Hybond

Poly(A) RNA was extracted from individual oocytes using Hybondmessenger affinity paper (Hybond-mAP; Amersham Pharmacia Biotech, Piscataway, NJ). Oocytes were incubated with a 3-to 4-mm 2 piece of Hybond-mAP for 2 h in guanidium isothiocyanate (GITC) lysis solution (4 M GITC, 0.1 M Tris-HCl, pH 7.4, 1 M beta-mercaptoethanol; all in DEPC-treated water). After incubation, the Hybond-mAP was placed on Whatman filter paper (Fischer Scientific, St. Louis, MO), and the aqueous contents of the vials was carefully spotted onto the membrane. The Hybond-mAP was then washed twice in 0.5 M NaCl plus 0.1 M Tris-HCl, pH 7.4, in DEPC-treated water, twice in 0.5 M NaCl in DEPC-treated water, and twice in 70% ethanol. The Hybond-mAP was then allowed to air dry for a few minutes and immediately used for reverse transcription (RT).
Because mammalian trp is expressed at high levels in ovarian tissues [19, 20] , total RNA was isolated from porcine ovaries to be used as a positive control for RT polymerase chain reaction (PCR). Ovaries were flash frozen in liquid nitrogen immediately after removal and stored at Ϫ70ЊC until processed. For RNA isolation, ovaries were removed from the liquid nitrogen, placed into 20 ml lysis buffer (STAT-60; Tel-Test, Inc., Friendswood, TX), and homogenized using a rotor-stator homogenizer. An additional 20 ml of lysis buffer was added to the homogenate followed by a 1/10 volume of bromo-chloro-propane. The mixture was then shaken vigorously for 30 sec and allowed to sit for 2-3 min. Following centrifugation at 10 000 ϫ g for 15 min, the supernatant was collected into a new tube and the RNA was precipitated by adding an equal volume of ice-cold isopropyl alcohol. The tube was shaken gently, stored at room temperature for 5 min, and centrifuged at 10 000 ϫ g for 15 min. The isopropyl alcohol was then poured off, the pellet was washed in ice-cold 80% ethanol, and the RNA was aliquoted in DEPC-treated water with 5 l/ml RNasin. Aliquots were stored at Ϫ70ЊC until use.

Bacterial Strain, Culture Conditions, and Preparation of Cell-free Extracts-A. brasilense ATCC29145 was cultivated aerobically for 48 h at 30 °C in a synthetic medium containing 37 mM L-arabinose as described previously (12) . The grown cells were harvested by centrifugation at 30,000 ϫ g for 20 min, washed with 20 mM potassium phosphate buffer, pH 7.5, containing 1 mM EDTA and 10 mM 2-mercaptoethanol (referred to as Buffer A), and stored at Ϫ35 °C until use. The washed cells were suspended in Buffer A, disrupted by sonication for 20 min with appropriate intervals on ice using ASTRASON Ultrasonic Liquid Processor XL2020 (Misonix Inc., New York), and then centrifuged at 108,000 ϫ g for 20 min at 4 °C to obtain the cell-free extracts.
PAGE-SDS-PAGE was performed as described by Laemmli (17) . Nondenaturing PAGE was performed by omitting SDS and 2-mercaptoethanol from the solution used in SDS-PAGE. Proteins on the gel were stained with Coomassie Brilliant Blue R-250 and destained with 7.5% (v/v) acetic acid in 25% methanol. Enzyme Activity Assay-␣KGSA dehydrogenase activity was assayed routinely in the direction of aldehyde oxidation by measuring the reduction of NAD(P) ϩ at 340 nm at 25 °C using a Jasco spectrophotometer model V-550 (Japan Spectroscopic Co., Ltd., Tokyo, Japan). The standard assay mixture contained appropriate aldehyde, 1 mM EDTA, and 1 mM 2-mercaptoethanol in 66.7 mM potassium phosphate buffer (pH 7.2). The reaction was started by the addition of 15 mM NAD(P) ϩ solution (100 l) with a final reaction volume of 1 ml. ␣KGSA was synthesized from potassium arabonate by sequential reactions with L-ar- abonate dehydratase and L-KDA dehydratase of A. brasilense. 3Potassium arabonate was prepared by the hypoiodite-in-methanol oxidization of L-arabinose (18) . The kinetic parameters, K m and k cat values, were calculated by Lineweaver-Burk plot. Protein concentrations were determined by the method of Lowry et al. (19) with bovine serum albumin as the standard.
Zymogram Staining Analysis-Cell-free extract or purified enzyme was separated on nondenaturing PAGE with 6% gel at 4 °C. The gels were then soaked in 10 ml of staining solution (20) consisting of 50 mM HEPES, pH 7.2, appropriate aldehyde, 0.25 mM nitroblue tetrazolium, 0.06 mM phenazine methosulfate, and 1.5 mM NAD(P) ϩ at room temperature for 15 min. Dehydrogenase activity appeared as a dark band.
Purification of ␣KGSA Dehydrogenase from A. brasilense-Ten mM glutaraldehyde was used as an aldehyde substrate for the purification of ␣KGSA dehydrogenase from A. brasilense cells. All purification steps were performed below 4 °C. The cell-free extract was loaded onto a column of HiPrep 16/10 Q FF (1.6 ϫ 10 cm; Amersham Biosciences) equilibrated with Buffer A and washed thoroughly with the same buffer. The column was developed with 300 ml of a linear gradient of 0 -1 M NaCl in Buffer A. The active fractions containing ␣KGSA dehydrogenase were combined and dialyzed against an excess volume of 5 mM potassium phosphate, pH 7.5, containing 1 mM EDTA and 10 mM 2-mercaptoethanol (referred to as Buffer B) overnight. The enzyme solution was applied to a column of ceramic hydroxyapatite type I (1.6 ϫ 5 cm; Bio-Rad), equilibrated with Buffer B, and washed thoroughly with the same buffer. The column was developed with 300 ml of a linear gradient of 5-200 mM potassium phosphate in Buffer B. The fractions with high enzymatic activity were combined and concentrated by ultrafiltration with Centriplus YM-30 (Millipore) at 18,000 ϫ g for ϳ2 h. The enzyme solution was loaded onto a column of HiLoad 16/60 Superdex 200 pg (1.6 ϫ 60 cm; Amersham Biosciences) equilibrated with Buffer A. The active fractions were pooled, concentrated, and dialyzed against an excess volume of Buffer A containing 1.3 M (NH 4 ) 2 SO 4 overnight. The solution was applied to a HiPrep 16/10 Butyl FF column (1.6 ϫ 10 cm; Amersham Biosciences) equilibrated with the dialyzing buffer and washed with the same buffer. Proteins were eluted using a reversed linear gradient of 1.3-0 M (NH 4 ) 2 SO 4 in Buffer A (300 ml). The active fractions containing ␣KGSA dehydrogenase were combined, concentrated, and dialyzed against Buffer C (20 mM potassium phosphate, pH 7.5, containing 1 mM EDTA, 1 mM dithiothreitol, and 50% (v/v) glycerol), and stored at Ϫ35 °C until use (referred to as sample I).
