Biomedical subjects

G Kunze

Publications and source records attributed to G Kunze.

At least 19 recordsLinked to original sources

PubMed2003

Non-conventional yeasts as producers of polyhydroxyalkanoates--genetic engineering of Arxula adeninivorans.

The non-conventional yeast Arxula adeninivorans was equipped with the genes phbA, phbB and phbC of the polyhydroxyalkanoate (PHA) biosynthetic pathway of Ralstonia eutropha, which encode beta-ketothiolase, NADPH-linked acetoacetyl-CoA reductase and PHA synthase, respectively. Arxula strains transformed solely with the PHA synthase gene (phbC) were able to produce PHA. However, the maximum content of the polymer detected in these strains was just 0.003% poly-3-hydroxybutyrate (PHB) and 0.112% poly-3-hydroxyvalerate (PHV). The expression of all three genes (phbA, phbB, phbC) resulted in small increases in the PHA content of the transgenic Arxula cells. However, under controlled cultivation conditions with minimal medium and ethanol as the carbon source, the recombinant yeast was able to accumulate up to 2.2% PHV and 0.019% PHB. Possible reasons for these differences are discussed.

Acetyl-CoA C-Acyltransferase↗

PubMed2003

The constitutive AHSB4 promoter--a novel component of the Arxula adeninivorans-based expression platform.

An Arxula adeninivorans-AHSB4 gene, encoding histone H4, was isolated and characterized. The gene includes a coding sequence of 363 bp disrupted by a 51-bp intron, similar to the situation in other fungal H4 genes. The identity of the gene was confirmed by the high degree of homology of the derived amino acid sequence with that of other H4 histones. The gene is strongly and constitutively expressed, maintaining this expression profile under salt-stress conditions. The AHSB4 promoter was tested for suitability in heterologous gene expression using genes encoding the intracellular green fluorescent protein and the secreted human serum albumin (HSA) for assessment. Plasmids incorporating respective expression cassettes were used to transform the host strain A. adeninivorans LS3, which forms budding cells at 30 degrees C, and strain 135, which forms mycelia under these conditions. Transformants of both types were found to harbor a single copy of the heterologous DNA. Strong constitutive expression was observed during culture in salt-containing and salt-free media, as expected from the expression profile of AHSB4. In 200-ml shake-flask cultures, maximal HSA levels of 20 mg l(-1) culture medium were achieved. This productivity could be increased to 50 mg l(-1 )in strains harboring two copies of the expression cassette. The AHSB4 promoter thus provides an attractive component for constitutive heterologous gene expression under salt-free and salt-stress conditions.

Amino Acid Sequence↗

PubMed2001

Regulation of the AEFG1 gene, a mitochondrial elongation factor G from the dimorphic yeast Arxula adeninivorans LS3.

Oxygen influences the synthesis of mitochondrial proteins by alteration of the expression of mitochondrial genes and several nuclear genes. One of the genes localised in the nucleus is the EFG1 gene that encodes the mitochondrial elongation factor G (MEF-G). This unique gene (AEFG1) has been isolated from the non-conventional dimorphic yeast, Arxula adeninivorans LS3. The AEFG1 gene comprises a ORF of 2,274 bp, which corresponds to 757 amino acids. In the present study, the regulation of AEFG1 has been analysed for different morphological stages of A. adeninivorans and various culture conditions. It was demonstrated that the transfer of aerobically growing cultures to anaerobic conditions resulted in an accumulation of AEFG1 transcript, correlating with an increase in AMEF-G protein concentration. Since this regulation occurred in budding-cell culture growing at 30 degrees C and in both of the mycelial cultures grown at 45 degrees C and 30 degrees C, respectively, it was the oxygen level (but not the cultivation temperature or the morphological stage) which influenced the AEFG1 regulation.

Chromosome Mapping↗

PubMed2001

IgG and IgE immune response against the surface glycoprotein gp200 of Saccharomyces cerevisiae in patients with atopic dermatitis.

