Vectors for rapid selection of integrants with different plasmid copy numbers in the yeast Hansenula polymorpha DL1

Agaphonov, Michael O.; Trushkina, Polina M.; Sohn, J.‐H.; Choi, E.‐S.; Rhee, S.‐K.; Ter‐Avanesyan, Michael D.

doi:10.1002/(sici)1097-0061(199905)15:7<541::aid-yea392>3.3.co;2-7

Cited by 16 publications

(30 citation statements)

References 24 publications

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“…The use of in vivo recombination diminished the problems usually encountered in library construction in H. polymorpha, such as variations in the copy number and the integration locus. Transformants from the circular vector with HARS36 usually showed over 50% multiple integrations (3). When the acceptor and the insert shared the 475-bp 3Ј-overlapping sequence in HARS36, the portion of the transformants with multiple gene integration was approximately 15%.…”

Section: Discussionmentioning

confidence: 99%

“…The combination of the ARS domain and the telomeric repeats of HARS36 greatly increases the potential of HARS36 for multiple gene integration into the chromosome. The expression level of foreign proteins, however, is diverse due to a difference in the integrated gene copy number (3,27). Thus, the integrants are not adequate for activity-based selection of a mutant library.…”

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confidence: 99%

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Efficient Library Construction by In Vivo Recombination with a Telomere-Originated Autonomously Replicating Sequence of Hansenula polymorpha

Kim

Sohn

Bae

et al. 2003

Appl Environ Microbiol

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A high frequency of transformation and an equal gene dosage between transformants are generally required for activity-based selection of mutants from a library obtained by directed evolution. An efficient library construction method was developed by using in vivo recombination in Hansenula polymorpha. Various linear sets of vectors and insert fragments were transformed and analyzed to optimize the in vivo recombination system. A telomere-originated autonomously replicating sequence (ARS) of H. polymorpha, reported as a recombination hot spot, facilitates in vivo recombination between the linear transforming DNA and chromosomes. In vivo recombination of two linear DNA fragments containing the telomeric ARS drastically increases the transforming frequency, up to 10-fold, compared to the frequency of circular plasmids. Direct integration of the one-end-recombined linear fragment into chromosomes produced transformants with single-copy gene integration, resulting in the same expression level for the reporter protein between transformants. This newly developed in vivo recombination system of H. polymorpha provides a suitable library for activity-based selection of mutants after directed evolution.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Efficient Library Construction by In Vivo Recombination with a Telomere-Originated Autonomously Replicating Sequence of Hansenula polymorpha

Kim

Sohn

Bae

et al. 2003

Appl Environ Microbiol

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“…Plasmid p27OPU8 was recovered from the H. polymorpha CBS4732 genomic library by complementation of the ret1-27 mutation. To construct the H. polymorpha genomic library, the 5-to 10-kb fraction of DNA fragments obtained by partial digestion of H. polymorpha chromosomal DNA with Sau3A was ligated with the SalI-digested AMIpSL1 vector (5). Two base pairs of the cohesive DNA ends was preliminarily filled in with Klenow fragment.…”

Section: Methodsmentioning

confidence: 99%

“…The pCOP1BE plasmid was constructed by insertion of the 1.9-kb BglII-Ecl136II fragment of the HpRET1 gene from p27OPU8 into the BamHI-Ecl136II sites of the pJJ282 plasmid. To construct the pMCOP1-L plasmid, the 5Ј part of the HpRET1 gene (the 882-bp EspI-BglII fragment of the p27OPU8 plasmid) was fused with the MOX promoter (the 0.9-kb StuI-ScaI fragment of pMMU [5]) and inserted together with the ScLEU2 selectable marker (the 1.6-kb BsrGI-SacI fragment of pJJ282) into the StuI-SacI sites of the pUK21 plasmid (49). The pH5 plasmid was constructed by insertion of the 1.3-kb NotI-EcoRV fragment of pCHLX (45) into the NotI and SmaI sites of the pE1 plasmid (29).…”