Sample I (100 g of protein) was run by native PAGE with 6% (w/v) gel. The gel was then stained in a zymogram staining solution containing 10 mM glutaraldehyde. The active band that appeared in the staining was cut off and broken in 20 mM Tris-HCl (pH 8.0) containing 1% (w/v) SDS. The protein was extracted from the gels by vigorous mixing overnight. A 4-fold volume of acetone chilled at Ϫ35 °C was then added to the extracts. After cooled for 1 h at Ϫ80 °C, the mixture was centrifuged at 39,120 ϫ g for 15 min at 4 °C (referred to as sample II). The sample was dissolved in a small volume of SDS-PAGE sample buffer (500 mM Tris-HCl (pH 6.8) containing 5% (w/v) SDS, 10% (v/v) glycerol, 0.25% (w/v) bromphenol blue, and 5% (v/v) 2-mercaptoethanol) and separated by SDS-PAGE with 10% (w/v) gel. This gave an enzyme preparation with a single band, which corresponded to a polypeptide with a subunit molecular mass of ϳ55-kDa in sample I (band with a black triangle in Fig. 3A).
Determination of N-terminal and Internal Amino Acid Sequences-To determine the N-terminal amino acid sequence of ␣KGSA dehydrogenase, sample II (ϳ50 g) was separated by SDS-PAGE with 10% (w/v) gel and then transferred to a Hybond TM -P (Amersham Biosciences) at 3 mA/cm 2 for 0.5 h in a transfer buffer (10 mM CAPS-NaOH buffer, pH 11, containing 10% (v/v) methanol) with a horizontal electrophoretic blotting system (model AE-7500, Atto Instruments). After staining and destaining the protein, an area of the membrane corresponding to the 55-kDa protein band was excised and analyzed with a Procise TM 492 HT protein sequencer (Applied Biosystems). Chemical digestion with CNBr was carried out to determine internal amino acid sequences (21) . Sample II (100 g) was digested chemically at room temperature in 70% (v/v) formic acid containing 1% (w/v) CNBr (100 l) in the dark and under N 2 over- night. The solution was diluted with 900 l of deionized water, frozen with liquid N 2 , and lyophilized. The sample was dissolved in SDS-PAGE sample buffer and separated by SDS-PAGE with 18% (w/v) gel. Peptide fragments on the gel were transferred to a Hybond TM -P membrane as described above. After staining and destaining, areas of the membrane corresponding to the four major peptide fragments from sample II were excised and sequenced.
Cloning of ␣KGSA Dehydrogenase Gene-As described under "Results," the 5Ј-and 3Ј-terminal DNA sequences of a putative ALDH gene of Burkholderia cepacia strain R18194 (GenBank TM accession No. ZP_00215432) were used to design genomic PCR primers for amplification of a DNA fragment of the ␣KGSA dehydrogenase gene as follows: KGSA U1 , 5Ј-ATGGCTA- ACGTGACTTATACGGATACGCAACTGCTGATCGACGG-3Ј; and KGSA D1 , 5Ј-TCAGACGGCCATCACCGTCACCGACT-TCGTGACGAGGTACGG-3Ј. A. brasilense genomic DNA was prepared using a DNeasy TM tissue kit (Qiagen). PCR was carried out using a PCR Thermal Cycler PERSONAL (Takara) for 30 cycles in 50 l of reaction mixture containing 10 pmol of primers, 1.25 unit of Ex Taq DNA polymerase (Takara), and 300 ng of A. brasilense genomic DNA under the following conditions: denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, and extension at 72 °C for 2 min, each for 30 cycles. A PCR single product with a length of ϳ1.5-kbp was purified, cloned into a pGEM-T vector (referred to as pGEM3 in Fig. 2), and sequenced using a Dual CyDye TM terminator sequencing kit (Veritas) and appropriate primers with Long-Read Tower, UBC DNA sequencer (Amersham Biosciences). For Southern hybridization, ϳ1.8 g of A. brasilense genomic DNA was digested with appropriate restriction enzymes, separated on 1% (w/v) agarose gel, and transferred to Hybond TM -N (Amersham Biosciences). Probe DNA, amplified by PCR with AraE-UP1 and AraE-DOWN1 primers and pGEM3 as a template, was labeled with digoxigenin-11-dUTP and hybridized using a DIG-High Prime DNA labeling and detection starter kit (Roche Applied Science). The membrane was visualized using a nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate reagent detection system (Roche Applied Science).
For the identification of 5Ј-and 3Ј-terminal nucleotide sequences of the ␣KGSA dehydrogenase gene, two synthetic oligonucleotide primers were designed from the flanking region of the ALDH gene of B. cepacia R18194 as follows: KGSA U2 , 5Ј-CGGCGGACGTCGCGAAACTCCGCAAGGCGACGGCG-3Ј; and KGSA D2 , 5Ј-GCGCGCACGCGATCAAGGTCCCCGCCA-GCACAACG-3Ј. A single product with a length of ϳ2.3-kbp obtained by A. brasilense genomic PCR was purified, cloned into a pGEM-T vector (referred to as pGEM4), and sequenced.
Functional Expression and Purification of His 6 -tagged ␣KGSA Dehydrogenase-To clone the ␣KGSA dehydrogenase gene into the expression plasmid vector, two synthetic oligonucleotide primers were designed as follows: KGSA U3 , 5Ј-caccat-ggatccGCTAACGTGACTTATACGGATACGCAACTGC-3Ј; and KGSA D3 , 5Ј-gcttggctgcagTCAGACGGCCATCACCGT-CACCGACTTCGTGACC-3Ј (small letters indicate additional bases for introducing digestion sites of BamHI and PstI (underlined letters), respectively). PCR was carried out using pGEM4 as a template and KGSA U3 and KGSA D3 as primers, and the amplified DNA fragment was then introduced into BamHI-PstI sites in pQE-80L (Qiagen), a plasmid vector for conferring N-terminal His 6 tag on the expressed proteins, to obtain pHIS KGSA .
E. coli DH5␣ harboring the pHIS KGSA plasmid was grown at 37 °C to a turbidity of 0.6 at 600 nm in Super Broth medium (pH 7.0, 12 g of tryptone, 24 g of yeast extract, 5 ml of glycerol, 3.81 g of KH 2 PO 4 , and 12.5 g of K 2 HPO 4 per liter) containing 50 mg/liter ampicillin. After the addition of 1 mM of isopropyl ␤-D-thiogalactopyranoside, the culture was further grown for 6 h to induce the expression of His 6 -tagged ␣KGSA dehydrogenase protein. Cells were harvested and resuspended in Buffer D (pH 8.0, 50 mM sodium phosphate containing 300 mM NaCl, 10 mM 2-mercaptoethanol, and 10 mM imidazole). The cells were then disrupted by sonication, and the solution was centrifuged. The supernatant was loaded onto a nickel-nitrilotriacetic acid spin column (Qiagen) equilibrated with Buffer D. The column was washed three times with Buffer E (pH 8.0, Buffer D containing 10% (v/v) glycerol and 50 mM imidazole instead of 10 mM imidazole). The enzymes were then eluted with Buffer F (pH 8.0, Buffer E containing 250 mM imidazole instead of 50 mM imidazole). The eluant was dialyzed against Buffer C and stored at Ϫ35 °C until used.
