1887

Microbiology Society logo

Volume 138, Issue 11

Chromosomal targeting of replicating plasmids in the yeast Free

Abstract

Using an optimized transformation protocol we have studied the possible interactions between transforming plasmid DNA and the genome. Plasmids consisting only of a pBR322 replicon, an antibiotic resistance marker for and the gene were shown to replicate autonomously in the yeast at an approximate copy number of 6 (copies per genome equivalent). This autonomous behaviour is probably due to an replicon-like sequence present on the gene fragment. Plasmids replicated as multimers consisting of monomers connected in a head-to-tail configuration. Two out of nine transformants analysed appeared to contain plasmid multimers in which one of the monomers contained a deletion. Plasmids containing internal or flanking regions of the genomic alcohol oxidase gene were shown to integrate by homologous single or double cross-over recombination. Both single- and multicopy (two or three) tandem integrations were observed. Targeted integration occurred in 1-22% of the cases and was only observed with plasmids linearized within the genomic sequences, indicating that homologous linear ends are recombinogenic in . In the cases in which no targeted integration occurred, double-strand breaks were efficiently repaired in a homology-independent way. Repair of double-strand breaks was precise in 50-68% of the cases. Linearization within homologous as well as nonhomologous plasmid regions stimulated transformation frequencies up to 15-fold.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
© Society for General Microbiology, 1992
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-138-11-2405
1992-11-01
2024-04-25
Loading full text...

Full text loading...

/deliver/fulltext/micro/138/11/mic-138-11-2405.html?itemId=/content/journal/micro/10.1099/00221287-138-11-2405&mimeType=html&fmt=ahah

