Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Aug;178(15):4420–4428. doi: 10.1128/jb.178.15.4420-4428.1996

A novel autonomously replicating sequence (ARS) for multiple integration in the yeast Hansenula polymorpha DL-1.

J H Sohn 1, E S Choi 1, C H Kim 1, M O Agaphonov 1, M D Ter-Avanesyan 1, J S Rhee 1, S K Rhee 1
PMCID: PMC178207  PMID: 8755868

Abstract

Several autonomously replicating sequences of Hansenula polymorpha DL-1 (HARSs) with the characteristics of tandem integration were cloned by an enrichment procedure and analyzed for their functional elements to elucidate the mechanism of multiple integration in tandem repeats. All plasmids harboring newly cloned HARSs showed a high frequency of transformation and were maintained episomally before stabilization. After stabilization, the transforming DNA was stably integrated into the chromosome. HARS36 was selected for its high efficiency of transformation and tendency for integration. Several tandemly repeated copies of the transforming plasmid containing HARS36 (pCE36) integrated into the vicinity of the chromosomal end. Bal 31 digestion of the total DNA from the integrants followed by Southern blotting generated progressive shortening of the hybridization signal, indicating the telomeric localization of the transforming plasmids on the chromosome. The minimum region of HARS36 required for its HARS activity was analyzed by deletion analyses. Three important regions, A, B, and C, for episomal replication and integration were detected. Analysis of the DNA sequences of regions A and B required for the episomal replication revealed that region A contained several AT-rich sequences that showed sequence homology with the ARS core consensus sequence of Saccharomyces cerevisiae. Region B contained two directly repeated sequences which were predicted to form a bent DNA structure. Deletion of the AT-rich core in region A resulted in a complete loss of ARS activity, and deletion of the repeated sequences in region B greatly reduced the stability of the transforming plasmid and resulted in retarded cell growth. Region C was required for the facilitated chromosomal integration of transforming plasmids.

