Cloning of the blasticidin S deaminase gene (BSD) from Aspergillus terreus and its use as a selectable marker for Schizosaccharomyces pombe and Pyricularia oryzae

Aspergillus terreus produces a unique enzyme, blasticidin S deaminase, which catalyzes the deamination of blasticidin S (BS), and in consequence confers high resistance to the antibiotic. A cDNA clone derived from the structural gene for BS deaminase (BSD) was isolated by transforming Escherichia coli with an Aspergillus cDNA expression library and directly selecting for the ability to grow in the presence of the antibiotic. The complete nucleotide sequene of BSD was determined and proved to contain an open reading frame of 393 bp, encoding a polypeptide of 130 amino acids. Comparison of its nulceotide sequence with that of bsr, the BS deaminase gene isolated from Bacillus cereus, indicated no homology and a large difference in codon usage. The activity of BSD expressed in E. coli was easily quantified by an assay based on spectrophotometric recording. The BSD gene was placed in a shuttle vector for Schizosaccharomyces pombe, downstream of the SV40 early region promoter, and this allowed direct selection with BS at high frequency, following transformation into the yeast. The BSD gene was also employed as a selectable marker for Pyricularia oryzae, which could not be transformed to BS resistance by bsr. These results promise that the BSD gene will be useful as a new dominant selectable marker for eukaryotes.     

Inheritance of black sigatoka disease resistance in plantain-banana (Musa spp.) hybrids

Black sigatoka (Mycosphaerella fijiensis Morelet), an airborne fungal leaf-spot disease, is a major constraint to plantain and banana (Musa spp.) production world-wide. Gaining further knowledge of the genetics of host-plant resistance will enhance the development of resistant cultivars, which is considered to be the most appropriate means to achieve stable production. Genetic analysis was conducted on 101 euploid (2x, 3x and 4x) progenies, obtained from crossing two susceptible triploid plantain cultivars with the resistant wild diploid banana Calcutta 4. Segregating progenies, and a susceptible reference plantain cultivar, were evaluated over 2 consecutive years. Three distinct levels of host response to black sigatoka were defined as follows: susceptible (< 8 leaves without spots), less susceptible (8–10) and partially resistant (> 10). Segregation ratios for resistance at the 2x level fitted a genetic model having one major recessive resistance allele (bs ₁) and two independent alleles with additive effects (bsr ₂ and bsr ₃). A similar model explains the results at the 4x level assuming that the favourable resistance alleles have a dosage effect when four copies of them are present in their respective loci (bs _i ⁴ ). The proposed model was further validated by segregation data of S ₁ progenies. Mechanisms of black sigatoka resistance are discussed in relation to the genetic model.     

Over expression of the selectable marker blasticidin S deaminase gene is toxic to human keratinocytes and murine BALB/MK cells

Background  

The blasticidin S resistance gene (bsr) is a selectable marker used for gene transfer experiments. The bsr gene encodes for blasticidin S (BS) deaminase, which has a specific activity upon BS. Therefore, its expression is supposed to be harmless in cells. The work reported on herein consisted of experiments to verify a possible toxicity of bsr on mammalian cells, which include several cell lines and primary cultures.     

Effects of the loss of clonal integration on four sedges that differ in ramet aggregation

Although clonal growth is a dominant mode of plant growth in wetlands, the importance of clonal integration, resource sharing among ramets, to individual ramet generations (mother and daughter) and entire clones of coexisting species has not been well investigated. This study evaluated the significance of clonal integration in four sedge species of varying ramet aggregations, from clump-forming species (Clumpers –Carex sterilis, Eleocharis rostellata), with tightly aggregated ramets (rhizomes<1cm), to runner species (Runners –Schoenoplectus acutus, Cladium mariscoides), with loosely aggregated ramets. We manipulated clonal integration by either severing connections between target mother and daughter ramets or leaving connections intact, and then planted them in an intact neighborhood of a fen in Michigan, USA. We measured growth parameters of original and newly produced ramets over two growing seasons and conducted a final biomass harvest, to address four hypotheses. First, we expected integrated clones to accumulate more biomass than severed clones. However, final clone-level biomass and ramet production were the same for both treatments in all species although severing initially stimulated ramet production by Schoenoplectus and produced a more compact ramet aggregation in Cladium. Second, we hypothesized that mother ramets would experience a cost of integration, through reduced ramet or biomass production, while daughters would experience a benefit, through increased resource availability from mothers. Mother ramets of Cladium suffered a cost from integration, while Schoenoplectus mothers suffered a slight cost and Carex daughters saw a slight benefit. Finally, we hypothesized that integration would be more active in runner species than in clumper species. Indeed, we documented more active integration in runners than clumpers, but none of the study species were dependent upon integration for growth or survival once daughter ramets were established with their own roots and shoots. This study demonstrates that integration between established ramets may not be the most important advantage to clonal growth in this wetland field site. The loss of integration elicited varied responses among coexisting species in their natural habitat, somewhat but not completely related to their growth form, suggesting that a combination of plant life history traits contributes to the dependence upon clonal integration among established ramets of clonal species.     

