' '
`
`· - - - ~ ·-
`.---
`·.
`. .... .
`..
`
`"
`
`'
`
`~-
`
`... :·"'
`
`.
`, .. ·-,
`'
`
`•
`
`_.,.
`
`~ ..
`
`an international journa~ ~.-oct1sing on gene
`·. .cloning and gene structure , ~nd function
`. - ,, .
`
`... . ,.,.
`
`.
`
`,:_.
`
`'l'\
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`;:. . ._,
`
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`
`ELSEVIER
`
`1995
`
`Completing this volume
`
`Volume 167 Nos. 1 and 2
`
`LCY Biotechnology Holding, Inc.
`Ex. 1065
`Page 1 of 14
`
`

`

`© 1995, Elsevier Sci ence B.V. All rights reserved .
`No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical,
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`
`LCY Biotechnology Holding, Inc.
`Ex. 1065
`Page 2 of 14
`
`

`

`gene
`
`an international journal focusing on gene
`cloning and gene structure and function
`
`EDITOR-IN-CHIEF: W. Szybalski, Madison, WI (U.S.A.)
`
`EDITORIAL BOARD
`
`M. Bagdasarian, Lansing, Ml
`F. Barany, New York, NY
`M. Belfort, Albany, NY
`M.J. Benedik, Houston, TX
`G. Bernardi, Paris
`F. Bolivar, Cuernavaca
`A.M . Campbell, Stanford, CA
`A.M. Chakrabarty, Chicago, IL
`K.F. Chater, Norwich
`R.W. Davies, Glasgow
`A. Dugaiczyk, Riverside, CA
`J.A. Engler, Birmingham , AL
`G.N. Godson, New York, NY
`R.M . Harshey, Austin, TX
`J.A. Hoch , La Jolla, CA
`Z. Hradecna, Brno
`C.M . Kane, Berkeley, CA
`J .R. Kinghorn, St. Andrews
`J.K.C. Knowles, Geneva
`S.R. Kushner, Athens, GA
`G.P. Livi, King of Prussia, PA
`
`Vol. 167
`
`P.A. Manning, Adelaide
`J. Marmur, New York, NY
`J. Messing, Piscataway, NJ
`A. Nakazawa, Ube
`V. Paces, Prague
`R. Padmanabhan, Kansas City, KS
`S.D.J. Pena, Belo Horizonte
`A .J. Podhajska, Gdansk
`J.P. Richardson, Bloomington, IN
`A .O. Riggs, Duarte, CA
`Y. Sakaki, Tokyo
`M. Salas, Madrid
`T. Sekiya, Tokyo
`P.F.G. Sims, Manchester
`A.-M . Skalka, Philadelphia, PA
`J .L. Slightom, Kalamazoo, Ml
`W.F . Stevens, Bangkok
`H. van Ormond!, Leiden
`J. Wild, Madison, WI
`R.E. Yasbin, Baltimore, MD
`H. Yoshikawa, lkoma City
`H.G. Zachau, Munich
`
`ELSEVIER
`AMSTERDAM - LAUSANNE - NEW YORK - OXFORD - SHANNON - TOKYO
`
`LCY Biotechnology Holding, Inc.
`Ex. 1065
`Page 3 of 14
`
`

`

`Gene, 167 (1995) 347- 352
`Elsevier
`
`AUTHOR INDEX VOLUME 167 (1995)
`
`Airenne, K.J. and Kulomaa, M.S., Rapid purification of recombinant proteins fused to chicken avidin
`Aitken, R., see Catherine Sinclair, M.
`Albani, D. and Robert, L.S., Coning and characterization of a Brassica napus gene encoding a homologue of the B subunit of
`a heteromeric CCAAT-binding factor
`Altamura, S., see Cabibbo, A.
`Amemura; A., see Fujita, M.
`Anton, D.L., see Payne, M.S.
`Arai, H., see Kawasaki, S.
`Arrecubieta, C., Garcia, E. and Lopez, R., Sequence and transcriptional analysis of a DNA region involved in the production
`of capsular polysaccharide in Streptococcus pneumoniae type 3
`
`Bakhshi, M .R., see Janel, N.
