The Hansenula polymorpha PDD7 gene is essential for macropexophagy and microautophagy

Hansenula polymorpha PDD genes are involved in the selective degradation of peroxisomes via macropexophagy. We have isolated various novel pdd mutants by a gene-tagging method. Here we describe the isolation and characterisation of PDD7, which encodes a protein with high sequence similarity (40% identity) to Saccharomyces cerevisiae Apg1p/Aut3p, previously described to be involved in random autophagy and the cytoplasm-to-vacuole targeting pathway. Our data indicate that HpPdd7p is essential for two processes that degrade peroxisomes, namely the highly selective process of macropexophagy and microautophagy, which occurs in H. polymorpha upon nitrogen starvation.     

Identification of intragenic mutations in the Hansenula polymorpha PEX6 gene that affect peroxisome biogenesis and methylotrophic growth

Two interacting AAA ATPases, Pex1p and Pex6p, are indispensable for peroxisome biogenesis in different organisms. Mutations affecting corresponding genes are the most common cause of the peroxisome biogenesis disorders in humans. By UV mutagenesis of the Hansenula polymorpha pex6 mutant, deficient in peroxisome biogenesis, we isolated a conditional cold-sensitive strain with restored ability to grow in methanol medium at 37 degrees C but not at 28 degrees C. Sequencing of the pex6 allele revealed a point mutation in the first AAA module of the PEX6 gene that leads to substitution of a conserved amino acid residue (G737E). An additional intragenic mutation identified in the cold-sensitive pex6 allele leads to a conserved amino acid substitution in the second AAA domain (R1000G). Electron microscopic analysis revealed restored peroxisomes in methanol-induced cold-sensitive pex6 cells at both permissive and restrictive temperatures. If separated, the secondary mutation did not affect methylotrophic growth. Our data suggest that H. polymorpha Pex6p may have a complex function in peroxisome biogenesis in which identified amino acid residues are involved.     

Atg21p is essential for macropexophagy and microautophagy in the yeast Hansenula polymorpha

ATG genes are required for autophagy-related processes that transport proteins/organelles destined for proteolytic degradation to the vacuole. Here, we describe the identification and characterisation of the Hansenula polymorpha ATG21 gene. Its gene product Hp-Atg21p, fused to eGFP, had a dual location in the cytosol and in peri-vacuolar dots. We demonstrate that Hp-Atg21p is essential for two separate modes of peroxisome degradation, namely glucose-induced macropexophagy and nitrogen limitation-induced microautophagy. In atg21 cells subjected to macropexophagy conditions, sequestration of peroxisomes tagged for degradation is initiated but fails to complete.     

Characterization of a higher plant herbicide-resistant phytoene desaturase and its use as a selectable marker

Three natural somatic mutations at codon 304 of the phytoene desaturase gene (pds) of Hydrilla verticillata (L. f. Royle) have been reported to provide resistance to the herbicide fluridone. We substituted the arginine 304 present in the wild-type H. verticillata phytoene desaturase (PDS) with all 19 other natural amino acids and tested PDS against fluridone. In in vitro assays, the threonine (Thr), cysteine (Cys), alanine (Ala) and glutamine (Gln) mutations imparted the highest resistance to fluridone. Thr, the three natural mutations [Cys, serine (Ser), histidine (His)] and the wild-type PDS protein were tested in vitro against seven inhibitors of PDS representing several classes of herbicides. These mutations conferred cross-resistance to norflurazon and overall negative cross-resistance to beflubutamid, picolinafen and diflufenican. The T3 generation of transgenic Arabidopsis thaliana plants harbouring the four selected mutations and wild-type pds had similar patterns of cross-resistance to the herbicides as observed in the in vitro assays. The Thr304 Hydrilla pds mutant proved to be an excellent marker for the selection of transgenic plants. Seedlings harbouring Thr304 pds had a maximum resistance to sensitivity (R/S) ratio of 57 and 14 times higher than that of the wild-type for treatments with norflurazon and fluridone, respectively. These plants exhibited normal growth and development, even after long-term exposure to herbicide. As Thr304 pds is of plant origin, it could become more acceptable than other selectable markers for use in genetically modified food.     

