Effect of de-phosphorylation of linearized pAN7-1 and of addition of restriction enzyme on plasmid integration in Penicillium paxilli

As part of a study to investigate the pathways of plasmid pAN7-1 integration in Penicillium paxilli, a molecular analysis of 90 different integration events was carried out. Twenty out of forty five integration events analyzed from transformants obtained without the addition of restriction enzyme to the transformation reaction mixture were single-copy integrations, whereas the remaining 25 were tandem-repeat integrations. The addition of restriction enzyme resulted in a shift in this ratio in favour of single-copy integration events. Analysis of the 33 tandem-repeat integration events showed that the orientation of the plasmid copies was not random, with 88% organized as tandem head-to-tail arrays. De-phosphorylation of linearized pAN7-1 did not affect the frequency with which multiple copies were integrated. This suggests that the predominant mechanism for the generation of tandem repeats in P. paxilli is by homologous recombination rather than in vivo ligation of linearized plasmids. Received: 11 March / 6 May 1997     

Influence of homology size and polymorphism on plasmid integration in the yeast CYC1 DNA region

We studied the influence of homology size and polymorphism on the integration of circular plasmids into the yeast CYC1 region. The plasmids used also contained the URA3 gene, and the proportion of Ura⁺ transformants resulting from plasmid integration into the CYC1 region was determined by Southern-blot analysis. A size-dependent decrease in integration into the CYC1 region was observed from 858 bp to 363 bp of homology. However, with a homology size of 321, 259 or 107 bp, about 2% of the transformants still contained plasmid molecules integrated in the CYC1 region. A single point mutation in the 858-bp fragment decreased the proportion of integrations to the CYC1 gene, but the presence of additional mutations did not have a cumulative effect. For plasmids isolated in a single-stranded (ss) form, the presence of two or six point mutations did not influence integration. These results were compared with those obtained in other assays designed to study substrate requirements for homologous recombination. Received: 18 October / 15 December 1999     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of the antitumor clavaric acid-producing basidiomycete <Emphasis Type="Italic">Hypholoma sublateritium</Emphasis>

The basidiomycete Hypholoma sublateritium produces clavaric acid, an antitumor isoprenoid compound. Arthrospores of this fungus were transformed by Agrobacterium tumefaciens-mediated conjugation. Five plasmids carrying different regulatory sequences to drive expression of the hph (hygromycin phosphotransferase) gene were tested. The promoter used was critically important in order to express heterologous genes in H. sublateritium. Constructions carrying the Agaricus bisporus glyceraldehyde-3-phosphate dehydrogenase promoter (Pgpd) showed a good transformation efficiency, whereas constructions with the gpd promoter from ascomycetes were ineffective. Transformant clones showed a random integration pattern of plasmid DNA. Most transformants showed a single integrated copy of the transforming plasmid, but about 1.5% showed double or multiple integrations. All the analyzed transformants were mitotically stable and maintained the integrated exogenous DNA in the absence of antibiotic. The green fluorescent protein gene was expressed from the A. bisporus gpd promoter, as shown by RT-PCR studies, but no significant fluorescence was observed. Transformation of H. sublateritium opens the way for the genetic manipulation of clavaric acid biosynthesis in this fungus.     

DNA repair genes of Saccharomyces cerevisiae: complementing rad4 and rev2 mutations by plasmids which cannot be propagated in Escherichia coli

Summary The RAD4 gene of yeast required for the incision step of DNA excision repair and the REV2 (= RAD5) gene involved in mutagenic DNA repair could not be isolated from genomic libraries propagated in E. coli regardless of copy number of the shuttle vector in yeast. Transformants with plasmids conferring UV resistance to a rad4-4 or a rev2-1 mutant were only recovered if yeast was transformed directly without previous amplification of the gene bank in E. coli. DNA preparations from these yeast clones yielded no transformants in E. coli but retransformation of yeast was possible. This lead to the isolation of a defective derivative of the rad4 complementing plasmid. The modified plasmid was now capable of transforming E. coli but still interfered significantly with its growth.Dedicated to Prof. Dr. Fritz Kaudewitz on the occasion of his 65th birthday     

Multiple copies of the amdS gene of Aspergillus nidulans cause titration of trans-acting regulatory proteins

