Cointegration of transforming DNAs in Aspergillus nidulans: a model using autonomously-replicating plasmids

Transforming DNAs form cointegrates in Aspergillus nidulans by homologous and non-homologous recombination as well as by end-to-end ligation of linear fragments. This process has been studied by means of a model in which the linkage of a marker gene to the origin of autonomous replication AMA1 was selected for. Recombinant plasmids were rescused into Escherichia coli and subjected to restriction mapping and sequence analysis. It was shown that circular DNA molecules recombined predominantly within homologous fragments. Linear DNA fragments integrated into circular plasmids by invasion of their ends into random non-homologous sites, but exhibited some bias in choice of a target sequence. Cointegrates of multiple plasmid copies were often observed. In some of the plasmids analysed, short duplications of the target sequence flanking an inserted linear DNA fragment have been revealed.     

Autonomously replicating plasmids carrying theAMA1 region inPenicillium chrysogenum

Plasmid vectors containing theAMA1 sequence transformed with high efficiency and replicated autonomously inPenicillium chrysogenum. The efficiency of transformation ofP. chrysogenum was related to the length of theAMA1 fragment used for constructing the different autonomously replicating plasmids. One of the two palindromic inverted repeats ofAMA1 (the 2.2-kbSalI-HindIII fragment) is sufficient to confer autonomous replication and a high transformation efficiency. Deletion of the 0.6-kb central fragment located between the inverted repeats did not affect either the ability of the plasmids to replicate autonomously or the efficiency of transformation, but did alter the mitotic stability and the plasmid copy number. Deletion of any fragment of the 2.2-kb repeat caused the loss of the ability to replicate autonomously and reduced the transformation efficiency. Most of the transformants retained the original plasmid configuration, as multimers and without reorganization, after several rounds of autonomous replication. TheAMA1 region works as an origin of replication inP. chrysogenum andA. nidulans but not apparently inAcremonium chrysogenum.     

New in-vivo cloning methods by homologous recombination in yeast

We have devised a new strategy to clone DNA sequences from an yeast autonomously-propagating plasmid into a non-autonomous integrative vector by in-vivo recombination. The method consists of a first step in which the replicative plasmid carrying the DNA fragment of interest forms a co-integrate with the non-replicative plasmid by an induced in-vivo reciprocal exchange accompanied by gene conversion. The dimeric plasmid obtained is then purified and cut with an appropriate restriction enzyme and ligated independently to obtain the two intact monomeric plasmids, the original autonomous plasmid plus the new non-autonomous plasmid carrying the subcloned DNA fragment. The dimeric co-integrate can also serve as substrate for a second in-vivo reciprocal exchange that produces new autonomous plasmids carrying the desired DNA fragment. The technique considerably expands the applications of in-vivo cloning in yeast by complementing three important characteristics of previously published methods: (1) it can be used to clone into non-propagating vectors; (2) co-transformation experiments are not required; and (3) the intermediate co-integrate can be used to generate new types of autonomously-propagating plasmids directly. These characteristics are independent of whether the DNA insert is flanked by appropriate restriction sites or whether it does, or does not, express a detectable phenotype in yeast. The method is particularly useful for the cloning of large DNA fragments and can be used for plasmids from organisms other than yeasts.     

LTR-directed homologous recombination of full-length HIV-1 provirus clone inrecA(−) bacteria

Summary During molecular cloning of full-length retroviral plasmid clones occurrence of homologous recombination (HR) between LTR regions is frequently observed. In order to evaluate appropriate host bacterial strains for cloning such HR-prone plasmids, we utilized a linearized template plasmid containing a full-length HIV-1 proviral sequence. The plasmid was linearized within the viral sequence so that plasmid transformed bacteria would grow only when the plasmid was circularized by HR. Using this genetic system for detecting HR, we evaluated the frequency of HR in variousrecA(–) bacterial strains which are commercially available and in somerecA-null strains in whichrecA-defective phenotype was constructed by P1 transduction. We found that HR occurred even inrecA-null strains although in lesser frequencies. The nucleotide sequence analysis at the junction of recombination revealed no loss, insertion or duplication of DNA sequence. It is suggested that recombination machinery other than theRecA system is involved.     

