Overexpression of rat long chain acyl-coa synthetase 1 alters fatty acid metabolism in rat primary hepatocytes

Long chain acyl-CoA synthetases (ACSL) activate fatty acids (FA) and provide substrates for both anabolic and catabolic pathways. We have hypothesized that each of the five ACSL isoforms partitions FA toward specific downstream pathways. Acsl1 mRNA is increased in cells under both lipogenic and oxidative conditions. To elucidate the role of ACSL1 in hepatic lipid metabolism, we overexpressed an Acsl1 adenovirus construct (Ad-Acsl1) in rat primary hepatocytes. Ad-ACSL1, located on the endoplasmic reticulum but not on mitochondria or plasma membrane, increased ACS specific activity 3.7-fold. With 100 or 750 mum [1-(14)C]oleate, Ad-Acsl1 increased oleate incorporation into diacylglycerol and phospholipids, particularly phosphatidylethanolamine and phosphatidylinositol, and decreased incorporation into cholesterol esters and secreted triacylglycerol. Ad-Acsl1 did not alter oleate incorporation into triacylglycerol, beta-oxidation products, or total amount of FA metabolized. In pulse-chase experiments to examine the effects of Ad-Acsl1 on lipid turnover, more labeled triacylglycerol and phospholipid, but less labeled diacylglycerol, remained in Ad-Acsl1 cells, suggesting that ACSL1 increased reacylation of hydrolyzed oleate derived from triacylglycerol and diacylglycerol. In addition, less hydrolyzed oleate was used for cholesterol ester synthesis and beta-oxidation. The increase in [1,2,3-(3)H]glycerol incorporation into diacylglycerol and phospholipid was similar to the increase with [(14)C]oleate labeling suggesting that ACSL1 increased de novo synthesis. Labeling Ad-Acsl1 cells with [(14)C]acetate increased triacylglycerol synthesis but did not channel endogenous FA away from cholesterol ester synthesis. Thus, consistent with the hypothesis that individual ACSLs partition FA, Ad-Acsl1 increased FA reacylation and channeled FA toward diacylglycerol and phospholipid synthesis and away from cholesterol ester synthesis.     

Mouse cardiac acyl coenzyme a synthetase 1 deficiency impairs Fatty Acid oxidation and induces cardiac hypertrophy

Long-chain acyl coenzyme A (acyl-CoA) synthetase isoform 1 (ACSL1) catalyzes the synthesis of acyl-CoA from long-chain fatty acids and contributes the majority of cardiac long-chain acyl-CoA synthetase activity. To understand its functional role in the heart, we studied mice lacking ACSL1 globally (Acsl1(T-/-)) and mice lacking ACSL1 in heart ventricles (Acsl1(H-/-)) at different times. Compared to littermate controls, heart ventricular ACSL activity in Acsl1(T-/-) mice was reduced more than 90%, acyl-CoA content was 65% lower, and long-chain acyl-carnitine content was 80 to 90% lower. The rate of [(14)C]palmitate oxidation in both heart homogenate and mitochondria was 90% lower than in the controls, and the maximal rates of [(14)C]pyruvate and [(14)C]glucose oxidation were each 20% higher. The mitochondrial area was 54% greater than in the controls with twice as much mitochondrial DNA, and the mRNA abundance of Pgc1α and Errα increased by 100% and 41%, respectively. Compared to the controls, Acsl1(T-/-) and Acsl1(H-/-) hearts were hypertrophied, and the phosphorylation of S6 kinase, a target of mammalian target of rapamycin (mTOR) kinase, increased 5-fold. Our data suggest that ACSL1 is required to synthesize the acyl-CoAs that are oxidized by the heart, and that without ACSL1, diminished fatty acid (FA) oxidation and compensatory catabolism of glucose and amino acids lead to mTOR activation and cardiac hypertrophy without lipid accumulation or immediate cardiac dysfunction.     