`Baralle, F.E., see Tisminetzky, S.
`Barbe, J., see Jordan, A.
`Barbier, G., see Bouyo ub, A.
`Barek, L., see Janel, N.
`Barlati, S., see Tisminetzky, S.
`Baud, E., see Schwaab, V.
`Bayles, K.W., see Domanski, T.L.
`Benitez, E., see Bosch, T.C.G.
`Bennett, J., Sun, D. and Karik6, K ., Sequence analysis of the 5.34-kb 5' flanking region of the human rhodopsin-encoding gene
`Bianchi, G., see Tisminetzky, S.
`Bosch, T.C.G., Benitez, E., Gellner, K ., Praetzel, G. and Salgado, L.M., Cloning ofa ras-related gene from Hydra which responds
`to head-specific signals
`Bouyoub, A., Barbier, G., Querellou, J. and Forterre, P., A putative SOS repair gene (dinF-like) in a hyperthermophilic archaeon
`Brickner, A.G., Gossage, D.L., Dusing, M.R. and Wiginton, D.A., Identification of a murine homolog of the human adenosine
`deaminase thymic enhancer
`
`Cabibbo, A., Sporeno, E., Toniatti, C., Altamura, S., Savino, R., Paonessa, G. and Ciliberto, G., Monovalent phage display of
`human interleukin (hIL)-6: selection of superbinder variants from a complex molecular repertoire in the hIL-6 D-helix
`Caricasole, A., see Ward, A.
`Carvajal, E., see McGuire, T.M.
`Chang, Y.C., Wickes, B.L. and Kwon-Chung, K.J., Further analysis of the CAP59 locus of Cryptococcus neoformans: structure
`defined by forced expression and description of a new ribosomal protein-encoding gene
`Chepelinsky, A.B., see Wang, X.Y.
`Chernoff, J., see Creasy, C.L.
`Chevallet, M ., see Dupuis, A.
`Ciliberto, G., see Cabibbo, A.
`Cole, G.T., see Pishko, E.J.
`Colombi, M., see Tisminetzky, S.
`Courtney, M.-A., see Simpson-Haidaris, P.J.
`Coyne Jr., M.J. and Goldberg, J.B., Cloning and characterization of the gene (rfc) encoding O-antigen polymerase of
`Pseudomonas aeruginosa PAOl
`Creasy, C.L. and Chernoff, J., Cloning and characterization of a member of the MST subfamily of Ste20-like kinases
`Cundliffe, E., see Thiara, A.S.
`
`Darrouzet, E., see Dupuis, A.
`Dashper, S., see Jackson, C.A.
`Delrot, S., see Montamat, F.
`Devescovi, G., see Tisminetzky, S.
`Dew-Jager, K., Yu, W.-Q. and Huang, W.M., The recA gene of Borrelia burgdorferi
`DiCosimo, R., see Payne, M.S.
`Dietel, M., see Holm, P.S.
`Domanski, T.L. and Bayles, K.W., Analysis of Staphylococcus aureus genes encoding penicillin-binding protein 4 and an ABC(cid:173)
`. type transporter
`Drevet, J.R., see Schwaab, V.
`Driscoll, J., see Rowland, B.
`
`347
`
`167 ( 1995)
`63
`167 ( 1995) 285
`
`167 ( 1995) 209
`167 (1995)
`41
`167 (1995)
`93
`167 (1995) 215
`167 ( 1995)
`87
`
`167 ( 1995)
`
`167 (1995) 291
`167 (1995) 313
`167 ( 1995)
`75
`167 ( 1995) 147
`167 (1995) 291
`167 (1995) 313
`167 (1995)
`25
`167 (1995) 111
`167 (1995) 191
`167 (1995) 317
`167 (1995) 313
`
`167 (1995) 191
`167 (1995) 147
`
`167 ( 1995) 261
`
`41
`167 ( 1995)
`167 ( 1995) 239
`167 (1995) 151
`
`167 ( 1995) 179
`167 ( 1995) 321
`167 ( 1995) 303
`167 (1995)
`99
`167 (1995)
`41
`167 ( 1995) 173
`167 ( 1995) 313
`167 ( 1995) 273
`
`167 ( 1995)
`81
`167 ( 1995) 303
`167 ( 1995) 121
`
`167 ( 1995) 99
`167 ( 1995) 127
`167 ( 1995) 197
`167 (1995) 313
`167 ( 1995) 137
`167 ( 1995) 215
`167 (1995) 221
`
`167 (1995) 111
`167 ( 1995)
`25
`167 ( 1995) 105
`
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`
`

`

`348
`
`Duarte, A., see Ward, A.
