cDNA Cloning, Genomic Structure, and Chromosomal Localization of Three Members of the Human Fatty Acid Desaturase Family

Genomics 66, 175–183 (2000) doi:10.1006/geno.2000.6196, available online at http://www.idealibrary.com on

cDNA Cloning, Genomic Structure, and Chromosomal Localization of Three Members of the Human Fatty Acid Desaturase Family Andreas Marquardt, Heidi Sto¨hr, Karen White, and Bernhard H. F. Weber 1 Institute of Human Genetics, University of Wu¨rzburg, 97074 Wu¨rzburg, Germany Received December 8, 1999; accepted March 7, 2000

The insertion of double bonds into specific positions of fatty acids is achieved by the action of distinct desaturase enzymes. Here we report the cloning and characterization of three members of the fatty acid desaturase (FADS) gene family in humans. Initially identified as cDNA fragments by direct cDNA selection within a defined 1.4-Mb region in 11q12– q13.1, fulllength fatty acid desaturase-1 (FADS1) and fatty acid desaturase-2 (FADS2) transcripts were obtained by EST sequence assembly. A third member, fatty acid desaturase-3 (FADS3), was identified in silico revealing 62 and 70% nucleotide sequence identity with FADS1 and FADS2, respectively. The three genes are clustered within 92 kb of genomic DNA located 2 kb telomeric to FEN1 and 50 kb centromeric to VMD2 and are likely to have arisen evolutionarily from gene duplication as they share a remarkably similar exon/ intron organization. Protein database searches identified FADS1, FADS2, and FADS3 as fusion products composed of an N-terminal cytochrome b5-like domain and a C-terminal multiple membrane-spanning desaturase portion. © 2000 Academic Press

INTRODUCTION

Unsaturated fatty acids are essential components of all biological systems. Their presence in cellular membranes greatly influences the dynamic properties of the lipid bilayer by modulating the fluidity and other chemical and physical characteristics of the membranes (Singer and Nicolson, 1972; Shinitzky, 1984; Stubbs and Smith, 1984). As major components of membrane phospholipids, unsaturated fatty acids modify the function of membrane-associated proteins involved in transport of substrates across the membrane and transmembrane communication (Stubbs Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession Nos. AF084558 (FADS1), AF084559 (FADS2), and AF084560 (FADS3). 1 To whom correspondence and reprint requests should be addressed at Institut fu¨r Humangenetik, Biozentrum, Am Hubland, D-97074 Wu¨rzburg, Germany. Telephone: (⫹)49-931-888-4062. Fax: (⫹)49-931-888-4069. E-mail: [email protected].

and Smith, 1984; Nishida and Murata, 1996). In addition to their vital role in membrane structure and function, unsaturated fatty acids are known to regulate gene transcription of proteins involved in lipid metabolism (Clarke et al., 1997) and to serve as precursors of biologically active molecules such as prostaglandins, thromboxanes, and leukotrienes (Cook, 1991). The availability of unsaturated fatty acids in the cell greatly depends on the activity of enzymes involved in fatty acid biosynthesis and metabolism. Importantly, the de novo unsaturation of fatty acids is regulated by individual desaturase enzymes that introduce double bonds between defined carbons of the fatty acyl chain (Jeffcoat and James, 1984). While desaturases are present in all living organisms, the repertoire of specific desaturase activities within bacteria, fungi, plants, and animals differs considerably. In contrast to higher animals that are capable of synthesizing double bonds only at the ⌬-9, ⌬-6, ⌬-5, and possibly ⌬-4 positions, insects and plants can insert additional double bonds at the ⌬-12 and ⌬-15 positions (for a review see Cook, 1991). As a consequence, fatty acids that contain unsaturated carbon bonds beyond the ⌬-9 position, such as linoleic acid and ␣-linolenic acid, need to be provided in the diet of higher animals (“essential fatty acids”). In animals, ⌬9-desaturase (e.g., Marzo et al., 1995), ⌬6-desaturase (e.g., Marzo et al., 1996), and ⌬5-desaturase (e.g., Rodriguez et al., 1998) activities have been well documented, whereas direct evidence for a distinct ⌬4-desaturase still remains elusive (Voss et al., 1991; Marzo et al., 1996). Thus far, cDNAs have been cloned for the ⌬9-desaturase, e.g., in rat (Thiede et al., 1986), mouse (Ntambi et al., 1988; Kaestner et al., 1989), and human (Zhang et al., 1999); the ⌬6-desaturase in Caenorhabditis elegans (Napier et al., 1998), rat (Aki et al., 1999), mouse, and human (Cho et al., 1999a); and the ⌬5-desaturase in C. elegans (Michaelson et al., 1998; Watts and Browse, 1999) and more recently in human (Cho et al., 1999b). Information on the genomic structure of these genes is confined to the ⌬5- and the ⌬6-desaturases in C. elegans (Watts and Browse, 1999)