Rat long-chain acyl-CoA synthetase mRNA, protein, and activity vary in tissue distribution and in response to diet

Distinct isoforms of long-chain acyl-CoA synthetases (ACSLs) may partition fatty acids toward specific metabolic cellular pathways. For each of the five members of the rat ACSL family, we analyzed tissue mRNA distributions, and we correlated the mRNA, protein, and activity of ACSL1 and ACSL4 after fasting and refeeding a 69% sucrose diet. Not only did quantitative real-time PCR analyses reveal unique tissue expression patterns for each ACSL isoform, but expression varied markedly in different adipose depots. Fasting increased ACSL4 mRNA abundance in liver, muscle, and gonadal and inguinal adipose tissues, and refeeding decreased ACSL4 mRNA. A similar pattern was observed for ACSL1, but both fasting and refeeding decreased ACSL1 mRNA in gonadal adipose. Fasting also decreased ACSL3 and ACSL5 mRNAs in liver and ACSL6 mRNA in muscle. Surprisingly, in nearly every tissue measured, the effects of fasting and refeeding on the mRNA abundance of ACSL1 and ACSL4 were discordant with changes in protein abundance. These data suggest that the individual ACSL isoforms are distinctly regulated across tissues and show that mRNA expression may not provide useful information about isoform function. They further suggest that translational or posttranslational modifications are likely to contribute to the regulation of ACSL isoforms.     

Long-chain acyl-CoA synthetase isoforms differ in preferences for eicosanoid species and long-chain fatty acids

Because the signaling eicosanoids, epoxyeicosatrienoic acids (EETs) and HETEs, are esterified to membrane phospholipids, we asked which long-chain acyl-CoA synthetase (ACSL) isoforms would activate these molecules and whether the apparent FA substrate preferences of each ACSL isoform might differ depending on whether it was assayed in mammalian cell membranes or as a purified bacterial recombinant protein. We found that all five ACSL isoforms were able to use EETs and HETEs as substrates and showed by LC-MS/MS that ACSLs produce EET-CoAs. We found differences in substrate preference between ACS assays performed in COS7 cell membranes and recombinant purified proteins. Similarly, preferences and Michaelis-Menten kinetics for long-chain FAs were distinctive. Substrate preferences identified for the purified ACSLs did not correspond to those observed in ACSL-deficient mouse models. Taken together, these data support the concept that each ACSL isoform exhibits a distinct substrate preference, but apparent substrate specificities depend upon multiple factors including membrane character, coactivators, inhibitors, protein interactions, and posttranslational modification.     

Revised nomenclature for the mammalian long-chain acyl-CoA synthetase gene family

By consensus, the acyl-CoA synthetase (ACS) community, with the advice of the human and mouse genome nomenclature committees, has revised the nomenclature for the mammalian long-chain acyl-CoA synthetases. ACS is the family root name, and the human and mouse genes for the long-chain ACSs are termed ACSL1,3-6 and Acsl1,3-6, respectively. Splice variants of ACSL3, -4, -5, and -6 are cataloged. Suggestions for naming other family members and for the nonmammalian acyl-CoA synthetases are made.     

Acyl-CoA synthetase 1 deficiency alters cardiolipin species and impairs mitochondrial function

Long-chain acyl-CoA synthetase 1 (ACSL1) contributes more than 90% of total cardiac ACSL activity, but its role in phospholipid synthesis has not been determined. Mice with an inducible knockout of ACSL1 (Acsl1^T−/−) have impaired cardiac fatty acid oxidation and rely on glucose for ATP production. Because ACSL1 exhibited a strong substrate preference for linoleate, we investigated the composition of heart phospholipids. Acsl1^T−/− hearts contained 83% less tetralinoleoyl-cardiolipin (CL), the major form present in control hearts. A stable knockdown of ACSL1 in H9c2 rat cardiomyocytes resulted in low incorporation of linoleate into CL and in diminished incorporation of palmitate and oleate into other phospholipids. Overexpression of ACSL1 in H9c2 and HEK-293 cells increased incorporation of linoleate into CL and other phospholipids. To determine whether increasing the content of linoleate in CL would improve mitochondrial respiratory function in Acsl1^T−/− hearts, control and Acsl1^T−/− mice were fed a high-linoleate diet; this diet normalized the amount of tetralinoleoyl-CL but did not improve respiratory function. Thus, ACSL1 is required for the normal composition of several phospholipid species in heart. Although ACSL1 determines the acyl-chain composition of heart CL, a high tetralinoleoyl-CL content may not be required for normal function.     