The heat-stable and soluble glycoprotein gp200 (molecular weight 200 kDa) is part of the cell wall of S. cerevisiae. Recently, an association was shown between IgA and IgG against gp200 and inflammation in Crohn's disease. Gp200 is able to induce a proliferation of human lymphocytes in vitro, together with a natural killer cell associated cytotoxicity. Specific IgE against Saccharomyces cerevisiae (baker's or brewer's yeast) may be detected in approximately 73%, against Candida albicans in 68% of those patients suffering from severe atopic dermatitis. The aim of this study was to elucidate the possible role of an anti-gp200 immune response for the pathogenesis of atopic dermatitis by immunoblot analysis. Anti-gp200 IgE was found in 55% of healthy individuals, in 67% of individuals with atopic predisposition without eczema, in 63% of the patients with mild atopic dermatitis, and in 86% of patients with severe atopic dermatitis, respectively. On the contrary, anti-gp200 IgG could be shown in 55% of healthy individuals, in 89% of individuals with atopic predisposition but without eczema, in 100% of patients with mild atopic dermatitis, and in 79% with severe atopic dermatitis, respectively. No immunoreactivity was found when an extract of Arxula adeninivorans was used as antigen. These results underline the specificity of the immunoblot results with gp200 from Saccharomyces cerevisiae. It can be concluded that occurrence of specific IgE against Saccharomyces cerevisiae cannot be explained by a cross reactivity, e.g., against Candida albicans allergens. Further investigations with the recombinant gp200 will give information on the role of this glycoprotein both in atopic dermatitis and Morbus Crohn.

Antigens, Fungal↗

PubMed2000

Morphology-related effects on gene expression and protein accumulation of the yeast Arxula adeninivorans LS3.

The dimorphism of the yeast Arxula adeninivorans LS3 is regulated by cultivation temperatures. Up to 42 degrees C the yeast grows as budding cells, which turn to mycelia at higher temperatures. To test whether the dimorphism is exclusively induced by high temperatures or also by other conditions, mutants were selected with an altered behaviour with respect to dimorphism. After mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, five of 25,000 colonies formed a very rough surface consisting of mycelia at 30 degrees C, in contrast to the wild-type. These mutants allow temperature-mediated and morphology-related effects on gene expression and protein accumulation to be distinguished. Budding cells and mycelia showed different expression of genes encoding secretory proteins at the same temperature. Mycelia secreted two-fold more protein than budding cells, including the enzymes glucoamylase and invertase. This indicated that morphology, rather than temperature, is the decisive factor in the analysed processes.

Ascomycota↗

PubMed2000

Genetic transformation and biotechnological application of the yeast Arxula adeninivorans.

The relatively unknown, non-pathogenic, dimorphic, haploid, ascomycetous yeast Arxula adeninivorans exhibits some unusual properties which are of biotechnological interest. The yeast is able to assimilate and ferment many compounds as sole source of carbon and/or nitrogen, it utilises n-alkanes and degrades starch efficiently. A. adeninivorans features such as thermo- and haloresistance as well as the yeast's uncommon growth and secretion behaviour should be especially emphasised. In media containing up to 20% NaCl, A. adeninivorans is able to grow at cultivation temperatures up to 48 degrees C. Additionally, the dimorphism of the yeast is unusual. Arxula grows at up temperatures of up to 42 degrees C as budding cells, which turn into mycelia at higher temperatures. This environmentally conditioned dimorphism is reversible and budding is reestablished when the cultivation temperature is decreased below 42 degrees C. Alteration of morphology correlates with changes in secretion behaviour. Mycelium cultures accumulate two-fold higher protein concentrations and contain two- to five-fold higher glucoamylase and invertase activities in the medium than budding cells. Based on these unusual properties, Arxula adeninivorans is used for heterologous gene expression and as a gene donor to construct more suitable yeasts for biotechnology. For example the Arxula glucoamylase gene was successfully expressed in Saccharomyces cerevisiae and Kluyveromyces lactis. Both transformed yeasts are able to assimilate and ferment starch as carbon source. A transformation system is used for heterologous gene expression which is based on integration of linearised DNA fragments in two to ten copies, e.g. into the 25S rDNA of A. adeninivorans by homologous recombination. The obtained transformants are mitotically stable. The expression of the lacZ gene from E. coli as well as the XylE gene from Pseudomonas putida indicates the suitability of A. adeninivorans as host for heterologous gene expression.

Biotechnology↗

PubMed2000

Amperometric measurement of copper ions with a deputy substrate using a novel Saccharomyces cerevisiae sensor.