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and the murine and human ⌬9-desaturases, which share a very similar organization consisting of six exons and five introns (Ntambi et al., 1988; Kaestner et al., 1989; Zhang et al., 1999). In addition, Zhang et al. (1999) mapped the human ⌬9-desaturase to chromosome 10 and described a transcriptionally inactive, fully processed pseudogene of the human ⌬9-desaturase mapping to chromosome 17. In an effort to establish a comprehensive transcript map spanning a 1.4-Mb region on chromosome 11q12– q13.1 (Sto¨hr et al., 1998), we identified a large number of partial cDNA sequences representing distinct genes. Here, we describe the further characterization of three transcripts including the cloning of the full-length cDNA sequences, the characterization of their exon/ intron organization, and the assessment of their expression profile in various human tissues. We provide strong evidence that we have cloned three members of the fatty acid desaturase family in humans and therefore, in accordance with the gene nomenclature guidelines, have designated the genes fatty acid desaturase-1 (FADS1), fatty acid desaturase-2 (FADS2), and fatty acid desaturase-3 (FADS3). MATERIALS AND METHODS cDNA cloning and genomic organization. The identification of directly selected cDNA contigs C97A-5, C97A-7, C97A-18/1, C97A18/2, and C97A-12 (GenBank Accession Nos. AF009755–AF009758 and AF009767) corresponding to FADS1 as well as clone C97A-8 (GenBank Accession No. AF009759) corresponding to FADS2 (Fig. 1) has been reported elsewhere (Sto¨hr et al., 1998). Starting with the cDNA contig sequences, consecutive nucleotide sequence homology searches in the dbEST database were performed (http://dot.imgen.bcm.tmc.edu:9331/seq-search/nucleic_acid-search.html). Only highquality EST sequences were assembled to generate contigs with overlapping EST clones revealing sequence identities at a level greater than 98%. To obtain the full-length cDNA of FADS2, the human retina cDNA library ␭gt10HRET (kindly provided by J. Nathans, University School of Medicine, Baltimore, MD) was screened with the [ 32P]dCTP-radiolabeled insert of IMAGE clone ym42c04 (GenBank Accession No. H17219). To extend the obtained sequence, 5⬘RACE was performed on reverse-transcribed human liver RNA using the 5⬘RACE kit, Version 2 (Gibco BRL). In a first-round PCR, the FADS2-specific reverse primer TUK8-F3 (5⬘-AAA TAC ATG GGG ATG AGC AG-3⬘) was used followed by two additional steps of nested PCR amplifications using reverse primers TU13-F5 (5⬘-AGT ATT CGT GCT GGT GAT TG-3⬘) and TU13-F4 (5⬘-TGT AGG GCA GGT ATT TCA GC-3⬘). The resulting PCR products were directly sequenced using the Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit (Amersham–USB). To verify the correct assembly of the cDNAs (Fig. 2A) and to confirm the nucleotide sequences, oligonucleotide primers were designed to PCR amplify the entire coding sequences of FADS1, FADS2, and FADS3 from first-strand cDNA prepared from human liver RNA. The primer sequences were as follows: FADS1, TU12Ex1-F2/TU12-F5 (5⬘-CGT CGC CAG GCC AGC TAT G-3⬘/5⬘-CTC AAG CTC CCC TCT GCC T-3⬘); FADS2, FADS2-F/TU13-1F (5⬘-CGT CAC AGT CGG CAG GCA GC-3⬘/5⬘-CCT CAG AAC AAA AGC CCA TC-3⬘); FADS3, TU19-Ex1-F/TU19-F (5⬘-AGG ACT CGT GCG TGC AGC AT-3⬘/5⬘-TGG TTG CTG GTG CCC TGA G-3⬘). Nucleotide sequence databases were searched at http://dot.imgen. bcm.tmc.edu:9331/seq-search/nucleic_acid-search.html to identify genomic sequences corresponding to FADS1, FADS2, and FADS3 cDNAs. To assign consensus donor and acceptor splice recognition