Expression of the blasticidin S deaminase gene (bsr) in tobacco: Fungicide tolerance and a new selective marker for transgenic plants

Summary Blasticidin S (BS), a fungicide of microbial origin, is used for the practical control of rice blast disease. It has broad antimicrobial activity but occasionally exhibits adverse phytotoxic effects on some dicot plants. An inactivating enzyme, BS deaminase, was discovered in the BS resistant strain, Bacillus cereus K55-S1, and the structural gene, bsr, for the enzyme has been cloned. We introduced the bsr gene into tobacco plants using the Ti plasmid vector system and demonstrated that the bsr gene conferred a BS resistant phenotype to the plants. Thus the bsr gene could be useful as a selective marker for plant transformation and provides an example for a new approach to the solution of phytotoxicity problems associated with the use of some types of fungicide.     

On the integration of subthreshold inputs from Perforant Path and Schaffer Collaterals in hippocampal CA1 pyramidal neurons

Using a realistic model of a CA1 hippocampal pyramidal neuron, we make experimentally testable predictions on the roles of the non-specific cation current, I _h , and the A-type Potassium current, I _A , in modulating the temporal window for the integration of the two main excitatory afferent pathways of a CA1 neuron, the Schaffer Collaterals and the Perforant Path. The model shows that the experimentally observed increase in the dendritic density of I _h and I _A could have a major role in constraining the temporal integration window for these inputs, in such a way that a somatic action potential (AP) is elicited only when they are activated with a relative latency consistent with the anatomical arrangement of the hippocampal circuitry.     

Use of the transposable element Ac/Ds in conjunction with Spm/dSpm for gene tagging allows extensive genome coverage with a limited number of starter lines: functional analysis of a four-element system in Arabidopsis thaliana

We have developed a novel four-element based gene tagging system in Arabidopsis to minimize the number of starter lines required to generate genome-wide insertions for saturation mutagenesis. In this system, the non-autonomous cassette, Ds(dSpm), comprises of both Ds and dSpm elements cloned one within the other along with appropriate selection markers to allow efficient monitoring of excision and re-integration of the transposons. Trans-activation of the outer borders (Ds) and selection against the negative selection marker (iaaH) linked to the cassette ensures unlinked spread of the Ds(dSpm) cassette from the initial site of integration of the T-DNA. This creates several launch pads within the genome from where the internal element (dSpm) can be subsequently mobilized to generate secondary insertions. In this study, starting from a single T-DNA integration we could spread the Ds(dSpm) cassette to 11 different locations over all the five chromosomes of Arabidopsis. The frequency of unlinked Ds transpositions in the F2 generation varied between 0.05 and 3.35%. Three of these lines were then deployed to trans-activate the internal dSpm element which led to the selection of 29 dSpm insertions. The study conclusively shows the feasibility of deploying Ds and the dSpm elements in a single construct for insertional mutagenesis.     

Site-directed integration system using a combination of mutant <Emphasis Type="Italic">lox</Emphasis> sites for <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

The engineering of Corynebacterium glutamicum is important for enhanced production of biochemicals. To construct an optimal C. glutamicum genome, a precise site-directed gene integration method was developed by using a pair of mutant lox sites, one a right element (RE) mutant lox site and the other a left element (LE) mutant lox site. Two DNA fragments, 5.7 and 10.2 kb-long, were successfully integrated into the genome. The recombination efficiency of this system compared to that obtained by single crossover by homologous recombination was 2 orders of magnitude higher. Moreover, the integrated DNA remained stably maintained on removal of Cre recombinase. The Cre/mutant lox system thus represents a potentially attractive tool for integration of foreign DNA in the course of the engineering of C. glutamicum traits.     

