Biomedical subjects

M O Agaphonov

Publications and source records attributed to M O Agaphonov.

8 recordsLinked to original sources

PubMed2001

Mutation of the homologue of GDP-mannose pyrophosphorylase alters cell wall structure, protein glycosylation and secretion in Hansenula polymorpha.

A Hansenula polymorpha mutant with enhanced ability to secrete a heterologous protein has been isolated. The mutation defines a gene, designated OPU24, which encodes a protein highly homologous to GDP-mannose pyrophosphorylase Psa1p/Srb1p/Vig9p of Saccharomyces cerevisiae and CaSrb1p of Candida albicans. The opu24 mutant manifests phenotypes similar to those of S. cerevisiae mutants depleted for GDP-mannose, such as cell wall fragility and defects in N- and O-glycosylation of secreted proteins. The influence of the opu24 mutation on endoplasmic reticulum-associated protein degradation is discussed. The GenBank Accession No. for the OPU24 sequence is AF234177.

Amino Acid Sequence↗

PubMed1999

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

Plasmids with different selectable markers were constructed and used to transform the Hansenula polymorpha strain DL1. It was shown that, depending on the host mutant strain, the use of these plasmids enables rapid selection of transformants with plasmids integrated in low (1-2), moderate (6-9) or high (up to 100) copy numbers. The vectors and mutant described are potentially useful for the construction of efficient producers of heterologous proteins in H. polymorpha.

Blotting, Southern↗

PubMed1999

A dominant selection system designed for copy-number-controlled gene integration in Hansenula polymorpha DL-1.

To facilitate the selection of multiple gene integrants in Hansenula polymorpha, a rapid and copy-number-controlled selection system was developed using a vector containing a telomeric autonomous replication sequence and the bacterial aminoglycoside 3-phosphotransferase (APH) gene. Direct use of the unmodified APH gene as a dominant selectable marker resulted in the extremely slow growth of transformants and the frequent selection of spontaneous resistance. For the proper performance of the APH gene, a set of deleted glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoters of H. polymorpha were fused to the APH gene. The fusion construct with the 578-bp GAPDH promoter conferred G418 resistance sufficient to allow rapid growth of transformants, and thus facilitated the selection of transformants with up to 15 tandem copies of the vector. To increase further the integration copy number within the gene-dose-dependent range, the GAPDH promoter was serially deleted down to the -61 nucleotide. With this weak expression cassette, the integration copy number could easily be controlled between 1 and 50. Tandemly integrated copies of plasmids near the end of the chromosome were mitotically stable over 150 generations. The dosage-dependent selection system of this study would provide a powerful tool for the development of H. polymorpha as an industrial strain to produce recombinant proteins.

Bacterial Proteins↗

PubMed1998

Sequences of Saccharomyces cerevisiae 2 microns DNA improving plasmid partitioning in Hansenula polymorpha.

Insertion of the HindIII-PstI fragment of Saccharomyces cerevisiae 2 microns DNA into the Hansenula polymorpha replicative plasmids decreases plasmid copy number and ensures their distribution to daughter cells at both mitotic and meiotic cell divisions. This suggests that the stabilization effect is caused by the improvement of plasmid partitioning. Deletion analysis revealed that the region of 2 microns DNA sequence responsible for the increase of mitotic stability of H. polymorpha plasmids involves the 2 microns STB locus and adjoining region. Further analysis demonstrated that the stabilization effect may depend on the number of 24-28 bp imperfect repeats which were found in several copies in the STB locus and adjoining region.

Base Sequence↗

PubMed1996

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

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.

Base Sequence↗

PubMed1995

Plasmid reorganization during integrative transformation in Hansenula polymorpha.

During studies of integrative transformation in Hansenula polymorpha, it was found that transformants with plasmids possessing the LEU2 gene of H. polymorpha were frequently unstable and lost plasmids while growing on non-selective medium. These transformants possessed reorganized plasmids capable of replication in H. polymorpha. Two such plasmids were isolated and characterized. It was shown that they contain additional DNA segments which were not present in the original plasmid used for transformation. Southern hybridization analysis carried out with labeled DNA probes derived from these segments showed that they consisted of H. polymorpha DNA. The hybridization patterns indicated that corresponding sequences were homologous to several chromosomal regions. These chromosomal DNA segments apparently carried H. polymorpha autonomous replicating sequences (HARS), since plasmids bearing them could transform H. polymorpha with high efficiency and were maintained in transformants in an autonomous state. Sequence analysis of one such captured chromosomal fragment revealed several eight- to ten-base AT-rich blocks similar to the presumed HARS sequence defined by Roggenkamp et al. (1986). Analogous reorganization was also observed with respect to integrative plasmids carrying the TRP3 and HIS3 genes of H. polymorpha and the ADE2 gene of Saccharomyces cerevisiae as selectable markers.

Base Sequence↗

PubMed1995

A disruption-replacement approach for the targeted integration of foreign genes in Hansenula polymorpha.

A system has been developed which allows the selection of integrative transformants with replacement of the Hansenula polymorpha methanol oxidase gene (MOX) with expression cassettes carrying heterologous gene under the control of the MOX promoter. The system is convenient for comparison of the expression levels of different constructs integrated into the same locus of the H. polymorpha genome. This system was used to compare the secretion levels of human urinary plasminogen activator, the secretion of which was directed by different signal sequences.

Alcohol Oxidoreductases↗

PubMed1994

Isolation and characterization of the LEU2 gene of Hansenula polymorpha.

A DNA fragment carrying the LEU2 gene of methylotrophic yeast Hansenula polymorpha was isolated by complementation of the leuB mutation of Escherichia coli. The nucleotide sequence of the isolated DNA fragment contains an open reading frame of 363 codons, coding for a protein 80% identical to the LEU2 gene product of Saccharomyces cerevisiae. Further downstream, there is a partial reading frame with no obvious similarity to known proteins. The LEU2 gene of H. polymorpha cannot complement the leu2 mutation of S. cerevisiae.

3-Isopropylmalate Dehydrogenase↗