Concurrent transfer and recombination between plasmids encoding for heat-stable enterotoxin and drug resistance in porcine enterotoxigenicEscherichia coli

The frequency of and genetic mechanisms for simultaneous transfer of genes encoding for tetracycline and sulpha-streptomycin resistance, heat-labile (LT) and heat-stable (ST-mouse) enterotoxin production in porcine enterotoxigenicEscherichia coli toEscherichia coli K12 were investigated. SevenE.coli strains of 0-group 149 were studied by conjugation and transformation experiments. All strains transferred tetracycline-resistance plasmids at a high frequency. No interaction was observed between these plasmids and those encoding for LT production. However, most tetracycline-resistant recipient cells were ST-mouse⁺ following recombination events between plasmids encoding for colicin B and ST-mouse production and plasmids encoding for tetracycline resistance. Alternatively, when selecting for sulpha or streptomycin resistance a majority of the transconjugants were also ST-mouse⁺, as plasmids coding for sulpha and streptomycin were mobilized by the colicin B and ST-mouse encoding plasmid. Since the simultaneous transfer of genes encoding for drug resistance, colicin B and ST-mouse production are common events in vitro, they might also occur frequently in vivo during antibiotic selective pressure.     

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     

The dynamics of mitochondrial plasmids in a Hawaiian population of Neurospora intermedia

We analysed the distribution of mitochondrial plasmids among 82 Neurospora intermedia isolates from Hawaii; 74% of the isolates carried the neutral circular plasmid Han-2, whereas 38% contained the linear senescence-causing plasmid kalDNA. The distributions of the two plasmids are independent. There is no significant difference between the Kauaian population of 1972 and that of 1976. To further examine the reasons for this frequency distribution we studied the transmission of both Hawaiian plasmids through the maternal parent in a large series of crosses using non-Kalilo isolates as conidial parents. Plasmids can be lost during the sexual cycle. The Han-2 plasmid is transmitted more efficiently than kalDNA. No clear cases of autonomous or non-autonomous plasmid suppression were observed, so loss can be considered accidental. One Kalilo strain proved to be ineffectual as a maternal parent, and this reduced its ability to transmit kalDNA to the next generation. The dynamic balance of plasmids in natural populations over time is probably a result of the interplay of many forces, including those described in this work and those from several other studies on Neurospora plasmids.     

Terminal segment of Kluyveromyces lactis linear DNA plasmid pGKL2 supports autonomou sreplication of hybrid plasmids in Saccharomyces cerevisiae

Summary By use of linear DNA plasmid pGKL2 from the yeast Kluyveromyces lactis we have constructed hybrid plasmids carrying a LEU2 gene of Saccharomyces cerevisiae as a selectable marker. The replication properties of hybrid plasmids in yeasts were investigated. We demonstrated that the insertion of a LEU2 gene into pGKL2 resulted in circularization of the hybrid plasmids and pGKL2 segment supported autonomous replication of the plasmids. Moreover, the hybrid plasmids propagated autonomously, independently of the presence of the natural pGKL2 plasmid.     

Heterologous gene expression on the linear DNA killer plasmid from Kluyveromyces lactis

Summary Linear hybrid plasmids based on the killer plasmid pGKL1 from Kluyveromyces lactis were obtained by in vivo recombination in Saccharomyces cerevisiae. Like pGKL1, the hybrids are located in the cytoplasm, have terminal inverted repeats (TIR) and possess covalently linked proteins at their 5 ends. The construction of cytoplasmic hybrid plasmids is based on the use of a pGKL1 promoter to control the marker gene used for recombination. Nuclear promoters are not recognised in the cytoplasm.     

A novel class of vector for yeast transformation

Summary We have constructed a set of hybrid yeast Escherichia coli vectors which utilise the site specific recombination function of the Saccharomyces cerevisiae 2 m plasmid to completely eliminate the bacterial moiety upon introduction into yeast. A number of these plasmids have been shown to exhibit high inheritable stability in both laboratory and industrial strains during non-selective growth. These plasmids are beneficial for the genetic modification of industrial yeast, particularly those used in the production of food and beverages, and are of benefit in the study of plasmid maintenance and heterologous gene expression.     