References

  1. Berardi E., Thomas D. Y. 1990; An effective transformation method for Hansenula polymorpha. Current Genetics 18:169–170
     [Google Scholar]
  2. Beburov M. Y., Zlochewsky M. L., Michailover V. M., Semenova V. D., Gracheva V. D., Lahtchev K. 1990 Transformation of methylotrophic yeast Hansenula polymorpha with linear DNA fragments. In The Fifteenth International Conference on Yeast Genetics and Molecular Biology, The Hague, The Netherlands. (book of abstracts), p.118 Edited by Oliver S. G. Chichester: John Wiley;
     [Google Scholar]
  3. Broach J. R., Strathern J. N., Hicks J. B. 1979; Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene 8:121–133
     [Google Scholar]
  4. Bruinenberg P. G., Evers M., Waterham H. R., Kuipers J., Arnberg A. C., Ab G. 1989; Cloning and sequencing of the peroxisomal amine oxidase gene from Hansenula polymorpha. Biochimica et Biophysica Acta 1008:157–167
     [Google Scholar]
  5. Cregg J.M., Barringer K. J., Hessler A. Y., Madden K. R. 1985; Pichia pastoris as a host system for transformations. Molecular and Cellular Biology 5:3376–3385
     [Google Scholar]
  6. Cregg J. M., Van Der Klei I. J., Sulter G. J., Veenhuis M., Harder W. 1990; Peroxisome deficient mutants of Hansenula polymorpha. Yeast 6:87–97
     [Google Scholar]
  7. Cregg J. M., Madden K. R. 1987 Development of yeast transformation systems and construction of methanol utilization defective mutants of Pichia pastoris by gene disruption. In Biological Research on Industrial Yeasts, II pp. 1–18 Edited by Stewart G. G., Russel I., Klein R. D., Hiebsch R. R. Boca Raton, FL: CRC Press;
     [Google Scholar]
  8. Dohmen R. J., Strasser W. M., Honer C. B., Hollenberg C. P. 1991; An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera. Yeast 1:691–692
     [Google Scholar]
  9. Dower W. J., Miller J. F., Ragsdale C. W. 1988; High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Research 16:6127–6145
     [Google Scholar]
  10. Eckart M. 1988 Klonierung und Charakterisierung der Gene fur Dihydroxyacetonesynthase und Methanoloxidase aus der Methylotrophen Hefen Hansenula polymorpha. PhD thesis University of Diisseldorf, FRG;
     [Google Scholar]
  11. Fellinger A. J., Verbakel J.M. A., Veale R. A., Sudbery P. E., Bom I. J., Overbeeke N., Verrips C. T. 1991; Expression of the a-galactosidase from Cyamopsis tetragonoloba (Guar) by Hansenula polymorpha. Yeast 1:463–473
     [Google Scholar]
  12. Fincham J. R. S. 1989; Transformation in fungi. Microbiological Reviews 53:148–170
     [Google Scholar]
  13. Gleeson M. A., Sudbery P. E. 1988; Genetic analysis in the methylotrophic yeast Hansenula polymorpha. Yeast 4:293–303
     [Google Scholar]
  14. Gleeson M.A., Ortori G. S., Sudbery P. E. 1986; Transformation of the methylotrophic yeast Hansenula polymorpha. Journal of General Microbiology 132:3459–3465
     [Google Scholar]
  15. Grimm C., KOHLI J. 1988; Observations on integrative transformation in Schizosaccharomyces pombe. Molecular and General Genetics 215:87–93
     [Google Scholar]
  16. Hanahan D. 1983; Studies on transformation of Escherichia coli with plasmids. Journal of Molecular Biology 166:557–580
     [Google Scholar]
  17. Hinnen A., Hicks J. B., Fink G. R. 1978; Transformation of yeast. Proceedings of the National Academy of Sciences of the United States of America 751929–1933
     [Google Scholar]
  18. Hodgkins M.A., Sudbery P. E., Kerry-Williams S., Goodey A. 1990 Secretion of human serum albumin from Hansenula polymorpha. In The Fifteenth International Conference on Yeast Genetics and Molecular Biology, The Hague, The Netherlands. (book of abstracts), p.435 Edited by Oliver S. G. Chichester: John Wiley;
     [Google Scholar]
  19. Hoffman C. S., Winston F. 1987; A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 51:267–272
     [Google Scholar]
  20. Holmes D.S., Quigley M. 1981; A rapid boiling method for the preparation of bacterial plasmids. Analytical Biochemistry 114:19–3
     [Google Scholar]
  21. Horinouchi H., Weisblum B. 1982a; Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide and streptogramin type B antibiotics. Journal of Bacteriology 150:804–814
     [Google Scholar]
  22. Horinouchi H., Weisblum B. 1982b; Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. Journal of Bacteriology 150:815–825
     [Google Scholar]
  23. Ish-Horowicz D., Burke F. J. 1979; Rapid and efficient cosmid cloning. Nucleic Acids Research 9:2989–2999
     [Google Scholar]
  24. Janowicz A. Z., Melber K., Merckelbach A., Jacobs E., Harford N., Comberbach M., Hollenberg C. P. 1991; Simultaneous expression of the S and L surface antigens of hepatitis B, and formation of mixed particles in the methylotrophic yeast, Hansenula polymorpha. Yeast 1:431–443
     [Google Scholar]
  25. Klebe R. J., Harriss J. V., Sharp Z. D., Douglas M. G. 1983; A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast. Gene 25:331–341
     [Google Scholar]
  26. Kunes S., Botstein D., Fox M. S. 1985; Transformation of yeast with linearized plasmid DNA, formation of inverted dimers and recombinant plasmid products. Journal of Molecular Biology 184:375–387
     [Google Scholar]
  27. Ledeboer A. M., Edens L., Maat J., Visser C., Bos J. W., Verrips C. T., Janowicz Z., Eckart M., Roggenkamp R., Hollenberg C. P. 1985; Molecular cloning and characterization of a gene coding for methanol oxidase in Hansenula polymorpha. Nucleic Acids Research 13:3063–3082
