Photocross-linking of the homing endonuclease PI-SceI to its recognition sequence

PI-SceI is an intein-encoded protein that belongs to the LAGLIDADG family of homing endonucleases. According to the crystal structure and mutational studies, this endonuclease consists of two domains, one responsible for protein splicing, the other for DNA cleavage, and both presumably for DNA binding. To define the DNA binding site of PI-SceI, photocross-linking was used to identify amino acid residues in contact with DNA. Sixty-three double-stranded oligodeoxynucleotides comprising the minimal recognition sequence and containing single 5-iodopyrimidine substitutions in almost all positions of the recognition sequence were synthesized and irradiated in the presence of PI-SceI with a helium/cadmium laser (325 nm). The best cross-linking yield (approximately 30%) was obtained with an oligodeoxynucleotide with a 5-iododeoxyuridine at position +9 in the bottom strand. The subsequent analysis showed that cross-linking had occurred with amino acid His-333, 6 amino acids after the second LAGLIDADG motif. With the H333A variant of PI-SceI or in the presence of excess unmodified oligodeoxynucleotide, no cross-linking was observed, indicating the specificity of the cross-linking reaction. Chemical modification of His residues in PI-SceI by diethylpyrocarbonate leads to a substantial reduction in the binding and cleavage activity of PI-SceI. This inactivation can be suppressed by substrate binding. This result further supports the finding that at least one His residue is in close contact to the DNA. Based on these and published results, conclusions are drawn regarding the DNA binding site of PI-SceI.     

Alterations in the directionality of lambda site-specific recombination catalyzed by mutant integrases in vivo.

Phage lambda integrative and excisive recombination normally proceeds by a pair of sequential strand exchanges. During the first exchange reaction, the "top" strand in each recombination site is cleaved, exchanged, and religated generating a Holliday junction intermediate. This intermediate DNA structure is resolved through a pair of reciprocal "bottom" strand exchanges, leading to recombinant products. The strict co-ordination of exchange reactions ensures religation between correct partner strands only. Here we show that the directionality of recombination is altered in vivo by two mutant integrases, Int-h (E174 K) and a double mutant Int-h/218 (E174 K/E218 K). This change in directionality leads to deletion instead of inversion on substrates that carry inverted attachment sites and, depending on the pair of target sites employed, requires the presence or absence of integration host factor. Neither Fis nor Xis is involved in deletion. Sequence analyses of deletion products reveal that the newly generated hybrid attachment site exhibits a reversed genetic polarity. We demonstrate that only one of two possible hybrid site configurations is generated and discuss two pathways leading to deletion. In the first, deletion results from a wrong alignment of the two recombination sites within the synaptic complex. In the second pathway, the unco-ordinated cleavage by the mutant integrases of all four DNA strands present in a conventional Holliday junction intermediate leads to two double-stranded breaks, whereby the subsequent rejoining between "wrong" partner strands appears restricted to only two strands.     

The monomeric homing endonuclease PI-SceI has two catalytic centres for cleavage of the two strands of its DNA substrate

The monomeric homing endonuclease PI-SceI cleaves the two strands of its DNA substrate in a concerted manner, which raises the question of whether this enzyme harbours one or two catalytic centres. If PI-SceI has only one catalytic centre, one would expect that cross-linking enzyme and substrate should prevent reorientation of the enzyme required to perform the second cut after having made the first cut: PI-SceI, however, when cross-linked to its substrate, is able to cleave both DNA strands. If PI-SceI has two catalytic centres, one would expect that it should be possible to inactivate one catalytic centre by mutation and obtain a variant with preference for a substrate nicked in one strand; such variants have been found. The structural homology between the catalytic domain of PI-SceI having a pseudo 2-fold symmetry, and I-CreI, a homodimeric homing endonuclease, suggests that in PI-SceI active site I, which attacks the top strand, comprises Asp218, Asp229 and Lys403, while Asp326, Thr341 and Lys301 make up active site II, which cleaves the bottom strand. Cleavage experiments with modified oligodeoxynucleotides and metal ion mapping experiments demonstrate that PI-SceI interacts differently with the two strands at the cleavage position, supporting a model of two catalytic centres.     

