RWTH Aachen. After the frozen tissue was pulverized with mortar and pestle, 3 volumes of TRIZOL® reagent (Invitrogen, Germany) were added and the suspension was vortexed at room temperature for 15 min. One volume of 1-chlor-3-brompropane (Applichem, Germany) was added and the mixture was incubated at room temperature for 10 min. After centrifugation at 14 000 × g and 4°C for 20 min, the RNA of the aqueous phase was precipitated by adding 1 volume isopropanol and incubating the mixture 15 min at room temperature. The RNA was sedimented by centrifugation at 14 000 × g and 4°C for 20 min. After washing with 70% ethanol, the sedimented RNA was dissolved in 100-300 μL distilled water. Integrity of isolated RNA was analyzed by agarose gel electrophoresis, and the concentration of nucleic acids was measured photometrically. mRNA preparation was carried out using Dynabeads® (Invitrogen, Germany).
Vector construction and yeast transformation
First strand cDNA synthesis of FAR sequences from mRNA of the uropygial glands was carried out using an AMV reverse transcriptase (Fermentas, Germany) and reverse primer of the respective sequences (Rev-FAR1, 5'-TCAGTATCTCATAGTACTGGAGG-3' or Rev-FAR2, 5'-TCAGTGCCTGAGGGTGCTGG-3'). PCR of FAR1 and FAR2 sequences was carried out with a Pfu-polymerase (Fermentas, Germany) and the primers For-FAR1, 5'-CACCATGGTTTCCATACCTGAATATTATG-3' and Rev-FAR1, or For-FAR2, 5'-CACCATGTCTTCAGTCTCAGCTTATTAC-3' and Rev-FAR2 (Eurofins MWG operon, Germany). Sequences were cloned into the Gateway®
entry vector pENTR-SD/D-TOPO (Invitrogen), transformed into Escherichia coli
TOP10 (Invitrogen). Vectors were re-isolated, sequenced (Fraunhofer IME, Aachen, Germany) and the LR-reaction was carried out with the Gateway®
Clonase Mix II™(Invitrogen) and the Gateway®
yeast expression vector pYES-DEST52 (Invitrogen) containing a galactose inducible promoter (GAL1). Yeast cells of the strain Saccharomyces cerevisiae
BY4741 Ɗdga1 Ɗlro1
(MATa, his3Ɗ1, leu2Ɗ0, met15Ɗ0, ura3Ɗ0, lro1–Ɗ::kanMX4, dga1-Ɗ::natMX4
] were transformed with the expression constructs or the empty vector as a control via electroporation. Transgenic yeast strains were grown in synthetic dropout (SD) medium containing 0.068% (w/v) amino acid supplement mixture (CSM) without uracil and leucine (MP Biomedicals, France), 0.5% (w/v) ammonium sulfate (Roth, Germany), 0.17% (w/v) yeast nitrogen base (MP Biomedicals, France), 0.01% (w/v) leucine (Roth, Germany), 2% (w/v) galactose or 2% (w/v) glucose at 28°C.
Lipid analysis of transgenic yeast cultures
SD-medium with 2% galactose was inoculated with a yeast preculture harboring one of the FAR constructs or the empty vector as a control and was incubated for 72 h at 28°C without or with supplementation of 250 μM fatty acids. Cells of 10 ml culture were harvested, washed twice with distilled water, dried and after adding of 125 nmol dodecanoic acid and dodecanol (Sigma-Aldrich, Germany) as internal standards the transmethylation of fatty acids was carried out with 2 mL 0.5 M H2
and 2% 2,2-dimethoxypropane in methanol at 80°C for 1 h. Fatty acid methyl esters (FAME) and fatty alcohols were extracted with 3 mL heptane and analyzed via gas chromatography. Derivatization of fatty alcohols was carried out with 1:1 (v/v) heptane/N, O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) (Roth, Germany) at 70°C for 1 h. Solvents were removed under a stream of nitrogen at room temperature, lipids were dissolved in heptane and analyzed via gas chromatography. Total lipids of transgenic yeast cells were extracted according to Bligh & Dyer [55
] and analyzed by TLC (silica gel plate, 60 Å, Merck; Germany) in heptane/diethylether/acetic acid (90:60:1, v/v/v). Lipids were visualized by staining with dichlorofluorescein and identified by comparison with external standards.
Gas chromatography (GC)
Gas chromatographic analysis was carried out with a HP 6890 gas chromatographic system equipped with the column OPTIMA-5MS (Macherey & Nagel, Germany) and a flame ionization detection (FID) system. The following temperature program was used for FAME and fatty alcohol analysis (2 min at 120°C, then 10°C per min up to 150°C; then 3°C per min up to 270°C; then 10°C per min up to 300°C, hold 1 min at 300°C; with a total column flow of 1.0 mL per min and 1 bar pressure; N2 as carrier gas). Identification of the analytes was carried out by comparison of the retention time with those of the external alcohol standards of different chain lengths and degrees of saturation (Sigma-Aldrich, Germany) and in addition by the derivatization of fatty alcohols with BSTFA that resulted in a shift of retention time compared to the free fatty alcohols.