Summary It has been established that a plasmid containing the amdS gene of Aspergillus nidulans may be used to transform amdS⁺ strains by selecting for increased utilization of acetamide as sole nitrogen source. Analysis of transformants has shown that multiple tandem copies of the plasmid can be integrated into the chromosome, commonly at sites other than the amdS locus. While the transformed phenotype was relatively stable through mitotic and meiotic divisions evidence was found for variation in plasmid copy number presumably due to unequal recombination events. Expression of the integrated amdS genes was related to copy number, and the amdS RNA produced was similar in size to wild-type RNA. Evidence for titration of the product of the regulatory gene amdR by multiple copies of amdS was found. No titration of the product of the areA gene was observed, and amdS expression was still dependent on areA function. Multiple copies of the amdI9 mutation resulted in poor growth on acetate. This was not observed in the case of the amdS⁺ gene. The cis-acting amdI9 mutation causes increased facB dependent acetate induction of amdS expression. Titration of the facB gene produce by amdI9 DNA, but not by amdS⁺ DNA, therefore suggested that the mutation results in increased affinity for the facB gene product.     

Multiple-copy integration of the α-galactosidase gene from Cyamopsis tetragonoloba into the ribosomal DNA of Kluyveromyces lactis

Summary We have developed a vector system for high-copy-number integration into the ribosomal DNA of the yeast Kluyveromyces lactis. This system is analogous to the pMIRY-system developed for Saccharomyces cerevisiae. Plasmids containing a portion of K. lactis rDNA for targeted homologous recombination, as well as the S. cerevisiae TRP1 gene with various promoter deletions, were constructed and, after transformation to K. lactis, analyzed for both copy number and stability. These plasmids were found to be present in about 60 copies per cell and were stably maintained during growth under non-selective conditions. Using this vector system, we expressed a fusion construct containing the S. cerevisiae GAL7 promoter, the SUC2 (invertase) signal sequence and the gene coding for -galactosidase from the plant Cyamopsis tetragonoloba. Although the maximum copy number of these integrated plasmids was only about 15, we nevertheless obtained a high level of -galactosidase production (250 mg/l) with a secretion efficiency of about 95%. When compared to extrachromosomal K. lactis vectors containing the same fusion construct, the multicopy integrants showed a much higher -galactosidase production level and a considerably higher stability under non-selective conditions.     

Gibberella pulicaris transformants: state of transforming DNA during asexual and sexual growth

A genetically fertile, trichothecene-producing plant pathogen, Gibberella pulicaris (Fusarium sambucinum), was transformed with three different vectors: cosHyg1, pUCH1, and pDH25. All three vectors carry hph (encoding hygromycin B phosphotransferase) as the selectable marker. Transformation frequency was 0.03 transformants per mg of DNA for pDH25 and 0.5 for pUCH1 or cosHyg1. The vector DNA sequences integrated at different sites into the fungal genome. Transformants were classified into three types based upon distinctive integration patterns: type A contained a single, intact copy of the vector at one site per genome; type B contained multiple tandem copies or a combination of single and multiple tandem copies at one or more sites per genome; type C contained a partial vector copy at one site per genome. While the transformants with cosHyg1 and pUCH1 were type A or B, type C was unique to pDH25 transformants. Type A and C transformants were both meiotically and mitotically stable. However, type B multiple inserts were unstable in mitosis and meiosis since: (1) multiple tandem copies were deleted: (2) rearrangements occurred during premeiosis; and (3) inserts in one of the type B transformants became methylated during premeiosis. Differential expression of transforming sequences between spore germination and mycelial growth was also observed among type B transformants. The ability to transform G. pulicaris with the resulting varied features of integration patterns and the behavior of transforming DNA during mitosis and meiosis provides a means to isolate, manipulate, and study cloned genes in this mycotoxin-producing plant pathogen.Mention of companies or products by name does not imply the endorsement by the U.S. Department of Agriculture over others not cited     

Shuttle vectors for <Emphasis Type="Italic">Candida albicans</Emphasis>: control of plasmid copy number and elevated expression of cloned genes

