Expression of a rice chitinase gene in transgenic banana ('Gros Michel', AAA genome group) confers resistance to black leaf streak disease.

Transgenic banana (Musa acuminata 'Gros Michel') integrating either of two rice chitinase genes was generated and its resistance to Black Leaf Streak disease caused by the fungus Mycosphaerella fijiensis was tested using a leaf disk bioassay. PCR screening indicated the presence of the hpt selectable marker gene in more than 90 % of the lines tested, whereas more than three quarters of the lines contained the linked rice chitinase gene resulting in a co-transformation frequency of at least 71.4 %. Further, a unique stable integration of the transgenes in each line revealed some false negative PCR results and the expected co-transformation frequency of 100 %. The transgene insert number per line ranged from 1 to 5 and single transgene insert lines (25 % of all) were identified. Considerable delay in disease development (up to 63 days post-incoculation) over a monitoring period of 108 days occurred in nine lines with extracellularly targeted chitinase out of 17 transgenic lines tested and their necrotic leaf area decreased by 73-94 % compared to the untransformed susceptible control line. Finally, correlation between symptom development and rice chitinase expression was confirmed in two lines by Western analysis. The potential of rice chitinase genes to enhance resistance against M. fijiensis in banana was demonstrated as well as the usefulness of the leaf disk bioassay for early disease screening in transgenic banana lines.

Characterization and isolation of a T-DNA tagged banana promoter active during in vitro culture and low temperature stress.

BACKGROUND: Next-generation transgenic plants will require a more precise regulation of transgene expression, preferably under the control of native promoters. A genome-wide T-DNA tagging strategy was therefore performed for the identification and characterization of novel banana promoters. Embryogenic cell suspensions of a plantain-type banana were transformed with a promoterless, codon-optimized luciferase (luc+) gene and low temperature-responsive luciferase activation was monitored in real time. RESULTS: Around 16,000 transgenic cell colonies were screened for baseline luciferase activity at room temperature 2 months after transformation. After discarding positive colonies, cultures were re-screened in real-time at 26 degrees C followed by a gradual decrease to 8 degrees C. The baseline activation frequency was 0.98%, while the frequency of low temperature-responsive luciferase activity was 0.61% in the same population of cell cultures. Transgenic colonies with luciferase activity responsive to low temperature were regenerated to plantlets and luciferase expression patterns monitored during different regeneration stages. Twenty four banana DNA sequences flanking the right T-DNA borders in seven independent lines were cloned via PCR walking. RT-PCR analysis in one line containing five inserts allowed the identification of the sequence that had activated luciferase expression under low temperature stress in a developmentally regulated manner. This activating sequence was fused to the uidA reporter gene and back-transformed into a commercial dessert banana cultivar, in which its original expression pattern was confirmed. CONCLUSION: This promoter tagging and real-time screening platform proved valuable for the identification of novel promoters and genes in banana and for monitoring expression patterns throughout in vitro development and low temperature treatment. Combination of PCR walking techniques was efficient for the isolation of candidate promoters even in a multicopy T-DNA line. Qualitative and quantitative GUS expression analyses of one tagged promoter in a commercial cultivar demonstrated a reproducible promoter activity pattern during in vitro culture. Thus, this promoter could be used during in vitro selection and generation of commercial transgenic plants.

Improved T-DNA vector for tagging plant promoters via high-throughput luciferase screening.

Transferred DNA (T-DNA) tagging is a powerful tool for tagging and in planta characterization of plant genes on a genome-wide scale. An improved promoter tagging vector is described here, which contains the codon-optimized luciferase (luc+) reporter gene 31 bp from the right border of the T-DNA. Compared to the wild-type luciferase gene, this construct provides significantly increased reporter gene expression and a 40 times higher tagging frequency. The utility of the construct is demonstrated in banana, a tropical monocot species, by screening embryogenic cell colonies and regenerated plants with an ultrasensitive charged-coupled device (CCD) camera. The improved vector resulted in a luciferase activation frequency of 2.5% in 19,000 cell colonies screened. Detailed molecular analysis of flanking DNA sequences in a tagged line revealed insertion of the luciferase tag in a novel gene with near-constitutive expression.

Influence of exonic polymorphisms in the gene for LDL receptor-related protein (LRP) on risk of coronary artery disease.