Overexpression of acyl-CoA synthetase-1 increases lipid deposition in hepatic (HepG2) cells and rodent liver in vivo

Accumulation of intracellular lipid in obesity is associated with metabolic disease in many tissues including liver. Storage of fatty acid as triglyceride (TG) requires the activation of fatty acids to long-chain acyl-CoAs (LC-CoA) by the enzyme acyl-CoA synthetase (ACSL). There are five known isoforms of ACSL (ACSL1, -3, -4, -5, -6), which vary in their tissue specificity and affinity for fatty acid substrates. To investigate the role of ACSL1 in the regulation of lipid metabolism, we used adenoviral-mediated gene transfer to overexpress ACSL1 in the human hepatoma cell-line HepG2 and in liver of rodents. Infection of HepG2 cells with the adenoviral construct AdACSL1 increased ACSL activity >10-fold compared with controls after 24 h. HepG2 cells overexpressing ACSL1 had a 40% higher triglyceride (TG) content (93 +/- 3 vs. 67 +/- 2 nmol/mg protein in controls, P < 0.05) after 24-h exposure to 1 mM oleate. Furthermore, ACSL1 overexpression produced a 60% increase in cellular LCA-CoA content (160 +/- 6 vs. 100 +/- 6 nmol/g protein in controls, P < 0.05) and increased [(14)C]oleate incorporation into TG without significantly altering fatty acid oxidation. In mice, AdACSL1 administration increased ACSL1 mRNA and protein more than fivefold over controls at 4 days postinfection. ACSL1 overexpression caused a twofold increase in TG content in mouse liver (39 +/- 4 vs. 20 +/- 2 mumol/g wet wt in controls, P < 0.05), and overexpression in rat liver increased [1-(14)C]palmitate clearance into liver TG. These in vitro and in vivo results suggest a pivotal role for ACSL1 in regulating TG synthesis in liver.     

Dissociation of Systemic Glucose Homeostasis from Triacylglyceride Accumulation by Reduced <Emphasis Type="Italic">Acsl6</Emphasis> Expression in Skeletal Muscle

Long chain acyl-CoA synthetase (ACSL) is an enzyme that activates fatty acids before they are further metabolized. ACSL6 is the one of main ACSL isoforms exclusively expressed in skeletal muscle, but the consequences of the suppression of this gene in systemic glucose homeostasis has yet to be reported. Hence, we investigated the roles of ACSL6 gene in glucose tolerance and TAG distribution in physiological conditions. Eight-week-old male C57BL/6J mice were administered with control or Acsl6 siRNAs and then fed with either AIN-93 control diet or high fat diet. At seven days after the first siRNA injection, oral glucose tolerance tests and TAG quantification were performed. In vivo administration of Acsl6 siRNA decreased Acsl6 expression only in skeletal muscle under AIN-93 or a high fat diet. However Acsl6 siRNA injection to animals increased TAG accumulation in the liver without the change of Acsl6 expression. Atelocollagen mediated Acsl6 suppression enhanced whole-body glucose tolerance coinciding with decreased TAG accumulation in skeletal muscle of mice fed an AIN-93 diet. However, the improved glucose tolerance by Acsl6 reduction was ablated by high fat diet. Moreover reduced Acsl6 did not alter the phosphorylation of insulin signaling proteins in skeletal muscle. These results suggest that Acsl6 reduction in skeletal muscle enhances glucose homeostasis and dissociates the insulin responses from TAG accumulation in skeletal muscle.     

Acyl-CoA synthetase isoforms 1, 4, and 5 are present in different subcellular membranes in rat liver and can be inhibited independently

Inhibition studies have suggested that acyl-CoA synthetase (ACS, EC ) isoforms might regulate the use of acyl-CoAs by different metabolic pathways. In order to determine whether the subcellular locations differed for each of the three ACSs present in liver and whether these isoforms were regulated independently, non-cross-reacting peptide antibodies were raised against ACS1, ACS4, and ACS5. ACS1 was identified in endoplasmic reticulum, mitochondria-associated membrane (MAM), and cytosol, but not in mitochondria. ACS4 was present primarily in MAM, and the 76-kDa ACS5 protein was located in mitochondrial membrane. Consistent with these locations, N-ethylmaleimide, an inhibitor of ACS4, inhibited ACS activity 47% in MAM and 28% in endoplasmic reticulum. Troglitazone, a second ACS4 inhibitor, inhibited ACS activity <10% in microsomes and mitochondria and 45% in MAM. Triacsin C, a competitive inhibitor of both ACS1 and ACS4, inhibited ACS activity similarly in endoplasmic reticulum, MAM, and mitochondria, suggesting that a hitherto unidentified triacsin-sensitive ACS is present in mitochondria. ACS1, ACS4, and ACS5 were regulated independently by fasting and re-feeding. Fasting rats for 48 h resulted in a decrease in ACS4 protein, and an increase in ACS5. Re-feeding normal chow or a high sucrose diet for 24 h after a 48-h fast increased both ACS1 and ACS4 protein expression 1.5-2.0-fold, consistent with inhibition studies. These results suggest that ACS1 and ACS4 may be linked to triacylglycerol synthesis. Taken together, the data suggest that acyl-CoAs may be functionally channeled to specific metabolic pathways through different ACS isoforms in unique subcellular locations.     