The first microbial biosensor to detect Cu2+ by an amperometric method has been developed. For this purpose, recombinant Saccharomyces cerevisiae strains are suitable as the microbial component. These strains contain plasmids with the Cu2+-inducible promoter of the CUP1-gene from Saccharomyces cerevisiae fused to the lacZ-gene from E. coli. On this sensor the CUP1 promoter is first induced by the Cu2+-containing probe and subsequently lactose is used as a deputy substrate to make the measurement. If Cu2+ is present in the sample, these recombinant strains are able to utilize lactose as a carbon source, which leads to alterations in the oxygen consumption of the cells. The sensor measured Cu2+ in a concentration range between 0.5 and 2 mM CuSO4. In addition, an indirect amperometric measurement principle was developed which allows the detection of samples containing Cu2+ and fast biodegradable substances.

Base Sequence↗

PubMed2000

Designing an amperometric thick-film microbial BOD sensor.

Thick film oxygen electrodes manufactured by screen print method have been used as a transducer for a biochemical oxygen demand (BOD) sensor. The kinetics of the immobilized yeast, Arxula adeninivorans (Arxula) has been studied. The apparent KM of immobilized Arxula (> 100 microM) is higher than free cells of Arxula (70 microM). The increase in KM caused by the effect of immobilization extends the linear range of the sensor. End-point measurement and quasi-kinetic measurement have been studied comparatively as measurement procedures with a good correlation. The Vmax for end-point measurement is 790.7 microM/s and that for quasi-kinetic measurement is 537.3 microM/s. The limit of detection is calculated 1.24 mg/l BOD. Using the quasi-kinetic measurement, instead of end-point measurements, the measuring time can be reduced from 5-30 min to 100 s. The sensor layer thickness or increase in the layer of covering gel can increase the KM that is accompanied with the extension of the linear range of the sensor. Nevertheless, increase in the layer of covering gel will not increase the saturation signal. Domestic wastewater was checked by the thick film BOD sensor and the results are satisfactory.

Bacteria↗

PubMed2000

Halotolerance of the yeast Arxula adeninivorans LS3.

The non-pathogenic, dimorphic, ascomycetous yeast Arxula adeninivorans LS3 is halotolerant. It can grow in a minimal medium containing up to 20% NaCl. The growth parameters are only weakly influenced by 10% NaCl. However, NaCl in a concentration higher than 10% causes a decrease in the specific growth rate, a longer adaptation phase and a lower cell count in the stationary growth phase. Concentrations of glycerol and trehalose, which differed 100-fold in magnitude in a salt free medium, are also influenced differently by salt. NaCl induces accumulation of intracellular glycerol in exponentially growing cells but a reduced concentration of intracellular trehalose in stationary cells. Transcripts of the genes ARFC3, encoding a component of the replication factor C, and GAA, encoding a secretory glucoamylase, can be detected only in cells cultured in media with NaCl concentrations below 10%. Furthermore, NaCl in high concentration reduces the level of secreted proteins including glucoamylase end invertase.

Adaptation, Biological↗

PubMed2000

HLA-A, B, Cw and DRB1, DRB3/4/5, DQB1, DPB1 frequencies in German immunoglobulin A-deficient individuals.

HLA class I and II frequencies and haplotype frequencies were determined in 80 German immunoglobulin (Ig)A-deficient individuals and 157 healthy controls with normal IgA levels using serological and DNA typing methods. For several alleles, significant associations were found, which could be explained mainly in the context of a positive association with three different extended haplotypes (HLA-B*08:DRB1*0301: DQB1*0201, HLA-B*14:DRB1*0102:DQB1*0501 and HLA-B*44:DRB1*0701:DQB1*0202) and a negative association with a fourth haplotype (HLA-B*07:DRB1*1501:DQB1*0602). Furthermore, for the first time this study reports a positive association of IgA deficiency with DPB1 alleles. Homozygosity rate for the gene loci DRB1 and DQB1 was increased in IgA deficiency. Further analysis suggested a different pattern of HLA associations depending on the degree of IgA deficiency and the gender of the IgA-deficient individuals.

Alleles↗

PubMed1999

Measurement of biodegradable substances using the salt-tolerant yeast Arxula adeninivorans for a microbial sensor immobilized with poly(carbamoyl)sulfonate (PCS). Part II: Application of the novel biosensor to real samples from coastal and island regions.

A microbial sensor for rapid measurement of the amount of biodegradable substances based on the salt-tolerant yeast Arxula adeninivorans LS3 has been developed especially for coastal and island regions. Our parameter, the so-called sensorBOD, that is available after only a few minutes, agrees with the 5-day value for the biochemical oxygen demand (BOD5) very well. We have employed the Arxula sensor in the short-time estimation and supervision of the BOD of both domestic and industrial wastewater with high salinity. The novel sensor makes it possible to monitor the different types of wastewater rapidly without pretreatment, and it can be used for an active process control of sewage treatment works. Compared to a commercially available sensor, the novel sensor achieves better agreement between sensorBOD and BOD5 measurements with salt containing samples.