sequences, assembled cDNAs and genomic sequences were aligned using MacVector Version 4.1.4 (Kodak, New Haven, CT). Computational amino acid sequence analyses. The conceptual amino acid sequences of FADS1, FADS2, and FADS3 were compared to known protein sequences deposited in public databases at http:// dot.imgen.bcm.tmc.edu:9331/seq-search/protein-search.html. Multiple amino acid sequence alignments were performed using the MultAlin program (http://www.toulouse.inra.fr/multalin.html) and shaded by version 3.2.1 of BOXSHADE (http://www.isrec.isb-sib.ch/ software/BOX_form.html). Protein motif analyses were carried out with TMpred (http://dot.imgen.bcm.tmc.edu:9331/seq-search/ struc-predict.html), ProDom (http://protein.toulouse.inra.fr/prodom/ prodom.html), and ProSite algorithms (http://dot.imgen.bcm. tmc.edu:9331/seq-search/protein-search.html). TREECON for Windows version 1.3b (http:⶿⶿bioc-www.uia.ac.be/u/yvdp/index.html) was utilized to assemble a phylogenetic tree based on the amino acid sequences of selected desaturases. Northern blot analysis. Total RNA from heart, liver, lung, uterus, and brainstem was isolated according to the guanidinium thiocyanate–phenol– chloroform extraction protocol (Chomczynski and Sacchi, 1987). RNAs were separated on a 1.2% agarose gel in the presence of 3-(N-morpholino)propanesulfonic acid (MOPS) and formaldehyde (Sambrook et al., 1989). The RNAs were then transferred to a Hybond-N⫹ membrane on a vacuum blotter (VacuGene XL, Pharmacia) and used for filter hybridization at 65°C in 0.5 mM sodium phosphate buffer, pH 7.2, 7% SDS, 1 mM EDTA. The hybridization probe was PCR amplified from the 3⬘-UTR of FADS3 with primers F3 (5⬘-GGC CTC AGC TAC GAA GTG AAG-3⬘) and R (5⬘-ACA GCT TTC CCC CAA TTC TC-3⬘). To test for equal loading and RNA integrity, the Northern blots were subsequently probed with human ␤-actin.

RESULTS

Assembly of FADS1, FADS2, and FADS3 cDNAs Repeated database searches were initiated with sequences from six cDNA contigs isolated by retina/RPEspecific direct cDNA selection (Sto¨hr et al., 1998) (Fig. 1). A large number of EST sequences were retrieved, of which 155 ESTs were clustered into a 4205-bp transcript representing the complete FADS1 cDNA (GenBank Accession No. AF084558). It contains an open reading frame (ORF) of 1410 bp starting 79 nucleotides downstream of the 5⬘-end of EST clone AA029030. This positions the tissue-selected cDNA contigs C97A-5, C97A-7, C97A-18/1, C97A-18/2, and C97A-12 to various regions of the 3⬘-UTR of FADS1 (Fig. 1). Extending the sequence of the tissue-selected cDNA fragment C97A-8 (Sto¨hr et al., 1998), an additional 76 EST clones were assembled into two nonoverlapping contigs with a gap between EST clones AI815395 and H17219 (Fig. 1). To link the two contigs, the retinal cDNA library ␭gt10HRET was screened with radiolabeled EST fragment H17219. This resulted in the identification of clone F2-␭gt10HRET, providing an extension of about 250 bp in the 5⬘-direction. The closure of the gap was eventually accomplished with PCR fragment AUAP-F4, generated by 5⬘-RACE with nested primers derived from the F2-␭gt10HRET sequence. The assembled 3149-bp FADS2 cDNA (GenBank Accession No. AF084559) contains an ORF of 1485 bp starting 151 bp downstream of the 5⬘-end and places the directly selected cDNA contig C97A-8 into the 3⬘UTR of this gene (Fig. 1).