Plasmids containing the inosine monophosphate dehydrogenase gene CaIMH3 from Candida albicans strain ATCC 32354 transform their host to resistance against mycophenolic acid (MPA). The transformants maintain the plasmids at a high copy number (20–40 per cell) and express the CaIMH3 gene at very high levels relative to untransformed controls. The plasmid copy number can be controlled by the concentration of MPA in the media. The transformation procedure is reproducible and the efficiency of transformation is high, up to 15,000 per microgram. Unrearranged plasmids are readily recovered by transforming total DNA from transformants back into Escherichia coli. C. albicans genes cloned into the plasmid are expressed at elevated levels relative to untransformed controls. A derivative vector containing the CaMAL2 promoter and termination sequences expresses the CaERG11 ORF at high levels and confers moderate resistance to fluconazole. These shuttle vectors should facilitate global genomics approaches in C. albicans that have been hampered by its diploid genome.     

DNA integration into recipient yeast chromosomes by trans-kingdom conjugation between Escherichia coli and Saccharomyces cerevisiae

Summary IncQ-derived conjugative shuttle vectors, which carried the yeast gene URA3 and/or the yeast autonomously replicating sequence (ARS1), were constructed. Both the ars-plus plasmid pAY205 and the ars-less plasmid pAY201 were successfully transmitted from E. coli to S. cerevisiae by the action of mob and tra. In this trans-kingdom conjugation, plasmid pAY205 could replicate and be retained in transconjugants. Plasmid pAY201 caused the formation of micro-colonies of abortive transconjugants due to its transient expression and rapid disappearance. Nevertheless, one per about 10³ colonies caused by transmitted pAY201 plasmids were uncurable by integration into the homologous region of a yeast chromosome. Analyses by restriction enzyme mapping and Southern hybridization indicate that this integration is primarily caused by a double crossover during conjugation and not by a single reciprocal recombination.     

The CDC8 gene product is required for transformation with episomal and integrative plasmids in Saccharomyces cerevisiae

Summary The product of the yeast CDC8 gene (thymidylate kinase), which is required for chromosomal, mitochondrial and 2 plasmid replication, also participates in plasmid transformation processes in S. cerevisiae. The thermosensitive cdc8-1 mutant strain was transformed with episomal pDQ9 and integrative pDQ9-1 plasmids both of which carry the CDC8 gene. The results suggest that thymidylate kinase is essential for the expression of genes carried on transforming episomal plasmid DNA (probably through its replication) and is also essential for homologous recombination between chromosomal and linearized integrative plasmid DNA.     

Ribosomal RNA gene organization in Cryptosporidium parvum

The cytoplasmic ribosomal RNA (rRNA) genes of the Apicomplexan protozoan parasite Cryptosporidium parvum have been analyzed with respect to size, copy number, organization and structure. The small and large subunit rRNAs are 1.7 and 3.6 kb, respectively. A 151 bp putative 5.8S rRNA gene was identified. The rDNA unit is 5′ small subunit rRNA–internal transcribed spacer 1–5.8S rRNA–internal transcribed spacer 2–large subunit rRNA 3′. There are five copies of the rDNA unit per haploid genome and they are not organized in a conventional head to tail tandem array with a conserved external transcribed spacer. The rDNA units are dispersed through the genome to at least three chromosomes. At least two of the rDNA units are single unlinked copies on different chromosomes. There are two structurally distinct types of rDNA unit, Type A and B, with marked differences in the internal transcribed spacer regions. There are four copies of the Type A rDNA unit and one copy of the Type B rDNA unit.     

Transformation of Sordaria macrospora to hygromycin B resistance: characterization of transformants by electrophoretic karyotyping and tetrad analysis

The ascomycete Sordaria macrospora was transformed using different plasmid molecules containing the bacterial hygromycin B resistance gene (hph) under the control of different expression signals. The highest transformation frequency was obtained with vector pMW1. On this plasmid molecule, expression of the hph gene is directed by the upstream region of the isopenicillin N synthetase gene (pcbC) from the deuteromycete Acremonium chrysogenum. Southern analysis suggests that the vector copies are integrated as tandem repeats into the S. macrospora chromosomes and that duplicated sequences are most probably not inactivated by methylation during meiosis. Furthermore, the hygromycin B resistance (hygR) is not correlated with the number of integrated vector molecules. Electrophoretic karyotyping was used to further characterize S. marcospora transformants. Five chromosomal bands were separated by pulsed-field gel electrophoresis (PFGE) representing seven chromosomes with a total genome size of 39.5 Mb. Hybridization analysis revealed ectopic integration of vector DNA into different chromosomes. In a few transformants, major rearrangements were detected. Transformants were sexually propagated to analyze the fate of the heterologous vector DNA. Although the hygR phenotype is stably maintained during mitosis, about a third of all lines tested showed loss of the resistance marker gene after meiosis. However, as was concluded from electrophoretic karyotyping, the resistant spores showed a Mendelian segregation of the integrated vector molecules in at least three consecutive generations. Our data indicate that heterologous marker genes can be used for transformation tagging, or the molecular mapping of chromosomal loci in S. macrospora     