Stable transformation of trypanosomatids through targeted chromosomal integration of the selectable marker gene encoding blasticidin S deaminase

The susceptibilities of the protozoan parasites Leishmania mexicana and Trypanosoma brucei to the nucleoside antibiotic blasticidin S were assessed. A concentration of 10 microg ml(-1) was sufficient to cause cell death within 72 h of L. mexicana promastigotes and bloodstream forms of T. brucei in vitro. The gene encoding blasticidin S deaminase (BSD) was therefore incorporated into cassettes for targeting to the cysteine proteinase C locus of L. mexicana (CPC::BSD) and the tubulin locus of T. brucei (tub::RAD51-BSR). Following transfection of mutant parasites that contained other well-established selectable marker genes (HYG, NEO, BLE, PAC and SAT), clones resistant to 10 microg ml(-1) blasticidin S were shown by PCR and Southern blotting to have integrated the cassettes by homologous recombination. The results confirm that BSD can be used as a selectable marker gene for targeted chromosomal integration during genetic manipulations of trypanosomatids.     

Cloning and characterization of the Pichia pastoris MET2 gene as a selectable marker

We describe the isolation and characterization of a new biosynthetic gene, MET2, from the methylotrophic yeast Pichia pastoris. The predicted product of PpMET2 is significantly similar to its Saccharomyces cerevisiae counterpart, ScMET2, which encodes homoserine-O-transacetylase. The ScMET2 was able to complement the P. pastoris met2 strain; however, the converse was not true. Expression vectors based on PpMET2 for the intracellular and secreted production of foreign proteins and corresponding auxotrophic strains were constructed and tested for use in heterologous expression. The expression vectors and corresponding strains provide greater flexibility when using P. pastoris for recombinant protein expression.     

Transformation of Brassica napus by using the aadA gene as selectable marker and inheritance studies of the marker genes

An Agrobacterium tumefaciens -mediated transformation system for Brassica napus has been improved. We investigated several marker genes for transformation of Brassica napus , and the aadA gene, which confers resistance to streptomycin and spectinomycin, was found to be the most suitable. Forty-three out of 193 putative transformants in the T₁ generation were investigated by Southern blot analysis. Transformants containing a range of 1 to 10 integrated T-DNA copies per genome were found. Total DNA from 35 plants showed hybridisation to both the aadA and the nptll marker gene probes, from 5 plants only to one marker gene probe and from 3 plants DNA did not hybridise to any of the gene probes. Furthermore, more complex integration patterns such as direct repeated copies of the T-DNA, both as tandem and inverted copies, were observed. Inheritance of the marker genes in the T₂ generation was studied in 37 of the plants. This revealed that 22% of the plants that contained both marker genes, segregated as one single locus (3:1) for both genes, while 46% of the plants gave a segregation pattern corresponding to one T-DNA locus for at least one of the marker genes. Moreover, these inheritance patterns appeared to be more or less independent of the number of genes seen in the Southern blot analysis of the T, generation. In this study we show that the introduced marker genes are inherited by the T; generation in a less predictable way than was earlier reported for B. napus .     

Use of bar as a selectable marker gene and for the production of herbicide-resistant rice plants from protoplasts

We have used the bar gene in combination with the herbicide Basta to select transformed rice (Oryza sativa L. cv. Radon) protoplasts for the production of herbicide-resistant rice plants. Protoplasts, obtained from regenerable suspension cultures established from immature embryo callus, were transformed using PEG-mediated DNA uptake. Transformed calli could be selected 2–4 weeks after placing the protoplast-derived calli on medium containing the selective agent, phosphinothricin (PPT), the active component of Basta. Calli resistant to PPT were capable of regenerating plants. Phosphinothricin acetyltransferase (PAT) assays confirmed the expression of the bar gene in plants obtained from PPT-resistant calli. The only exceptions were two plants obtained from the same callus that had multiple copies of the bar gene integrated into their genomes. The transgenic status of the plants was varified by Southern blot analysis. In our system, where the transformation was done via the protoplast method, there were very few escapes. The efficiency of co-transformation with a reporter gene gusA, was 30%. The T_o plants of Radon were self-fertile. Both the bar and gusA genes were transmitted to progeny as confirmed by Southern analysis. Both genes were expressed in T₁ and T₂ progenies. Enzyme analyses on T₁ progeny plants also showed a gene dose response reflecting their homozygous and heterozygous status. The leaves of T_o plants and that of the progeny having the bar gene were resistant to application of Basta. Thus, the bar gene has proven to be a useful selectable and screenable marker for the transformation of rice plants and for the production of herbicide-resistant plants.     