The low density lipoprotein (LDL) receptor-related protein (LRP) is a multifunctional receptor involved in numerous biological processes relevant to vascular biology including lipoprotein metabolism. Several polymorphisms in the LRP gene have been described and in this study we examined their influence on coronary artery disease (CAD). We compared the frequencies of the exon 3 (C766T), exon 6 (C663T), exon 22 (C200T), and four rarer and more recently described polymorphisms in approximately 600 Caucasian subjects aged <50 years with angiographic CAD and approximately 700 similarly aged subjects without symptomatic CAD randomly selected from the community. We found the distribution of exon 22 C200T genotypes to differ significantly between the CAD (CC: 52%, CT: 39%, TT: 9%) and control subjects (CC: 43%, CT: 46%, TT: 11%, P=0.005), with the CC genotype conferring an odds ratio (OR) for CAD of 1.5 (95% CI: 1.2-1.8, P=0.001) despite a lack of significant influence on plasma cholesterol or triglyceride. The other LRP polymorphisms were less common. Two showed an association with CAD; for the exon 3 C766T polymorphism the TT genotype was significantly lower (1.0 vs. 2.7%; OR: 0.36; P=0.04) and, for the exon 6 C663T polymorphism, the heterozygote frequency was higher (6.2 vs. 3.4%; OR: 1.9; P=0.03) in CAD subjects. In conclusion, LRP gene polymorphisms, particularly the relatively common exon 22 C200T polymorphism, are a significant risk factor for premature CAD in Caucasians.

Functional expression of murine LRP1 requires correction of Lrp1 cDNA sequences.

Here, we describe the reconstruction of a functional 14 kbp full-length murine Lrp1 cDNA from overlapping partial cDNAs, which were described before [Biochim. Biophys. Acta 1173 (1993) 71]. The reconstructed full-length cDNA needed sequence correction (by mutagenesis) due to nucleotide errors present in the underlying partial cDNAs. These mistakes compromised the proteolytical maturation of the LRP precursor (4545 aa) into its alpha- and beta-subunits. To identify these mistakes initially, detailed sequence analyses and comparison of genomic and cDNA sequences of different murine strains proved to be necessary to obtain correct wild-type sequences. Comparison of Lrp1 cDNA sequences of CBA mice with Lrp1 genomic exon sequences of 129P3/J mice (like in man 89 exons) revealed only 24 nucleotide differences within about 14.8 kbp. Only 1 out of 23 nucleotide differences in the protein coding region affected an amino acid residue: Thr versus Ala at amino acid residue position 2642 in 129P3/J and CBA, respectively. After correction by mutagenesis, both a 129P3/J and a CBA-based version of a full-length wild-type Lrp1 cDNA were functionally expressed in an LRP-deficient mutant CHO cell line. Transient expression showed the expected maturation of the LRP precursor into its two subunits. Furthermore, stable transfection restored the sensitivity to exposure to Pseudomonas aeruginosa toxin A (PEA). Since LRP is the unique receptor for this toxin, this indicates that the toxin could enter the cells after binding to and endocytosis by its genuine receptor. This murine LRP expression system will be instrumental in future experimental dissection of this multifunctional receptor.

Neuronal deficiency of presenilin 1 inhibits amyloid plaque formation and corrects hippocampal long-term potentiation but not a cognitive defect of amyloid precursor protein [V717I] transgenic mice.

In the brain of Alzheimer's disease (AD) patients, neurotoxic amyloid peptides accumulate and are deposited as senile plaques. A major therapeutic strategy aims to decrease production of amyloid peptides by inhibition of gamma-secretase. Presenilins are polytopic transmembrane proteins that are essential for gamma-secretase activity during development and in amyloid production. By loxP/Cre-recombinase-mediated deletion, we generated mice with postnatal, neuron-specific presenilin-1 (PS1) deficiency, denoted PS1(n-/-), that were viable and fertile, with normal brain morphology. In adult PS1(n-/-) mice, levels of endogenous brain amyloid peptides were strongly decreased, concomitant with accumulation of amyloid precursor protein (APP) C-terminal fragments. In the cross of APP[V717I]xPS1 (n-/-) double transgenic mice, the neuronal absence of PS1 effectively prevented amyloid pathology, even in mice that were 18 months old. This contrasted sharply with APP[V717I] single transgenic mice that all develop amyloid pathology at the age of 10-12 months. In APP[V717I]xPS1 (n-/-) mice, long-term potentiation (LTP) was practically rescued at the end of the 2 hr observation period, again contrasting sharply with the strongly impaired LTP in APP[V717I] mice. The findings demonstrate the critical involvement of amyloid peptides in defective LTP in APP transgenic mice. Although these data open perspectives for therapy of AD by gamma-secretase inhibition, the neuronal absence of PS1 failed to rescue the cognitive defect, assessed by the object recognition test, of the parent APP[V717I] transgenic mice. This points to potentially detrimental effects of accumulating APP C99 fragments and demands further study of the consequences of inhibition of gamma-secretase activity. In addition, our data highlight the complex functional relation of APP and PS1 to cognition and neuronal plasticity in adult and aging brain.