`Dubin, R.A., Kazmi, M.A. and Ostrer, H., Inverted repeats are necessary for circularization of the mouse testis Sry transcript
`Dufaure, J.-P., see Schwaab, V.
`Dupuis, A., Peinnequin, A., Chevallet, M., Lunardi, J., Darrouzet, E., Pierrard, B., Procaccio, V. and Issartel, J.-P., Identification
`of five Rhodobacter capsulatus genes encoding the equivalent of ND subunits of the mitochondrial NADH-ubiquinone
`oxidoreductase
`Dusing, M .R., see Brickner, A.G.
`Dyer, D.W., see Giardina, P.C.
`
`Earnest, B.J., see Simpson-Haidaris, P.J.
`
`Faerman, M., Filon, D., Kahila, G., Greenblatt, C.L., Smith, P. and Oppenheim, A., Sex identification of archaeological human
`remains based on amplification of the X and Y amelogenin alleles
`Feltens, R., see Pfeiffer, T.
`Filon, D., see Faerman, M.
`Forterre, P., see Bouyoub, A.
`Foster, L.-A., see Giardina, P.C.
`Frandsen, N., see Guerout-Fleury, A.-M.
`Frye, J.G., see Stamm, L.V.
`Fujita; M ., Hanaura, Y. and Amemura, A., Analysis of the rpoD gene encoding the principal sigma factor of Pseudomonas putida
`
`Garcia, E., see Arrecubieta, C.
`Gautvik, K .M., see Mathavan, S.
`Gautvik, V.T., see Mathavan, S.
`Gavagan, J.E., see Payne, M.S.
`Gellner, K., see Bosch, T.C.G.
`Ghyselinck, N., see Schwaab, V.
`Giancotti, V., see Manfioletti, G.
`Giardina, P.C., Foster, L.-A., Toth, S.I., Roe, B.A. and Dyer, D.W., Identification of alcA , a Bordetella bronchiseptica gene
`necessary for alcaligin production
`Gibert, I., see Jordan, A.
`Gigliotti, F ., see Wright, T.W.
`Goffeau, A., see McGuire, T.M.
`Goldberg, J.B., see Coyne Jr., M.J.
`Golightly, E.J., see Thompson, S.A.
`Golin, J., see McGuire, T.M.
`Goodwin, G.H., see Manfioletti, G.
`Gossage, D.L., see Brickner, A.G.
`Greenblatt, C.L., see Faerman, M .
`Grenet, J., see Li, H.
`Guerout-Fleury, A.-M., Shazand, K ., Frandsen, N. and Stragier, P., Antibiotic-resistance cassettes for Bacillus subtilis .
`Guilfoyle, T.J., see Ulmasov, T.
`Guilloton, M ., see Montamat, F.
`
`Haas, H ., see Marx, F.
`Haas, R., see Kahrs, A.F.
`Habib, G .M., see Shi, Z.-Z.
`Haidaris, C.G., see Wright, T.W.
`Hanaura, Y., see Fujita, M.
`Hansen, K .R., Resta, R., Webb, C.F. and Thompson, L.F., Isolation and characterization of the promoter of the human
`5'-nucleotidase (CD73)-encoding gene
`Hardham, J.M., see Stamm, L.V.
`Hartmann, R.K., see Pfeiffer, T.
`Hashimoto-Gotoh, T., Yasojima, K. and Tsujimura, A., Plasmids with a kanamycin-resistance gene for site-directed muta'.genesis
`using the oligodeoxyribonucleotide-directed dual amber method
`Hastie, N.D., see Ward, A.