Isolation and sequence analysis of a beta-tubulin gene from Aspergillus flavus and its use as a selectable marker

An altered beta-tubulin gene that confers resistance to benomyl [whose active ingredient is 2-(methoxycarbonylamino)benzimidazole (MBC)] was isolated from a DNA library of Aspergillus flavus and used as a selectable marker for transformation. The beta-tubulin gene was cloned into a plasmid vector containing the pyr-4 gene of Neurospora crassa, and transformants were selected either for uracil prototrophy or MBC resistance. Transformants selected for uracil prototrophy were of three phenotypic classes: sensitive, intermediate, and resistant to MBC. Transforming DNA appeared to integrate at several sites in the genome, with the more resistant phenotypes having more copies of the altered beta-tubulin gene than the sensitive and intermediate phenotypes. Transformants were also selected on medium containing MBC. The average frequency of transformation (1 to 3 transformants per micrograms of transforming DNA) was lower than that obtained by selection for uracil prototrophy, presumably because of failure to select transformants that contained few copies of the altered beta-tubulin gene. The sequence of the beta-tubulin gene was determined and compared with the published sequence of the benA gene of A. nidulans; the beta-tubulin gene was found to be highly conserved between the two Aspergillus species. Notable differences were that the beta-tubulin gene of A. flavus lacks intron 6 present in benA and has an additional leucine at position 148. This is the first gene sequence reported from an aflatoxin-producing fungus and adds to the growing body of knowledge of the beta-tubulin genes and their use as selectable markers for transformation of filamentous fungi.     

Inactivation of the Hansenula polymorpha PMR1 gene affects cell viability and functioning of the secretory pathway

In yeast, functions of the endoplasmic reticulum (ER) depend on the Golgi apparatus Ca2+ pool, which is replenished by the medial-Golgi ion pump Pmr1p. Here, to dissect the role of the Golgi Ca2+ pool in protein folding and elimination of unfolded proteins in the ER, the manifestations of the pmr1 mutation in yeast Hansenula polymorpha were studied. The PMR1 gene was disrupted in a H. polymorpha diploid strain. Haploid segregants of this diploid bearing the disruption allele were viable, though they showed a severe growth defect on synthetic medium and rapidly died during storage at low temperature. Disruption of H. polymorpha PMR1 led to defects of the Golgi-hosted protein glycosylation and vacuolar protein sorting. This mutation increased the survival rate of H. polymorpha cells upon treatment with the proapoptotic drug amiodarone. Unlike Saccharomyces cerevisiae, the H. polymorpha pmr1 mutant was not hypersensitive to chemicals that induce the accumulation of unfolded proteins in the ER, indicating that the elimination of unfolded proteins from the ER was not essentially affected. At the same time, the pmr1 mutation improved the secretion of human urokinase and decreased its intracellular aggregation, indicating an influence of the mutation on the protein folding in the ER.     

The application of the mutated acetolactate synthase gene from rice as the selectable marker gene in the production of transgenic soybeans

We investigated selective culturing conditions for the production of transgenic soybeans. In this culturing system, we used the acetolactate synthase (ALS)-inhibiting herbicide-resistance gene derived from rice (Os-mALS gene) as a selectable marker gene instead of that derived from bacteria, which interfered with the cultivation and practical usage of transgenic crops. T₁ soybeans grown from one regenerated plant after selection of the ALS-targeting pyrimidinyl carboxy (PC) herbicide bispyribac-sodium (BS) exhibited herbicide resistance, and the introduction and expression of the Os-mALS gene were confirmed by genetic analysis. The selective culturing system promoted by BS herbicide, in which the Os-mALS gene was used as a selectable marker, was proved to be applicable to the production of transgenic soybeans, despite the appearance of escaped soybean plants that did not contain the Os-mALS transgene.     

Engineering of an endogenous phytoene desaturase gene as a dominant selectable marker for Chlamydomonas reinhardtii transformation and enhanced biosynthesis of carotenoids

A phytoene desaturase (PDS) gene was cloned and characterized from the unicellular green microalga Chlamydomonas reinhardtii. Functional complementation analysis revealed C. reinhardtii PDS (CrPDS) catalyzes the conversion of phytoene to the colored carotenoid ζ-carotene. A single amino acid substitution, L505F, enhanced its desaturation activity by 29%, as indicated by an in vitro enzymatic assay. In addition, CrPDS-L505F exhibited 27.7-fold higher resistance to the herbicide norflurazon. Glass bead-mediated delivery displayed a high transformation efficiency of C. reinhardtii with CrPDS-L505F, demonstrating clearly that the engineered endogenous CrPDS is a dominant selectable marker for C. reinhardtii and possibly for other green algae. Furthermore, the expression of PDS could enhance the intracellular carotenoid accumulation of transformants, opening up the possibility of engineering the carotenogenic pathway for improved carotenoid production in microalgae.     

Molecular characterization of the actin-encoding gene and the use of its promoter for a dominant selection system in the methylotrophic yeast Hansenula polymorpha

The actin gene (ACT) from the methylotrophic yeast Hansenula polymorpha was cloned and its structural feature was characterized. In contrast to the actin genes of other ascomycetous yeasts, which have only one large intron, the H. polymorpha ACT gene was found to be split by two introns. The H. polymorpha ACT introns were correctly processed in the heterologous host Saccharomyces cerevisiae despite appreciable differences in the splice site sequences. The promoter region of H. polymorpha ACT displayed two CCAAT motifs and two TATA-like sequences in a configuration similar to that observed in the S. cerevisiae actin promoter. A set of deleted H. polymorpha ACT promoters was exploited to direct expression of the bacterial hygromycin B resistance (hph) gene as a dominant selectable marker in the transformation of H. polymorpha. The resistance level of H. polymorpha transformants to the antibiotic was shown to be dependent on the integration copy number of the hph cassette. The selectivity of the hygromycin B resistance marker for transformants of higher copy number was remarkably increased with the deletion of the upstream TATA-like sequence, but not with the removal of either CCAAT motif, from the H. polymorpha promoter. The dosage-dependent selection system developed in this study should be useful for genetic manipulation of H. polymorpha as an industrial strain to produce recombinant proteins.     

Cloning and characterization of the gene for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker.

We cloned the beta-tubulin gene of Neurospora crassa from a benomyl-resistant strain and determined its nucleotide sequence. The gene encodes a 447-residue protein which shows strong homology to other beta-tubulins. The coding region is interrupted by six introns, five of which are within the region coding for the first 54 amino acids of the protein. Intron position comparisons between the N. crassa gene and other fungal beta-tubulin genes reveal considerable positional conservation. The mutation responsible for benomyl resistance was determined; it caused a phenylalanine-to-tyrosine change at position 167. Codon usage in the beta-tubulin gene is biased, as has been observed for other abundantly expressed N. crassa genes such as am and the H3 and H4 histone genes. This bias results in pyrimidines in the third positions of 96% of the codons in codon families in which there is a choice between purines and pyrimidines in this position. Bias is also evident by the absence of 19 of the 61 sense codons. We demonstrated that benomyl resistance is due to the cloned beta-tubulin gene of strain Bml511(r)a and that this gene can be used as a dominant selectable marker in N. crassa transformation.     

Mutant acetolactate synthase gene is an efficient in vitro selectable marker for the genetic transformation of Brassica juncea (oilseed mustard)

We report in this study, the successful deployment of a double mutant acetolactate synthase gene (ALSdm, containing Pro 197 to Ser and Ser 653 to Asn substitutions) as an efficient in vitro selection marker for the development of transgenic plants in Brassica juncea (oilseed mustard). The ALS enzyme is inhibited by two categories of herbicides, sulfonylureas (e.g. chlorsulfuron) and imidazolinones (e.g. imazethapyr), while the mutant forms are resistant to the same. Three different selection agents (kanamycin, chlorsulfuron and imazethapyr) were tested for in vitro selection efficiency in two B. juncea cultivars, RLM198 and Varuna. For both the cultivars, higher transformation frequencies were obtained using chlorsulfuron (3.8 +/- 0.6% and 4.6 +/- 0.9% for RLM198 and Varuna, respectively) and imazethapyr (10.2 +/- 0.7% for RLM198 and 7.8 +/- 1.2% for Varuna) as compared to that obtained on kanamycin (3.1 +/- 0.2% and 2.8 +/- 0.5% for RLM198 and Varuna, respectively). Additionally, transformation frequencies were higher on imazethapyr than on chlorsulfuron for both the cultivars indicating that imidazolinones are better selective agents than sulfonylureas for the selection of mustard transgenics.     

Use of the GUS gene as a selectable marker for Agrobacterium-mediated transformation of Rubus

A transformation system was established for red raspberry, blackberry and blackberry x raspberry hybrids, utilizing the binary vector system of Agrobacterium tumefaciens. Leaf discs or internodal stem segments were inoculated with Agrobacterium strain LBA4404 containing the binary vectors PBI121.X, which has the -glucuronidase (GUS) marker gene, or Bin 19, which has the neomycin phosphotransferase II (NPT II) gene. Regenerants were produced on media containing MS salts, 20 gl^-1 sucrose, 7 gl^-1 agar, 100 mgl^-1 inositol, 0.5 mgl^-1 nicotinic acid, 0.5 mgl^-1 pyridoxine-HCl, 0.1 mgl^-1 thiamine, and either 0.1 mgl^-1 IBA and 2 mgl^-1 BAP for leaf discs, or 0.2 mgl^-1 BAP and 0.2 mgl^-1 2,4-D for stem segments. Kanamycin sulphate, which was used as a selective agent for the NPT II gene, inhibited organogenesis at 50 mgl^-1 and was therefore unsuitable for use as a selectable marker gene in Rubus. All regenerants were assayed utilizing the fluorogenic assay procedure to determine if the GUS gene had been transferred into the material and could therefore cleave the substrate 4-methyl-umbelliferyl--D-glucuronide. Seven GUS-positive plantlets were obtained which confirmed that this marker gene had been transferred into Rubus. A dot blot assay was carried out on GUS-positive plant material to establish if the NPT II gene had also been transferred to the plant material.     

Comparison of selective agents for use with the selectable marker gene bar in maize transformation

The effectiveness of four phosphinothricin (PPT)-based selective agents were evaluated for use in maize transformation: glufosinate, bialaphos, Basta^® and Herbiace^®. Glufosinate and its commercial formulation, Basta^®, were less effective in controlling growth of non-transgenic corn callus than the tripeptide, bialaphos, or its commercial formulation, Herbiace^®. Addition of 25 mM l-proline had no significant effect on selection when using bialaphos. However, when l-proline was included with the selective agent glufosinate, selection was inhibited and callus growth was enhanced. At four weeks, callus growth on 0.3, 1.0 and 3.0 mg l^-1 glufosinate in the presence of proline was 76, 43, and 21% of control growth, respectively, and in the absence of proline was only 32, 9, and 6% of control growth. Optimized selection protocols for Basta^® and bialaphos yielded comparable numbers of transformants. Using these protocols, fertile transgenic plants were regenerated from transformed callus cultures.Abbreviations AA amino acid - GS glutamine synthetase - PAT phosphinothricin acetyl transferase - PPT 2-amino-(methylphosphinyl)-butanoic acid (phosphinothricin) - 2,4-d 2,4-dichlorophenoxyacetic acid     

Cloning and sequencing of the ura3 locus of the methylotrophic yeast Hansenula polymorpha and its use for the generation of a deletion by gene replacement

The ura3 gene of Hansenula polymorpha was cloned, sequenced and used to generate a ura3 mutant from the wild-type strain of this yeast via integrative mutagenesis. The Tn5 neomycin-resistance marker (neo) under control of the ADH1 promoter from Saccharomyces cerevisiae served as a transformation marker. The results show that gene replacement can be achieved in H. polymorpha, a yeast with a high level of non-homologous integration. Correspondence to: C. P. Hollenberg