Diminished Acyl-CoA Synthetase Isoform 4 Activity in INS 832/13 Cells Reduces Cellular Epoxyeicosatrienoic Acid Levels and Results in Impaired Glucose-stimulated Insulin Secretion

Glucose-stimulated insulin secretion (GSIS) in pancreatic beta-cells is potentiated by fatty acids (FA). The initial step in the metabolism of intracellular FA is the conversion to acyl-CoA by long chain acyl-CoA synthetases (Acsls). Because the predominantly expressed Acsl isoforms in INS 832/13 cells are Acsl4 and -5, we characterized the role of these Acsls in beta-cell function by using siRNA to knock down Acsl4 or Acsl5. Compared with control cells, an 80% suppression of Acsl4 decreased GSIS and FA-potentiated GSIS by 32 and 54%, respectively. Knockdown of Acsl5 did not alter GSIS. Acsl4 knockdown did not alter FA oxidation or long chain acyl-CoA levels. With Acsl4 knockdown, incubation with 17 mm glucose increased media epoxyeicosatrienoic acids (EETs) and reduced cell membrane levels of EETs. Further, exogenous EETs reduced GSIS in INS 832/13 cells, and in Acsl4 knockdown cells, an EET receptor antagonist partially rescued GSIS. These results strongly suggest that Acsl4 activates EETs to form EET-CoAs that are incorporated into glycerophospholipids, thereby sequestering EETs. Exposing INS 832/13 cells to arachidonate or linoleate reduced Acsl4 mRNA and protein expression and reduced GSIS. These data indicate that Acsl4 modulates GSIS by regulating the levels of unesterified EETs and that arachidonate controls the expression of its activator Acsl4.     

Expression and characterization of recombinant rat Acyl-CoA synthetases 1, 4, and 5. Selective inhibition by triacsin C and thiazolidinediones

Inhibition by triacsins and troglitazone of long chain fatty acid incorporation into cellular lipids suggests the existence of inhibitor-sensitive and -resistant acyl-CoA synthetases (ACS, EC ) that are linked to specific metabolic pathways. In order to test this hypothesis, we cloned and purified rat ACS1, ACS4, and ACS5, the isoforms present in liver and fat cells, expressed the isoforms as ACS-Flag fusion proteins in Escherichia coli, and purified them by Flag affinity chromatography. The Flag epitope at the C terminus did not alter the kinetic properties of the enzyme. Purified ACS1-, 4-, and 5-Flag isoforms differed in their apparent K(m) values for ATP, thermolability, pH optima, requirement for Triton X-100, and sensitivity to N-ethylmaleimide and phenylglyoxal. The ACS inhibitor triacsin C strongly inhibited ACS1 and ACS4, but not ACS5. The thiazolidinedione (TZD) insulin-sensitizing drugs and peroxisome proliferator-activated receptor gamma (PPARgamma) ligands, troglitazone, rosiglitazone, and pioglitazone, strongly and specifically inhibited only ACS4, with an IC(50) of less than 1.5 microm. Troglitazone exhibited a mixed type inhibition of ACS4. alpha-Tocopherol, whose ring structure forms the non-TZD portion of troglitazone, did not inhibit ACS4, indicating that the thiazolidine-2,4-dione moiety is the critical component for inhibition. A non-TZD PPARgamma ligand, GW1929, which is 7-fold more potent than rosiglitazone, inhibited ACS1 and ACS4 poorly with an IC(50) of greater than 50 microm, more than 100-fold higher than was required for rosiglitazone, thereby demonstrating the specificity of TZD inhibition. Further, the PPARalpha ligands, clofibrate and GW4647, and various xenobiotic carboxylic acids known to be incorporated into complex lipids had no effect on ACS1, -4, or -5. These results, together with previous data showing that triacsin C and troglitazone strongly inhibit triacylglycerol synthesis compared with other metabolic pathways, suggest that ACS1 and ACS4 catalyze the synthesis of acyl-CoAs used for triacylglycerol synthesis and that lack of inhibition of a metabolic pathway by triacsin C does not prove lack of acyl-CoA involvement. The results further suggest the possibility that the insulin-sensitizing effects of the thiazolidinedione drugs might be achieved, in part, through direct interaction with ACS4 in a PPARgamma-independent manner.     

