©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning and Expression of Rat Squalene Epoxidase (*)

(Received for publication, August 25, 1994; and in revised form, October 26, 1994)

Jun Sakakibara (1)(§) Rikio Watanabe (1) Yoshinori Kanai (1) (2) Teruo Ono (1)

From the  (1)Department of Biochemistry, Niigata University School of Medicine, Niigata 951 and the (2)Department of Agricultural Chemistry, Faculty of Agriculture, Niigata University, Niigata 950-21, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Squalene epoxidase (SE) (EC 1.14.99.7) catalyzes the first oxygenation step in sterol biosynthesis and is suggested to be one of the rate-limiting enzymes in this pathway. Rat SE cDNA was isolated by selecting yeast transformants expressing rat cDNA in the presence of terbinafine, an inhibitor specific for fungal SE. The expression of rat SE in the isolated terbinafine-resistant clone was confirmed by its survival in the presence of either terbinafine or an inhibitor specific for mammalian SE, NB-598, but not in the presence of both terbinafine and NB-598. Rat SE polypeptide deduced from the nucleotide sequence contains 573 amino acids, and its molecular weight is 63,950 Da. The amino acid sequence reveals one potential transmembrane domain, a hydrophobic segment (Leu to Tyr) in the NH(2)-terminal region. This region also contains a beta1-alphaA-beta2 motif, which is the consensus sequence for an FAD binding domain, suggesting that SE is a flavoenzyme. This deduced rat SE sequence is 30.2% identical to the ERG 1 gene, which encodes SE from an allylamine-resistant Saccharomycescerevisiae mutant. Expression of a full-length rat SE protein in Escherichia coli confirms this polypeptide as a functional SE. This is the first report of the molecular cloning of mammalian SE.


INTRODUCTION

Cholesterol biosynthesis is a complex biological process(1, 2) , and it provides a therapeutic target for the reduction of low density lipoprotein cholesterol, a key risk factor in coronary heart disease (3) . Recent studies have revealed a post-mevalonate pathway of cholesterol biosynthesis that plays an important role in the supply of obligate precursors for dolichol, ubiquinone and isopentenyl tRNA synthesis, and for polyprenylation of p21(4) and small GTP-binding proteins(5) , which function in the regulation of normal cellular processes and in oncogenesis(1) . As a result, three enzymes which are located beyond branch point in this pathway, namely squalene synthase, squalene epoxidase (SE), (^1)and oxidosqualene cyclase, have evoked considerable interest as potential targets for the design of hypercholesterolemic chemotherapeutics(6, 7) . SE appears to be an important rate-limiting enzyme in this cascade, as it has an extremely low specific activity compared to 3-hydroxy-3-methylglutaryl-coenzyme A reductase or squalene synthase (8, 9) . Furthermore, the addition of exogenous cholesterol to human renal cancer cells results in the accumulation of squalene(10) .

SE (EC 1.14.99.7) catalyzes the first oxygenation step in the sterol biosynthetic pathway, the conversion of squalene to 2,3(S)-oxidosqualene. It has been reported, however, that not only 2,3(S)-oxidosqualene but also 2,3(S);22(S),23-squalene diepoxide is accumulated in liver homogenates (11, 12) and in cultured mammalian cells (13) in the presence of an oxidosqualene cyclase inhibitor. 2,3(S);22(S),23-Squalene diepoxide is cyclized in rat liver homogenates to 24(S),25-epoxylanosterol(14) , which is then converted to 24(S),25-epoxycholesterol. 24(S),25-Epoxycholesterol was identified as one of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase repressor activities in the cultured cells(15) . Consequently, it would be interesting to know whether SE is involved in the synthesis of such oxysterols via squalene dioxide formation(16) .