The Hermes element from Musca domestica can transpose in four families of cyclorrhaphan flies

Transgenic insect technology will provide opportunities to explore the basic biology of a broad range of insect species in ways that will prove insightful and important. It is also a technology that will provide opportunities to manipulate the genotypes of insects of practical significance to the health and welfare of humans. TheHermes transposable element from the housefly,Musca domestica, is a short inverted repeat-type element related tohobo fromDrosophila melanogaster, Ac fromZea mays, andTam3 fromAntirrhinum majus. It has potential to become a versatile and efficient broad host-range insect transformation vector. The ability ofHermes to transpose when introduced into five species of diptera from four divergent families was tested using anin vivo, interplasmid transpositional recombination assay.Hermes was capable of transposing in all species tested, demonstrating thatHermes has a broad host-range. In addition, the rates of transposition were sufficiently high in all species tested to suggest thatHermes will be an efficient gene transfer vector in a wide range of insect species. TheHermes element also revealed a pattern of integration into the target substrate that permitted factors determining integration site selection to be identified. Primary nucleotide sequence of the integration site played a role as did proximity to preferred integration sites and the nucleosomal organization of the target.     

Unmarked gene deletion and host–vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus

Sulfolobus islandicus is being used as a model for studying archaeal biology, geo-biology and evolution. However, no genetic system is available for this organism. To produce an S. islandicus mutant suitable for genetic analyses, we screened for colonies with a spontaneous pyrEF mutation. One mutant was obtained containing only 233 bp of the original pyrE sequence in the mutant allele and it was used as a host to delete the β-glycosidase (lacS) gene. Two unmarked gene deletion methods were employed, namely plasmid integration and segregation, and marker replacement and looping out, and unmarked lacS mutants were obtained by each method. A new alternative recombination mechanism, i.e., marker circularization and integration, was shown to operate in the latter method, which did not yield the designed deletion mutation. Subsequently, Sulfolobus–E. coli plasmid shuttle vectors were constructed, which genetically complemented ΔpyrEFΔlacS mutation after transformation. Thus, a complete set of genetic tools was established for S. islandicus with pyrEF and lacS as genetic markers.     

Transposon Tn21 encodes a RecA-independent site-specific integration system

Summary The IncW plasmid R388 and the DNA region of Tn21 containing the Sm^r and the Su^r genes are capable of RecA-independent recombination. This recombination occurs at a relatively high frequency (up to 10^-4 recombinants per recipient molecule) and results in integration of the two plasmids. No detectable repeats are formed in the process. The crossover points have been confined to a 0.4-kb homologous segment in both plasmids which contains a 59-bp DNA sequence presumably involved in the acquisition of new genes by Tn21 and its relatives (Cameron et al. 1986). It is likely that the recombination occurs precisely at this point. At least one trans-acting function (an integrase) is required for the site-specific recombination. It has been localized to a 1456-bp BstEII-BamHI fragment of Tn21 and can efficiently complement the integration of plasmids containing the integration site.     

Phylogeny of <Emphasis Type="Italic">Banana Streak Virus</Emphasis> Reveals Recent and Repetitive Endogenization in the Genome of Its Banana Host (<Emphasis Type="Italic">Musa</Emphasis> sp.)

Banana streak virus (BSV) is a plant dsDNA pararetrovirus (family Caulimoviridae, genus badnavirus). Although integration is not an essential step in the BSV replication cycle, the nuclear genome of banana (Musa sp.) contains BSV endogenous pararetrovirus sequences (BSV EPRVs). Some BSV EPRVs are infectious by reconstituting a functional viral genome. Recent studies revealed a large molecular diversity of episomal BSV viruses (i.e., nonintegrated) while others focused on BSV EPRV sequences only. In this study, the evolutionary history of badnavirus integration in banana was inferred from phylogenetic relationships between BSV and BSV EPRVs. The relative evolution rates and selective pressures (d_N/d_S ratio) were also compared between endogenous and episomal viral sequences. At least 27 recent independent integration events occurred after the divergence of three banana species, indicating that viral integration is a recent and frequent phenomenon. Relaxation of selective pressure on badnaviral sequences that experienced neutral evolution after integration in the plant genome was recorded. Additionally, a significant decrease (35%) in the EPRV evolution rate was observed compared to BSV, reflecting the difference in the evolution rate between episomal dsDNA viruses and plant genome. The comparison of our results with the evolution rate of the Musa genome and other reverse-transcribing viruses suggests that EPRVs play an active role in episomal BSV diversity and evolution.     

