Summary
The Aspergillus nidulans pyruvate kinase gene was isolated by heterologous hybridization using the corresponding yeast gene as a probe. A 2.9 kb EcoRI/BamHI fragment, which exclusively hybridized to the yeast gene, was subcloned in pBR322. This clone was used to transform an A. nidulans pkiA deletion mutant to PKI+. The analysis of transformants with respect to the kind of integration revealed about 80% homologous integration − 55% by a double cross-over event (type III integration), 25% by a single cross-over event (type I integration). Type II transformants (20%) that arise by non-homologous integration have not been further characterized with respect to the sites of integration.
A direct correlation between the number of copies of the gene integrated into the genome and the measured pyruvate kinase activity was found after growth on a glycolytic carbon source. From this, it was concluded that the 2.9 kb EcoRI/BamHI fragment contains the complete pyruvate kinase structural gene, including the promoter region.
However, after growth on a gluconeogenic carbon source, the regulation of gene expression was found to be disturbed. On acetate an increase in activity per gene copy (0.2 IU) was found in the transformants, as compared with wild-type levels. It is suggested that the pyruvate kinase gene is regulated by negative control, and that some sequences involved in this regulation are missing in the cloned fragment.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- ATP:
-
Adenosinetriphosphate
- MM:
-
Minimal medium
- PEP:
-
Phosphoenolpyruvate
References
Ballance DJ, Turner G (1985) Gene 36:321–331
Ballance DJ, Turner G (1986) Mol Gen Genet 202:271–275
Benton WD, Davis RW (1977) Science 196:180–182
Burke L, Tekamp-Olson P, Najarian R (1983) J Biol Chem 258:2193–2201
Buxton FP, Gwynne DI, Davies RW (1985) Gene 37:207–214
Clements JM, Roberts CF (1985) Curr Genet 9:293–298
Clements JR, Roberts CF (1986) Gene 44:97–105
Goosen T, Bloemheuvel G, Gysler Ch, de Bie DM, van den Broek HWJ, Swart K (1987) Curr Genet 11:499–503
Hinnen A, Hicks JB, Fink GR (1978) Proc Natl Acad Sci USA 75:1929–1933
Hitzemann RA, Hagie FE, Hayflick JS, Chen CY, Seeburg PH, Derynck R (1982) NAR 10:7791–7808
John MA, Peberdy JF (1984) Enzyme Microb Technol 6:386–389
Johnstone IL (1985) Microbiol Sci 10:307–311
Johnstone IL, Hughes SG, Clutterbuck AJ (1985) EMBO J 4:1307–1311
Kapoor M, Tronsgaard TM (1972) Can J Microbiol 18:805–815
Kapoor M (1975) Can J Biochem 53:109–119
Kawasaki G, Fraenkel DG (1982) Biochem Biophsy Res Comm 108:1107–1112
Kelly JM, Hynes M (1985) EMBO J 4:475–479
Lockington RA, Scaly-Lewis HM, Scazzocchio C, Davies RW (1985) Gene 33:137–149
Lonberg N, Gilbert W (1983) Proc Natl Acad Sci USA 80:3661–3665
Lonberg N, Gilbert W (1985) Cell 40:81–90
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (A laboratory manual)
Meixner-Monori B, Kubicek CP, Rohr M (1984) Can J Microbiol 30:16–22
Payton MA, Roberts CF (1976) FEBS Lett 66:73–76
Penttilla ME, Nevalainen KMH, Raynal A, Knowles JKC (1984) Mol Gen Genet 194:494–499
Pontecorvo G, Roper JA, Hemmons LJ, MacDonald KD, Buxton AWJ (1953) Adv Genet 5:141–238
Slater RJ (1985) In: Walker JM (ed) Methods in molecular biology, vol 2, nucleic acids. Humana Press, Cliftar New Jersey, pp 101–108
Stone EM, Rothblurn KN, Schwartz RJ (1985) Nature 313:498–500
Terenzi HF, Roselino E, Passeron S (1971) Eur J Biochem 18:342–350
Tilburn J, Scazzocchio C, Taylor GG, Zabicky-Zissmann JH, Lockington A, Davies RA (1983) Gene 26:205–221
Tso JY, Sun XH, Wu R (1985a) J Biol Chem 260:8220–8228
Sun XH, Reece KS, Wu R (1985b) NAR 13:2485–2502
Uitzetter JHAA (1982) Thesis Agricultural University Wageningen, The Netherlands
Wernars K, Goosen T, Wennekes LMJ, Visser J, Bos CJ, van den Brook HWJ, van Gorcom RFM, van den Hondel CAMJJ (1985) Curr Genet 9:361–368
Yelton MM, Hamer JE, De Souza ER, Mullaney EJ, Timberlake WE (1983) Proc Natl Acad Sci USA 80:7576–7580
Yelton MM, Hamer JE, Timberlake WE (1984) Proc Natl Acad Sci USA 81:1470–1474
Yelton MM, Timberlake WE, van den Hondel CAMJJ (1985) Proc Natl Acad Sci USA 82:834–838
Yoshino M, Murakami K (1985) J Biol Chem 260:4729–4732
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
de Graaff, L., van den Broek, H. & Visser, J. Isolation and transformation of the pyruvate kinase gene of Aspergillus nidulans . Curr Genet 13, 315–321 (1988). https://doi.org/10.1007/BF00424425
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00424425