SE is believed to be a microsomal membrane-bound protein. Rat SE requires FAD, NADPH-cytochrome P-450 reductase (EC 1.6.2.4), NADPH, and a supernatant protein factor for its activity(17, 18, 19, 20) . Triton X-100 can be substituted for the supernatant protein factor(19) . The fact that this enzyme is not inhibited by CO(17, 21) , as well as the product analysis of a porphyrin mutant of Saccharomyces cerevisiae(22, 23) , indicates that SE is not a cytochrome P-450 isozyme. It is the only non-cytochrome P-450 enzyme that epoxidizes a double bond in an alkyl chain(24) . Although much less is known about fungal SE, the ERG 1 gene recently cloned from an allylamine-resistant S. cerevisiae mutant was shown to encode a 55,190-Da SE protein consisting of 496 amino acids(25) . In this report, we describe the molecular cloning of rat SE by a new inhibitor selection method, its structure, and its expression in prokaryotic cells.


EXPERIMENTAL PROCEDURES

Yeast Strain and Materials

The Schizosaccharomyces pombe strain JY266 (h, leu1) was kindly donated by Dr. Y. Kimura and Dr. S. Matsumoto, The Tokyo Metropolitan Institute of Medical Science. The SE inhibitors terbinafine and NB-598 were gifts from Dr. T. Kamei of Banyu Pharmaceutical Co., Ltd. The rat kidney-derived cell line (NRK) cDNA library (cDNAs were inserted into pcD2) and the plasmid pAL17 were kindly provided by Dr. H. Okayama and Dr. A. Nagata, University of Tokyo Faculty of Medicine.

Cloning of Rat SE cDNA

Yeast transformation was performed according to the method of Okazaki et al.(26) , except for the following modifications. JY266 was incubated for 2 days on YEA plates at 32 °C. About 10^8 cells were inoculated into 100 ml of MB medium supplemented with 150 µg/ml leucine and grown to a density of 5 times 10^6/ml with moderate shaking at 23 °C. Cells were collected by centrifugation at 1,000 times g for 5 min at room temperature. The cells were washed with distilled water, and 10^8 cells were suspended in 100 µl of 0.1 M lithium acetate (pH 4.9) for 1 h at 23 °C. Fifteen µl of 10 mM Tris-HCl (pH 7.5), containing 2 µg of rat cDNA library and 1 µg of PstI-digested pAL17, and 290 µl of 50% polyethylene glycol 4000 were mixed and incubated for 1 h at 30 °C. The cells were then heat-shocked by incubating for 15 min at 43 °C and centrifuged at 4,000 times g for 2 min. The cell pellet was suspended in 1/2YEL medium and incubated for 2 h at 23 °C. Cells were pelleted again by centrifugation, suspended in MB medium (not supplemented with leucine), and shaken at 23 °C overnight. The cells were then spread on MMA plates containing 12.5 µg/ml terbinafine in the absence of leucine and incubated for 1 week at 32 °C. Terbinafine-resistant clones were isolated and plasmids were recovered according to the method of Okazaki et al.(26) .

DNA Sequencing and Computer Analysis

The cloned plasmid DNA was digested with BamHI, and the recovered insert was ligated into the BamHI site of pUC18 (pUCTb1). pUCTb1 was sequenced in both directions using a Sequenase kit (U.S. Biochem Corp.). DNA sequence data were organized and analyzed using the program Gene Works (IntelliGenetics, Inc.)

Expression of Recombinant Rat SE in E. coli

We synthesized two primers, 5`-GAATTCCATATGTGGACTTTTCTTGGAAT-3` and 5`-GAATTCGGATCCTCAGTGAACCAGATACTTCA-3`, which contain the sequences corresponding to amino acids 1-6 and 568-573 of rat SE, respectively. Polymerase chain reaction (PCR) was carried out using these primers and pUCTb1 as template. The PCR product was digested with BamHI and NdeI, and this fragment was ligated into the BamHI-NdeI large fragment of pET3a(27) . This expression vector (pETRSE) was sequenced and introduced into E. coli BL21(DE3). Transformants containing control vector (pET3a) or pETRSE were grown at 37 °C in LB medium to an optical density of 0.5 at 600 nm and isopropyl-beta-D-thiogalactopyranoside was added to 0.4 mM for induction. After a 1.5-h incubation, cells were harvested and SE enzymatic activity was assayed.