The native molecular weight of ␣KGSA dehydrogenase was estimated by gel filtration. The purified enzyme was loaded onto a HiLoad 16/60 Superdex 200 pg column equilibrated with Buffer A containing 1 mM dithiothreitol instead of 10 mM 2-mercaptoethanol. High and low molecular weight gel filtration calibration kits (Amersham Biosciences) were used as molecular markers.
Western Blot Analysis of His 6 -tagged ␣KGSA Dehydrogenase-For Western blot analysis, purified ␣KGSA dehydrogenase from A. brasilense and/or recombinant His 6 -tagged ␣KGSA dehydrogenase from E. coli were separated by SDS-PAGE, and the proteins on the gels were transferred onto a nitrocellulose membrane (Hybond TM -ECL; Amersham Biosciences). Western blot analysis was carried out using the ECL TM Western blotting analysis system (Amersham Biosciences) and RGS⅐His horseradish peroxidase antibody, a horseradish peroxidase-fused mouse monoclonal antibody against Arg-Gly-Ser-His 6 in the N-terminal additional peptide of the expressed recombinant proteins (Qiagen).
Product Identification by HPLC-The product of the dehydrogenation reaction of ␣KGSA was identified by HPLC with a Multistation LC-8020 model II system (Tosoh). A solution containing 66.7 mM potassium phosphate, pH 7.2, 10 mM ␣KGSA or succinic semialdehyde, 1 mM EDTA, and 1 mM 2-mercaptoethanol, 10 mM NAD ϩ , and the purified His 6 -tagged enzyme (10 g) was incubated at 25 °C, and 100 l of this solution was then analyzed. Samples were applied at 35 °C to an Aminex HPX-87H Organic Analysis column (300 ϫ 7.8 mm; Bio-Rad) monitored with an RID-8020 refractive index detector (Tosoh) and eluted with 5 mM H 2 SO 4 at a flow rate of 0.6 ml/min.
Disruptant Construction-For the construction of a disputant strain of the ␣KGSA dehydrogenase gene, pHIS KGSA was digested with SalI to remove the 0.5-kbp DNA fragment within the ␣KGSA dehydrogenase gene, and a 5.7-kbp DNA fragment was purified. The resulting DNA fragment was ligated with the Tn5-derived 1.3-kb SalI kanamycin resistance (Km r ) cassette of pUC4K (Amersham Biosciences) to yield pHIS KGSA::Km . To introduce the restriction site for MfeI at the 5Ј-and 3Ј-end of the DNA fragment containing the Km r cassette in the ␣KGSA dehydrogenase gene, PCR was carried out using pHIS KGSA::Km as a template and the following two primers (lowercase letters indicate additional bases for introducing digestion sites of MfeI (underlined letters)): 5Ј-caccatcaattgATGGCTAACGTGAC-TTATACGGATACGCAAC-3Ј (MfeI-up) and 5Ј-gcttggcaattg-TCAGACGGCCATCACCGTCACCGACTTCGTGAC-3Ј (MfeI-down). The 2.2-kbp MfeI DNA fragment was subcloned into EcoRI site in the chloramphenicol resistance (Cm r ) cassette of the suicide vector pSUP202 (Amp r , Tet r , and Cm r ) (22) to yield pSUP KGSA::Km (Amp r , Tet r , Cm S , and Km r ).
Escherichia coli S17-1 ( 22) was transformed with pSUP KGSA::Km , and then the transformant was further mobilized to A. brasilense by biparental mating. The transconjugants were selected on a minimal medium agar plate supplemented with 5 g of sodium malate and 25 g of kanamycin per liter using Km r (the presence of Km r cassette) and Tc S (loss of pSUP202) phenotypes. The construction was confirmed by genomic PCR. One of the three resulting disruptants of A. brasilense was named ⌬ARA0512 and was used in this study.
Northern Blot Analysis-A. brasilense cells were cultured at 30 °C to the mid-log phase (A 600 ϭ 0.6 -0.8) in minimal medium supplemented with 37 mM appropriate carbon source (L-arabinose, D-glucose, D-glucarate, or D-galactarate) or nutrient medium (12) and harvested by centrifugation. Total RNA preparation and Northern hybridization were performed as described previously (12) . The same labeling probe as used in Southern blot analysis was used for the detection of mRNA of the ␣KGSA dehydrogenase gene.
Amino Acid Sequence Alignment and Phylogenetic Analysis-For phylogenetic analysis, 145 ALDH sequences were obtained from a website devoted to ALDHs (see Ref. 16 ). Furthermore, several ALDH sequences, which had been recently identified, were added. The sequences were aligned using the program, ClustalW, distributed by DNA Data Bank of Japan (DDBJ). The phylogenetic tree was produced using the TreeView 1.6.1. program.

Bacterial Strain, Culture Conditions, and Preparation of Cell-free Extracts-A. brasiliense ATCC29145 was purchased from RIKEN BioResource Center (Saitama, Japan) and cultured aerobically with vigorously shaking at 30 °C for 24 h in a minimal medium (13) (pH 6.8), containing 4.0 g of KH 2 PO 4 , 6.0 g of K 2 HPO 4 , 0.2 g of MgSO 4 ⅐H 2 O, 0.1 g of NaCl, 0.026 g of CaSO 4 ⅐2H 2 O, 1.0 g of NH 4 Cl, 0.01 g of FeCl 3 ⅐6H 2 O, 0.002 g of NaMoO 4 ⅐2H 2 O, and 0.0001 g of biotin/liter supplemented with 37 mM L-arabinose. L-Arabinose was sterilized separately by filtration and added to the medium. The grown cells were harvested by centrifugation at 30,000 ϫ g for 20 min, washed with 20 mM potassium phosphate buffer (pH 7.0), containing 2 mM MgCl 2 and 10 mM 2-mercaptoethanol (referred to as Buffer A), and stored at Ϫ35 °C until used. The washed cells were suspended in Buffer A, disrupted by sonication for 20 min with appropriate intervals on ice using ASTRASON Ultrasonic Liquid Processor XL2020 (Misonix Inc., New York), and then centrifuged at 108,000 ϫ g for 20 min at 4 °C to obtain cell-free extracts.
PAGE-SDS-PAGE was performed as described by Laemmli (29) . Nondenaturing PAGE was performed by omitting SDS and 2-mercaptoethanol from the solution used in SDS-PAGE. Proteins on the gel were stained with Coomassie Brilliant Blue R-250 and destained with 7.5% (v/v) acetic acid in 25% methanol.
Enzyme Activity Assay-L-Arabinose 1-dehydrogenase activity was assayed routinely in the direction of L-arabinose oxidation by measuring the reduction of NAD(P) ϩ at 340 nm at 30 °C using Jasco spectrophotometer model V-550 (Japan Spectroscopic Co., Ltd., Tokyo, Japan). The standard assay mixture contained 10 mM L-arabinose in 100 mM Tris-HCl (pH 9.0) buffer. The reaction was started by the addition of 10 mM NAD(P) ϩ solution (100 l) with a final reaction volume of 1 ml. The kinetic parameters, K m and k cat values, were calculated by Lineweaver-Burk plot. Protein concentrations were determined by the method of Lowry et al. (30) with bovine serum albumin as a standard.