Ascomycota↗

PubMed1999

The green fluorescent protein targets secretory proteins to the yeast vacuole

The green fluorescent protein (GFP) was used as a marker to study the intracellular transport of vacuolar and secretory proteins in yeast. Therefore, the following gene constructs were expressed in Saccharomyces cerevisiae under control of the GAL1 promoter: GFP N-terminally fused to the yeast secretory invertase (INV-GFP), the plant vacuolar chitinase (CHN-GFP) and its secretory derivative (CHNDeltaVTP-GFP), which did not contain the vacuolar targeting peptide (VTP), both chitinase forms (CHN and CHNDeltaVTP), GFP without any targeting information and two secretory GFP variants with and without the VTP of chitinase (N-GFP-V and N-GFP). Whereas chitinase without VTP is accumulated in the culture medium the other gene products are retained inside the cell up to 48 h of induction. Independently of a known VTP they are transported to the vacuole, so far as they contain a signal peptide for entering the endoplasmic reticulum. This was demonstrated by confocal laser scanning microscopy, immunocytochemical analysis and subcellular fractionation experiments as well. The transport of the GFP fusion proteins is temporary delayed by a transient accumulation in electron-dense structures very likely derived from the ER, because they also contain the ER chaperone Kar2p/Bip. Our results demonstrate that GFP directs secretory proteins without VTP to the yeast vacuole, possibly by the recognition of an unknown vacuolar signal and demonstrates, therefore, a first limitation for the application of GFP as a marker for the secretory pathway in yeast.

Journal Article↗

PubMed1999

Molecular cloning and expression of the ARFC3 gene, a component of the replication factor C from the salt-tolerant, dimorphic yeast Arxula adeninivorans LS3.

The yeast replication factor C (RF-C) is a multi-polypeptide complex with five sub-units involved in chromosomal DNA replication. This factor, encoded by five genes, is well characterized for the yeast Saccharomyces cerevisiae. However, RF-C components from other yeast species have not been analysed yet. Here we report the cloning and characterization of ARFC3 from the dimorphic and osmo-tolerant yeast Arxula adeninivorans. This gene encodes one subunit of the RF-C complex. It is localized on chromosome 1 of the four Arxula chromosomes and comprises a coding region of 1014 bp, which corresponds to 338 amino acids. Two introns are contained within this gene. The ARFC3 transcript level is influenced by both salt and temperature. The latter also influences the morphological state (budding cells, mycelium). High salt concentration and high temperature result in a rapid decrease of the ARFC3 mRNA.

Amino Acid Sequence↗

PubMed1999

Measurement of biodegradable substances using the salt-tolerant yeast Arxula adeninivorans for a microbial sensor immobilized with poly(carbamoyl) sulfonate (PCS) Part I: Construction and characterization of the microbial sensor.

A microbial biosensor based on the yeast Arxula adeninivorans LS3 has been developed for measurement of biodegradable substances. Arxula is immobilized in the hydrogel poly(carbamoyl) sulfonate (PCS). The immobilized yeast membrane is placed in front of an oxygen electrode with -600 mV versus Ag/AgCl. Arxula is salt tolerant; it can give a stable signal up to 2.5 M NaCl in sample (120 mM in measuring cell). The sensor's measurements are highly correlated to BOD5 measurements. It has a very high stability which can last for 40 day without any decrease in signal. The linear range of the sensor is up to a corresponding BOD value of 550 mg/l.

Biodegradation, Environmental↗

PubMed1998

The peptide HDEF as a new retention signal is necessary and sufficient to direct proteins to the endoplasmic reticulum.

The key feature of tomato RNase LX localised solely outside the vacuole is the C-terminal peptide HDEF which is very similar to known endoplasmic reticulum (ER) retention signals. For functional testing of the ER-targeting ability of HDEF, different constructs including the complete RNase LX, two truncated forms without HDEF and the truncated chitinase FB7-1deltaVTP C-terminally flanked by HDEF, were expressed in Saccharomyces cerevisiae. The majority of RNase and chitinase, both containing HDEF, accumulates within the ER. However, the truncated constructs without the peptide are released into the medium. We provide compelling evidence that peptide HDEF at the C-terminus of secretory plant proteins is a new ER retention signal in yeast and most likely in plants.