Enzymatic Assay for SE

S and P fractions from E. coli lysates were prepared as follows. Cells collected from a 250-ml culture were suspended in 12.5 ml of buffer A (50 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 0.4 M NaCl, 5 mM MgCl(2), 5% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM DTT) containing 1 mg/ml lysozyme for 1 h on ice. This suspension was frozen and thawed twice and adjusted to a final concentration of 1 mM EDTA and 0.5% Triton X-100. Next the suspension was sonicated and centrifuged at 5,000 times g. The supernatant was used as the S fraction. The pellet was dissolved in 5 ml of urea buffer (50 mM Tris-HCl, pH 8.0, 1% Triton X-100, 6 M urea, 5% glycerol, 50 mM phenylmethylsulfonyl fluoride, 1 mM DTT), and incubated on ice for 2 h. The suspension was then centrifuged at 100,000 times g for 2 h, and the supernatant dialyzed with buffer A and used as the P fraction in the following assay. Assay mixtures contained 20 mM Tris-HCl, pH 7.5, 0.01 mM FAD, 0.2 unit of NADPH-cytochrome c (P-450) reductase, 0.01 mM [^14C]squalene (dispersed with the aid of 20 µl of Tween 80), 0.2% Triton X-100, and 1 mM NADPH in a total volume of 0.5 ml. The NADPH was added to initiate the reaction. Mixtures were incubated at 37 °C for 30 min. The nonsaponifiable lipids were analyzed by silica gel TLC developed with ethyl acetate/benzene (0.5:99.5, v/v) as described previously(20) .

Immunoblotting Analysis of SE

Crude extracts from rat liver microsomes and a partially purified SE fraction from the extract were prepared as described previously(28) . Antibodies against rat SE were produced by immunizing rabbits with a bacterially produced His(6)-tagged protein consisting of amino acids 100-573 of rat SE. The fusion protein was constructed in the pET3a vector. An initiator methionine and 6 consecutive histidine residues were placed at the NH(2)-terminal and COOH-terminal ends, respectively, of the PCR-generated rat SE sequence. The protein was expressed in E. coli and purified on a Ni-NTA-agarose column according to the instructions supplied by the manufacturer (Qiagen). Antiserum against rat SE was used at a 1:1000 dilution after antibodies against bacterial proteins were absorbed by bacterial proteins conjugated to Affi-Gel 10 (Bio-Rad).

For immunoblot analysis, the antibody was visualized with horseradish peroxidase-conjugated goat anti-rabbit IgG (Life Technologies, Inc.), using Renaissance Western blot chemiluminescence reagent (DuPont NEN) according to the instructions of the manufacturer. Gels were calibrated with prestained low range molecular weight markers (Bio-Rad).


RESULTS AND DISCUSSION

Isolation of Rat SE cDNA

Our cloning strategy for rat SE employed a trans-complementation method in yeast. The yeast SE is specifically inhibited by the antimycotic agent terbinafine, so that yeast do not survive in its presence (Fig. 1). Mammalian SE is scarcely inhibited by terbinafine. In order to express the mammalian cDNA library in S. pombe, we used a method developed by Okazaki et al.(26) . A rat NRK cDNA library constructed with the pcD2 vector, which is driven by an SV40 promoter, was introduced into S. pombe. S. pombe allows transcription, splicing, and polyadenylation of inserted mammalian cDNAs. To suppress recombination and stabilize the plasmid in yeast, S. pombe was co-transformed with the cDNA library and PstI-digested pAL17, which contains an S. pombe-derived autonomously replicating sequence and the LEU2 gene. The two plasmids form a co-polymer DNA in the transformed cells. After screening 7 times 10^5 colonies of transformants in the presence of terbinafine, we isolated two terbinafine-resistant clones. To examine whether the terbinafine resistance was due to exogenous plasmid DNA or to mutations in the S. pombe chromosome, the plasmids were recovered from the clones and again transformed into S. pombe. Transformants containing one of the two plasmids survived again in the presence of terbinafine. The ability of transformants to grow in the presence of either terbinafine and/or NB-598, specific inhibitors of fungal and mammalian SE, respectively, was then tested (Fig. 2). The transformants survived in the presence of either terbinafine or NB598 but not in the presence of both terbinafine and NB-598. They must express, therefore, not only fungal SE but also rat SE.