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FIG. 1. Assembly of FADS1, FADS2, and FADS3 cDNAs. Thick lines represent cDNA fragments isolated by the cDNA selection technique (Sto¨hr et al., 1998). These cDNA fragments represent segments corresponding to the 3⬘-UTRs of FADS1 (GenBank Accession Nos. AF009755–AF009758, AF009767) and FADS2 (GenBank Accession No. AF009759). Based on the selected cDNAs, repeated dbEST database searches facilitated the assembly of a minimal set of EST clones (thin lines) covering the complete FADS cDNAs. cDNA clones F2-␭gt10HRET and AUAP-F4 were isolated by cDNA library screening and 5⬘-RACE experiments, respectively (medium lines). The first nucleotide positions of the start (ATG) and stop (TGA) codons as well as of the polyadenylation signals are given in parentheses. Also shown are the products and positions of RT-PCR amplified cDNAs spanning the entire coding sequences of FADS1, FADS2, and FADS3.

Sixty-two human ESTs demonstrated a high level of sequence similarity but could not be assigned to either FADS1 or FADS2. This suggested the presence of at least one additional member of the FADS gene family. Subsequent assembly of the EST sequences was achieved with matches of overlapping segments greater than 98%. This resulted in a third distinct transcript, designated FADS3 (GenBank Accession No. AF084560), with a 1757-bp cDNA sequence and an ORF of 1468 bp (Fig. 1). All three transcripts contain the first potential inframe translation initiation codon, ATG, in a sequence context meeting the criteria of the Kozak consensus sequence for translation initiation (Kozak, 1996). Further computational analysis of the sequence revealed potential polyadenylation signals in the 3⬘-UTRs of all three genes. In addition, the 3⬘-UTR of FADS1 contains repetitive elements of the SINE/Alu class of repeats (AluSg/x at nucleotide position ⫹1702 to ⫹1869; AluJb at nucleotide position ⫹3394 to ⫹3566). The assembled ORFs of all three genes were verified by RT-PCR amplification (Fig. 2A) and subsequent sequencing.

The putative coding sequences of the three genes share a high degree of nucleotide sequence identity, ranging from 62 to 70%. Homology searches with the assembled full-length cDNA sequences of FADS1, FADS2, and FADS3 failed to identify additional unassigned human EST clones, making the existence of additional, closely related, family members unlikely. Expression Analysis of FADS1, FADS2, and FADS3 The expression pattern of FADS1 and FADS2 has previously been reported as that of TU12 (FADS1) and TU13 (FADS2) (Sto¨hr et al., 1998). By Northern blot analysis, single transcripts of approximately 4.0 kb (FADS1) and 3.1 kb (FADS2) in length were detected in lung, cerebellum, retina, and the RPE cell line ARPE-19 with almost equal abundance. Here, we performed Northern blot analysis of FADS3 with probe F3-R derived from the 3⬘-UTR of the gene and identified two transcripts of 1.8 and 1.2 kb in heart, liver, lung, uterus, and brainstem (Fig. 2B). While the larger transcript corresponds well to the length of the assembled FADS3 cDNA, the presence of the smaller tran-

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FIG. 2. Analysis of transcription. (A) Amplification, as well as subsequent sequencing, of the entire coding sequences of FADS1, FADS2, and FADS3 from human liver RNA verifies the correct cDNA assembly of the three genes. Note that no differentially spliced products are evident in the three desaturase transcripts. (B) Northern blot analysis of FADS3 in various human tissues including heart, liver, lung, uterus, and brainstem with a probe derived from the 3⬘-UTR of the gene. Two transcripts of 1.8 and 1.2 kb are present in all tissues with the smaller transcript in greater abundance in heart, liver, and brainstem. The nature of the 1.2-kb transcript remains unknown.