Zymogram Staining Analysis-The cell-free extracts or purified enzyme were separated on nondenaturing PAGE with 12% gel at 4 °C. The gels were then soaked in 10 ml of staining solution (31) consisting of 100 mM Tris-HCl (pH 9.0), 100 mM L-arabinose, 0.25 mM nitro blue tetrazolium, 0.06 mM phenazine methosulfate, and 1 mM NAD(P) ϩ at room temperature for 15 min. The dehydrogenase activity appeared as a dark band.
Purification of L-Arabinose 1-Dehydrogenase-All purification steps were performed below 4 °C. The cell-free extracts were fractionated between 50 and 60% saturation of (NH 4 ) 2 SO 4 . The precipitate was dissolved in a small volume of Buffer A, and the solution was then dialyzed against a large volume of Buffer A containing 1.3 M (NH 4 ) 2 SO 4 overnight. All chromatography was carried out using an ⌬KTA purifier system (Amersham Biosciences). After insoluble materials were removed by centrifugation, the supernatant was applied to a HiPrep 16/10 Butyl FF column (1.6 ϫ 10 cm, Amersham Biosciences) equilibrated with Buffer A containing 1.3 M (NH 4 ) 2 SO 4 and washed with the same buffer. Proteins were eluted using a reversed linear gradient of 1.3-0 M (NH 4 ) 2 SO 4 in Buffer A (300 ml). The fractions with high enzymatic activity were pooled and dialyzed overnight against a large volume of Buffer A. The enzyme solution was loaded onto a column of HiPrep 16/10 Q FF (1.6 ϫ 10 cm, Amersham Biosciences) equilibrated with Buffer A and washed thoroughly with the same buffer. The column was developed with 300 ml of linear gradient 0 -1 M NaCl in Buffer A. The fractions containing L-arabinose 1-dehydrogenase activity were combined and dialyzed against a large volume of 5 mM potassium phosphate (pH 7.0), containing 10 mM 2-mercaptoethanol (referred to as Buffer B) overnight. The enzyme solution was applied to a column of Ceramic Hydroxyapatite Type I (1.6 ϫ 5 cm, Bio-Rad), equilibrated with Buffer B. The column was washed thoroughly with the same buffer and developed with 200 ml of linear gradient 0.005-0.5 M potassium phosphate in Buffer B. The fractions with high enzymatic activity were combined and concentrated by ultrafiltration with Centriplus YM-30 (Millipore) at 18,000 ϫ g for ϳ2 h. The enzyme solution was loaded onto a column of HiLoad 16/60 Superdex 200 pg column (1.6 ϫ 60 cm, Amersham Biosciences) equilibrated with Buffer A. The active fractions were pooled, concentrated, and re-loaded onto the same column. Proteins in the fractions containing high activity L-arabinose 1-dehydrogenase were analyzed by SDS-PAGE, and the fractions containing a single protein were collected, concentrated, dialyzed against Buffer C (100 mM Tris-HCl (pH 9.0), containing 2 mM MgCl 2 , 10 mM L-arabinose, 1 mM dithiothreitol, and 50% (v/v) glycerol), and stored at Ϫ35 °C until used.
The native molecular mass of L-arabinose 1-dehydrogenase was estimated by gel filtration. The purified enzyme was loaded onto a HiLoad 16/60 Superdex 200 pg column equilibrated with 20 mM potassium phosphate (pH 7.0), containing 2 mM MgCl 2 and 1 mM dithiothreitol. High and low molecular weight gel filtration calibration kits (Amersham Biosciences) were used as molecular markers.
Product Identification by HPLC-The product of the dehydrogenation reaction of L-arabinose was identified by HPLC with a Multistation LC-8020 model II system (Tosoh). L-Arabino-␥-lactone was chemically synthesized with boiling potassium arabonate in 0.2 M HCl for 5 min. Potassium arabonate was prepared by the hypoiodite-in-methanol oxidization of L-arabinose (32) . A solution containing 100 mM Tris-HCl (pH 9.0), 10 mM L-arabinose, 10 mM NADP ϩ , and the purified enzyme (10 g) was incubated for 30 min at 30 °C, and 100 l of this solution was then analyzed. Samples were applied at 30 °C into an Aminex HPX-87H Organic Analysis column (300 ϫ 7.8 mm, Bio-Rad) linked to an RID-8020 refractive index detector (Tosoh) and eluted with 5 mM H 2 SO 4 at a flow rate of 0.6 ml/min.
Determination of N-terminal and Internal Amino Acid Sequences-To determine the N-terminal amino acid sequence of L-arabinose 1-dehydrogenase, the purified enzyme was separated by SDS-PAGE with 12% (w/v) gel, and then transferred to Hybond TM -P (Amersham Biosciences) at 3 mA/cm 2 for 0.5 h in a transfer buffer (10 mM CAPS (pH 11) containing 10% (v/v) methanol) with a horizontal electrophoretic blotting system (model AE-7500, Atto). After staining and destaining the protein, an area of the membrane corresponding to the protein band of L-arabinose 1-dehydrogenase was excised and analyzed with a Procise TM 492 HT protein sequencer (Applied Biosystems).
Chemical digestion with cyanogen bromide (CNBr) was carried out to determine internal amino acid sequences (33) . The purified L-arabi- nose 1-dehydrogenase (100 g) was dialyzed overnight against deionized water and lyophilized. The enzyme protein was digested chemically at room temperature in 70% (v/v) formic acid containing 1% (w/v) CNBr (100 l) in the dark and under N 2 overnight. The solution was diluted with 900 l of deionized water, frozen with liquid N 2 , and lyophilized. The sample was dissolved in SDS-PAGE sample buffer (500 mM Tris-HCl (pH 6.8), containing 5% (w/v) SDS, 10% (v/v) glycerol, 0.25% (w/v) bromphenol blue, and 5% (v/v) 2-mercaptoethanol) and separated by SDS-PAGE with 18% (w/v) gel. Peptide fragments on the gel were trans-ferred to a Hybond TM -P membrane as described above. After staining and destaining, areas of the membrane corresponding to the two peptide fragments from the L-arabinose 1-dehydrogenase (see in Fig. 2B) were excised and sequenced.
