Target Plasmid Research Articles

Bcl2 Suppresses DNA Repair by Enhancing c-Myc Transcriptional Activity

Bcl2 and c-Myc are two major oncogenic proteins that can functionally promote DNA damage, genetic instability, and tumorigenesis. However, the mechanism(s) remains unclear. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most potent carcinogen contained in cigarette smoke that induces cellular DNA damage. Here we found that Bcl2 potently suppresses the repair of NNK-induced abasic sites of DNA lesions in association with increased c-Myc transcriptional activity. The Bcl2 BH4 domain (amino acids 6-31) was found to bind directly to c-Myc MBII domain (amino acids 106-143), and this interaction is required for Bcl2 to enhance c-Myc transcriptional activity and inhibit DNA repair. In addition to mitochondria, Bcl2 is also expressed in the nucleus, where it co-localizes with c-Myc. Expression of nuclear-targeted Bcl2 enhances c-Myc transcriptional activity with suppression of DNA repair but fails to prolong cell survival. Depletion of c-Myc expression from cells overexpressing Bcl2 significantly accelerates the repair of NNK-induced DNA damage, indicating that c-Myc may be essential for the Bcl2 effect on DNA repair. It is known that apurinic/apyrimidinic endonuclease (APE1) plays a crucial role in the repair of abasic sites of DNA lesions. That overexpression of Bcl2 results in up-regulation of c-Myc and down-regulation of APE1 suggests APE1 may function as the downstream target of Bcl2/c-Myc in the DNA repair machinery. Thus, Bcl2, in addition to its survival function, may also suppress DNA repair in a novel mechanism involving c-Myc and APE1, which may lead to an accumulation of DNA damage in living cells, genetic instability, and tumorigenesis.

Regulation of Intracellular Accumulation of Mutant Huntingtin by Beclin 1

Intracellular accumulation of mutant Huntingtin with expanded polyglutamine provides a context-dependent cytotoxicity critical for the pathogenesis of Huntington disease (Everett, C. M., and Wood, N. W. (2004) Brain 127, 2385-2405). Here we demonstrate that the accumulation of mutant Huntingtin is highly sensitive to the expression of beclin 1, a gene essential for autophagy. Moreover, we show that the accumulated mutant Huntingtin recruits Beclin 1 and impairs the Beclin 1-mediated long lived protein turnover. Thus, sequestration of Beclin 1 in the vulnerable neuronal population of Huntington disease patients might further reduce Beclin 1 function and autophagic degradation of mutant Huntingtin. Finally, we demonstrate that the expression of beclin 1 decreases in an age-dependent fashion in human brains. Because beclin 1 gene is haploid insufficient in regulating autophagosome function (Qu, X., Yu, J., Bhagat, G., Furuya, N., Hibshoosh, H., Troxel, A., Rosen, J., Eskelinen, E. L., Mizushima, N., Ohsumi, Y., Cattoretti, G., and Levine, B. (2003) J. Clin. Invest. 112, 1809-1820; Yue, Z., Jin, S., Yang, C., Levine, A. J., and Heintz, N. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 15077-15082), we propose that the age-dependent decrease of beclin 1 expression may lead to a reduction of autophagic activity during aging, which in turn promotes the accumulation of mutant Htt and the progression of the disease.

Murine Thrombin Lacks Na+ Activation but Retains High Catalytic Activity

Human thrombin utilizes Na+ as a driving force for the cleavage of substrates mediating its procoagulant, prothrombotic, and signaling functions. Murine thrombin has Asp-222 in the Na+ binding site of the human enzyme replaced by Lys. The charge reversal substitution abrogates Na+ activation, which is partially restored with the K222D mutation, and ensures high activity even in the absence of Na+. This property makes the murine enzyme more resistant to the effect of mutations that destabilize Na+ binding and shift thrombin to its anticoagulant slow form. Compared with the human enzyme, murine thrombin cleaves fibrinogen and protein C with similar k(cat)/K(m) values but activates PAR1 and PAR4 with k(cat)/K(m) values 4- and 26-fold higher, respectively. The significantly higher specificity constant toward PAR4 accounts for the dominant role of this receptor in platelet activation in the mouse. Murine thrombin can also cleave substrates carrying Phe at P1, which potentially broadens the repertoire of molecular targets available to the enzyme in vivo.

Multiplexed Detection of Anthrax-Related Toxin Genes

Simultaneous analysis of three targets in three colors on any real-time polymerase chain reaction (PCR) instrument would increase the flexibility of real-time PCR. For the detection of Bacillus strains that can cause inhalation anthrax-related illness, this ability would be valuable because two plasmids confer virulence, and internal positive controls are needed to monitor the testing in cases lacking target-specific signals. Using a real-time PCR platform called MultiCode-RTx, multiple assays were developed that specifically monitor the presence of Bacillus anthracis-specific virulence plasmid-associated genes. In particular for use on LightCycler-1, two triplex RTx systems demonstrated high sensitivity with limits of detection nearing single-copy levels for both plasmids. Specificity was established using a combination of Ct values and correct amplicon melting temperatures. All reactions were further verified by detection of an internal positive control. For these two triplex RTx assays, the analytical detection limit was one to nine plasmid copy equivalents, 100% analytical specificity with a 95% confidence interval (CI) of 9%, and 100% analytical sensitivity with a CI of 2%. Although further testing using clinical or environmental samples will be required to assess diagnostic sensitivity and specificity, the RTx platform achieves similar results to those of probe-based real-time systems.

