Zinc improves gene transfer mediated by DNA/cationic polymer complexes

Background

The weak efficiency of plasmid transfer into the cytosol remains one of the major limiting factors to achieve an efficient transfection with DNA/cationic polymer complexes. We found that divalent metal Zn²⁺ can improve the polyfection efficiency, especially with DNA/histidylated polylysine (His‐pLK) complexes.

Methods and results

The supplementation of the transfection medium with 250 µM ZnCl₂ increased the polyfection of human hepatocarcinoma (HepG2) cells with a plasmid encoding EGFP complexed with pLK, polyethyleneimine and His‐pLK. Zn²⁺ is more efficient on DNA/His‐pLK complexes: the number of EGFP‐positive cells increased from 1% to more than 40%. This phenomenon is selective to Zn²⁺ because no effect was obtained with other divalent cations. The effect of zinc varies from cell to cell. The binding of Zn²⁺ to histidyl residues might increase zinc endosomal concentration favoring membrane fusion. Flow cytometry and confocal microscopy studies clearly indicate that with His‐pLK, the plasmid is better delivered in the cytosol as well as in the cell nucleus in zinc‐treated cells. An investigation conducted with the histidine‐rich peptide H5WYG showed that zinc inhibits membrane permeabilization but promotes membrane fusion as evidenced by resonance energy transfer.

Conclusions

Data reported here imply that the addition of zinc ions in the transfection medium can trigger an increase of the fusion of endosomes containing polyplexes which is more effective in the presence of histidine‐rich molecules. Consequently, the amount of plasmid in the cytosol available to reach the nucleus is increased leading to an improvement of polyfection. Copyright © 2002 John Wiley & Sons, Ltd.

     

A novel PCR-mediated method for one-tube generation of a gene disruption construct

A novel PCR-based method is reported for generating a gene disruption construct which requires no purification of PCR fragments and enables the whole procedure to be completed in one tube very rapidly. The procedure starts with PCR amplification of both the 5 and 3 regions of a particular gene in one tube. Then, exonuclease I is added to the tube to remove the residual primers. After heat inactivation of the enzyme, a marker cassette DNA fragment is added and fusion PCR is performed to build up a gene disruption construct. The gene disruption construct is subsequently amplified with the outermost primers in the amount necessary for transformation. In order to distinguish the gene disruption construct from the remaining intact gene allele, the outermost primers are designed to have GC-rich tag sequences that anneal at a higher temperature, ensuring the specific amplification of the gene disruption construct.     

A novel phospholipid-binding protein from the yeast Saccharomyces cerevisiae with dual binding specificities for the transport GTPase Ypt7p and the Sec1-related Vps33p

The gene product of the Saccharomyces cerevisiae open reading frame YDR229w (named IVY1 for: Interacting with Vps33p and Ypt7p) was found to interact with both the GTPase Ypt7p and the Sec1-related Vps33 protein. While deletion of IVY1 does not lead to any recognized change in phenotype, overexpression of Ivy1p leads to fragmentation of the vacuole, missorting of the vacuolar enzyme carboxypeptidase Y (CPY) to the exterior of the cell, and an accumulation of multivesicular bodies inside the cell. All effects caused by the overexpression of Ivy1p can be reset by simultaneously raising the amount of Vps33p. This suppression activity of Vps33p suggests that Ivy1p and Vps33p at least partially counteract the action of each other in the cell. The intracellular level of Ivy1p increases in cells approaching stationary growth phase at which part of the protein is located at the rim of the vacuole. In addition to its specific interactions with members of two regulatory protein families, Ivy1p in vitro shows a marked propensity for binding phospholipids with high affinity.     