Active recombination of pKD1-derived vectors with resident pKD1 in Kluyveromyces lactis transformation

Summary The host specificity of the 2-like circular plasmid pKD 1 is such that this plasmid replicates stably in several species of Kluyveromyces yeasts, but not in Saccharomyces cerevisiae. pKD 1-derived plasmids containing various parts of the pKD 1 sequence were capable of transforming Kluyveromyees lactis with high efficiency. When such vectors were introduced into host strains that contained resident pKD1 plasmid, the input DNA frequently recombined with it to produce high proportions of additive recombinant molecules that replicate stably. Recombination events were shown to occur with vectors differing for the presence or absence of the putative origin of replication and of the inverted repeats. Structure, stability and copy number of the recombination products were analyzed for various types of vectors.     

Gene replacement and ectopic integration in the zygomycete Mucor circinelloides

Transformation of Mucor circinelloides with plasmid pTL42 led to the identification of different integrative events. Analysis of eight transformed strains allowed the detection of three classes of transformants: integrative types derived from either homologous (exclusively at the leuA locus) or heterologous recombination and a class which originated by gene replacement. No autonomous replication of the vector was observed.     

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.     

Transformation of Gibberella fujikuroi: effect of the Aspergillus nidulans AMA1 sequence on frequency and integration

Summary A stable and reproducible transformation selection system for Gibberella fujikuroi protoplasts based on the Aspergillus nidulans arg B gene, encoding ornithine transcarbamylase, has been developed. Inclusion into the vector of the A. nidulans DNA fragment (AMA1), which permits plasmid autonomous replication in A. nidulans, A. niger and A. oryzae, appeared to permit autonomous replication of G. fujikuroi although the transformation frequency was increased by only two-fold. Transformation was also achieved using the bacterial hygromycin B resistance gene under the control of G. pulicaris and A. nidulans promoters.     

In-vitro recombination in rad and rnc mutants of Saccharomyces cerevisiae

Summary Extracts of S. cerevisiae cells can catalyze homologous recombination between plasmids in vitro. Extracts prepared from rad50, rad52 or rad54 disruption mutants all have reduced recombinational activity compared to wild-type. The rad52 and rad54 extracts are more impaired in the recombination of plasmids containing double-strand breaks than of intact plasmids, whereas rad50 extracts are deficient equally for both types of substrate. The nuclease RhoNuc (previously designated yNucR), encoded by the RNC1 (previously designated NUC2) gene and regulated by the RAD52 gene, is not required for recombination when one substrate is single-stranded but is essential for the majority of recombination events when both substrates are double-stranded. Furthermore, elimination of this nuclease restores recombination in rad52 extracts to levels comparable to those in wild-type extracts.     

Genetic analysis of the 3' early region transformation and replication functions of bovine papillomavirus type 1

Cell transformation by BPV-1 is dependent on sequences confined to a region comprising only 69% of the viral DNA. Analysis of this subgenomic fragment of the viral genome has revealed a composite of functions. To further study this region of the BPV-1 genome, we constructed mutants affecting the 3' half of the transforming region. These mutations have revealed a plasmid maintenance function for the E2 ORF and a requirement that the 3' half of E5 be intact for in vitro mouse cell transformation. Cotransfection experiments revealed that phenotypic complementation (wild type transformation efficiency and autonomous replication of viral DNA) resulted in recombination between input plasmids in the majority of cases. However, rare cases were encountered where the wild type phenotype was observed with no evidence of recombination between input plasmids. These data suggest trans-acting functions of the E2 and E5 ORFs in viral DNA replication and the E5 ORF in cell transformation.     