Cloning of L-Arabinose 1-Dehydrogenase Gene-The N-terminal and internal peptide sequences were used to design PCR primers for amplification of a partial DNA fragment of the L-arabinose 1-dehydrogenase gene. Eight upstream primers (U1-U8, 26-mer) were designed from (M)SDQVSLGV, the N-terminal amino acid sequence, as follows: 5Ј-ATG(TCN/AGY)GAYCARGTN(TCN/AGY) (CTN/TTR)GGN-GT-3Ј. Two downstream primers (D1 and D2, 26-mer) were designed from the internal amino acid sequence (M)LEKPPGAT, as follows: 5Ј-GTNGCNCCNGGNGGYTTYTC(NAG/YAA)CAT-3Ј. A. brasiliense genomic DNA was prepared using a DNeasy TM tissue kit (Qiagen). PCR was carried out using a PCR Thermal Cycler-Personal (Takara) for 30 cycles in a 50-l reaction mixture containing 10 pmol of primers, 1.25 units of Ex Taq DNA polymerase (Takara), and 300 ng of A. brasiliense genomic DNA under the following conditions: denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, and extension at 72 °C for 30 s, each for 30 cycles. Based on the results of genomic PCR using each set of primers, U6 and D1 were chosen for cloning. The sequences of U6 and D1 were 5Ј-ATGAGYGAYCARGTNTCNTTRGGNGT-3Ј and 5Ј-GTNGCNCCNGGNGGYTTYTCNAGCAT-3Ј, respectively. A single PCR product with a length of ϳ300 bp was purified, cloned into a pGEM-T vector (Promega) (referred to as pGEM1), and sequenced using a Dual CyDye TM terminator sequencing kit (Veritas) and appropriate primers with Long-Read Tower, UBC DNA sequencer (Amersham Biosciences). The inserted fragment was amplified with U6 and D1 primers and with pGEM1 as a template DNA, and the PCR product was purified and utilized as a probe for Southern and Northern blot analysis and colony hybridization (34) .
For Southern blot analysis, ϳ1.8 g of A. brasiliense genomic DNA was digested with six restriction enzymes, EcoRI, HindIII, NotI, PstI, SalI, and XbaI, separated on 1% (w/v) agarose gel and blotted to Hybond TM -N (Amersham Biosciences) by capillary transfer using 10ϫ SSC as a transfer buffer (1ϫ SSC is 15 mM sodium citrate (pH 7.0), and 0.15 M NaCl). The blotted filter was cross-linked in an UV cross-linker CX-2000 (Ultra-Violet Products, Ltd.). A double-stranded probe DNA was labeled with digoxigenin-11-dUTP and hybridized using a DIG-High Prime DNA labeling and detection starter kit (Roche Applied Science). Membrane was visualized using a nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate reagent detection system (Roche Applied Science).
A. brasiliense partial genomic library was prepared with genomic DNA with NotI based on the results of Southern blot analysis. The DNA fragments corresponding to a positive band in size (ϳ2.0 kbp of the length) were ligated to a plasmid pBluescript SK(Ϫ) (Stratagene). Colony hybridization was carried out under the same conditions as Southern blot analysis except for the use of nylon membranes for colony and plaque hybridization (Roche Applied Science). The plasmid from a positive clone (referred to as pBS1) was purified, and the inserted A. brasiliense genome fragment was sequenced.
Northern Blot Analysis-A. brasiliense cells were cultured at 30 °C to the mid-log phase (A 600 ϭ 0.6 -0.8) in minimal medium supplemented with 37 mM appropriate sugar (D-glucose, L-arabinose, D-galactose, or D-xylose) or nutrient medium (10 g of peptone, 10 g of meat extract, and 5.0 g of NaCl (pH 7.0 -7.2)) and harvested by centrifugation. Total RNAs from A. brasiliense were prepared with an RNeasy mini kit (Qiagen) and subsequently treated with RNase-free DNase I. The isolated RNA (4 g) was subjected to electrophoresis on 1.2% (w/v) agarose gel containing 0.66 M formaldehyde. The subsequent steps were performed using the same methods as for Southern blot analysis.
Cloning of the L-Arabinose 1-Dehydrogenase Gene into Expression Plasmid Vector-To introduce the restriction site for BglII at the 5Ј-end and PstI at the 3Ј-end of the L-arabinose 1-dehydrogenase gene, PCR was carried out using pBS1 as a template and the following two primers (lowercase letters indicate additional bases for introducing digestion sites of BglII and PstI (underlined letters)): 5Ј-caccatagaTCTGATCAG-GTTTCGCTGGGTG-3Ј (HIS BglII ) and 5Ј-gcttggctgcagTCAGCGGC-CGAACGCGTCG-3Ј (HIS PstI ). The amplified DNA fragment was introduced into BamHI-PstI sites in pQE-80L (Qiagen), a plasmid vector for conferring N-terminal His 6 tag on the expressed proteins, to obtain pHIS WT .
Site-directed Mutagenesis-The following sense and antisense primers were designed to introduce the mutations into the L-arabinose 1-dehydrogenase gene (the mutated regions are underlined): to substitute Ala for Asp 168 (D168A), 5Ј-CGGCGTGTTCGCGCCGGGCATC-3Ј (D168A S ) and 5Ј-GATGCCCGGCGCGAACACGCCG-3Ј (D168A AS ); to substitute Ala for Asn 172 (N172A), 5Ј-CCCGGGCATCGCG-GCGCTGTCG-3Ј (N172A S ) and 5Ј-CGACAGCGCCGCGATGC-CCGGG-3Ј (N172A AS ). The mutations were introduced by sequential steps of PCR (35) with small modifications. In the first round, two reactions, I and II, were performed with the appropriate primers and pHIS WT as a template: reaction I, HIS BglII and one of the antisense primers containing the mutations; and reaction II, one of the sense primers containing the mutations and HIS PstI . In the final amplification step, purified overlapping PCR products were used as templates and HIS BglII and HIS PstI as primers. The final PCR products were cloned into pQE-80L to obtain plasmids pHIS D168A and pHIS N172A , respectively. The coding region of the mutated genes was confirmed by subsequent sequencing in both directions.
Functional Expression and Purification of His 6 -tagged L-Arabinose 1-Dehydrogenase-E. coli DH5␣ harboring the expression plasmid for the His 6 -tagged wild-type and mutated enzymes was grown at 37 °C to a turbidity of 0.6 at 600 nm in Super Broth medium (12 g of tryptone, 24 g of yeast extract, 5 ml of glycerol, 3.81 g of KH 2 PO 4 , and 12.5 g of K 2 HPO 4 /liter (pH 7.0)) containing 50 mg/liter ampicillin. After the addition of 1 mM of isopropyl-␤-D-thiogalactopyranoside, the culture was further grown for 6 h to induce the expression of His 6 -tagged L-arabinose 1-dehydrogenase protein. Cells were harvested and resuspended in Buffer D (50 mM sodium phosphate containing 2 mM MgCl 2 , 300 mM NaCl, 1 mM L-arabinose, 10 mM 2-mercaptoethanol, and 10 mM imidazole (pH 8.0)). The cells were then disrupted by sonication, and the solution was centrifuged. The supernatant was loaded onto a nickelnitrilotriacetic acid spin column (Qiagen) equilibrated with Buffer D. The column was washed three times with Buffer E (Buffer D containing 10% (v/v) glycerol and 50 mM imidazole instead of 10 mM imidazole (pH 8.0)). The enzymes were then eluted with Buffer F (Buffer E containing 250 mM imidazole instead of 50 mM imidazole (pH 8.0)). The elutant was dialyzed against Buffer C and stored at Ϫ35 °C until use.
