Abstract
Using an EcoRI-BglII fragment of the G unit of the rDNA of Y. lipolytica and a set of 11 deletions in the URA3 promoter, we have constructed several plasmids to test gene amplification in the rDNA. These plasmids contain the rDNA fragment for integration, defective versions of the URA3 gene, the XPR2 gene encoding alkaline extracellular protease (AEP) as a reporter gene, and part of the pBR322 plasmid for selection and replication in E. coli. Among these plasmids, one corresponds to a deletion which allows multiple integration into the rDNA (plasmid pINA773). Two other plasmids (pINA767 and pINA772) give multiple integration only with a mutated URA3 gene. Transformants carrying these three plasmids were tested for copy number, stability, chromosomal localization and AEP secretion. Transformants containing plasmids pINA767, 772 and 773 displayed an average copy number of 5, 12 and 25–60 copies respectively of the plasmid, as estimated by PCR and DNA hybridization. Integrations occurred in only one chromosome except for transformants containing 60 copies where copies were observed at least in two different chromosomes. Multiple integrations were found both as tandem repeats and as dispersed copies. Plasmid copy number was stable in both minimum and rich media, for strains containing less than ten copies per cells. However, for higher copy number, multiple integrations were stable only when AEP synthesis was not induced, while in inducing medium stability of the multiple integrations was dramatically affected.
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References
Bergkamp RJM, Kool IM, Geerse RH, Planta RJ (1992) Curr Genet 21:365–370
Chen XJ, Bianchi MM, Suda K, Fukuhara H (1989) Curr Genet 16:95–98
Davidow LS, Apostolakos D, O'Donnell MM, Proctor AR, Ogrydziak DM, Wing RA, Stasko I, De Zeuuw JR (1985) Curr Genet 10:39–48
Davidow LS, Kaczmarek FS, De Zeeuw JR, Conlon SW, Lauth MR, Pereira DA, Franke AE (1987) Curr Genet 11:377–388
Erhardt E, Hollenbeirg CP (1983) J Bacteriol 156:625–635
Fabre E, Nicaud JM, Lopez MC, Gaillardin C (1991) J Biol Chem 266:3782–3790
Fabre E, Tharaud C, Gaillardin C (1992) J Biol Chem 267:15049–15055
Fournier C, Gaillardin C, Persuy MA, Klootwijk J, van Heerikhuizen H (1986) Gene 42:273–282
Fournier P, Guyaneux L, Chasles M, Gaillardin C (1991) Yeast 7:25–36
Fournier P, Abbas H, Chasles M, Kudla B, Ogrydziak DM, Yaver D, Xuan JW, Peito A, Ribet AM, Feynerol C, He F, Gaillardin C (1993) Proc Natl Acad Sci USA 90:4912–4916
Franke AE, Kaczmarek FS, Eisenhard ME, Geoghegan KF, Danley DE, De Zeuuw JR, O'Donnell MM, Gollaher MG, Davidow LS (1988) Dev Ind Microbiol 29:43–57
Gaillardin C, Ribet AM (1987) Curr Genet 11:369–375
Gaillardin C, Ribet AM, Heslot H (1985) Curr Genet 10:49–58
Gussow D, Clackson T (1989) Nucleic Acids Res 17:4000
Heslot H (1990) Adv. Biochem. Eng./Biotech 43:43–73
Kingsman SM, Kingsman AJ, Mellor J (1987) Trends Biochem Sci 5:53–57
Lopes TS, Klootwijk J, Veenstra AE, Van der Aar PC, van Heerikhuizen H, Raué HA, Planta RJ (1989) Gene 79:199–206
Lopes TS, Hakkaart GJ, Koerts BL, Raué HA, Planta RJ (1991) Gene 105:83–90
Matoba S, Fukuyama J, Wing RA, Ogrydziak DM (1988) Mol Cell Biol. 8:4904–4916
Naumova E, Naumov G, Fournier P, Nguyen HV, Gaillardin C (1993) Curr. Genet 23:450–454
Nicaud JM, Fabre E, Beckerich JM, Fournier P, Gaillardin C (1989) J Biotechnol 12:285–298
Nicaud JM, Fournier P, La Bonnardière C, Chasles M, Gaillardin C (1991) J Biotechnol 19:259–270
Ogrydziak DM, Mortimer RK (1977) Genetics 87:621–632
Ogrydziak DM, Scharf SJ (1982) J Gen Microbiol 128:1225–1234
Romanos M, Scorer C, Clarc J (1992) Yeast 8:423–488
Sakai A, Ozawa F, Higashizaki T, Shimizu Y, Hishinuma F (1991) Bio/Technology 9:1382–1385
Sanger FS, Nicklen S, Coulson AR (1977) Proc Natl Acad Sci USA 74:5463–5468
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Sherman F, Fink GR, Hicks J (1986) Methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Tharaud C, Ribet AM, Costes C, Gaillardin C (1992) Gene 121:111–119
Treton BY, Le Dall MT, Gaillardin C (1992) Curr Genet 22:345–355
Van Heerikhuizen H, Ykema A, Klootwijk J, Gaillardin C, Ballas C, Fournier P (1985), Gene 42:273–282
Xuan JW, Fournier P, Gaillardin C (1988) Curr Genet 14:15–21
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Communicated by C. P. Hollenberg
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Le Dall, MT., Nicaud, JM. & Gaillardin, C. Multiple-copy integration in the yeast Yarrowia lipolytica. Curr Genet 26, 38–44 (1994). https://doi.org/10.1007/BF00326302
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DOI: https://doi.org/10.1007/BF00326302