Plasmids in Gram negatives: molecular typing of resistance plasmids

A plasmid is defined as a double stranded, circular DNA molecule capable of autonomous replication. By definition, plasmids do not carry genes essential for the growth of host cells under non-stressed conditions but they have systems which guarantee their autonomous replication also controlling the copy number and ensuring stable inheritance during cell division. Most of the plasmids confer positively selectable phenotypes by the presence of antimicrobial resistance genes. Plasmids evolve as an integral part of the bacterial genome, providing resistance genes that can be easily exchanged among bacteria of different origin and source by conjugation. A multidisciplinary approach is currently applied to study the acquisition and spread of antimicrobial resistance in clinically relevant bacterial pathogens and the established surveillance can be implemented by replicon typing of plasmids. Particular plasmid families are more frequently detected among Enterobacteriaceae and play a major role in the diffusion of specific resistance genes. For instance, IncFII, IncA/C, IncL/M, IncN and IncI1 plasmids carrying extended-spectrum beta-lactamase genes and acquired AmpC genes are currently considered to be "epidemic resistance plasmids", being worldwide detected in Enterobacteriaceae of different origin and sources. The recognition of successful plasmids is an essential first step to design intervention strategies preventing their spread.     

Vaccinia virus DNA ligase is nonessential for virus replication: Recovery of plasmids from virus-infected cells

The essentiality of the vaccinia virus DNA ligase gene, SalF 15R, for virus growth was tested by insertional mutagenesis. A plasmid containing E. coli gpt inserted within a large deletion in the DNA ligase gene was transfected into vaccinia virus-infected cells and recombinant viruses selected by three cycles of plaque purification in the presence of mycophenolic acid (MPA). Surprisingly, in some isolates, which replicated in a manner indistinguishable from wild type (WT) virus, the WT gene was replaced by the gpt allele, demonstrating that the DNA ligase gene is nonessential for growth in cultured cells. In other isolates the entire plasmid was integrated into the virus genome by a single crossover event and a functional copy of the DNA ligase was retained. Southern blot analyses of the latter, drug-resistant viruses indicated extra DNA fragments, of sizes inconsistent with predicted viral structures, which represent the plasmid products of homologous recombination. Hirt extracts from cells infected with such multiply plaque purified virus isolates yielded plasmids that produced ampicillin-resistant colonies after transformation of E. coli. These plasmids were of two structures, representing either the original plasmid used for transfection, or a plasmid containing the WT ligase gene rescued by recombination with the virus genome. Similarly, insertional mutagenesis of the vaccinia virus thymidine kinase (TK) gene with gpt yielded plasmids containing mutant or wild type TK alleles when recombinant viruses were selected in MPA. Such plasmids were not isolated when TK minus viruses were selected in 5-bromodeoxyuridine (BUdR).     

Co-transformation with autonomously-replicating helper plasmids facilitates gene cloning from an Aspergillus nidulans gene library

Autonomously-replicating, marker-less helper plasmids were added to transformations of Aspergillus nidulans with plasmids which normally transform by chromosomal integration. This resulted in as much as a 200-fold increase in transformation efficiency. Recovery of autonomously-replicating plasmid co-integrates indicated that co-transformation involves recombination between integrating and helper plasmids, which occurs at a high frequency. Increasing DNA sequence-homology between pairs of plasmids used in simultaneous transformations enhanced co-transformation efficiency. Using helper plasmids and an A. nidulans gene library in a normally-integrating vector, the genes adC and adD were cloned as part of such a co-integrate. In effect, the addition of helper plasmid converts an integrating into an autonomously-replicating gene library in vivo.     

High frequency FLP-independent homologous DNA recombination of 2 μ plasmid in the yeast Saccharomyces cerevisiae

Summary The purpose of this work is to identify and quantitate in vivo 2 plasmid FLP-independent recombination in yeast, using a nonselective assay system for rapid detection of phenotypic expression of the recombination events. A tester plasmid was constructed such that in vivo recombination between 2 direct repeat sequences produces the resolution of the plasmid into two circular DNA molecules. This recombinational event is detected as a phenotypic shift from red to white colonies, due to the mitotic loss of the plasmid portion containing the yeast ADE8 gene in a recipient ade1 ade2 ade8 genetic background. In the absence of the 2 FLP recombinase and/or its target DNA sequence, recombination is not abolished but rather continues at a high frequency of about 17%. This suggests that the FLP-independent events are mediated by the chromosomally-encoded general homologous recombination system. We therefore conclude that the totality of 2 DNA recombination events occurring in FLP⁺ cells is the contribution of both FLP-mediated and FLP-independent events.