Amino Acid Sequence↗

PubMed1998

Correct targeting of a vacuolar tobacco chitinase in Saccharomyces cerevisiae--post-translational modifications are dependent on the host strain.

The chitinase gene FB7-1 of Nicotiana tabacum cv. samsun line 5 was expressed in the two Saccharomyces cerevisiae strains, INVSC2 and H4, under the control of the GAL1 promoter from S. cerevisiae and a multicopy plasmid vector. Both yeast strains express the plant gene as enzymatic active proteins. In transformants of the strain INVSC2, 94% of the total plant chitinase is contained inside the cells, probably within the vacuole which has been confirmed by subcellular fractionation as well as immunohistochemical experiments. This retention inside the cells is due to the C-terminally located 7 amino acids long vacuolar targeting peptide of the prochitinase. When this sequence was removed, chitinase was transported into the culture medium. Pulse-chase experiments revealed that during translation in transformants of both yeast strains one chitinase polypeptide can be immunoadsorbed with specific antibodies. In the case of INVSC2-transformants, newly formed chitinase is modified in a 60 min chase to slightly increase its molecular mass, whereas in H4-transformants the molecular mass constantly remained 32 kDa. By Western blot analysis two chitinase corresponding polypeptides of 32 and 37 kDa were accumulated in the culture medium of both transformants carrying the chitinase gene without the vacuolar targeting sequence. The larger one was very likely O-glycosylated. Whereas, both polypepitdes were also detected in cell extracts of the H4-transformant, only the smaller one was found in the INVSC2-transformant. The plant chitinase passed through the endoplasmic reticulum on its way to the vacuole. The N-terminal signal peptide responsible for the uptake into the endoplasmic reticulum is cleaved correctly. However, cleavage of the vacuolar targeting peptide located at the C-terminus, to give the mature chitinase is obviously influenced by the genetic background of the host strain. In INVSC2-transformants chitinase accumulates in its mature form whereas both the polypeptides of H4-transformants retain their vacuolar targeting peptide. Our results demonstrate that in the case of plant class I chitinase, the plant sorting signal is recognized in yeast cells but post-translational modifications are influenced by the host strain.

Amino Acid Sequence↗

PubMed1998

AILV1 gene from the yeast Arxula adeninivorans LS3--a new selective transformation marker.

The ILV1 gene of the yeast Arxula adeninivorans LS3 (AILV1) has been cloned from a genomic library, characterized and used as an auxotrophic selection marker for transformation of plasmids into this yeast. One copy of the gene is present in the Arxula genome, comprising 1653 bp and encoding 550 amino acids of the threonine deaminase. The protein sequence is similar (60.55%) to that of the threonine deaminase from Saccharomyces cerevisiae encoded by the gene ILV1. The protein is enzymatically active during the whole period of cultivation, up to 70 h. Maximal activities, as well as protein concentrations of this enzyme, were achieved after cultivation times of 20-36 h. The AILV1 gene is a suitable auxotrophic selection marker in transformation experiments using an Arxula adeninivorans ilv1 mutant and a plasmid containing this gene, which is fused into the 25S rDNA of Arxula adeninivorans. One to three copies of the linearized plasmid were integrated into the 25S rDNA by homologous recombination. Transformants resulting from complementation of the ilv1 mutation can be easily and reproducibly selected and in addition are mitotically stable. Therefore, the described system is preferred to the conventional selection for hygromycin B resistance.

Amino Acid Sequence↗

PubMed1998

Integrative transformation of the dimorphic yeast arxula adeninivorans LS3 based on hygromycin B resistance.

A transformation system has been developed for the dimorphic yeast Arxula adeninivorans based on a stable integration of the donor DNA into ribosomal DNA. For this purpose a cassette was constructed which contains the E. coli hph gene, conferring hygromycin B resistance, fused to the 5' expression signals of the A. adeninivorans TEF1 gene, encoding the translation elongation factor EF-1alpha, and the transcription termination region of the Saccharomyces cerevisiae PHO5 gene. This cassette was fused into the 25S rDNA of A. adeninivorans. Linearization of this vector was required for high transformation frequencies. The vector was integrated in multiple copies into the 25S rDNA by homologous recombination. Copy number was not altered even after the growth of transformants for 15 generations under non-selective growth conditions. Microscopical analyses revealed that integration of the transformed plasmid did not influence the dimorphism, which is triggered at 42 degrees C for both transformed and non-transformed cells.

Anti-Bacterial Agents↗