     [Google Scholar]
  28. Mead D. A., Szczesna-Skorupa E., Kemper B. 1986; Single-stranded DNA ’blue’ T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Protein Engineering 1:67–74
     [Google Scholar]
  29. Meissner P. S., Sisk W. P., Berman M. L. 1987; Bacteriophage cloning system for the construction of directional cDNA libraries. Proceedings of the National Academy of Sciences of the United States of America 844171–4175
     [Google Scholar]
  30. Orr-Weaver T. L., Szostak J. W. 1983; Yeast recombination: the association between double strand gap repair and crossing-over. Proceedings of the National Academy of Sciences of the United States of America 804417–4421
     [Google Scholar]
  31. Orr-Weaver T. L., Szostak J. W., Rothstein R. J. 1981; Yeast transformation: a model system for the study of recombination. Proceedings of the National Academy of Sciences of the United States of America 786354–6358
     [Google Scholar]
  32. Orr-Weaver T. L., Szostak J. W., Rothstein R. J. 1983; Genetic applications of yeast transformation with linear and gapped plasmids. Methods in Enzymology 101:228–245
     [Google Scholar]
  33. Perera J. R., Glasunov A. V., Glaser V. M., Boreiko A. V. 1988; Repair of double-strand breaks in plasmid DNA in the yeast Saccharomyces cerevisiae. Molecular and General Genetics 213:421–424
     [Google Scholar]
  34. Roggenkamp R., Hansen H., Eckart M., Janowicz Z., Hollenberg C. P. 1986; Transformation of the methylotrophic yeast Hansenula polymorpha by autonomous replication and integration vectors. Molecular and General Genetics 202:302–308
     [Google Scholar]
  35. Romanos M.A., Rayment F., Beesley K. M., Clare J. J. 1990 Expression of the Bordetella pertussis (whooping cough) P.69 antigen in Saccharomyces cerevisiae and Pichia. In The Fifteenth International Conference on Yeast Genetics and Molecular Biology, The Hague, The Netherlands (book of abstracts), p.428 Edited by Oliver S. G. Chichester: John Wiley;
     [Google Scholar]
  36. Sakaguchi J., Yamamoto M. 1982; Cloned ural locus of Schizosaccharomyces pombe propagates autonomously in this yeast assuming a polymeric form. Proceedings of the National Academy of Sciences of the United States of America 197819–7823
     [Google Scholar]
  37. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning, a La,boratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  38. Shen S. H., Bastien L., Nguyen T., Fung M., Slilaty S. N. 1989; Synthesis and secretion of hepatitis B middle surface antigen by the methylotrophic yeast Hansenula polymorpha. Gene 84:303–309
     [Google Scholar]
  39. Sherman F., Fink G. R., Hicks J. B. 1986 Methods in Yeast Genetics, a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  40. Sierkstra L. N., Verbakel J. M. A., Verrips C. T. 1991; Optimisation of a host/vector system for heterologous gene expression by Hansenula polymorpha. Current Genetics 19:81–87
     [Google Scholar]
  41. Southern E. M. 1975; Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98:50–3
     [Google Scholar]
  42. Sudbery P. E., Gleeson M. A., Veale R. A., Ledeboer A. M., Zoetmulder M. C. M. 1988; Hansenula polymorpha as a novel yeast system for expression of heterologous genes. Biochemical Society Transactions 16:1081–1083
     [Google Scholar]
  43. Tikhomirova L. P., Ikonomova R. N., Kuznetsova E. N. 1986; Evidence for autonomous replication and stabilization of recombinant plasmids in the transformants of yeast Hansenula polymorpha. Current Genetics 10:741–747
     [Google Scholar]
  44. Tikhomirova L. P., Ikonomova R. N., Kuznetsova E. N., Fodor I. I., Bystrykh L. V., Aminova L. R., Trotsenko Y. A. 1988; Transformation of methylotrophic yeast Hansenula polymorpha: cloning and expression of genes. Journal of Basic Microbiology 28:343–351
     [Google Scholar]
  45. Van Der Klei I. J., Harder W., Veenhuis M. 1991a; Biosynthesis and assembly of alcohol oxidase, a peroxisomal matrix protein in methylotrophic yeasts: a review. Yeast 1:195–209
     [Google Scholar]
  46. Van Der Klei I. J., Harder W., Veenhuis M. 1991b; Methanol metabolism in a peroxisome-deficient mutant of Hansenula polymorpha: a physiological study. Archives of Microbiology 156:15–23
     [Google Scholar]
  47. Veale R. A., Giusseppin M. L. F., Van Eijk H. M. J., Sudbery P. E., Verrips C. T. 1992; Development of a strain of Hansenula polymorpha for the efficient expression of guar cx-galactosidase. Yeast 8:361–372
     [Google Scholar]
  48. Veenhuis M., Van Dijken J. P., Harder W. 1983; The significance of peroxisomes in the metabolism of one-carbon compounds in yeast. Advances in Microbiological Physiology 24:1–82
     [Google Scholar]
  49. Veenhuis M., Harder W. 1991 Microbodies. In The Yeasts, 4, 2nd edn. pp. 601–653 Edited by Rose A. H., Harrison J. S. London: Academic Press;
     [Google Scholar]
  50. Yanisch-Perr0N C., Vieira J., Messing J. 1985; Improved M13 phage cloning vectors and host strains: nucleotide sequences of Ml3mpl8 and pUC19 vectors. Gene 33:103–119
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-138-11-2405
Loading
Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha
Microbiology 138, 2405 (1992); https://doi.org/10.1099/00221287-138-11-2405
/content/journal/micro/10.1099/00221287-138-11-2405
/content/journal/micro/10.1099/00221287-138-11-2405
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error