Section: Methodsmentioning

confidence: 99%

C-Terminal Truncation of α-COP Affects Functioning of Secretory Organelles and Calcium Homeostasis in Hansenula polymorpha

et al. 2004

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In eukaryotic cells, COPI vesicles retrieve resident proteins to the endoplasmic reticulum and mediate intra-Golgi transport. Here, we studied the Hansenula polymorpha homologue of the Saccharomyces cerevisiae RET1 gene, encoding ␣-COP, a subunit of the COPI protein complex. H. polymorpha ret1 mutants, which expressed truncated ␣-COP lacking more than 300 C-terminal amino acids, manifested an enhanced ability to secrete human urokinase-type plasminogen activator (uPA) and an inability to grow with a shortage of Ca 2؉ ions, whereas a lack of ␣-COP expression was lethal. The ␣-COP defect also caused alteration of intracellular transport of the glycosylphosphatidylinositol-anchored protein Gas1p, secretion of abnormal uPA forms, and reductions in the levels of Pmr1p, a Golgi Ca 2؉ -ATPase. Overexpression of Pmr1p suppressed some ret1 mutant phenotypes, namely, Ca 2؉ dependence and enhanced uPA secretion. The role of COPI-dependent vesicular transport in cellular Ca 2؉ homeostasis is discussed.In eukaryotic cells, secretory proteins synthesized at the endoplasmic reticulum (ER) pass through multiple distinct membrane-bound organelles comprising the secretory pathway. Transport of proteins and lipids between membrane compartments of the early secretory system is mediated by COPIand COPII-coated vesicles that capture cargo, bud from the donor membrane, and then target, dock, and fuse with an appropriate acceptor compartment (40). Both the COPI and COPII protein coat complexes employ a GTP switch mechanism for coating and uncoating. The COPII complex mediates selective protein export from the ER, while COPI-coated vesicles retrieve resident proteins to the ER and mediate intraGolgi transport (8,38).The Golgi apparatus is composed of distinct cisternal regions, namely, the cis, medial, trans-Golgi, and trans-Golginetwork regions (13). Secretory cargo proteins passing through the Golgi apparatus are modified by the unique sets of resident enzymes of different subcompartments. Two alternative models for the origin of the Golgi subcompartments suggest different roles for COPI vesicles in secretory cargo transport and the function of the Golgi apparatus. In the stable-compartments model, anterograde COPI vesicles carry secretory cargo forward and exclude resident Golgi proteins. In the cisternalmaturation model, entire cisternae carry secretory cargo forward, and retrograde COPI vesicles recycle resident Golgi proteins to younger cisternae. Thus, the distinction between the two models centers on the contents of COPI vesicles and the directionality of their transport. However, recent experimental data indicate that intra-Golgi COPI traffic has a bidirectional character (for reviews, see references 23 and 38). Moreover, the dynamics of cisternal progression and vesicular traffic are different in higher eukaryotes versus yeasts. Progression of the tightly stacked Golgi cisternae of animal cells occurs at a significantly lower rate than the traffic of most cargoes through the secretory pathway. COPI vesicles mediating cargo tra...

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“…However, plasmids with the HpLEUz-d marker tend to integrate as single copies in strains bearing ku2 point mutation via homologous recombination with the chromosomal Ieu2 gene. Nevertheless, multiple integration is also possible if some molecules of the autonomously replicating plasmid have integrated into other loci prior to the integration into the Ieu2 locus (Agaphonov et al 1999). The presence of HARSj6 plasmid provides a high probability of such events due to the efficient recombination at telomeres of several chromosomes (Sohn et al i999a).…”

Section: Multiple Integration Systems Based On Complementation Of Auxmentioning

confidence: 99%

Development of Expression Systems for the Production of Recombinant Proteins in Hansenula polymorpha DL‐1

Kang

Sohn

Agaphonov

et al. 2002

Hansenula Polymorpha

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Vectors for rapid selection of integrants with different plasmid copy numbers in the yeast Hansenula polymorpha DL1

Cited by 16 publications

References 24 publications

Efficient Library Construction by In Vivo Recombination with a Telomere-Originated Autonomously Replicating Sequence of Hansenula polymorpha

Efficient Library Construction by In Vivo Recombination with a Telomere-Originated Autonomously Replicating Sequence of Hansenula polymorpha

C-Terminal Truncation of α-COP Affects Functioning of Secretory Organelles and Calcium Homeostasis in Hansenula polymorpha

Development of Expression Systems for the Production of Recombinant Proteins in Hansenula polymorpha DL‐1

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