Figure 1: Strategy for rat SE cDNA cloning. Tb stands for terbinafine.




Figure 2: Sensitivities of S. pombe transformants for inhibitors of SE. Transformants were grown on leucine-deficient MMA plates supplemented with nothing (Control), terbinafine (+Tb), NB598 (+NB), and both terbinafine and NB598 (+Tbbullet+NB). Terbinafine and NB 598 concentration was 12.5 µg/ml.



Structure of Rat SE

We determined the nucleotide sequence of the rat SE cDNA. The 2199-bp cDNA contains a 5` non-coding region of 199 bp, an open reading frame of 1719 bp and a 3` non-coding region of 281 bp. A polyadenylation signal precedes the 3`-terminal poly(A) sequence by 20 bp. The long 5` non-coding sequence and the presence of a poly(A) tail suggest that this clone contains almost the entire cDNA sequence. An in-frame stop codon is located 3 nucleotides upstream of the first ATG in the open reading frame. The sequence surrounding this ATG matches well the Kozak consensus translation initiation sequence (29) , strongly suggesting that this ATG functions as the initiation codon. The deduced protein sequence consists of 573 amino acids with a molecular mass of 63,950 Da (Fig. 3).


Figure 3: Comparison of the amino acid sequences of the predicted rat liver and yeast SE. Amino acid residues are numbered on the left. R and Y stand for rat and yeast (S. cerevisiae) SE, respectively. Residues identical in rat and yeast SE are boxed. The hydrophobic region Leu-Tyr, which is predicted to be a membrane associated domain, is underlined with a singleline. The putative FAD binding domain is underlined with a doubleline.



The hydropathy plot, using a standard algorithm of Kyte and Doolittle (30) , revealed one possible transmembrane domain in the NH(2)-terminal portion (Leu-Tyr) of the protein. This domain consists of 70% hydrophobic residues, which could be sufficient to anchor the protein in the membrane of the endoplasmic reticulum(31, 32) .

As shown in Fig. 4, there is a beta1-alphaA-beta2 motif (33) in both rat SE (Val-Glu) and S. cerevisiae SE (Ala-Glu) that demonstrates significant homology with the FAD binding domains of human erythrocyte glutathione reductase (33) and Pseudomonas fluorescensp-hydroxybenzoic acid hydroxylase(34) . The presence of a consensus FAD binding domain sequence is consistent with data suggesting that rat SE requires exogenous FAD for its activity (19) and strongly suggests that SE is directly associated with FAD.


Figure 4: Putative FAD binding site sequences of SE. The highly conserved beta1-alphaA-beta2 motifs of the yeast and rat SE and two flavoenzymes are depicted. Completely conserved amino acid residues are boxed with boldlines, hydrophobic residues in which conservative changes are tolerated are boxed with lightlines.



Comparison of the Rat SE Sequence with Yeast SE

The nucleotide sequence of rat SE cDNA is 38.3% (659/1719) identical to that of allylamine-resistant S. cerevisiae SE(25) . The FASTA search algorithm of the GenPept data bank showed that the deduced protein sequence of rat SE is 30.2% (173/573) identical to that of allylamine-resistant S. cerevisiae SE (Fig. 3). The rat SE contains 77 additional amino acids compared to the S. cerevisiae SE. In addition, the amino acid composition of rat SE is considerably more basic than yeast SE. The S. cerevisiae SE sequence begins at Glu of the rat SE, but includes a stretch of 31 amino acids (Lys-Asp) not present in the rat SE. It is interesting that S. cerevisiae SE lacks the putative transmembrane domain located in the NH(2)-terminal region of rat SE. This suggests that S. cerevisiae and rat SE are associated with the endoplasmic reticulum in different manners.