script is currently unaccounted for and requires further investigation. It should be noted that RT-PCR amplification of the entire ORF of FADS3 does not reveal any evidence of an alternatively spliced transcript (Fig. 2A). A survey of the 293 EST sequences used to assemble the FADS1, FADS2, and FADS3 transcripts showed that for each of the three genes, the EST clones were derived from a large number of different cDNA libraries including brain, heart, testis, kidney, prostate, ovary, breast, adipose tissue, melanocytes, colon, and others. Interestingly, a high proportion of EST clones representing FADS1, FADS2, and FADS3 were derived from human brain, suggesting a high level of expression of the three genes in neuronal tissue. Refined Localization and Genomic Organization of FADS1, FADS2, and FADS3 Querying the nucleotide sequence database with full-length FADS1, FADS2, and FADS3 cDNAs identified significant matches with genomic sequences within PAC clone dJ466a11 (GenBank Accession No. AC003025) and BAC clone CIT-HSP-311e8 (GenBank Accession No. AC004770). As well as further confirming our transcript assembly, the alignment of the cDNA sequences with genomic DNA localizes the three genes in a well-characterized chromosome region on 11q12– q13.1. Both clones are part of the highly redundant 1.4-Mb PAC/BAC contig used to isolate partial cDNA fragments of FADS1 and FADS2 (Cooper et al., 1997; Sto¨hr et al., 1998). The assembly of overlapping genomic sequences of dJ466a11 and 311e8 results in 290 kb of contiguous genomic DNA sequence contain-

ing the gene loci FEN1 and FTH1, which anchor the genomic sequence within the genetic framework map (Fig. 3A). Within this region on chromosome 11q12– q13.1, FADS1 is located 2.4 kb telomeric to and in tail-to-tail orientation with the flap endonuclease FEN1 gene (Fig. 3A). Downstream of the FADS1 locus, FADS2 follows head-to-head at a distance of 11.3 kb. The third family member, FADS3, is located tail-to-tail at a distance of 6.0 kb telomeric to FADS2. Toward the long arm telomere, the VMD2 gene locus follows at a distance of approximately 55 kb. Alignment of the cDNAs to the genomic sequences enabled us to determine the genomic exon/intron organization of the three FADS genes. They consist of 12 exons each and span 17.2 kb (FADS1), 39.1 kb (FADS2), and 17.9 kb (FADS3) of genomic DNA (Fig. 3B). While the lengths of the intervening sequences of the FADS genes vary considerably, the exon/intron structure is remarkably similar. In each gene, exons 2– 4, exons 6 –11, and the coding part of the last exon 12 are identical in length, while exons 1 and 5 differ in length by only three and six nucleotides, respectively. Moreover, the exon/intron boundaries disrupt the coding sequence at identical nucleotide positions within highly conserved codons in every exon of each of the three genes. The splice acceptor and donor sequences for each gene are summarized in Tables 1 to 3. Computational Analysis of FADS1, FADS2, and FADS3 The conceptual translation products of both the FADS1 and the FADS2 transcripts code for 444-aminoacid peptides with estimated molecular masses of 52.0 and 52.3 kDa, respectively. The putative coding region of FADS3 encodes 445 amino acids, resulting in a calculated molecular mass of 51.1 kDa. Alignment of the putative polypeptide sequences exhibits a high degree of sequence identity between FADS1 and FADS2 (61%), FADS1 and FADS3 (52%), and FADS2 and FADS3 (62%) (Fig. 4). Amino acid sequence comparisons of FADS1, FADS2, and FADS3 to known proteins in the database indicate significant homology to the desaturase enzymes. In particular, three histidine motifs, HDxGH, HFQHH, and QIEHH, characteristic of membranebound desaturases (Los and Murata, 1998; Shanklin et al., 1994), are shared by FADS1, FADS2, and FADS3 (Fig. 4). Up to four transmembrane domains are predicted for FADS1, FADS2, and FADS3, strongly supporting a membrane association of these proteins. Furthermore, FADS1, FADS2, and FADS3 contain an N-terminal cytochrome b5-like domain, which is a common structural feature in a subset of desaturases from plants, animals, and yeast (Dailey and Strittmatter, 1980; Mitchell and Martin, 1995; Sayanova et al., 1997; Napier et al., 1998). All cytochrome b5-like domains contain the highly conserved sequence motif “HPGG,”