Western Blot Analysis of His 6 -tagged L-Arabinose 1-Dehydrogenase-For Western blot analysis, the purified L-arabinose 1-dehydrogenase from A. brasiliense and/or recombinant His 6 -tagged L-arabinose 1-dehydrogenase from E. coli was separated by SDS-PAGE, and the proteins on the gels were transferred onto a nitrocellulose membrane (Hybond TM -ECL; Amersham Biosciences). Western blot analysis was carried out using the ECL TM Western blotting analysis system (Amersham Biosciences) and RGS⅐His horseradish peroxidase antibody, a horseradish peroxidase-fused mouse monoclonal anti-body against Arg-Gly-Ser-His 6 in the N-terminal additional peptide of the expressed recombinant proteins (Qiagen).
Disruptant Construction-The overall scheme of the plasmid construction for disruption of the L-arabinose 1-dehydrogenase gene is shown in Fig. 9A. The Tn5-derived 1.3-kb BamHI kanamycin resistance (Km r ) cassette of pUC4K (Amersham Biosciences) was inserted into the single BamHI site in the coding sequence of the L-arabinose 1-dehydrogenase gene of pHIS WT to yield pHIS WT::Km . To introduce the restriction site for MfeI at the 5Ј-and 3Ј-end of the DNA fragment containing the Km r gene in the L-arabinose 1-dehydrogenase gene, PCR was carried out using pHIS WT::Km as a template and the following two primers (lowercase letters indicate additional bases for introducing digestion sites of MfeI (underlined letters)): 5Ј-caccatcaattgGATCAGGTTTCGCTGG-GTGTCGTCGGCATCG-3Ј (MfeI-up) and 5Ј-gcttggcaattgTCAGCG-GCCGAACGCGTCGGTCTGCACGCGC-3Ј (MfeI-down). The 2.3-kbp MfeI DNA fragment was subcloned into the EcoRI site in the chloramphenicol resistance (Cm r ) cassette of the suicide vector pSUP202 (36) to yield pSUP WT::Km .
E. coli S17-1 (36) was transformed with pSUP WT::Km , and then the transformant was further mobilized to A. brasiliense by biparental mating. The transconjugants were selected on a minimal medium agar plate supplemented with 5 g of sodium malate and 25 g of kanamycin/liter using Km r (the presence of Km r cassette) and Tc S (loss of pSUP202) phenotypes. The construction was confirmed by genomic PCR and Southern hybridization on total DNA digested with NotI. One of the resulting disruptants of A. brasiliense was named ⌬ARA5034 and was used in this study.
Amino Acid Sequence Alignment and Phylogenetic Analysis-Protein sequence of L-arabinose 1-dehydrogenase from A. brasiliense was analyzed using the Protein-BLAST and ClustalW program distributed by DDBJ (www.ddbj.nig.ac.jp). The phylogenetic tree was produced using the TreeView 1.6.1. program.

General Methods-Total RNA and mRNA were isolated using a guanidium-based method (11) and the Micro Poly(A) Pure mRNA purification kit (Ambion Inc.), respectively. All proteins blotted from SDSpolyacrylamide gel for Western analysis were transferred to Hybond-P membrane (Amersham Pharmacia Biotech), and the secondary antibody (HRP-conjugated IgG) was detected using one-step 3,3Ј,5,5Ј-tetramethylbenzidine (Pierce).
Baculovirus Expression and Purification of Juvenile Hormone Esterase-Recombinant JHE and mutants JHE K29R, JHE K524R, and JHE K29R/K524R were produced by infection of Spodoptera frugiperda cells (12) with recombinant baculoviruses (10, 13) in serum-free medium (14) . Recombinant enzymes were purified by loading JHE-containing medium (300 ml) onto Q-Sepharose columns (25-ml column volume; Amersham Pharmacia Biotech) and eluting in 10-ml fractions with a sodium chloride step gradient (85-90 mM in 50 mM Tris-HCl, 2 mM EDTA, and 0.02% sodium azide, pH 7.5). Fractions containing JHE activity, identified using 3 H-labeled JH-III as described (15, 16) , were concentrated using Centricon 30 filters (Amicon, Inc.) and subjected to SDS-PAGE. Purity was assessed by Coomassie Blue and silver staining of the SDS-polyacrylamide gels.
Construction of the cDNA Phage Display Vector pBJuFo-Plasmid pBJuFo is shown in Fig. 1. A DNA fragment encoding a Jun leucine zipper domain fused to fd phage coat protein gene III (GenBank TM /EBI accession number J02448) and a leader sequence fused to the Fos leucine zipper domain was a generous gift from R. Crameri (17, 18) . EcoRV and NotI sites were added to the 5Ј-and 3Ј-ends, respectively, by PCR using the primers JF5ЈRV (5Ј-GGGATATCTTCTATTCAAG-GAGACAGTCATAG-3Ј) and JF3ЈNot (5Ј-CCGCGGCCGCACCACCG-CAACCACCGTGTGCCGCC-3Ј) prior to cloning into pCR2.1TOPO (Invitrogen). The resulting insert was isolated by digestion with EcoRV and NotI and cloned into pcDNA2.1 (Invitrogen), which had previously been digested with KpnI, blunt-ended by end filling with Klenow, and digested with NotI. The sequence encoding the gene III leader was constructed using overlapping oligonucleotides and inserted 5Ј to the jun leucine zipper region at the HindIII site. This step replaced the pelB leader sequence that was present in the original fragment with the gene III leader sequence. Next, a V5 epitope tag with a small 3Ј-multiple cloning site was constructed using the same technique and inserted downstream from the fos leucine zipper sequence into the NotI site to produce pBJuFo (see Fig. 1). All constructs were confirmed by sequencing.
Construction and Enrichment of the Phage Display Library-Pericardial cell complexes (pericardial cells and associated dorsal aortas) were dissected from 50 M. sexta larvae at the fifth instar (day 2 or day 3). Total RNA and mRNA were extracted (see "General Methods"), and cDNA was synthesized (Smart PCR cDNA synthesis kit, CLONTECH). First-strand synthesis was conducted using reverse transcriptase (Promega) with the oligo(T) NotI primer (Invitrogen). Second-strand synthesis was conducted using the Capswitch primer (CLONTECH) for synthesis of full-length cDNAs and the Advantage PCR kit (CLON-TECH). The PCR products were treated with T7 DNA polymerase; ligated to BstXI adaptors (Invitrogen); size-selected for Ͼ400 bp (on Size-Sep400 spin columns, Amersham Pharmacia Biotech); digested with NotI; and ligated into the phage display vector pBJuFo, which had previously been restricted with BstXI and NotI. Escherichia coli strain XL-1 Blue (Stratagene) was transformed with the ligation mixture. An aliquot of the recombinant E. coli cells was plated on Luria broth/ ampicillin plates for overnight incubation. Recombinant plasmids were isolated and restricted with EcoRI and NotI to show the range of cDNA insert sizes in pBJuFo. The recombinant E. coli cells were then infected with the helper phage vector cloning system M13 (Stratagene) to generate a large-scale recombinant phage expression library, which was at Ϫ70 °C. The phage display library was enriched by biopanning as described (18) . One well of a polystyrene 24-well microtiter plate (Falcon) was coated with JHE (3 g in 300 l of 0.1 M sodium bicarbonate, pH 8.6), and recombinant phage (ϳ2.5 ϫ 10 7 plaque-forming units in 250 l) were added. After binding of phage and removal of unbound phage by washing with TBST (25 mM Tris, 3 mM KCl, 150 mM NaCl, and 0.01% Tween 20, pH 7.4), bound phage were eluted. For the first three rounds of screening, phage were eluted in acidic buffer (300 l of 50 mM HCl/glycine, pH 2.2, per well). For the fourth round of screening, phage were eluted with JHE (7.5 g of JHE in 150 l of PBS for 15 min). Fifty l of recombinant phage eluted after the fourth round of enrichment were used to infect E. coli cells. After overnight incubation on Luria broth/ampicillin plates, individual colonies were picked to test binding of specific recombinant phage to JHE on 96-well plates by enzymelinked immunosorbent assay.