`Heuermann, D., see Kahrs, A.F.
`Hill, K., see Rowland, B.
`Holm, P.S., Dietel, M. and Krupp, G., Similar cleavage efficiencies of an oligoribonucleotide substrate and an mdrl mRNA
`segment by a hammerhead ribozyme
`Horikoshi, M., see Umehara, T.
`Huang, W.M ., see Dew-Jager, K.
`Hui, Z., see Simpson-Haidaris, P.J.
`
`167 (1995) 239
`167 ( 1995) 245
`167 ( 1995) 25
`
`167 (1995) 99
`167 (1995) 261
`167 ( 1995) 133
`
`167 ( 1995) 273
`
`167 ( 1995) 327
`167 ( 1995) 141
`167 ( 1995) 327
`167 ( 1995) 147
`167 (1995) 133
`167 (1995) 335
`167 ( 1995) 339
`167 ( 1995)
`93
`
`1
`167 (1995)
`33
`167 (1995)
`167 ( 1995)
`33
`167 ( 1995) 215
`167 ( 1995) 191
`167 ( 1995) 25
`167 ( 1995) 249
`
`167 ( 1995) 133
`167 (1995)
`75
`167 ( 1995) 185
`167 (1995) 151
`167 (1995)
`81
`167 (1995) 337
`167 (1995) 151
`167 ( 1995) 249
`167 (1995) 261
`167 (1995) 327
`167 (1995) 341
`167 (1995) 335
`16_7 (1995) 203
`167 ( 1995) 197
`
`167 ( 1995) 167
`53
`167 ( 1995)
`167 ( 1995) 233
`167 (1995) 185
`167 ( 1995) 93
`
`167 (1995) 307
`167 ( 1995) 339
`167 ( 1995) 141
`
`167 (1995) 333
`167 ( 1995) 239
`53
`167 ( 1995)
`167 ( 1995) 105
`
`167 ( 1995) 221
`167 (1995) 297
`167(1995) 137
`167 (1995) 273
`
`LCY Biotechnology Holding, Inc.
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`Page 5 of 14
`
`

`

`lgarashi, Y., see Kawasaki, S.
`Issartel, J.-P., see Dupuis, A.
`Ito, M ., see Izuhara, M .
`Izuhara, M., Ito, M. and Takagi, Y., Transcription of the rat liver uricase-encoding gene is regulated via a cis-acting element
`responsive to cAMP
`
`Jackson, C.A., Kirszbaum, L., Dash per, S. and Reynolds, E.C., Cloning, expression and sequence analysis of the genes encoding
`the heterodimeric methylmalonyl-CoA mutase of Porphyromonas gingivalis W50
`Janel, N., Schwachtgen, J.-L., Bakhshi, M .R., Barek, L., Meyer, D. and Kerbiriou-Nabias, D., Comparison of the 5'-flanking
`sequences of the human and bovine von Willebrand factor-encoding genes reveals alternation of highly homologous domains
`with species-specific Alu-type repeats
`Jannatipour, M., see Labbe, J.-C.
`Jaworski, C.J., see Wang, X.Y.
`Jorcke, D, see Pfeiffer, T.
`Jordan, A., Gibert, I. and Barbe, J., Two different operons for the same function: comparison of the Salmonella typhimurium
`nrdAB and nrdEF genes
`
`Kahila, G., see Faerman, M.
`Kahrs, A.F., Odenbreit, S., Schmitt, W., Heuermann, D., Meyer, T.F. and Haas, R., An improved TnMax mini-transposon
`system suitable for sequencing, shuttle mutagenesis and gene fusions
`Kareem, B.N., see Mathavan, S.
`Karik6, K., see Bennett, J.
`Karst, F., see Montamat, F.
`Katzmann, D., see McGuire, T.M.
`Kawasaki, S., Arai, H., lgarashi, Y. and Kodama, T., Sequencing and characterization of the downstream region of the genes
`encoding nitrite reductase and cytochrome c-551 (nirSM) from Pseudomonas aeruginosa: identification of the gene necessary
`for biosynthesis of heme dl
`Kazmi, M.A., see Dubin, R.A.