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FIG. 3. Chromosomal localization and genomic organization of FADS1, FADS2 and FADS3. (A) The three genes are clustered within a 92-kb region of 290 kb of sequenced genomic DNA (dJ466a11, CIT-HSP-311e8) in chromosomal band 11q12– q13.1 and are flanked on the centromeric side by FEN1 (flap endonuclease-1) and on the telomeric side by the genes VMD2 (vitelliform macular dystrophy-2) and FTH1 (ferritin heavy chain-1). The sizes of the genomic loci of the genes are given in kilobase pairs below the respective gene symbols. (B) Exons of FADS1, FADS2, and FADS3 are indicated by vertical bars. The 5⬘- and 3⬘-untranslated regions are symbolized by gray bars. The exonic and intronic sequences of FADS1, FADS2, and FADS3 are drawn to scale.

which is also present in FADS1 (at amino acid positions 52–55), FADS2 (53–56), and FADS3 (55–58) (Fig. 4). DISCUSSION

Within human chromosomal band 11q12– q13.1 we have cloned and characterized three genes encoding putative membrane-anchored proteins with several features characteristic of the family of fatty acid desaturases. FADS1, FADS2, and FADS3 can be assigned to the class of cytochrome b5 fusion proteins (Napier et al., 1997). Their conceptual translation products consist of an N-terminal heme-binding domain highly homologous to the electron carrier cytochrome b 5 and a C-terminal desaturase portion. Specifically, the heme-binding histidine residues in

cytochrome b 5, His 44 and His 68, are at the same relative positions in the newly identified desaturases. Moreover, the His 44 residue is embedded in a highly conserved motif, HPGG, predicted to facilitate accessibility to heme iron (Lederer, 1994). In addition, the desaturase domains of FADS1, FADS2, and FADS3 share three histidine-rich regions that are generally found in plant and animal desaturases and are thought to bind nonheme iron required for enzymatic activity (Shanklin et al., 1994). A phylogenetic tree was constructed on the basis of sequence alignments between the predicted protein sequences of FADS1, FADS2, and FADS3 and 22 selected desaturases with known enzymatic activity from cyanobacteria, fungi, plants, and animals (Fig. 5). The topology of the tree reflects an overall sequence identity of 22–27% between the human FADS proteins and

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Size Exon (bp) 1 2 3 4 5 6 7 8 9 10 11 12

282 a 111 198 102 129 61 77 98 97 80 126 2819 b

TABLE 1

TABLE 3

Exon/Intron Boundaries of FADS1

Exon/Intron Boundaries of FADS3

5⬘ Splice donor

Intron size (bp)

..TGCCACGgtgagcg.. ..CAAGAATgtaagac.. ..AGTTCAGgtgagag.. ..CCTGAAGgtcagtg.. ..TGTGGAGgtgcgtg.. ..TTCCTAAgtgagtg.. ..GTGGGTGgtgagta.. ..TAGTCAGgtagtat.. ..CACCCAGgtaaggg.. ..AGCACCAgtgagta.. ..TCATCCAgtgagta..

3202 574 1395 94 4026 1902 917 210 251 105 338

3⬘ Splice acceptor

..cttgcagGATCCCT.. ..tctgcagAAAGAGC.. ..cacctagGCCCAGG.. ..tctgtagGGGGCCC.. ..ttcacagCTTGGGA.. ..tccccagTTGGGCC.. ..actatagGACTTGG.. ..ctaacagGTTCCTG.. ..ccgccagCTCCAGG.. ..cctaaagTCTTTTT.. ..cctgcagCTCACTA..

a Size of the exon refers to the most 5⬘ cDNA sequence of zk09h08.r1 (AA029030) and includes a 5⬘-UTR of 78 bp. b The exon includes a 3⬘-UTR of 2792 bp.

the various ⌬5, ⌬6, and ⌬8 desaturases. The ⌬9 desaturases from a wide range of plant and animal species are more similar to one another than to the known ⌬5, ⌬6, and ⌬8 desaturases or to the FADS desaturases, thus forming a more distinct group within the desaturase family (Fig. 5). Consistent with these data is the finding that the recently cloned human ⌬9 desaturase (Zhang et al., 1999) does not show significant amino acid identity to any of the three human FADS proteins, suggesting that none of the desaturases identified in this study should possess ⌬9 desaturase activity. While the isolation of the FADS transcripts and their genomic characterization were in progress, Cho et al. (1999a) reported the cloning of human and mouse ⌬6 desaturase transcripts and subsequently the cloning of a human ⌬5 desaturase cDNA (Cho et al., 1999b). Comparison of the human ⌬6 desaturase nucleotide sequence to FADS2 showed that the two transcripts TABLE 2 Exon/Intron Boundaries of FADS2 Size Exon (bp) 1 2 3 4 5 6 7 8 9 10 11 12