Screening of the Phage Display Library-JHE (1 g in 100 l of 0.1 M sodium bicarbonate, pH 8.6 per well) was adsorbed to the solid phase of alternate rows on 96-well microtiter plates. Recombinant phage isolated from individual E. coli colonies were added to adjacent wells with or without recombinant JHE and incubated (3-4 h). Unbound phage were removed by washing in TBST, and bound phage were detected by enzyme-linked immunosorbent assay using anti-M13 antiserum (Amersham Pharmacia Biotech) conjugated to HRP. HRP activity on the substrate ABTS (2,2Ј-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt; Amersham Pharmacia Biotech) was quantified at 412 nm. Wells precoated with monoclonal anti-M13 antiserum or skimmed milk were used as positive and negative controls, respectively. Wells with optical density readings of Ͼ2ϫ background levels were considered to be positive, and these clones were screened by enzyme-linked immunosorbent assay a second time. Plasmids were then isolated from positive clones and subjected to restriction analysis with NotI, EcoRI, and HindIII. Clones with dissimilar restriction enzyme fragments were selected for DNA sequencing using Applied Biosystems 377 automated DNA sequencing technology. Sequences were compared with those in GenBank TM /EBI Data Bank using BLAST (19) . Protein motifs were identified, and the isoelectric point was determined from the deduced amino acid sequence using the program MOTIF (Genome Net). Preparations of pericardial cell protein-gene III fusions were isolated from the periplasm of selected clones, separated by SDS-PAGE, transferred to membrane, and probed with anti-V5 antibody (Invitrogen; see "General Methods") to confirm the presence of larger proteins (rather than peptides) that bind JHE.
Expression and Purification of Recombinant Juvenile Hormone Esterase-binding Proteins-The insert from a selected clone (pBJuFo.56) was restricted with BstXI and NotI, directionally cloned into the T7 polyhistidine expression vector pRSET-JF (Invitrogen) to produce pR-SET-JF.56, and transformed into E. coli BL21(DE3). Transformants were induced for 3 h (0.5 mM isopropyl-␤-D-thiogalactopyranoside), harvested, and lysed, and the recombinant protein was bound to a nickel column. Protein was then eluted in 50 -200 mM imidazole according to the manufacturer's directions (Invitrogen). The purified recombinant binding protein (P29) was separated by SDS-PAGE, electroblotted onto membrane, and detected with anti-polyhistidine primary antibody (anti-Xpress, Invitrogen; see "General Methods"). Purified P29 was used for production of polyclonal antisera in mice as described (20) .
Analysis of JHE Binding by P29 in Vitro-Purified JHE and P29 were labeled with biotin (biotin labeling kit, Roche Molecular Biochemicals), column-purified on streptavidin to eliminate non-biotinylated protein, and quantified (Bio-Rad protein assay). Biotinylated proteins were separated by SDS-PAGE, transferred to Hybond-P membrane, and examined using streptavidin-HRP conjugate and the ECL chemiluminescence substrate luminol (Amersham Pharmacia Biotech). Fluorescence was detected by film exposure (Eastman Kodak Co.).
The binding of JHE to pericardial cell proteins and recombinant P29 was examined by ligand blotting. Pericardial cell complexes were dissected from larvae of M. sexta (fifth instar, day 3); homogenized in PBS, pH 7.4, supplemented with 10 mM EDTA and 10 mM phenylmethylsulfonyl fluoride; and centrifuged at 5200 ϫ g for 10 min. The supernatant was used for ligand blot analysis. E. coli samples from recombinant BL21(DE3) cells transformed with pRSET-JF.56 were sonicated for 2 min in PBS, pH 7.4, supplemented with 10 mM EDTA and 10 mM phenylmethylsulfonyl fluoride and centrifuged at 5200 ϫ g for 10 min. Protein concentrations were determined (Bio-Rad), and proteins were separated by SDS-PAGE and electroblotted onto Hybond-P membrane. Blots were incubated for 4 h with biotin-labeled JHE (2 g/ml) in PBS, washed with PBS and 0.1% Tween 20, and then blocked with skimmed milk prior to detection with streptavidin-HRP conjugate and one-step 3,3Ј,5,5Ј-tetramethylbenzidine.
For immunoprecipitation experiments, biotin-labeled JHE (50 l, 3.3 g) and biotin-labeled P29 (50 l, 1.4 g) were mixed and incubated at 37 °C for 2 h. Anti-JHE or anti-Xpress antiserum (2 l) was added; the reaction was incubated on ice for 2 h; and Affi-Gel-protein A (200 l: Bio-Rad) was added to precipitate immune complexes. The immune complexes were washed (2 ml of PBS); pelleted by centrifugation at 10,600 ϫ g for 10 min; and then treated with 0.1 M sodium citrate, pH 3.0, to release proteins from the affinity gel. Samples were pelleted at 10,600 ϫ g for 5 min, and proteins in the supernatant were separated by SDS-PAGE (12% gel) and transferred to Hybond-P membrane. Biotinylated proteins were detected as described above. For positive controls, purified JHE was immunoprecipitated with anti-JHE antiserum, and P29 was immunoprecipitated with anti-Xpress antiserum. In negative control reactions, immunoprecipitation reactions contained JHE with anti-Xpress antiserum or P29 with anti-JHE antiserum.
Analysis of Expression and JHE Binding of P29 in Vivo-Pericardial cell and fat body proteins were separated by SDS-PAGE, transferred to membrane, and probed with primary antiserum raised against P29 (see "General Methods"). Poly(A) ϩ mRNAs (see "General Methods") from third, fourth, and fifth instar larvae of M. sexta were separated on a 2.2 M formaldehyde-containing 1% agarose gel; transferred to nitrocellulose; and probed with a biotinylated P29 coding sequence under high stringency conditions (21) . The 1.3-kilobase biotin-labeled probe was prepared from pBJuFo.56 template by PCR with primers flanking the P29 coding sequence (forward primer PhD, 5Ј-GCGGCACACGGTGGT-TGC-3Ј; and reverse primer T7, 5Ј-AATACGACTCACTATAG-3Ј). As a negative control, a second probe was amplified by PCR using the T7 and PhD primers and an irrelevant cDNA insert (including a poly(A) tail) in pBJuFo. Biotinylated probe bound to mRNA on the membrane was detected using streptavidin-HRP with the ECL chemiluminescence substrate.