Factors influencing the copy number of F-like plasmids in Escherichia coli and Salmonella typhimurium

The copy numbers of Flac, four F-like plasmids and pLT2 were estimated in two strains of Salmonella typhimurium and (for all except pLT2) one strain of Escherichia coli. For organisms grown in casamino acids minimal medium, the plasmids spanned a 7–8 fold range of copy number with ColB-K98 having the highest copy number in each strain and R124 the lowest. The copy number of ColB-K98 was substantially greater than 1 in each of the strains tested. There was no clear relation between the plasmid size and copy number, although the plasmids studied spanned only a narrow size range. The copy number of individual plasmids was slightly reduced or not affected at all by the presence of a second plasmid in the same strain. Derivatives harbouring each of the plasmids were grown in three different media to ascertain how plasmid copy number responds to changes in growth rate. For each plasmid, the copy number increased with decreasing growth rate. Extracts from each of the three strains harbouring ColB-K98 contained two distinct plasmid species. One appeared to be about twice as large as the other and both were absent from Col^? segregants.     

Repair of plasmid DNA used for transformation of <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> by gene conversion

Techniques for genetic manipulation of the filamentous fungus Rhizopus have been hampered due to a lack of understanding regarding the recombination and replication mechanisms that affect the fate of introduced DNA. The ability to target chromosomal integration of a plasmid has been difficult because DNA transformed into Rhizopus rarely integrates and is autonomously replicated in a high molecular weight concatenated arrangement (i.e., series or chain). Linearization of the plasmid prior to transformation at a site having homology with the genomic DNA yields the highest frequency of integration, but repair of the double-strand break by end-joining is still the predominant event. We recently attempted to circumvent replication of the plasmid by introducing frameshift mutations in pyrG, the R. oryzae orotidine-5-monophosphate decarboxylase gene used for selection of the vector. It was hypothesized that autonomous replication of the mutated plasmids would be incapable of restoring prototrophic growth, since the genomic pyrG also contained a mutation. However, homologous integration of the plasmid results in duplication of the pyrG gene, which can create a functional copy of pyrG if both the genomic and plasmid mutations are paired on the same duplicate copy. While this event was detected in one of the isolates, it represented less than 8% of the total transformants. The majority of transformants contained plasmid replicating autonomously in a concatenated arrangement. Sequence analysis showed that prototrophic growth was restored by repairing the non-functional pyrG sequence in the plasmid, while the genomic pyrG gene was unaltered. Frequent transfer of the genomic pyrG mutation to the plasmid suggests that gene conversion is likely occurring by recombination pathways involving break-induced replication or synthesis-dependent strand annealing.USDA: Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Regulated overproduction of α-amylase by transformation of the amylolytic yeast Schwanniomyces occidentalis

Summary High frequency transformation of a Schwanniomyces occidentalis mutant defective in the last step of tryptophan synthesis was achieved with plasmids containing the tryptophan synthetase gene (TRP5) of Saccharomyces cerevisiae and an autonomous replication sequence from S. occidentalis, which we called SwARS1. The SwARS1 fragment is also functional in S. cerevisiae. The average copy number of the plasmids in both yeast species was 5–10 per cell under selective conditions. S. occidentalis cells that were transformed with an autonomously replicating plasmid carrying the cloned -amylase gene from S. occidentalis secreted about five times more -amylase than cells without additional copies of the -amylase gene. Both the chromosomal copy and the plasmid-carried copies of the -amylase gene were repressed in the presence of glucose. This transformation system provides a possibility to improve starch degradation by S. occidentalis.