`Kerbiriou-Nabias, D., see Janel, N .
`Kida, S., see Umehara, T.
`Kidd, V.J., see Li, H.
`Kimura, H., see Takizawa, N.
`Kirkland, T.N., see Pishko, E.J.
`Kirszbaum, L., see Jackson, C.A.
`Kodama, T., see Kawasaki, S.
`Kohler, C., see Liang, X.
`Kortt, A.A., see Riffkin, M.C.
`Krueger, K.E., see Oberto, A.
`Krupp, G., see Holm, P.S.
`Kulomaa, M.S., see Airenne, K.J.
`Kwon-Chung, K.J., see Chang, Y.C.
`
`Labbe, J.-C., Jannatipour, M. and Rokeach, L.A., The Caenorhabditis elegans rop-1 gene encodes the homologue of the human
`60-kDa Ro autoantigen
`Larkin, R.M., see Ulmasov, T.
`Lebovitz, R.M., see Shi, Z.-Z.
`Lee, C.Y., Association of staphylococcal type-1 capsule-encoding genes with a discrete genetic element
`Lee, N.S., see Valinluck, B.
`Li, H., Grenet, J. and Kidd, V.J., Structure and gene expression of avian cyclin D2
`Li, K., see Liang, X.
`Liang, X., Oono, Y., Shen, N.F., Kohler, C., Li, K., Scolnik, P.A. and Theologis, A., Characterization of two members (ACSJ
`and ACS3) of the 1-aminocyclopropane-1-carboxylate synthase gene family of Arabidopsis thaliana
`Lieberman, M.W., see Shi, Z.-Z.
`Liras, P ., see Rodriguez-Garcia, A.
`Longone, P., see Oberto, A.
`Lopez, R., see Arrecubieta, C.
`Lottspeich, F., see Marx, F.
`Lunardi, J., see Dupuis, A.
`
`Maeda, S., see Mathavan, S.
`Manfioletti, G., Rustighi, A., Mantovani, F., Goodwin, G.H. and Giancotti, V., Isolation and characterization of the gene
`coding for murine high-mobility-group protein HMGI-C
`Mantovani, F., see Manfioletti, G.
`Martin, J.F., see Rodriguez-Garcia, A.
`
`349
`
`167 (1995)
`87
`167 ( 1995) 99
`167 ( 1995) 267
`
`167 ( 1995) 267
`
`167 ( 1995) 127
`
`167 ( 1995) 291
`167 ( 1995) 227
`167 (1995) 321
`167 ( 1995) 141
`
`167 ( 1995)
`
`75
`
`167 (1995) 327
`
`167 ( 1995)
`53
`167 (1995)
`33
`167 ( 1995) 317
`167 ( 1995) 197
`167 (1995) 151
`
`167 ( 1995)
`87
`167 (1995) 245
`167 ( 1995) 291
`167 ( 1995) 297
`167 ( 1995) 341
`167 (1995) 343
`167 (1995) 173
`167 (1995) 127
`167 ( 1995) 87
`167 (1995)
`17
`167 (1995) 279
`167 ( 1995) 255
`167 ( 1995) 221
`167 (1995)
`63
`167 ( 1995) 179
`
`167 ( 1995) 227
`167 ( 1995) 203
`167 ( 1995) 233
`167 ( 1995) 115
`167 ( 1995)
`59
`167 (1995) 341
`167 (1995)
`17
`
`167 ( 1995) 17
`167 (1995) 233
`167 ( 1995)
`9
`167 ( 1995) 255
`167 (1995)
`167 ( 1995) 167
`167 ( 1995) 99
`
`167 ( 1995)
`
`33
`
`167 ( 1995) 249
`167 (1995) 249
`167 (1995)
`9
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`350
`
`Marx, F., Haas, H., Reindl, M., Stoffler, G., Lottspeich, F. and Red!, B., Cloning, structural organization and regulation of
`expression of the Penicillium chrysogenum paf gene encoding an abundantly secreted protein with antifungal activity
`Masuda, Y., Park, S.M., Ohta, A. and Takagi, M., Cloning and characterization of the POX2 gene in Candida maltosa
`Mathavan, S., Gautvik, V.T., Rokkones, E., Olstad, O.K., Kareem, B.N., Maeda, S. and Gautvik, K.M., High-level production
`of human parathyroid hormone in Bombyx mori larvae and BmN cells using recombinant baculovirus
`Mattei, M.-G., see Schwaab, V.
