Intra-chromosomal rearrangements generated by Cre-lox site-specific recombination.

Chromosomal rearrangements are useful genetic and breeding tools but are often difficult to detect and characterize. To more easily identify and define chromosome deletions and inversions, we have used the bacteriophage P1 Cre-lox site-specific recombination system to generate these events in plants. This involves three steps: (i) the introduction of two lox sites into one locus in a plant genome, including one site within a modified Ds transposon; (ii) Ac transposase-mediated transposition of the Ds-lox element to a new locus on the same chromosome; (iii) Cre-mediated site-specific recombination between the two lox sites that bracket a chromosome segment. We report the production of a deletion and three inversion events in tobacco. The utility of chromosomal segments bracketed by lox sites for targeted manipulation and cloning is discussed.     

Aspergillus fumigatus chsE: A Gene Related to CHS3 of Saccharomyces cerevisiae and Important for Hyphal Growth and Conidiophore Development but Not Pathogenicity

The Aspergillus fumigatus chsE (AfchsE) gene was isolated from an A. fumigatus DNA library on the basis of hybridization to a segment of Saccharomyces cerevisiae CHS3 (ScCHS3). The amino acid sequence derived from AfchsE is 28% identical with ScCHS3 and 80% identical with the product of Aspergillus nidulans chsD (AnchsD). A mutant strain constructed by disruption of AfchsE has reduced levels of mycelial chitin, periodic swellings along the length of hyphae, and a block in conidiation that can be partially restored by growth in osmotic stabilizer. This phenotype is different from that reported for an AnchsD mutant, in which germinating conidia and hyphal tips undergo lysis and the colonial growth rate is significantly reduced. Despite the defects associated with the AfchsE- strain, its virulence was not significantly reduced when compared with the wild-type parental strain in a mouse model of pulmonary aspergillosis.     

A fission yeast gene for mitochondrial sulfide oxidation

A cadmium-hypersensitive mutant of the fission yeast Schizosaccharomyces pombe was found to accumulate abnormally high levels of sulfide. The gene required for normal regulation of sulfide levels, hmt2(+), was cloned by complementation of the cadmium-hypersensitive phenotype of the mutant. Cell fractionation and immunocytochemistry indicated that HMT2 protein is localized to mitochondria. Sequence analysis revealed homology between HMT2 and sulfide dehydrogenases from photosynthetic bacteria. HMT2 protein, produced in and purified from Escherichia coli, was soluble, bound FAD, and catalyzed the reduction of quinone (coenzyme Q2) by sulfide. HMT2 activity was also detected in isolated fission yeast mitochondria. We propose that HMT2 functions as a sulfide:quinone oxidoreductase. Homologous enzymes may be widespread in higher organisms, as sulfide-oxidizing activities have been described previously in animal mitochondria, and genes of unknown function, but with similarity to hmt2(+), are present in the genomes of flies, worms, rats, mice, and humans.     

Thermal acclimation of leaf respiration but not photosynthesis in Populus deltoides x nigra

Dark respiration and photosynthesis were measured in leaves of poplar Populus deltoides x nigra ('Veronese') saplings to investigate the extent of respiratory and photosynthetic acclimation in pre-existing and newly emerged leaves to abrupt changes in air temperature. The saplings were grown at three temperature regimes and at high and low nitrogen availabilities. Rates of photosynthesis and dark respiration (R(d)) were measured at the initial temperature and the saplings were then transferred to a different temperature regime, where the plants remained for a second and third round of measurements on pre-existing and newly emerged leaves. Acclimation of photosynthesis was limited following transfer to warmer or cooler growing conditions. There was strong evidence of cold and warm acclimation of R(d) to growth temperature, but this was limited in pre-existing leaves. Full acclimation of R(d )was restricted to newly emerged leaves grown at the new growth temperature. These findings indicate that the extent of thermal acclimation differs significantly between photosynthesis and respiration. Importantly, pre-existing leaves in poplar were capable of some respiratory acclimation, but full acclimation was observed only in newly emerged leaves. The R(d)/A(max) ratio declined at higher growth temperatures, and nitrogen status of leaves had little impact on the degree of acclimation.     