Mac1, a fission yeast transmembrane protein localizing to the poles and septum,is required for correct cell separation at high temperatures

Schizosaccharomyces pombe represents a genetic model system for studying cell polarity and division in eukaryotes. We report here the identification of Mac1, a novel fission yeast protein that localized predominantly to the cell tips and septum. Sequences corresponding to roughly the first 180 amino acids of Mac1, which exhibited weak homology to the transmembrane domains of the Aspergillus Pall protein [Mol. Microbiol. 30 (1998) 259], were found to specify localization to the cell periphery. The other 574 amino acids of Mac1 localized to the cytoplasm when expressed alone, thus suggesting that the N-terminal part of Mac1 functions as a plasma membrane anchor for the rest of the protein. In pom1 null mutant cells, which never switch from unipolar to bipolar growth but, instead, grow exclusively at the randomly chosen end [Genes Dev. 12 (1998) 1356], Mac1 was, nevertheless, found at both poles, thus suggesting that Mac1 does not specifically localize to the sites of growth. mac1 null mutant cells had no overt phenotype at 22-32 degrees C, but, nevertheless, displayed a marked decrease in viability at 34-36 degrees C, accompanied by severe separation defects. Overexpression of mac1 resulted in similar defects. Our data suggest that a correct dosage of Mac1 is needed for correct cell separation at elevated temperatures of growth.     

(T2AG3)n Telomeric Sequence Hybridization Indicating Centric Fusion Rearrangements in the Karyotype of the Rodent Oryzomys Subflavus

Chromosome preparations of 30 specimens of Oryzomys subflavus trapped in eight Brazilian localities were C-, and G-banded and analyzed by fluorescence in situ hybridization (FISH). Two karyotypes were found, 2n=50/FN=64, at three coastal localities of the Atlantic Forest domain, and 2n=58/FN=70 at two sites located in the Cerrado biome, Brazil Central. Two fluorescence in situ hybridization (FISH) patterns of the telomeric sequence (T₂AG₃)_n were observed: in both karyotypes the probes hybridized to the telomeres of all chromosomes and also a hybridization signal in the centromeric regions of two autosome pairs was seen in the 2n=50 karyotype. These results, together with the occurrence of other diploid numbers described in the literature, suggest that O. subflavus is a complex species, bearing fusion/fission rearrangements proper to the different biomes which it inhabits.     

Episodic chromosomal evolution in Planipapillus (Onychophora: Peripatopsidae): a phylogenetic approach to evolutionary dynamics and speciation

Planipapillus, a clade of onychophorans from southeastern Australia, exhibits substantial chromosomal variation. In the context of a robust phylogeny based on nuclear and mitochondrial sequence data, we evaluate models of chromosomal evolution and speciation that differ in the roles assigned to selection, mutation, and drift. Permutation tests suggest that all chromosome rearrangements in the clade have been centric fusions and, on the basis of parsimony and maximum-likelihood methods with independent estimates of branch lengths, we conclude that at least 31 centric fusions have been fixed in Planipapillus. A likelihood-ratio test approach, which is independent of our point estimates of ancestral states, rejects an evolutionary model in which the mutation rate is constant and centric fusions are effectively neutral. In contrast to the nucleotide sequence data, which are consistent with neutrality and rate constancy, centric fusions in Planipapillus are underdominant, spontaneous fusion rates vary among lineages, or both. We predict an inverse relationship between rates of chromosomal evolution and historical population size. Chromosomal evolution may play a role in speciation in Planipapillus, both by interactions between centric fusions with monobrachial homology and by the accumulation of multiple weakly underdominant fusions.     

Production of wild-type and peptide fusion cutinases by recombinant Saccharomyces cerevisiae MM01 strains