Full Text

The Full Text of this article is available as a PDF (471.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Agaphonov M. O., Poznyakovski A. I., Bogdanova A. I., Ter-Avanesyan M. D. Isolation and characterization of the LEU2 gene of Hansenula polymorpha. Yeast. 1994 Apr;10(4):509–513. doi: 10.1002/yea.320100410. [DOI] [PubMed] [Google Scholar]
  2. Boeke J. D., Xu H., Fink G. R. A general method for the chromosomal amplification of genes in yeast. Science. 1988 Jan 15;239(4837):280–282. doi: 10.1126/science.2827308. [DOI] [PubMed] [Google Scholar]
  3. Bogdanova A. I., Agaphonov M. O., Ter-Avanesyan M. D. Plasmid reorganization during integrative transformation in Hansenula polymorpha. Yeast. 1995 Apr 15;11(4):343–353. doi: 10.1002/yea.320110407. [DOI] [PubMed] [Google Scholar]
  4. Caddle M. S., Calos M. P. Specific initiation at an origin of replication from Schizosaccharomyces pombe. Mol Cell Biol. 1994 Mar;14(3):1796–1805. doi: 10.1128/mcb.14.3.1796. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cannon R. D., Jenkinson H. F., Shepherd M. G. Isolation and nucleotide sequence of an autonomously replicating sequence (ARS) element functional in Candida albicans and Saccharomyces cerevisiae. Mol Gen Genet. 1990 Apr;221(2):210–218. doi: 10.1007/BF00261723. [DOI] [PubMed] [Google Scholar]
  6. Della Seta F., Treich I., Buhler J. M., Sentenac A. ABF1 binding sites in yeast RNA polymerase genes. J Biol Chem. 1990 Sep 5;265(25):15168–15175. [PubMed] [Google Scholar]
  7. Foss M., McNally F. J., Laurenson P., Rine J. Origin recognition complex (ORC) in transcriptional silencing and DNA replication in S. cerevisiae. Science. 1993 Dec 17;262(5141):1838–1844. doi: 10.1126/science.8266071. [DOI] [PubMed] [Google Scholar]
  8. Fournier P., Abbas A., Chasles M., Kudla B., Ogrydziak D. M., Yaver D., Xuan J. W., Peito A., Ribet A. M., Feynerol C. Colocalization of centromeric and replicative functions on autonomously replicating sequences isolated from the yeast Yarrowia lipolytica. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):4912–4916. doi: 10.1073/pnas.90.11.4912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gellissen G., Janowicz Z. A., Merckelbach A., Piontek M., Keup P., Weydemann U., Hollenberg C. P., Strasser A. W. Heterologous gene expression in Hansenula polymorpha: efficient secretion of glucoamylase. Biotechnology (N Y) 1991 Mar;9(3):291–295. doi: 10.1038/nbt0391-291. [DOI] [PubMed] [Google Scholar]
  10. Gellissen G., Weydemann U., Strasser A. W., Piontek M., Janowicz Z. A., Hollenberg C. P. Progress in developing methylotrophic yeasts as expression systems. Trends Biotechnol. 1992 Dec;10(12):413–417. doi: 10.1016/0167-7799(92)90288-7. [DOI] [PubMed] [Google Scholar]
  11. Hinnen A., Buxton F., Chaudhuri B., Heim J., Hottiger T., Meyhack B., Pohlig G. Gene expression in recombinant yeast. Bioprocess Technol. 1995;22:121–193. [PubMed] [Google Scholar]
  12. Hodgkins M., Mead D., Ballance D. J., Goodey A., Sudbery P. Expression of the glucose oxidase gene from Aspergillus niger in Hansenula polymorpha and its use as a reporter gene to isolate regulatory mutations. Yeast. 1993 Jun;9(6):625–635. doi: 10.1002/yea.320090609. [DOI] [PubMed] [Google Scholar]
  13. Inoue H., Nojima H., Okayama H. High efficiency transformation of Escherichia coli with plasmids. Gene. 1990 Nov 30;96(1):23–28. doi: 10.1016/0378-1119(90)90336-p. [DOI] [PubMed] [Google Scholar]
  14. Janowicz Z. A., Eckart M. R., Drewke C., Roggenkamp R. O., Hollenberg C. P., Maat J., Ledeboer A. M., Visser C., Verrips C. T. Cloning and characterization of the DAS gene encoding the major methanol assimilatory enzyme from the methylotrophic yeast Hansenula polymorpha. Nucleic Acids Res. 1985 May 10;13(9):3043–3062. doi: 10.1093/nar/13.9.3043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Janowicz Z. A., Melber K., Merckelbach A., Jacobs E., Harford N., Comberbach M., Hollenberg C. P. Simultaneous expression of the S and L surface antigens of hepatitis B, and formation of mixed particles in the methylotrophic yeast, Hansenula polymorpha. Yeast. 1991 Jul;7(5):431–443. doi: 10.1002/yea.320070502. [DOI] [PubMed] [Google Scholar]