Cytoplasmic expression of a thermostable invertase from <Emphasis Type="Italic">Thermotoga maritima</Emphasis> in <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Objectives

To evaluate the secretory and cytoplasmic expression of a thermostable Thermogata maritima invertase in Lactococcus lactis.

Results

The thermostable invertase from T. maritima was cloned with and without the USP45 secretory peptide into the pNZ8148 vector for nisin-inducible expression in L. lactis. The introduction of an USP45 secretion peptide at the N-terminal of the enzyme led to a loss of protein solubility. Computational homology modeling and hydrophobicity studies indicated that the USP45 peptide exposes a stretch of hydrophobic amino acids on the protein surface resulting in lower solubility. Removal of the USP45 secretion peptide allowed a soluble and functional invertase to be expressed intracellularly in L. lactis. Immobilized metal affinity chromatography purification of the cell lysate with nickel-NTA gave a single protein band on SDS-PAGE, while E. coli-expressed invertase consistently co-purified with an additional band. The yields of the purified invertase from E. coli and L. lactis were 14.1 and 6.3 mg/l respectively.

Conclusions

Invertase can be expressed in L. lactis and purified in a functional form. L. lactis is a suitable host for the production of food-grade invertase for use in the food and biotechnology industries.

     

Recombinase-directed plant transformation for the post-genomic era

Plant genomics promises to accelerate genetic discoveries for plant improvements. Machine-driven technologies are ushering in gene structural and expressional data at an unprecedented rate. Potential bottlenecks in this crop improvement process are steps involving plant transformation. With few exceptions, genetic transformation is an obligatory final step by which useful traits are engineered into plants. In addition, transgenesis is most often needed to confirm gene function, after deductions made through comparative genomics, expression profiles, and mutation analysis. This article reviews the use of recombinase systems to deliver DNA more efficiently into the plant genome.     

Cre-lox site-specific recombination between Arabidopsis and tobacco chromosomes

To create hybrid chromosomes, we tested the Cre-lox system to mediate recombination between Arabidopsis thaliana and Nicotiana tabacum chromosomes. Protoplasts of the two plants were fused to allow site-specific recombination to join a promoter from tobacco to a hygromycin resistance coding-region from Arabidopsis. The expected recombination junction was detected in hygromycin-resistant calli. Analysis of one hybrid suspension cell line revealed the presence of markers corresponding to the north arm of Arabidopsis chromosome III, but not markers from other chromosome arms. However, these markers were not detected in regenerated plants. With a second hybrid cell line we obtained a single hygromycin-resistant progeny from approximately 18 000 self-fertilized seeds of one regenerated plant. Molecular analysis of this hybrid indicated that a small portion of the north arm of Arabidopsis chromosome V is present in the tobacco genome. However, neither the recombination junction nor Arabidopsis DNA was detected in tissue from the plant grown without selection or in the subsequent generation. Thus interspecies transfer of a chromosome arm between plant cells is possible, but maintenance of the hybrid chromosome in a plant is unlikely. The feasibility of site-specific recombination between genomes of different species offers new possibilities for engineering hybrid chromosomes that may be maintained in cell culture.     

GM maize from site-specific recombination technology,what next?

The term plant genetic engineering has long conveyed a highly efficient and precise process for the manipulation of plant genomes. For nearly two decades, research on recombinase-based applications has steadily advanced the surgical capabilities of plant genome rearrangements. Once considered interesting laboratory exercises, a first crop plant derived from this type of DNA acrobatics is heading to market. Originally configured for a specific application, to remove a selectable marker, it could be the first of more to come - and not just market-free plants.     

PhiC31 recombination system demonstrates heritable germinal transmission of site-specific excision from the <Emphasis Type="Italic">Arabidopsis</Emphasis> genome

Background  

The large serine recombinase phiC31 from broad host range Streptomyces temperate phage, catalyzes the site-specific recombination of two recognition sites that differ in sequence, typically known as attachment sites attB and attP. Previously, we characterized the phiC31 catalytic activity and modes of action in the fission yeast Schizosaccharomyces pombe.