Expression of Rat SE cDNA in Escherichia coli

We selected E. coli for expression of the isolated cDNA, since this bacterium does not synthesize 2,3-oxidosqualene or sterols in general. The expression vector pETRSE, which contains the entire coding region of rat SE downstream of the bacteriophage T7 promoter, was used to transform E. coli BL21(DE3). S and P fractions (see ``Experimental Procedures'') of E. coli lysates were prepared from control vector (pET3a) or pETRSE containing transformants, and assayed for SE activity. As shown in Fig. 5A, negligible activity was detected in the S and P fractions from a pET3a containing transformant (A, lanes 1 and 2), but significant SE activity was observed in the S fraction from a pETRSE transformant (A, lane3). These data clearly demonstrate that the isolated cDNA encodes a functional SE and, therefore, that rat SE is encoded by a single polypeptide chain.


Figure 5: Identification of SE activity of recombinant rat SE and immunoblotting analysis of recombinant and native rat SE. A, cell extracts were prepared as described under ``Experimental Procedures.'' Partially purified rat liver SE and cell extracts were incubated with [^14C]squalene for 30 min at 37 °C in the presence of 0.2 unit of NADPH-cytochrome c reductase, 1 mM NADPH, 0.01 mM FAD, and 0.3% Triton X-100. Total nonsaponifiable substances were applied to thin-layer chromatography plates, which were developed with 0.5% ethyl acetate/benzene and detected autoradiographically. The migration of standard compounds on the plate is indicated by arrows. The samples shown are: lanes1 and 2, S and P fractions, respectively, from control vector pET3a-transformed E. coli; lanes3 and 4, S and P fractions, respectively, from rat SE expression vector pETRSE-transformed E. coli. B, 20 µg of fractions, described in panel A, were separated on a 10% SDS-polyacrylamide gel in the same lanes as in panel A. They were blotted electrophoretically onto nitrocellulose membrane and analyzed immunochemically with anti-recombinant rat SE antiserum as mentioned under ``Experimental Procedures.'' C, 30 µg of partially purified SE fraction from rat liver (lane1) and crude extract from rat liver microsomes (lane2) were analyzed immunochemically as in panelB. The samples were prepared as described previously(28) .



In order to confirm the expression of rat SE protein in the transformants, we performed an immunoassay using an antiserum against the purified recombinant Glu-His fragment of rat SE expressed in E. coli. Although this fragment excluded the plausible membrane binding domain, it did contain the SE activity. As shown in Fig. 5B, a 64-kDa immunoreactive protein was present in both the S and P fractions of a pETRSE containing transformant (B, lanes 3 and 4). We also detected a truncated protein in the P fraction and small fragments in the S fraction. In spite of the existence of the immunoreactive protein, the P fraction did not show any SE activity (Fig. 5A, lane 4). This might be caused by insufficient renaturation by dialysis after solubilization of particulates with urea buffer.

Only a single immunoreactive protein, with relative molecular mass of 64 kDa, was detected in rat liver microsomes (Fig. 5C, lane2). Although the partially purified SE fraction from rat liver also contained an immunoreactive protein of 64 kDa, two other immunoreactive proteins of 57 and 51 kDa were detected. These results suggest that the molecular mass of rat SE is 64 kDa, and that a 51-kDa protein previously identified as rat SE (28) is most likely a truncated form of the enzyme produced by proteolysis during the purification procedure.