Identification of a novel function of hepatic long-chain acyl-CoA synthetase-1 (ACSL1) in bile acid synthesis and its regulation by bile acid-activated farnesoid X receptor

Long-chain acyl-CoA synthetase 1 (ACSL1) plays a pivotal role in fatty acid β‑oxidation in heart, adipose tissue and skeletal muscle. However, key functions of ACSL1 in the liver remain largely unknown. We investigated acute effects of hepatic ACSL1 deficiency on lipid metabolism in adult mice under hyperlipidemic and normolipidemic conditions. We knocked down hepatic ACSL1 expression using adenovirus expressing a ACSL1 shRNA (Ad-shAcsl1) in mice fed a high-fat diet or a normal chow diet. Hepatic ACSL1 depletion generated a hypercholesterolemic phenotype in mice fed both diets with marked elevations of total cholesterol, LDL-cholesterol and free cholesterol in circulation and accumulations of cholesterol in the liver. Furthermore, SREBP2 pathway in ACSL1 depleted livers was severely repressed with a 50% reduction of LDL receptor protein levels. In contrast to the dysregulated cholesterol metabolism, serum triglycerides, free fatty acid and phospholipid levels were unaffected. Mechanistic investigations of genome-wide gene expression profiling and pathway analysis revealed that ACSL1 depletion repressed expressions of several key enzymes for bile acid biosynthesis, consequently leading to reduced liver bile acid levels and altered bile acid compositions. These results are the first demonstration of a requisite role of ACSL1 in bile acid biosynthetic pathway in liver tissue. Furthermore, we discovered that Acsl1 is a novel molecular target of the bile acid-activated farnesoid X receptor (FXR). Activation of FXR by agonist obeticholic acid repressed the expression of ACSL1 protein and mRNA in the liver of FXR wild-type mice but not in FXR knockout mice.     

Rat long chain acyl-CoA synthetase 5, but not 1, 2, 3, or 4, complements Escherichia coli fadD

Long chain fatty acids are converted to acyl-CoAs by acyl-CoA synthetase (fatty acid CoA ligase: AMP forming, E.C. 6.2.1.3; ACS). Escherichia coli has a single ACS, FadD, that is essential for growth when fatty acids are the sole carbon and energy source. Rodents have five ACS isoforms that differ in substrate specificity, tissue expression, and subcellular localization and are believed to channel fatty acids toward distinct metabolic pathways. We expressed rat ACS isoforms 1-5 in an E. coli strain that lacked FadD. All rat ACS isoforms were expressed in E. coli fadD or fadDfadR and had ACS specific activities that were 1.6-20-fold higher than the wild type control strain expressing FadD. In the fadD background, the rat ACS isoforms 1, 2, 3, 4 and 5 oxidized [(14)C]oleate at 5 to 25% of the wild type levels, but only ACS5 restored growth on oleate as the sole carbon source. To ensure that enzymes of beta-oxidation were not limiting, assays of ACS activity, beta-oxidation, fatty acid transport, and phospholipid synthesis were also examined in a fadD fadR strain, thereby eliminating FadR repression of the transporter FadL and the enzymes of beta-oxidation. In this strain, fatty acid transport levels were low but detectable for ACS1, 2, 3, and 4 and were nearly 50% of wild type levels for ACS5. Despite increases in beta-oxidation, only ACS5 transformants were able to grow on oleate. These studies show that although ACS isoforms 1-4 variably supported moderate transport activity, beta-oxidation, and phospholipid synthesis and although their in vitro specific activities were greater than that of chromosomally encoded FadD, they were unable to substitute functionally for FadD regarding growth. Thus, membrane composition and protein-protein interactions may be critical in reconstituting bacterial ACS function.     