  16. Ledeboer A. M., Edens L., Maat J., Visser C., Bos J. W., Verrips C. T., Janowicz Z., Eckart M., Roggenkamp R., Hollenberg C. P. Molecular cloning and characterization of a gene coding for methanol oxidase in Hansenula polymorpha. Nucleic Acids Res. 1985 May 10;13(9):3063–3082. doi: 10.1093/nar/13.9.3063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lopes T. S., Klootwijk J., Veenstra A. E., van der Aar P. C., van Heerikhuizen H., Raúe H. A., Planta R. J. High-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae: a new vector for high-level expression. Gene. 1989 Jul 15;79(2):199–206. doi: 10.1016/0378-1119(89)90202-3. [DOI] [PubMed] [Google Scholar]
  18. Marahrens Y., Stillman B. A yeast chromosomal origin of DNA replication defined by multiple functional elements. Science. 1992 Feb 14;255(5046):817–823. doi: 10.1126/science.1536007. [DOI] [PubMed] [Google Scholar]
  19. Matsuoka M., Matsubara M., Daidoh H., Imanaka T., Uchida K., Aiba S. Analysis of regions essential for the function of chromosomal replicator sequences from Yarrowia lipolytica. Mol Gen Genet. 1993 Mar;237(3):327–333. doi: 10.1007/BF00279435. [DOI] [PubMed] [Google Scholar]
  20. Newlon C. S. Yeast chromosome replication and segregation. Microbiol Rev. 1988 Dec;52(4):568–601. doi: 10.1128/mr.52.4.568-601.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rao H., Marahrens Y., Stillman B. Functional conservation of multiple elements in yeast chromosomal replicators. Mol Cell Biol. 1994 Nov;14(11):7643–7651. doi: 10.1128/mcb.14.11.7643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Romanos M. A., Scorer C. A., Clare J. J. Foreign gene expression in yeast: a review. Yeast. 1992 Jun;8(6):423–488. doi: 10.1002/yea.320080602. [DOI] [PubMed] [Google Scholar]
  23. Rowley A., Dowell S. J., Diffley J. F. Recent developments in the initiation of chromosomal DNA replication: a complex picture emerges. Biochim Biophys Acta. 1994 Apr 6;1217(3):239–256. doi: 10.1016/0167-4781(94)90283-6. [DOI] [PubMed] [Google Scholar]
  24. Ryder K., Silver S., DeLucia A. L., Fanning E., Tegtmeyer P. An altered DNA conformation in origin region I is a determinant for the binding of SV40 large T antigen. Cell. 1986 Mar 14;44(5):719–725. doi: 10.1016/0092-8674(86)90838-x. [DOI] [PubMed] [Google Scholar]
  25. Sasnauskas K., Jomantienè R., Lebedienè E., Lebedys J., Januska A., Janulaitis A. Molecular cloning and analysis of autonomous replicating sequence of Candida maltosa. Yeast. 1992 Apr;8(4):253–259. doi: 10.1002/yea.320080403. [DOI] [PubMed] [Google Scholar]
  26. Snyder M., Buchman A. R., Davis R. W. Bent DNA at a yeast autonomously replicating sequence. Nature. 1986 Nov 6;324(6092):87–89. doi: 10.1038/324087a0. [DOI] [PubMed] [Google Scholar]
  27. Sudbery P. E. The non-Saccharomyces yeasts. Yeast. 1994 Dec;10(13):1707–1726. doi: 10.1002/yea.320101305. [DOI] [PubMed] [Google Scholar]
  28. Sweder K. S., Rhode P. R., Campbell J. L. Purification and characterization of proteins that bind to yeast ARSs. J Biol Chem. 1988 Nov 25;263(33):17270–17277. [PubMed] [Google Scholar]
  29. Theis J. F., Newlon C. S. Domain B of ARS307 contains two functional elements and contributes to chromosomal replication origin function. Mol Cell Biol. 1994 Nov;14(11):7652–7659. doi: 10.1128/mcb.14.11.7652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tikhomirova L. P., Ikonomova R. N., Kuznetsova E. N. Evidence for autonomous replication and stabilization of recombinant plasmids in the transformants of yeast Hansenula polymorpha. Curr Genet. 1986;10(10):741–747. doi: 10.1007/BF00405096. [DOI] [PubMed] [Google Scholar]
  31. Trifonov E. N. Sequence-dependent deformational anisotropy of chromatin DNA. Nucleic Acids Res. 1980 Sep 11;8(17):4041–4053. doi: 10.1093/nar/8.17.4041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Umek R. M., Linskens M. H., Kowalski D., Huberman J. A. New beginnings in studies of eukaryotic DNA replication origins. Biochim Biophys Acta. 1989 Jan 23;1007(1):1–14. doi: 10.1016/0167-4781(89)90123-1. [DOI] [PubMed] [Google Scholar]
  33. Williams J. S., Eckdahl T. T., Anderson J. N. Bent DNA functions as a replication enhancer in Saccharomyces cerevisiae. Mol Cell Biol. 1988 Jul;8(7):2763–2769. doi: 10.1128/mcb.8.7.2763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Williamson D. H. The yeast ARS element, six years on: a progress report. Yeast. 1985 Sep;1(1):1–14. doi: 10.1002/yea.320010102. [DOI] [PubMed] [Google Scholar]
  35. Zahn K., Blattner F. R. Direct evidence for DNA bending at the lambda replication origin. Science. 1987 Apr 24;236(4800):416–422. doi: 10.1126/science.2951850. [DOI] [PubMed] [Google Scholar]
  36. Zakian V. A. Structure and function of telomeres. Annu Rev Genet. 1989;23:579–604. doi: 10.1146/annurev.ge.23.120189.003051. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES