Transformation of Penicillium chrysogenum using dominant selection markers and expression of an Escherichia coli lacZ fusion gene

An industrial Penicillium chrysogenum strain was transformed using two dominant selection markers, namely the bacterial gene for phleomycin resistance (ble) fused to a fungal promoter, and the acetamidase (amdS) gene from Aspergillus nidulans. Transformation frequencies of up to 20 transformants per microgram of DNA were obtained with the ble system. With the amdS marker the frequency was up to 120 transformants. Cotransformation was very efficient when using amdS as a selection marker. The introduction of pAN5-41B, a plasmid carrying the Escherichia coli lacZ gene fused to the strong glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter from A. nidulans, resulted in the formation of blue colonies on XGal plates indicating expression of the lacZ fusion gene in P. chrysogenum. A more detailed analysis of expression levels in several transformants showed that up to 6% of the total amount of soluble protein consists of the beta-galactosidase fusion protein.     

Optimized vectors and selection for transformation of Neurospora crassa and Aspergillus nidulans to bleomycin and phleomycin resistance.

To provide a dominant selectable marker for transformation of Neurospora crassa strains lacking specific auxotrophic mutations, we have engineered the bleomycin (Bm) resistance-encoding gene (ble) from the bacterial transposon Tn5 for expression in N. crassa. The coding region of the ble gene was fused to the promoter and terminator regions of the N. crassa am gene. In some vectors, multiple cloning sites were placed flanking the ble gene to provide a versatile ble cassette. When introduced into N. crassa, the hybrid ble gene conferred resistance to greater than 15 micrograms Bm/ml. Under optimal conditions, the levels of Bm required (2.5 micrograms/ml) make even large-scale transformation experiments very economical. Aspergillus nidulans could also be efficiently transformed to Bm resistance using the N. crassa ble gene fusion. Since the ble gene functions in both N. crassa and A. nidulans, the gene should be useful as a transformation marker for the many other filamentous fungi which are sensitive to Bm.     

Sequence and expression characteristics of a shuttle chloramphenicol-resistance marker for Saccharomyces cerevisiae and Escherichia coli

An efficiently transforming chloramphenicol-resistance (CmR) shuttle marker for Saccharomyces cerevisiae and Escherichia coli has been characterized in terms of its primary structure and expression characteristics. The complete nucleotide (nt) sequence of the CmR marker is given, with details on restriction sites, apparent expression signals for both organisms, and translation of the Cm acetyltransferase (CAT)-coding sequence. SDS-polyacrylamide gel electrophoresis and Western blotting have confirmed that the marker produced an identical CAT protein in yeast and E. coli. Each copy of the marker, whether present in multiple copies or as a single copy, gave rise to approx. 0.1% of the total soluble protein as CAT in haploid yeast cells. When compared with homologous expression of alcohol dehydrogenase (ADH-I) by the same ADC1 promoter, this represents a 27-fold reduction for CAT expression, which is typical of heterologous gene expression in yeast. When the marker was on a multicopy plasmid in yeast, up to 2.1% of the total soluble cell protein was produced as CAT, but this did not adversely affect the growth of host cells. Increase of the Cm concentration in the medium did not result in an increase in the number of plasmids nor the amount of CAT protein produced, showing that plasmid copy number and marker expression are regulated independently of the selection pressure. In E. coli, the ADC1 yeast-promoter DNA was found to contain both forwards and backwards promoter activity. The level of expression provided by these promoters was equivalent to that of an average E. coli gene.     

Efficient targeted mutagenesis in Borrelia burgdorferi

Genetic studies in Borrelia burgdorferi have been hindered by the lack of a nonborrelial selectable marker. Currently, the only selectable marker is gyrB(r), a mutated form of the chromosomal gyrB gene that encodes the B subunit of DNA gyrase and confers resistance to the antibiotic coumermycin A(1). The utility of the coumermycin-resistant gyrB(r) gene for targeted gene disruption is limited by a high frequency of recombination with the endogenous gyrB gene. A kanamycin resistance gene (kan) was introduced into B. burgdorferi, and its use as a selectable marker was explored in an effort to improve the genetic manipulation of this pathogen. B. burgdorferi transformants with the kan gene expressed from its native promoter were susceptible to kanamycin. In striking contrast, transformants with the kan gene expressed from either the B. burgdorferi flaB or flgB promoter were resistant to high levels of kanamycin. The kanamycin resistance marker allows efficient direct selection of mutants in B. burgdorferi and hence is a significant improvement in the ability to construct isogenic mutant strains in this pathogen.     

Construction of an Arxula adeninivorans host-vector system based on trp1 complementation

A host/vector expression system based on an Arxula adeninivorans Delta atrp1 gene disruption mutant has been constructed. For this purpose the ATRP1 gene encoding a phosphoribosyl anthranilate isomerase was isolated from the yeast A. adeninivorans and its genome locus was characterized. The Delta atrp1 mutant was generated applying an amplified DNA fragment containing the ALEU2m gene flanked by ATRP1 gene sequences of some 750 bp. The generated auxotrophic host strain was transformed with the plasmid pAL-ATRP1-amyA, which contains the ATRP1 gene as selection marker and the 25S rDNA for targeting. For expression assessment, the plasmid was equipped with an expression cassette consisting of the Bacillus amyloliquefaciens-derived amyA gene fused to the constitutive A. adeninivorans-derived TEF1 promoter and Saccharomyces cerevisiae-derived PHO5 terminator. Transformants contained a single chromosomal copy of the heterologous DNA and were found to be mitotically stable. In initial fermentation trials on a 200 ml shake flask scale maximal alpha-amylase product levels of ca. 300 nkat ml(-1) were observed after 72 h of cultivation with more than 95% of the recombinant alpha-amylase accumulated in the culture medium.     

Phleomycin resistance encoded by the ble gene from transposon Tn 5 as a dominant selectable marker in Saccharomyces cerevisiae

Summary Phleomycin, a water-soluble antibiotic of the bleomycin family is as effective against Saccharomyces cerevisiae cells as against Escherichia coli cells. The ble gene of transposon Tn5, which confers resistance to phleomycin, was inserted in place of the iso-1-cytochrome C (CYC1) gene on an autonomously replicative multicopy E. coli-yeast shuttle plasmid. Higher resistance levels are obtained in S. cerevisiae when the region immediately upstream from the initiation codon conforms to the nucleotide sequence stringencies observed in almost every yeast gene. The expected regulation pattern of the whole CYC1 promoter confers different phleomycin resistance levels to the cell under varying physiological conditions. Partial deletions in the CYC1 promoter lead to changes in the resistance level of cells which are mostly accounted for by the removal of known positive and negative regulatory elements. Some of the vector constructions allow direct selection of phleomycin-resistant transformants on rich media.     

Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes

A general purpose transformation vector, designated pPha-T1, was constructed for use with the diatom Phaeodactylum tricornutum Bohlin. This vector harbors the sh ble cassette for primary selection on medium containing the antibiotic zeocin, and a multiple cloning site flanked by the P. tricornutum fcp A promoter. pPha-T1 was used to establish the utility of three selectable marker genes and two reporter genes for P. tricornutum transformation. The nat and sat-1 genes confer resistance to the antibiotic nourseothricin, and npt II confers resistance to G418. Each of these genes was effective as a selectable marker for identifying primary transformants. These markers could also be used for dual selections in combination with the sh ble gene. The reporter genes uid A and gfp were also introduced into P. tricornutum using pPha-T1. Gus expression in some transformants reached 15 μg·μg⁻¹ of total soluble protein and permitted excellent cell staining, while GFP fluorescence was readily visible with standard fluorescence microscopy. The egfp gene, which has optimal codon usage for expression in human cells, was the only version of gfp that produced a strong fluorescent signal in P. tricornutum. The codon bias of the egfp gene is similar to that of P. tricornutum genes. This study suggests that codon usage has a significant effect on the efficient expression of reporter genes in P. tricornutum. The results presented here demonstrate that a variety of selectable markers and reporter genes can be expressed in P. tricornutum , enhancing the potential of this organism for exploring basic biological questions and industrial applications.     

The Saccharomyces cerevisiae MGT1 DNA repair methyltransferase gene: its promoter and entire coding sequence, regulation and in vivo biological functions.

We previously cloned a yeast DNA fragment that, when fused with the bacterial lacZ promoter, produced O6-methylguanine DNA repair methyltransferase (MGT1) activity and alkylation resistance in Escherichia coli (Xiao et al., EMBO J. 10,2179). Here we describe the isolation of the entire MGT1 gene and its promoter by sequence directed chromosome integration and walking. The MGT1 promoter was fused to a lacZ reporter gene to study how MGT1 expression is controlled. MGT1 is not induced by alkylating agents, nor is it induced by other DNA damaging agents such as UV light. However, deletion analysis defined an upstream repression sequence, whose removal dramatically increased basal level gene expression. The polypeptide deduced from the complete MGT1 sequence contained 18 more N-terminal amino acids than that previously determined; the role of these 18 amino acids, which harbored a potential nuclear localization signal, was explored. The MGT1 gene was also cloned under the GAL1 promoter, so that MTase levels could be manipulated, and we examined MGT1 function in a MTase deficient yeast strain (mgt1). The extent of resistance to both alkylation-induced mutation and cell killing directly correlated with MTase levels. Finally we show that mgt1 S.cerevisiae has a higher rate of spontaneous mutation than wild type cells, indicating that there is an endogenous source of DNA alkylation damage in these eukaryotic cells and that one of the in vivo roles of MGT1 is to limit spontaneous mutations.     

Development of molecular markers using MFLP linked to a gene conferring resistance to Diaporthe toxica in narrow-leafed lupin ( Lupinus angustifolius L.)

Phomopsis stem blight (PSB) caused by Diaporthe toxica is a major disease in narrow-leafed lupin ( Lupinus angustifolius L.). The F(2) progeny and the parental plants from a cross between a breeding line 75A:258 (containing a single dominant resistance gene Phr1 against the disease) and a commercial cultivar Unicrop (susceptible to the disease) were used for development of molecular markers linked to the disease resistance gene. Two pairs of co-dominant DNA polymorphisms were detected using the microsatellite-anchored fragment length polymorphism (MFLP) technique. Both pairs of polymorphisms were isolated from the MFLP gels, re-amplified by PCR, sequenced, and converted into co-dominant, sequence-specific and PCR-based markers. Linkage analysis by MAPMAKER suggested that one marker (Ph258M2) was 5.7 centiMorgans (cM) from Phr1, and the other marker (Ph258M1) was 2.1 cM from Ph258M2 but further away from Phr1. These markers are suitable for marker-assisted selection (MAS) in lupin breeding.     

The global DNA methylation surrogate LINE-1 methylation is correlated with MGMT promoter methylation and is a better prognostic factor for glioma

Gliomas are the most frequently occurring primary brain tumor in the central nervous system of adults. Glioblastoma multiformes (GBMs, WHO grade 4) have a dismal prognosis despite the use of the alkylating agent, temozolomide (TMZ), and even low grade gliomas (LGGs, WHO grade 2) eventually transform to malignant secondary GBMs. Although GBM patients benefit from promoter hypermethylation of the O(6)-methylguanine-DNA methyltransferase (MGMT) that is the main determinant of resistance to TMZ, recent studies suggested that MGMT promoter methylation is of prognostic as well as predictive significance for the efficacy of TMZ. Glioma-CpG island methylator phenotype (G-CIMP) in the global genome was shown to be a significant predictor of improved survival in patients with GBM. Collectively, we hypothesized that MGMT promoter methylation might reflect global DNA methylation. Additionally in LGGs, the significance of MGMT promoter methylation is still undetermined. In the current study, we aimed to determine the correlation between clinical, genetic, and epigenetic profiles including LINE-1 and different cancer-related genes and the clinical outcome in newly diagnosed 57 LGG and 54 GBM patients. Here, we demonstrated that (1) IDH1/2 mutation is closely correlated with MGMT promoter methylation and 1p/19q codeletion in LGGs, (2) LINE-1 methylation levels in primary and secondary GBMs are lower than those in LGGs and normal brain tissues, (3) LINE-1 methylation is proportional to MGMT promoter methylation in gliomas, and (4) higher LINE-1 methylation is a favorable prognostic factor in primary GBMs, even compared to MGMT promoter methylation. As a global DNA methylation marker, LINE-1 may be a promising marker in gliomas.     

Positioning of a Nucleosome on Mouse Satellite DNA Inserted into a Yeast Plasmid Is Determined by Its DNA Sequence and an Adjacent Nucleosome

It has earlier been shown that multiple positioning of nucleosomes on mouse satellite DNA is determined by its nucleotide sequence. To clarify whether other factors, such as boundary ones, can affect the positionings, we modified the environment of satellite DNA monomer by inserting it into a yeast plasmid between inducible GalCyc promoter and a structural region of the yeast FLP gene. We have revealed that the positions of nucleosomes on satellite DNA are identical to those detected upon reconstruction in vitro. The positioning signal (GAAAAA sequence) of satellite DNA governs nucleosome location at the adjacent nucleotide sequence as well. Upon promoter induction the nucleosome, translationally positioned on the GalCyc promoter, transfers to the satellite DNA and its location follows the positioning signal of the latter. Thus, the alternatives of positioning of a nucleosome on satellite DNA are controlled by its nucleotide sequence, though the choice of one of them is determined by the adjacent nucleosome.