Assessment of Nematode Resistance in Wheat Transgenic Plants Expressing Potato Proteinase Inhibitor (<Emphasis Type="Italic">PIN2</Emphasis>) Gene

Serine proteinase inhibitors (IP’s) are proteins found naturally in a wide range of plants with a significant role in the natural defense system of plants against herbivores. The question addressed in the present study involves assessing the ability of the serine proteinase inhibitor in combating nematode infestation. The present study involves engineering a plant serine proteinase inhibitor (pin2) gene into T. durum PDW215 by Agrobacterium-mediated transformation to combat cereal cyst nematode (Heterodera avenae) infestation. Putative T₀ transformants were screened and positive segregating lines analysed further for the study of the stable integration, expression and segregation of the genes. PCR, Southern analysis along with bar gene expression studies corroborate the stable integration pattern of the respective genes. The transformation efficiency is 3%, while the frequency of escapes was 35.71%. χ² analysis reveals the stable integration and segregation of the genes in both the T₁ and T₂ progeny lines. The PIN2 systemic expression confers satisfactory nematode resistance. The correlation analysis suggests that at p < 0.05 level of significance the relative proteinase inhibitor (PI) values show a direct positive correlation vis-à-vis plant height, plant seed weight and also the seed number.     

Molecular analysis of <Emphasis Type="Italic">Agrobacterium</Emphasis> T-DNA integration in tomato reveals a role for left border sequence homology in most integration events

Studies in several plants have shown that Agrobacterium tumefaciens T-DNA can integrate into plant chromosomal DNA by different mechanisms involving single-stranded (ss) or double-stranded (ds) forms. One mechanism requires sequence homology between plant target and ssT-DNA border sequences and another double-strand-break repair in which preexisting chromosomal DSBs “capture” dsT-DNAs. To learn more about T-DNA integration in Solanum lycopersicum we characterised 98 T-DNA/plant DNA junction sequences and show that T-DNA left border (LB) and right border transfer is much more variable than previously reported in Arabidopsis thaliana and Populus tremula. The analysis of seven plant target sequences showed that regions of homology between the T-DNA LB and plant chromosomal DNA plays an important role in T-DNA integration. One T-DNA insertion generated a target sequence duplication that resulted from nucleolytic processing of a LB/plant DNA heteroduplex that generated a DSB in plant chromosomal DNA. One broken end contained a captured T-DNA that served as a template for DNA repair synthesis. We propose that most T-DNA integrations in tomato require sequence homology between the ssT-DNA LB and plant target DNA which results in the generation of DSBs in plant chromosomal DNA.     

Site-specific integration and excision of pMEA100 in Nocardia mediterranei

Summary Nocardia mediterranei strain LBG A3136 contains the 23.7 kb element pMEA100 in a chromosomally integrated form as well as in the free state (Moretti et al. 1985). The integrated form of this element can be excised precisely from the Nocardia chromosome without any accompanying rearrangements in flanking chromosomal DNA. After transfer into plasmid-free mutant strains, pMEA100 reintegrates site specifically into its original chromosomal locus. The exact mapping of the pMEA100 integration site was accomplished by restriction analysis and DNA sequencing. The attachment site of pMEA100, the junctions of its integrated form and plasmid-free chromosomal DNA of N. mediterranei contain an identical 47 bp long sequence which is probably required for site-specific recombination connected with integration and excision of pMEA100. Only one such sequence was found in the chromosome of pMEA100-free N. mediterranei derivatives as suggested by the single integration locus.     

Site-specific integration of plasmid pSAM2 in Streptomyces lividans and S. ambofaciens

Summary Streptomyces ambofaciens strain ATCC23877 contains the 11.1 kb plasmid pSAM2 stably integrated into its chromosome. This plasmidic sequence is able to loop out and to be transferred at high frequency to S. lividans where it is found simultaneously as both free and integrated plasmid. When a UV derivative of strain ATCC23877 (strain ATCC15154) is used, the resident copy of pSAM2 can be transferred to S. lividans, but only the integrated form is found in this strain. In both cases, the integration occurs at a unique chromosomal region through the same plasmidic integration site as that in strain ATCC23877. The resident copy of strain ATCC15154 can also be transferred at low frequency to S. ambofaciens DSM40697 (devoid of any pSAM2 sequence). In this case, as several copies of pSAM2 are integrated, the integration pattern is complicated. Integration of a complete pSAM2 sequence in this strain occurs in a region that hybridizes with the integration zones of S. lividans and of S. ambofaciens strain ATCC23877. Comparison of the cloned integration zone of S. lividans before and after the integration event showed that the restriction pattern of the resident pSAM2 in strain ATCC15154 is similar to that of the free form of pSAM2 found naturally in another UV derivative of strain ATCC23877 (strain JI3212).     