Both the recombinant rat SE and the partially purified SE from rat liver lost activity when Triton X-100 was absent during purification. Recombinant rat SE behaves as a multimeric aggregate during gel filtration in the absence of Triton X-100. Enzymatic activation by Triton X-100 at a concentration greater than its critical micelle concentration, in the presence of 1 mM DTT, suggests that the monomer is the active form of the enzyme. However, there are a number of alternative explanations for the Triton X-100 activation, such as a conformational change of the enzyme, an increased rate of enzyme diffusion or association of enzyme and substrate, and effects on enzyme kinetic parameters.

Until now mammalian liver SE had not been purified, due to its poor stability, and little is known of its structure or properties. Our new cloning strategy using a species-specific inhibitor of the enzyme demonstrates the utility of this trans-complementation cloning method, and has enabled us to produce the first purified SE. The availability of this cloned DNA will now permit detailed studies on the structure and properties of rat SE, and should also aid in the development of more effective inhibitors of SE. In addition, more precise studies of the regulation of the enzyme will now be feasible. Although it has been suggested that SE is regulated by sterols(8, 9) , the mechanism of this regulation is not known. The availability of a rat SE clone, and antibodies to the SE protein, will allow us to determine whether this regulation by sterols occurs at the level of mRNA synthesis, SE translation, or increased SE activity. Finally, we should now be able to determine if SE is involved in oxysterol formation.


FOOTNOTES

*
This work was supported by Grants-in-aid 06770080 and 06557135 for Scientific Research from the Ministry of Education, Science and Culture, Japan. Financial support was also provided by the Cosmetology Research Foundation, by ONO Medical Research Foundation, and by the Chiyoda Mutual Life Foundation, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D37920[GenBank].

§
To whom correspondence should be addressed: Dept. of Biochemistry, Niigata University School of Medicine, 1-757 Asahimachidori, Niigata 951, Japan. Tel.: 81-25-223-6161; Fax: 81-25-222-4599.

(^1)
The abbreviations used are: SE, squalene epoxidase; PCR, polymerase chain reaction; DTT, dithiothreitol; bp, base pair(s).


ACKNOWLEDGEMENTS

We thank Drs. J. Stein and J. Storch for critical review of the manuscript and valuable comments. We also thank Drs. H. Okayama and A. Nagata for the rat NRK cDNA library and pAL17, Drs. Y. Kimura and S. Matsumoto for JY266, and Dr. T. Kamei for the SE inhibitors.