Larvae of M. sexta (fifth instar, day 3) were cooled on ice and injected with 10 g of biotinylated JHE or 10 g of bovine serum albumin, and pericardial cell and fat body tissues were dissected 1 h after injection. Tissues were homogenized on ice in 20 mM Tris-HCl, pH 6.8, 150 mM NaCl, 1 mM EDTA, and 10 mM phenylmethylsulfonyl fluoride and centrifuged at 5200 ϫ g for 5 min. Anti-P29 antiserum (1 l) was added to proteins in the supernatant, followed by immunoprecipitation with Affi-Gel-protein A. Proteins in the immunoprecipitate were separated by SDS-PAGE and transferred to membrane for detection of biotinlabeled JHE. For tissue samples from insects injected with bovine serum albumin (n ϭ 3), precipitated native JHE was detected by radiochemical assay (15) .
Binding of P29 to JHE Mutants-The degree of biotinylation of JHE K29R, JHE K524R, and JHE K29R/K524R (purified and biotinylated as described above) was quantified by colorimetric assay at 412 nm in a microtiter plate using streptavidin-HRP conjugate with ABTS substrate. All assays (50 l of 2 g of enzyme/ml of stock per well) were replicated four times. Data were analyzed by one-way ANOVA.
A competition experiment was conducted to quantify the extent of binding of the JHE mutants to P29. Purified P29 was attached to a microtiter plate at different concentrations (0.75, 1.5, and 3 g/well).
Biotinylated JHE or mutant JHE (200 ng) in PBS, pH 7.4, was added. Bound enzyme was detected using streptavidin-HRP with ABTS at 412 nm. Five replicate assays were carried out, and data were analyzed by one-way ANOVA and Tukey's test for pairwise comparisons.

Materials-Restriction enzymes, T4 polynucleotide kinase, and Taq DNA polymerase were purchased from Fisher. Hybond-Nϩ nylon mem-branes were purchased from Amersham Pharmacia Biotech. The Ultraspec RNA II isolation system and Hybrisol II were obtained from Cinna Biotecx and Intergen, respectively. The zebrafish genomic library was purchased from Stratagene. The Thermoscript reverse transcriptase system and ThermoSequenase Radiolabeled Terminator cycle sequencing kit were obtained from Life Technologies, Inc. and U. S. Biochemical Corp., respectively. The pEGFP-1 plasmid and microcapillaries were purchased from CLONTECH and World Precision Instruments, Inc., respectively.
Southern Blotting-DNA was fractionated on 1% agarose gels and transferred to Hybond-Nϩ positively charged nylon membranes by downward alkaline capillary transfer (25) . Rod opsin cDNA probes were isolated by restriction digestion and labeled by random primers. Membranes were hybridized with probe in Church's buffer (1% bovine serum albumin, 1 mM EDTA, 0.5 M NaHPO 4 (pH 7.2), 7% SDS) at 65 °C for 16 hours and then washed twice under high stringency conditions (1 mM EDTA/40 mM NaHPO 4 (pH 7.2)/1% SDS) at 66 °C for 30 min prior to autoradiography.
Northern Blotting-Total RNA was isolated using the Ultraspec RNA II isolation system. RNA aliquots (Ϸ18 g) were electrophoresed in 1% agarose gels containing 6.6% formaldehyde, blotted to Hybond-N nylon membranes in 20ϫ SSC, and UV cross-linked. Membranes were hybridized with a probe in Hybrisol II containing 35% (v/v) formamide at 55 °C. Final high stringency washing conditions were 0.08ϫ SSC, 0.1% SDS at 65 °C for 30 min twice.
Genomic Library Screens-Approximately 10 7 recombinant phage in a Fix II zebrafish genomic library were screened with a radiolabeled zebrafish rod opsin cDNA (26) . Escherichia coli strain XL1-MRAP2 was infected with titrated phage and plated on 150-mm NZY Petri dishes. Plaques were lifted and UV-crosslinked onto Hybond-Nϩ nylon membranes (27) . Hybridization conditions were as described under "Southern Blotting."
Polymerase Chain Reaction (PCR) Mapping-Genomic, phage, and plasmid DNA templates were PCR-amplified using primers spanning the zebrafish rod opsin cDNA. PCR parameters were 30 cycles of denaturation at 94 °C for 30 s, anneal primers at 65-67 °C for 30 s, and extension at 72 °C for 5 min using Taq DNA polymerase.
Primer Extension-A primer complementary to the 5Ј end of the zebrafish rod opsin cDNA (RR1) was end-labeled with 32 P using T4 polynucleotide kinase. 12 g of zebrafish eye or body total RNA were hybridized with the labeled primer for 1 h at either 60 or 42 °C (in 50% formamide). The annealed primer was extended using the Thermoscript reverse transcriptase system supplemented with 50 ng/l actinomycin D. Products were extracted with phenol/chloroform/isoamyl alcohol, ethanol-precipitated, and electrophoresed on a 6% polyacrylamide-urea sequencing gel. The ThermoSequenase Radiolabeled Terminator cycle sequencing kit was used for DNA sequencing.
Embryo Microinjections-A 1.2-kbp (EcoRI/XbaI) promoter fragment of the zebrafish rod opsin gene was subcloned upstream of the EGFPcoding sequence in pEGFP-1, creating pZOP-EGFP. This vector was linearized and resuspended in water plus India ink at 50 ng/l. Zebrafish embryos were microinjected with linearized vector at the 1-to 2-cell developmental stage (28) . Injection needles were pulled from 1-mm (inner diameter) microcapillaries on a Sutter Instrument Co. model P-97 Flaming/Brown micropipette puller. Embryos positioned on an agarose injection chamber (28) were injected using air pressure on a Narishige U. S. A., Inc. micromanipulator. Some fish were reared in water treated with 0.003% 1-phenyl-2-thiourea to inhibit melanin formation (28) .
Screening for Transgenic Fish-Fish were anesthetized and placed on a depression slide for fluorescence microscopy using a Carl Zeiss, Inc. Axiovert 100 microscope and cooled charge-couple deviced camera under a ϫ10 objective. Fish were viewed using a Carl Zeiss, Inc. narrow bandpass fluorescein isothiocyanate filter set and compared with a rhodamine filter set to differentiate between a true EGFP signal and autofluorescence. Fish exhibiting EGFP expression were isolated and pooled for long term characterization. Caudal and anal fins were clipped from adult founders, and genomic DNA was isolated (28) . These samples were screened for the presence of the EGFP cassette using the EGFP-F (5Ј-GCCACAAGTTCAGCGTGTCC) and EGFP-R (5Ј-GATGC-CCTTCAGCTCGATGC) primers and the PCR parameters described above.
Whole-mount and Retina Section Antibody Labeling-Larval and juvenile transgenic G 1 fish were fixed in 4% paraformaldehyde, 5% sucrose, PBS pH 7.4. Antibody labeling of whole-mount and frozen sections (10 m) was performed as described using polyclonal antirhodopsin diluted 1:5,000 (29) .