Serum-free cryopreservation of porcine hepatocytes

The use of porcine hepatocytes in xenotransplantation, bioartificial liver support or pharmacological approaches demands serum-free cryopreservation protocols yielding high quality, viable, functional hepatocytes. Here, primary porcine hepatocytes were frozen without serum in liquid nitrogen by the use of a computer-assisted freezing device. After thawing, more than 90% of the initial hepatocytes were lost, in part because of damage to genomic DNA. When cryoprotectants were used, the loss was lowered to 70% of the initial cell number; 90% of the remaining cells excluded trypan blue indicating a high degree of viability. Cells were seeded serum-free onto collagen-coated plastic dishes to determine proliferation and retainment of specific functions representing prominent features of hepatocytes in vivo. Whereas no cells adhered to the substratum effectively in conventional culture medium, the addition of conditioned medium derived from hepatic non-parenchymal cells improved attachment. Cells proliferated, retained hepatocyte-specific functions, such as urea production and cytochrome P450 activity, and expressed liver-specific genes to levels observed in non-cryopreserved hepatocytes. Thus, serum-free cryopreserved primary porcine hepatocytes may serve as a valid source of cells for downstream applications. The cells seem to function adequately when an appropriate environment is chosen for recovery after cryopreservation, an ultimate demand for the clinical application of human hepatocytes.     

Mapping genes for resistance to<Emphasis Type="Italic"> Verticillium albo-atrum</Emphasis> in tetraploid and diploid potato populations using haplotype association tests and genetic linkage analysis

Verticillium wilt disease of potato is caused predominantly by Verticillium albo-atrum and V. dahliae. StVe1 —a putative QTL for resistance against V. dahliae —was previously mapped to potato chromosome 9. To develop allele-specific, SNP-based markers within the locus, the StVe1 fragment from a set of 30 North American potato cultivars was analyzed. Three distinct and highly diverse haplotypes can be distinguished at the StVe1 locus. These were detected in 97%, 33%, and 10% of the cultivars analyzed. We tested for haplotype association and for genetic linkage between the StVe1 haplotypes and resistance of tetraploid potato to V. albo-atrum. Moreover, field resistance was assessed in diploid populations with known molecular linkage maps in order to identify novel QTLs. Resistance QTLs against V. albo-atrum were detected on four chromosomes (2, 6, 9, and 12) at the diploid level, with one QTL on chromosome 2 contributing over 40% to the total phenotypic variation of the trait. At the tetraploid level, a significant association between the StVe1-839-C haplotype and susceptibility to the disease was detected, suggesting that resistance-related genes directed against V. albo-atrum and V. dahliae are located in the same genomic region of chromosome 9. However, on the basis of the present analysis, we cannot determine whether these genes are closely linked or if a single gene provides resistance against both Verticillium species. To assess the usefulness of the StVe1-839-C haplotype for marker-assisted selection, we subjected the resistance data to Bayesian analysis, and calculated positive (0.65) and negative (0.75) predictive values, and overall predictive accuracy (0.72). Our results indicate that tagging of additional genes for resistance to Verticillium with molecular markers will be required for efficient marker-assisted selection.Communicated by M.-A. Grandbastien     

Inhibitors of gap junctions attenuate myogenic tone in cerebral arteries

The effects of two structurally distinct inhibitors of gap junction communication were studied by using three different forms of vasoconstriction in pressurized rat middle cerebral arteries. The sensitivity of myogenic tone (at 60 mmHg), vasopressin-induced tone (10 nM, at 20 mmHg), and depolarizing solution-induced tone (80 mM K(+), at 20 mmHg) to inhibition by heptanol (1.0 microM to 3.0 mM) or 18alpha-glycyrrhetinic acid (18alpha-GA, 1.0 to 50 microM) were determined. Pressure-induced myogenic tone was inhibited by heptanol (IC(50) = 0.75 +/- 0.09 mM) and 18alpha-GA ( approximately 30 microM). Vasopressin-induced vasoconstriction was also inhibited by heptanol (IC(50) = 0.4 +/- 0.3 mM) and 18alpha-GA (>1 microM). Depolarizing solution-induced vasoconstriction was less sensitive to inhibition by heptanol compared to vasopressin (P < 0.01) or pressure-induced constriction (P < 0.05). However, 18alpha-GA did not inhibit depolarization-induced constriction. Sharp microelectrode experiments on isolated arteries revealed stable membrane potentials, with no detectable effect of heptanol (1 mM) or 18alpha-GA (20-30 microM) on the average membrane potential at 20 mmHg. However, approximately 20% of impaled cells (5 of 28) exhibited uncharacteristic oscillations in membrane potential after pharmacological uncoupling. At 60 mmHg a approximately 7- to 9-mV hyperpolarization and corresponding vasodilation (approximately 50%) was observed, and the frequency of membrane potential oscillations doubled (9 of 23 cells). These data indicate that gap junctions play an important role in the maintenance and modulation of membrane potential and tone in cerebral resistance arteries.