357 a 111 198 102 126 61 77 98 97 80 126 1698 b

3⬘ Splice acceptor

..tctccagGATGCCT.. ..tcaacagTCAAAGA.. ..tccccagGCCCAAG.. ..cccacagGGTGCCT.. ..tcctcagTACGGCA.. ..cctgcagTTGGGCC.. ..cccgcagGACCTGG.. ..cttgcagGTTCCTG.. ..ctgacagCTGACAG.. ..tcctcagCCTCTTC.. ..tttgcagGTCCCTG..

5⬘ Splice donor

Intron size (bp)

..TGCAACGgtaaggg.. ..CAAGAACgtaagtc.. ..CTCTCAGgtgaggc.. ..CTTAAAGgtaagtg.. ..CATCGAGgtacgac.. ..TTCCTGAgtgagtg.. ..CTGGGTGgtgagtt.. ..TCATCAGgtgcctg.. ..TAGCCAGgtaggga.. ..AGCACCAgtgagcg.. ..TCATCAGgtgaggg..

9158 2445 92 7432 8730 382 5451 212 328 1365 360

a Size of the exon refers to the most 5⬘ cDNA sequence of au42b11.y1 (AI815395) and includes a 5⬘-UTR of 150 bp. b The exon includes a 3⬘-UTR of 1664 bp.

Size Exon (bp) 1 2 3 4 5 6 7 8 9 10 11 12

346 a 111 198 102 123 61 77 98 97 80 126 325 b

3⬘ Splice acceptor

..ccttcagGATGCCT.. ..atggcagGCGCAGC.. ..tccccagGCTCAGT.. ..tccacagGGCTTCT.. ..tccacagTATGGCA.. ..cttgcagTCGGCCC.. ..tctgcagGATTTGC.. ..cccacagGGTCCTG.. ..ccggcagCTGGCAG.. ..cccccagCCTCTTC.. ..ctcccagGTCCCTG..

5⬘ Splice donor

Intron size (bp)

..CGCCACGgtaagga.. 11,015 ..CCTGAATgtgagcc.. 531 ..CTCTCAGgtgaccc.. 464 ..GCTAAAGgtgaggg.. 111 ..CGTCGAGgtgggtg.. 296 ..TTCCTGAgtgagtg.. 567 ..GTGGGCGgtgagtg.. 547 ..CTGTCAGgtatggc.. 410 ..CTCTCAGgtgggca.. 168 ..AGCACCAgtgagtg.. 134 ..TCGTCAGgtgaggc.. 1,977

a Size of the exon refers to the most 5⬘ cDNA sequence of tg24a08.x1 (AI394672) and includes a 5⬘-UTR of 133 bp. b The exon includes a 3⬘-UTR of 286 bp.

are identical except that the assembled FADS2 cDNA extends approximately 140 bp further upstream into the 5⬘-UTR. Consequently, alignments of amino acid sequences of FADS2 with cloned ⌬6 desaturases from mouse (Cho et al., 1999a) and rat (Aki et al., 1999) reveal strong evolutionary conservation with sequence identities of up to 90% (Figs. 4 and 5). Further comparison demonstrated that the human ⌬5 desaturase and FADS1 share identical nucleotide sequences in the ORF with the exception of six alterations, three of which result in amino acid discrepancy (V6L, P15L, and K255N). These could represent polymorphic changes or be the result of previous sequencing errors. Taken together, these findings provide evidence that we have characterized the human ⌬5- and ⌬6-desaturases. Interestingly, FADS3 shares sequence identities with FADS1 and FADS2 of only 52 and 62%, respectively, and is unlikely to represent a second human ⌬5 or ⌬6 desaturase. Currently, cell line expression assays are in progress to assess the specific desaturase activity of FADS3. In C. elegans two functional ⌬5- and ⌬6-desaturases were identified and found to share an amino acid identity of 45% (Napier et al., 1998; Michaelson et al., 1998; Watts and Browse, 1999). Similar to the human FADS gene organization described here, they are located in a headto-tail fashion within a kilobase of each other on chromosome IV. In human, the FADS gene cluster has likely arisen during evolution through gene duplication as the exon organization is nearly identical in the three family members with each gene consisting of 12 exons and splice donor and acceptor sites interrupted at identical nucleotide positions within highly conserved codons. In recent years, there has been increasing interest in the role of polyunsaturated fatty acids in the pathophysiology of a number of chronic conditions such as coronary and peripheral vascular disease (Horrobin, 1995), acute and chronic inflammatory immune re-