Preparation of total membrane fractions of transgenic yeast strains
200 mL of induced transgenic yeast cultures were incubated for 16 h at 28°C. Cells were harvested and washed in 20 mL buffer TH (50 mM TRIS/H2
, pH 7.4) and subsequently frozen at -20°C for 10 min. Glass beads (0.75-1 mm diameter) and 2 mL buffer were added and cells were vortexed for 5 min, centrifuged at 1300 × g and 4°C for 5 min. The supernatant was transferred into a new tube and the latter steps were repeated two times. The supernatants were pooled and sonicated twice for 30 s on ice. Cell debris were sedimented at 2500 × g and 4°C for 15 min and the resulting supernatant was centrifuged at 140 000 × g and 4°C for 1 h. Pelletized yeast membranes were resuspended in buffer TH, quick-frozen in liquid nitrogen, and stored at -80°C. Protein concentration was determined [56
Synthesis of [1-14C]-labeled branched-chain fatty acids
C]2-Methyltetradecanoic, 2-methylhexadecanoic and 2-methyloctadecanoic acids were prepared by α-methylation of the corresponding [1-14
C]-labeled fatty acids via the sequence carboxylic acid → acyl chloride → diazoketone → chloroketone → 2-methylcarboxylic acid essentially as described [57
]. Purification by reversed-phase HPLC (solvent system, acetonitrile/water/acetic acid 85:15:0.01, v/v/v) afforded > 98% pure materials having a specific radioactivity of 0.622 GBq/mmol.
[1-14C]3,7,11,15-Tetramethylhexadecanoic acid (phytanic acid) was synthesized starting with 2,6,10,14-tetramethylpentadecanoic acid (pristanic acid; Lipidox Co., Stockholm, Sweden) by the sequence carboxylic acid → primary alcohol → bromide → 14C-nitrile → 14C-carboxylic acid. The material was purified by reversed-phase HPLC (solvent system, acetontrile/acetic acid 100:0.01, v/v) to afford > 98% pure material having a specific radioactivity of 0.622 GBq/mmol.
In vitro FAR assays
FAR assays were routinely carried out with [1-14C]-labeled acyl-CoA thioesters namely 14:0-CoA, 2.04 GBq/mmol; 18:0-CoA, 2.04 GBq/mmol (Biotrend, Germany); 16:0-CoA, 2,22 GBq/mmol (Perkin Elmer, Germany); 2-methyl-branched-CoAs, 0.62 GBq/mmol, 3,7,11,15-tetramethylhexadecanoyl-CoA, 0.17 GBq/mmol, 10:0-CoA, 0.08 GBq/mmol, 12:0-CoA, 0.7 GBq/mmol (acyl-CoA synthesis carried out by Prof. Sten Stymne and his work group). Reaction mixture contained, in a total volume of 50 μl, 2-10 μg membrane protein, 20 μM acyl-CoA, 5 mM NADPH (Sigma-Aldrich, Germany), 1 mM MgCl2, 16 μM BSA, 25 mM sodium-phosphate-buffer, pH 6.5 (FAR1) or 25 mM sodium-citrate-buffer, pH 5.5 (FAR2). After 10 min incubation at 37°C reaction products were extracted with 250 μL chloroform/methanol (1:1, v/v) and 100 μL 0.9% (w/v) NaCl solution. The suspension was mixed and, after a brief centrifugation, 80 μL of the chloroform phase were analyzed by TLC. [14C]-labeled reaction products were visualized by the phosporimager system FLA3000 (Fujifilm), identified by external standards and quantified with the multi-purpose scintillation counter LS 6500 (Beckman Coulter).
FAR assays with non-labeled substrates were carried out in the similar way but 20 μM [1-14C]-labeled acyl-CoA was substituted by 20 μM of 12:0-CoA, 14:0-CoA, 16:0-CoA, 18:0-CoA, (Sigma-Aldrich, Germany) and 20:0-CoA (Avanti, USA) and incubation time was extended to 4 h and the assay volume was increased (500 μL). Extracted reaction products were transmethylated and analyzed by GC.
Semi-quantitative expression analysis of GgFAR1 and GgFAR2 in chicken
1 μg of total RNA isolated from tissues of pectoral muscles, liver, uropygial gland, brain, heart and adipose tissue of chicken were digested with DNase I (Fermentas, Germany) and reverse transcription was carried out with AMV reverse transcriptase (Fermentas) using an oligo-(dT)-primer. PCR was conducted with 1 μL first strand cDNA as template, Taq-polymerase (Genecraft, Germany) and primers for GgFAR1 (Forward: 5'-GACACCAGAAGCACGGATAG-3', Reverse: 5'-TCCAGTTCAGGCTGTGTAAG-3', 126 bp fragment), GgFAR2 (Forward: 5'-CTCCTGCCATACTCTATGAC-3', Reverse: 5'-GACTGGGTGGAGAAATACTG-3', 118 bp fragment) and β-actin (Forward: 5'-ACCTGAGCGCAAGTACTCTG-3', Reverse: 5'-ACAATGGAGGGTCCGGA-3', 114 bp fragment). Reactions without reverse transcriptase were carried out to exclude DNA contamination. Amplified fragments were analyzed by agarose gel electrophoresis.