REFERENCES

  1. Goldstein, J. L., and Brown, M. S. (1990) Nature 343, 425-430 [CrossRef][Medline] [Order article via Infotrieve]
  2. Rudney, H., and Panini, R. (1993) Curr. Opin. Lipidol. 4, 230-237
  3. Steinberg, D. (1988) Atheroscler. Rev. 18, 1-23
  4. Glomset, J. A., Gelb, M. H., and Farnsworth, C. C. (1993) Trends Biochem. Soc. 4, 230-237
  5. Gibbs, J. B. (1991) Cell 65, 1-4 [Medline] [Order article via Infotrieve]
  6. Ryder, N. S., and Dupont, M. C. (1985) Biochem. J. 230, 765-770 [Medline] [Order article via Infotrieve]
  7. Horie, M., Tsuchiya, Y., Hayashi, M., Iida, Y., Iwasawa, Y., Nagata, Y., Sawasaki, Y., Fukuzumi, H., Kitani, K., and Kamei, T. (1990) J. Biol. Chem. 265, 18075-18078 [Abstract/Free Full Text]
  8. Hidaka, Y., Satoh, T., and Kamei, T. (1990) J. Lipid Res. 31, 2087-2094 [Abstract]
  9. Satoh, T., Hidaka, Y., and Kamei, T. (1990) J. Lipid Res. 31, 2095-2101 [Abstract]
  10. Gonzalez, R., Carlson, J. P., and Dempsey, M. E. (1979) Arch. Biochim. Biophys. 196, 574-580 [Medline] [Order article via Infotrieve]
  11. Nelson, J. A., Czarny, M. R., Spencer, T. A., Limanek, J. S., McCrae, K. R., and Chang, T. Y. (1978) J. Am. Chem. Soc. 100, 4900-4902
  12. Chang, T. Y., Schiavoni, E. S., Jr., McCrae, E. S., Nelson, J. A., and Spencer, T. A. (1979) J. Biol. Chem. 254, 11258-11263 [Medline] [Order article via Infotrieve]
  13. Popjak, G., Meenan, A., and Nes, W. D. (1978) Proc. R. Soc. Lond. B Biol. Sci. 232, 273-287
  14. Nelson, J. A., Steckbeck, S. R., and Spencer, T. A. (1981) J. Biol. Chem. 256, 1067-1078 [Abstract/Free Full Text]
  15. Saucier, S. E., Kandutsch, A. A., Taylor, F. R., Spencer, T. A., Phirwa, S., and Gayen, A. K. (1985) J. Biol. Chem. 260, 14571-14579 [Abstract/Free Full Text]
  16. Spencer, T. A. (1994) Acc. Chem. Res. 27, 83-90
  17. Yamamoto, S., and Bloch, K. (1970) J. Biol. Chem. 245, 1670-1674 [Abstract/Free Full Text]
  18. Tai, H. H., and Bloch, K. (1972) J. Biol. Chem. 247, 3767-3773 [Abstract/Free Full Text]
  19. Ono, T., and Bloch, K. (1975) J. Biol. Chem. 250, 1571-1579 [Abstract]
  20. Ono, T., and Imai, Y. (1985) Methods Enzymol. 110, 375-380 [Medline] [Order article via Infotrieve]
  21. Ono, T., Okayasu, T., Kameda, K., and Imai, Y. (1979) in Biochemical Aspects of Nutrition (Yagi, K. ed) pp. 115-123, Japan Scientific Societies Press, Tokyo
  22. Gollub, E. G., Trocha, P., Lilu, P. K., and Sprinson, D. (1974) Biochem. Biophys. Res. Commun. 56, 471-477 [Medline] [Order article via Infotrieve]
  23. Gollub, E. G., Lilu, K. P., Dayan, J., Aldersberg, M., and Sprinson, D. (1977) J. Biol. Chem. 252, 2846-2854 [Abstract]
  24. Richard, D. S., Applebaum, S. W., Sliter, T. J., Baker, F. C., Schooley, D. A., Reuters, C. C., Henrich, V, C., and Gilbert, L. I. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1421-1425 [Abstract]
  25. Jandrositz, A., Turnowsky, F., and Hogenauer, G. (1991) Gene (Amst.) 107, 155-160 [CrossRef][Medline] [Order article via Infotrieve]
  26. Okazaki, K., Okazaki, N., Kume, K., Jinno, S., Tanaka, K., and Okayama, H. (1991) Nucleic Acids Res. 18, 6485-6489 [Abstract]
  27. Rosenberg, A. H., Lade, B. N., Chui, D. S., Lin, S. W., Dunn, J. J., and Studier, F. W. (1987) Gene (Amst.) 56, 125-135 [CrossRef][Medline] [Order article via Infotrieve]
  28. Ono, T., Nakazono, K., and Kosaka, H. (1982) Biochim. Biophys. Acta 709, 84-90 [Medline] [Order article via Infotrieve]
  29. Kozak, M. (1987) Nucleic Acids Res. 15, 8125-8131 [Abstract]
  30. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 175, 105-132
  31. Bretscher, M. S., and Munro, S. (1993) Science 261, 1280-1281 [Medline] [Order article via Infotrieve]
  32. Engelman, D. M., Steitz, T. A., and Goldman, A. (1986) Annu. Rev. Biophys. Biophys. Chem. 15, 321-353 [CrossRef][Medline] [Order article via Infotrieve]
  33. Wierenga, R. K., Terpstra, P., and Hol, W. G. J. (1986) J. Mol. Biol. 187, 101-107 [Medline] [Order article via Infotrieve]
  34. Schulz, G. E., Schrimer, R. H., and Pai, E. F. (1982) J. Mol. Biol. 160, 287-308 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.