Differentially localized acyl-CoA synthetase 4 isoenzymes mediate the metabolic channeling of fatty acids towards phosphatidylinositol

The acyl-CoA synthetase 4 (ACSL4) has been implicated in carcinogenesis and neuronal development. Acyl-CoA synthetases are essential enzymes of lipid metabolism, and ACSL4 is distinguished by its preference for arachidonic acid. Two human ACSL4 isoforms arising from differential splicing were analyzed by ectopic expression in COS cells. We found that the ACSL4_v1 variant localized to the inner side of the plasma membrane including microvilli, and was also present in the cytosol. ACSL4_v2 contains an additional N-terminal hydrophobic region; this isoform was located at the endoplasmic reticulum and on lipid droplets. A third isoform was designed de novo by appending a mitochondrial targeting signal. All three ACSL4 variants showed the same specific enzyme activity. Overexpression of the isoenzymes increased cellular uptake of arachidonate to the same degree, indicating that the metabolic trapping of fatty acids is independent of the subcellular localization. Remarkably, phospholipid metabolism was changed by ACSL4 expression. Labeling with arachidonate showed that the amount of newly synthesized phosphatidylinositol was increased by all three ACSL4 isoenzymes but not by ACSL1. This was dependent on the expression level and the localization of the ACSL4 isoform. We conclude that in our model system exogenous fatty acids are channeled preferentially towards phosphatidylinositol by ACSL4 overexpression. The differential localization of the endogenous isoenzymes may provide compartment specific precursors of this anionic phospholipid important for many signaling processes.     

Characterization of recombinant long-chain rat acyl-CoA synthetase isoforms 3 and 6: identification of a novel variant of isoform 6

The metabolism of long-chain fatty acids in brain and their incorporation into signaling molecules such as diacylglycerol and LPA and into structural components of membranes, including myelin, requires activation by long-chain acyl-CoA synthetase (ACSL). Because ACSL3 and ACSL6 are the predominant ACSL isoforms in brain, we cloned and characterized these isoforms from rat brain and identified a novel ACSL6 clone (ACSL6_v2). ACSL6_v2 and the previously reported ACSL6_v1 represent splice variants that include exon 13 or 14, respectively. Homologue sequences of both of these variants are present in the human and mouse databases. ACSL3, ACSL6_v1, and ACSL6_v2 with Flag-epitopes at the C-termini were expressed in Escherichia coli and purified on Flag-affinity columns. The three recombinant proteins were characterized. Compared to ACSL4, another brain isoform, ACSL3, ACSL6_v1, and ACSL6_v2 showed similarities in kinetic values for CoA, palmitate, and arachidonate, but their apparent Km values for oleate were 4- to 6-fold lower than for ACSL4. In a direct competition assay with palmitate, all the polyunsaturated fatty acids tested were strong competitors only for ACSL4 with IC50 values of 0.5 to 5 microM. DHA was also strongly preferred by ACSL6_v2. The apparent Km value for ATP of ACSL6_v1 was 8-fold higher than that of ACSL6_v2. ACSL3 and the two variants of ACSL6 were more resistant than ACSL4 to heat inactivation. Despite the high amino acid identity between ACSL3 and ACSL4, rosiglitazone inhibited only ACSL4. Triacsin C, an inhibitor of ACSL1 and ACSL4, also inhibited ACSL3, but did not inhibit the ACSL6 variants. These data further document important differences in the closely related ACSL isoforms and show that amino acid changes near the consensus nucleotide binding site alter function in the two splice variants of ACSL6.     

Positional cloning of a novel Fanconi anemia gene, FANCD2

Fanconi anemia (FA) is a genetic disease with birth defects, bone marrow failure, and cancer susceptibility. To date, genes for five of the seven known complementation groups have been cloned. Complementation group D is heterogeneous, consisting of two distinct genes, FANCD1 and FANCD2. Here we report the positional cloning of FANCD2. The gene consists of 44 exons, encodes a novel 1451 amino acid nuclear protein, and has two protein isoforms. Similar to other FA proteins, the FANCD2 protein has no known functional domains, but unlike other known FA genes, FANCD2 is highly conserved in A. thaliana, C. elegans, and Drosophila. Retroviral transduction of the cloned FANCD2 cDNA into FA-D2 cells resulted in functional complementation of MMC sensitivity.