This study focused on the growth of Saccha-romyces cerevisiae MM01 recombinant strains and the respective production of three extracellular heterologous cutinases: a wild-type cutinase and two cutinases in which the primary structure was fused with the peptides (WP)(2) and (WP)(4), respectively. Different cultivation and strategies were tested in a 2-L shake flask and a 5-L bioreactor, and the respective cell growth and cutinase production were analyzed and compared for the three yeast strains. The highest cutinase productions and productivities were obtained in the fed-batch culture, where wild-type cutinase was secreted up to a level of cutinase activity per dry cell weight (specific cell activity) of 4.1 Umg(-1) with activity per protein broth (specific activity) of 266 Umg(-1), whereas cutinase-(WP)(2) was secreted with a specific cell activity of 2.1 Umg(-1) with a specific activity of 200 Umg(-1), and cutinase-(WP)(4) with a specific cell activity of 0.7 Umg(-1) with a specific activity of 15 Umg(-1). The results indicate that the fusion of hydrophobic peptides to cutinase that changes the physical properties of the fused protein limits cutinase secretion and subsequently leads to a lower plasmid stability and lower yeast cell growth. These effects were observed under different cultivation conditions (shake flask and bioreactor) and cultivation strategies (batch culture versus fed-batch culture).     

Hyperpolarization, but not depolarization, increases intracellular Ca(2+) level in cultured chick myoblasts.

Ca(2+) influx appears to be important for triggering myoblast fusion. It remains, however, unclear how Ca(2+) influx rises prior to myoblast fusion. The present study examines a possible involvement of the voltage-dependent Ca(2+) influx pathways. Treatment with the L-type Ca(2+) channel blockers, diltiazem, and nifedipine did not alter cytosolic Ca(2+) levels. Depolarization with high K(+) solution and activation of Ca(2+) channel with Bay K 8644, and agonist of voltage dependent Ca(2+) channels, failed to elicit increases intracellular Ca(2+) level, indicating the absence of depolarization-operated mechanisms. In contrast, phloretin, an agonist of Ca(2+)-activated potassium (K(Ca)) channels, was able to hyperpolarize membrane potential and promoted Ca(2+) influx. These effects were completely abolished by treatment of charybdotoxin, a specific inhibitor of K(Ca) channels. In addition, gadolinium, a potent stretch-activated channel (SAC) blocker, prevented the phloretin-mediated Ca(2+) increase, indicating the involvement of SACs in Ca(2+) influx. Furthermore, phloretin stimulated precocious myoblast fusion and this effect was blocked with gadolinium or charybdotoxin. Taken together, these results suggest that induced hyperpolarization, but not depolarization increases Ca(2+) influx through stretch-activated channels, and in turn triggers myoblast fusion.     

Clathrin and plant endocytosis

Endocytosis requires the coordinated interaction of a plethora of cytosolic and membrane proteins. In mammalian cells, clathrin plays a crucial role in this process as a scaffolding protein underlying the invaginating plasma membrane and surrounding the primary endocytic vesicle. Despite great similarities at the morphological level, the cargo of endocytic clathrin-coated vesicles in plant cells remains to be elucidated. Thus, the role of endocytosis in the plant cell is difficult to ascertain. This review will present important discoveries on putative endosomal compartments and on the functions of plasma membrane-derived plant clathrin-coated vesicles, but will also emphasize the striking similarities of the clathrin-, network- and vesicle fusion-machineries between plant and animal cells.     

The protein coat in membrane fusion: lessons from fission

Multiple cell biological processes involve two opposite rearrangements of membrane configuration, referred to as fusion and fission. While membrane intermediates in protein-mediated fusion have been studied in some detail, the global force which drives sequential stages of the fusion reaction from early local intermediates to an expanding fusion pore remains unknown. Fusion proceeds via stages, which are analogous but in the opposite direction to that of membrane budding-off and fission driven by protein coats. On the basis of this analogy, we propose that an interconnected coat formed by membrane-bound activated fusion proteins surrounding the membrane contact zone generates the driving force for fusion. This fusion protein coat has a strongly curved intrinsic shape opposite to that of the protein coat driving fission. To relieve internal stresses, the fusion protein coat spontaneously bends out of the initial shape of the membrane surface. This bending produces elastic stresses in the underlying lipid bilayer and drives its fusion with the apposing membrane. The hypothesis that 'bystander' proteins (i.e. fusion proteins outside the contact zone) generate the driving force for fusion offers a new interpretation for a number of known features of the fusion reaction mediated by the prototype fusion protein, influenza hemagglutinin, and might bring new insights into mechanisms of other fusion reactions.