`McGuire, T.M.,- Carvajal, E., Katzmann, D., Wagner, M., Moye-Rowley, W.S., Goffeau, A. and Golin, J., Analysis of second-
`site mutations that suppress the multiple drug resistance phenotype of the yeast PDRl-7 allele
`Menzel, R., see Taylor, S.T.
`Meyer, D., see Janel, N.
`Meyer, T.F., see Kahrs, A.F.
`Miller, P., see Rowland, B.
`Miyagawa, K., see Ward, A.
`Montamat, F., Guilloton, M., Karst, F. and Delrot, S., Isolation and characterization of a cDNA encoding Arabidopsis thaliana
`3-hydroxy-3-methylglutaryl-coenzyme A synthase
`Moro, L., see Tisminetzky, S.
`Moye-Rowley, W.S., see McGuire, T.M.
`Muro, A., see Tisminetzky, S.
`
`Neroni, L.A., see Simpson-Haidaris, P.J.
`
`Oberto, A., Longone, P. and Krueger, K.E., Identification of three transcriptional regulatory elements in the rat mitochondrial
`benzodiazepine receptor-encoding gene
`Odenbreit, S., see Kahrs, A.F.
`Ohta, A., see Masuda, Y.
`Ohtaka-Maruyama, C., see Wang, X.Y.
`Olstad, O.K., see Mathavan, S.
`Oono, Y., see Liang, X.
`Oppenheim, A., see Faerman, M .
`Ostrer, H., see Dubin, R.A.
`
`Pannekoek, H., see van Zonneveld, A.-J.
`Paonessa, G., see Cabibbo, A.
`Park, S.M., see Masuda, Y.
`Payne, M.S., Petrillo, K.L., Gavagan, J.E., Wagner, L.W., DiCosimo, R. and Anton, D.L., High-level production of spinach
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`167 (1995)
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`167 ( 1995)
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`Gene, 167 (1995) 157- 161
`© 1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/ $09.50
`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`157
`
`GENE09360
`
`Cloning and characterization of the POX2 gene in Candida maltosa
`
`(Acyl~CoA oxidase; ~-oxidation; n-alkane; gene disruption; yeast)
`
`Yutaka Masuda, Sui;i Mee Park, Akinori Ohta and Masamichi Takagi
`
`Department of Agricultural Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan
`
`Received by G .P . Livi: 28 February 1995; Revised/ Accepted: 11 September/ 12 September 1995; Received at publishers: 14 September 1995
`
`SUMMARY
`
`To study the function of acyl-CoA oxidase in an n-alkane-assimilating yeast, Candida maltosa, we isolated the POX2
`gene which is a member of the acyl-CoA oxidase gene family. POX2 had a 2172-bp open reading frame (ORF) encoding
`an approx. 84-kDa polypeptide (724 amino acids (aa)) and was contiguous to POX4, another member of the acyl-CoA
`oxidase gene family on the same chromosomal DNA in a convergent arrangement. Northern blot analysis revealed that
`the expression of POX2 was induced in cells grown on oleic acid, n-tetradecanol and n-tetradecane. By using a gene(cid:173)
`disruption technique, we constructed strains (termed P2DD and P4DD) in which both alleles of POX2 and POX4 were
`disrupted. The P2DD strain was normal in assimilation of various hydrophobic carbon sources, such as n-tetradecane,
`n-tetradecanol and oleic acid. In contrast, the P4DD strain was defective in its ability to grow on such hydrophobic
`carbon sources.