Generation of trangenic Xenopus laevis using the Sleeping Beauty transposon system

Using the Sleeping Beauty (SB) transposon system, we have developed a simple method for the generation of Xenopus laevis transgenic lines. The transgenesis protocol is based on the co-injection of the SB transposase mRNA and a GFP-reporter transposon into one-cell stage embryos. Transposase-dependent reporter gene expression was observed in cell clones and in hemi-transgenic animals. We determined an optimal ratio of transposase mRNA versus transposon-carrying plasmid DNA that enhanced the proportion of hemi-transgenic tadpoles. The transgene is integrated into the genome and may be transmitted to the F1 offspring depending on the germline mosaicism. Although the transposase is necessary for efficient generation of transgenic Xenopus, the integration of the transgene occurred by an non-canonical transposition process. This was observed for two transgenic lines analysed. The transposon-based technique leads to a high transgenesis rate and is simple to handle. For these reasons, it could present an attractive alternative to the classical Restriction Enzyme Mediated Integration (REMI) procedure.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

A new Escherichia coli gene, fdrA, identified by suppression analysis of dominant negative FtsH mutations

An Escherichia coli membrane protein, FtsH, has been implicated in several cellular processes, including integration of membrane proteins, translocation of secreted proteins, and degradation of some unstable proteins. However, how it takes part in such diverse cellular events is largely unknown. We previously isolated dominant negative ftsH mutations and proposed that FtsH functions in association with some other cellular factor(s). To test this proposal we isolated multicopy suppressors of dominant negative ftsH mutations. One of the multicopy suppressor clones contained an N-terminally truncated version of a new gene that was designated fdrA. The FdrA fragment suppressed both of the phenotypes — increased abnormal translocation of a normally cytoplasmic domain of a model membrane protein and retardation of protein export — caused by dominant negative FtsH proteins. The intact fdrA gene (11.9 min on the chromosome) directed the synthesis of a 60 kDa protein in vitro.     

Agrobacterium-mediated transformation of cauliflower: optimization of protocol and development of Bt-transgenic cauliflower

A number of factors that are known to influence genetic transformation were evaluated to optimizeAgrobacterium-mediated transformation of hypocotyl explants of cauliflower variety Pusa Snowball K-1. The binary vector p35SGUSINT mobilized intoAgrobacterium strain GV2260 was used for transformation and transient GUS expression was used as the basis for identifying the most appropriate conditions for transformation. Explant age, preculture period, bacterial strain and density were found to be critical determinants of transformation efficiency. Using the optimized protocol, the syntheticcryIA(b) gene was mobilized into cauliflower. Molecular analyses of transgenics established the integration and expression of the transgene. Insect bioassays indicated the effectiveness of the transgene against infestation by diamondback moth (Plutella xylostella) larvae.     

Linear kalilo DNA is a Neurospora mitochondrial plasmid that integrates into the mitochondrial DNA

Summary The linear autonomous form of kalilo DNA (previously called AR-kalDNA) is shown to be resident within mitochondria rather than nuclei, as had been suggested by previous experiments. This form has been renamed mtAR-kalDNA, to signify its mitochondrial location. Experiments are described that illustrate the inheritance and somatic transmission patterns of the mitochondrial kalilo plasmid and the mitochondrial inserted form of kalilo DNA (mtlS-kalDNA). Progeny of a cross with a pre-senescent subculture as the female parent inherited mtAR-ka1DNA only; mtIS-kalDNA was not transmitted sexually. During somatic propagation of the ascospore cultures, novel kalilo DNA inserts appeared and most of them persisted until death. We propose that these inserts originated from de novo integration of mtAR-kalDNA into the mitochondrial DNA. In two of the ascopore-derived series analyzed, the first inserts detected were seen only transiently and inserts appearing subsequent to the transient inserts were retained until death. We propose that these enduring inserts originated either from rearrangements of the transient inserts or from novel integration events, either from mtAR-kalDNA or from transposition of the transient inserts.