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FIG. 4. Alignment of the deduced amino acid sequences from FADS1, FADS2, and FADS3 to ⌬-6-desaturases from mouse (GenBank Accession No. AF126798), rat (GenBank Accession No. AB021980), and C. elegans (GenBank Accession No. AF031477) as well as the ⌬5-desaturase from C. elegans (GenBank Accession No. AF078796) and the human cytochrome b5 (GenBank Accession No. M22865). Identical amino acids are highlighted in black while structurally and/or functionally similar amino acids are shaded. Gaps were introduced to maximize alignment and are indicated by a dot. Double-headed arrows indicate the four predicted transmembrane domains in FADS1, FADS2, and FADS3. The two highly conserved histidine positions in the N-terminal cytochrome b5 domain are indicated by open arrows. The “HPGG” motif, characteristic of cytochrome b5 and cytochrome b5-fusion proteins, is boxed while three conserved histidine motifs (HDXGH, HFQHH, and QIEHH) are represented by shaded boxes.

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FIG. 5. Phylogenetic relationship between FADS1, FADS2, FADS3 and the putative amino acid sequences of fatty acid desaturases with defined functional activities from various species. The amino acid sequences were derived from coding sequences deposited with GenBank under the following accession numbers (in parentheses): human ⌬5- (AF199596), ⌬6- (AF126799), and ⌬9- (Y13647) desaturases; ⌬6- and ⌬9-desaturases of Mus musculus (AF126798; M26270), ⌬6- and ⌬9-desaturases of Rattus norvegicus (AB021980; J02585); ⌬6- and ⌬9desaturases of Spirulina platensis (X87094; AJ002065); ⌬5- and ⌬9-desaturases of Mortierella alpina (AF067654; AB015611); ⌬6- and ⌬5-desaturases of Caenorhabditis elegans (AF031477; AF078796); ⌬8- and ⌬9-desaturases of Arabidopsis thaliana (AJ224161; D88537); ⌬6-desaturases of Physcomitrella patens (AJ222980), Borago officinalis (U79010), and Synechocystis (U38467); ⌬5-desaturase of Dictyostelium discoideum (AB022097); ⌬8-desaturase of Brassica napus (AJ224160); ⌬9-desaturases of Ovis aries (AJ001048.1), Sus scrofa (Z97186), Saccharomyces cerevisiae (J05676), and Tetrahymena thermophila (D83478).

sponses (Calder, 1998; Fan and Chapkin, 1998; Grimble and Tappia, 1998), cutaneous abnormalities (Horrobin, 1989; Grattan et al., 1990), essential hypertension (Russo et al., 1997; Chi and Gupta, 1998), diabetes mellitus (Mori et al., 1997), asthma (Leichsenring et al., 1995; Villani et al., 1998; Hodge et al., 1998), and rheumatoid arthritis (James and Cleland, 1997; ArizaAriza et al., 1998; Grimble and Tappia, 1998). To better understand lipid-related function in human health and disease, additional research into fatty acid biosynthesis and metabolism is required. In particular, we need to understand the pharmacological properties, the mechanisms of action, and the tissue-specific regulation of the composition of the polyunsaturated fatty acids and their metabolites. This will provide additional insight into the role of the polyunsaturated fatty acids in various chronic disease states and will make it feasible to focus pharmacogenomic research on drug design and evaluation. As a prerequisite, the genes and their gene products involved in the above-mentioned processes need to be identified and characterized. With the cloning of three members of the human desaturase gene family a first step toward this goal has been accomplished. ACKNOWLEDGMENT This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG) (We1259/2-5 and We1259/13-1).

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