`
`INTRODUCTION
`
`The transfer of Candida maltosa (Cm) cells from glu(cid:173)
`cose-medium to n-alkane medium causes the induction
`of various enzymes and proteins (Gallo et al., 1973;
`Sunairi et al., 1984) and alteration in the amounts of
`endoplasmic reticulum and peroxisomes in which these
`enzymes are located. This yeast provides a good model
`to investigate the mechanism of regulation of expression
`of genes involved in assimilation of hydrophobic com(cid:173)
`pounds. We have already cloned several genes of Cm
`involved in the initial step of n-alkane assimilation such
`
`Correspondence to: Dr. Y. Masuda at his present address: Department
`of Biological Chemistry, School of Pharmaceutical Sciences, Showa
`University, Hatanodai, Shinagawa-ku, Tokyo 142, Japan. Tel. (81-3)
`3784-8 218; Fax (81-3) 3784-8219; e-mail: masuda@pharm.showau.ac.jp
`
`Abbreviations: aa, amino acid(s); bp, base pair(s); C., Candida; Cm, C.
`maltosa; Ct, C. tropicalis; DD, double disruptant; E., Escherichia; kb,
`kilobase(s) or 1000 bp; nt, nucleotide(s); ORF, open reading frame;
`POX, gene(s) encoding PXP; PXP, peroxisomal polypeptide(s); S.,
`Saccharomyces; wt, wild type; : :, novel junction (fusion or insertion) .
`
`. SSDI 0378-1119(95)00655-9
`
`as cytochrome P-450 species (Takagi et al., 1989;
`Ohkuma et al., 1991).
`Peroxisomes of the yeast C. tropicalis (Ct) are responsi(cid:173)
`ble for ~-oxidation of fatty acids (Kawamoto et al., 1978)
`and more than 18 different proteins (PXPs) are induced
`in the presence of oleic acid (Kamiryo et al., 1982).
`Among these polypeptides, PXP2, PXP4 and PXP5 were
`identified as distinct long-chain acyl-CoA oxidases and
`the genes encoding these polypeptides were also cloned
`(Okazaki et al., 1986; 1987). Recently, Picataggio et al.
`( 1991) described the sequential disruption of acyl-CoA
`oxidase genes (POX4 and POX5) in Ct to show the genes
`are essential to utilize alkanes or fatty acids as a carbon
`sources. They also reported an efficient accumulation of
`dicarboxylic acid from a substrate (alkane or fatty acid)
`in these disruptants (Picataggio et al., 1992). In contrast
`to the importance of POX4 and POX5 in alkane assimila(cid:173)
`tion, the properties and function of POX2 gene are largely
`unknown. The present study describes the molecular
`cloning and characterization of Cm POX2 and POX4
`genes. We also examined the importance of these genes
`in n-tetradecane, n-tetradecanol and oleic acid assimila(cid:173)
`tion in Cm by using a gene-disruption technique. ·
`
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`158
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`EXPERIMENTAL AND DISCUSSION
`
`(a) Cloning and nt sequence of Cm POX2
`We screened the genomic library of Cm using the 1.5-kb
`BamHI-Sall fragment which contains the 3' half of Ct
`POX 4 gene as a hybridization probe (Okazaki et al.,
`1986). From a positive clone, we identified 2.4-kb and
`4.4-kb Dral genomic DNA fragments that had high sim(cid:173)
`ilarity to the Ct POX2 and POX4 genes, respectively.
`The complete nt sequence of Cm POX 4 gene was
`already reported by Hill et al. ( 1988), so we performed
`complete sequencing of the POX2 gene of Cm (Fig. 1). A
`typical TATA consensus sequence, TATAAA, was present
`around nt -120 relative to the ATG start codon. A single
`2172-nt ORF was shown and a polypeptide of 84 kDa
`(724 aa) was predicted. The POX2 structural gene had
`82% identity on the nt level and 85% identity on the aa
`level to its Ct counterpart. The POX4 and POX5 genes
`of Ct exhibited 52% and 48% identity on the deduced
`amino acid level to the Cm POX2 gene.
`
`(b) Arrangement of POX genes and expression on vario _,
`carbon sources
`In Ct, there were three genes (POX2, POX4 and POX ~)
`encoding a