A novel transposon-based method for elimination of large bacterial plasmids

Elimination or modification of large plasmids of bacteria is often an essential step in functional analysis of these replicons. However, the conventional plasmid-curing procedures such as ethidium bromide and heat treatment are insufficient in many cases. For instance, curing of the large virulence plasmid of Salmonella Enteritidis 2102 has failed when these treatments were applied. To overcome the difficulties, a two-step transposon-based curing method has been developed. First, a Tn 10-based transposable unit carrying a Km R marker gene and the joined IS 30 ends transposes from a replication deficient conjugative plasmid into the target replicon. Then, the inducible IS 30 transposase, using the highly reactive joined IS 30 ends, mediates deletions or gives rise to the loss of the target plasmid. The efficiency of the method has been monitored by the frequency of Km S colonies after induction of IS 30 transposase, and it was shown that the Km S phenotype often accompanied the complete loss of the virulence plasmid or the formation of deletion derivatives. The procedure has been successfully applied also in removing the large virulence plasmid from enterotoxigenic Escherichia coli (ETEC O147), suggesting that the transposon-based method can be a useful tool for eliminating native plasmids in several bacteria.

Characterization of IS1474, an insertion sequence of the IS21 family isolated from Pseudomonas alcaligenes NCIB 9867.

A new insertion sequence (IS) designated IS1474 was isolated from Pseudomonas alcaligenes NCIB 9867 (P25X). IS1474 is a 2632 bp element which showed a characteristic IS structure with 12 bp inverted repeats (IRs) flanking a 2608 bp central region. IS1474 contained four open reading frames (ORF1-ORF4), two in each orientation. Similarities were detected between ORF1 and ORF2 and the putative transposases of the IS21 family. Sequences upstream from IS1474 were found to display up to 89% homology with IS53 from Pseudomonas syringae suggesting that IS1474 had inserted into another related IS element designated IS1475. An open reading frame, ORF5, located at the junction of IS1474 and IS1475, showed similarities with the IstB protein of IS21 and could possibly be the transposase subunit of IS1475. Transposition assays showed that IS1474 transposed at a relatively low frequency leading to cointegration with target plasmids. Hybridization studies showed that IS1474 is present in at least 13 copies in the chromosome of P25X and one copy on its endogenous plasmid.

Gene disruption in Cryptococcus neoformans and Cryptococcus gattii by in vitro transposition

Cryptococcus neoformans and Cryptococcus gattii are basidiomycetous fungi that infect immunocompromised and immunocompetent people. We developed an insertional mutagenesis strategy for these species based on in vitro transposition and we tested the method by disrupting the URA5 gene in a strain of C. neoformans and the CAP10 gene in three strains of C. gattii. We targeted plasmid DNA containing the URA5 gene or plasmid DNA containing the CAP10 gene from genomic libraries from the shotgun sequencing project for the C. gatti strain WM276. In the latter case, the availability of the end sequences of the clones from the assembled genomic sequence allows rapid selection of target genes for disruption. Modified transposons containing the nourseothricin (NAT) or neomycin (Neo) resistance cassettes were randomly inserted into the target DNA by in vitro transposition. The disrupted genes were used for biolistic transformation and homologous integration was subsequently confirmed by PCR and Southern blot analysis. These results demonstrate that the emerging genomic resources, combined with in vitro transposition into plasmid DNAs from shotgun sequencing libraries or cloned PCR products, will facilitate high-throughput genetic analysis in Cryptococcus species.

Defining a Link with Autosomal-Dominant Polycystic Kidney Disease in Mice with Congenitally Low Expression of Pkd1

Mouse models for autosomal-dominant polycystic kidney disease (ADPKD), derived from homozygous targeted disruption of Pkd1 gene, generally die in utero or perinatally because of systemic defects. We introduced a loxP site and a loxP-flanked mc1-neo cassette into introns 30 and 34, respectively, of the Pkd1 locus to generate a conditional, targeted mutation. Significantly, before excision of the floxed exons and mc1-neo from the targeted locus by Cre recombinase, mice homozygous for the targeted allele appeared normal at birth but developed polycystic kidney disease with a slower progression than that of Pkd-null mice. Further, the homozygotes continued to produce low levels of full-length Pkd1-encoded protein, suggesting that slight Pkd1 expression is sufficient for renal cyst formation in ADPKD. In this viable model, up-regulation of heparin-binding epidermal growth factor-like growth factor accompanied increased epidermal growth factor receptor signaling, which may be involved in abnormal proliferation of the cyst-lining epithelia. Increased apoptosis in cyst epithelia was only observed in the later period that correlated with the cyst regression. Abnormalities in Na(+)/K(+)-ATPase, aquaporin-2, and vasopressin V2 receptor expression were also identified. This mouse model may be suitable for further studies of progression and therapeutic interventions of ADPKD.

515. Sequence Requirements for Alveolar Epithelial Cell-Specific Plasmid Nuclear Import

Top of pageAbstract Non-viral DNA gene delivery has many advantages over viral vectors, including potential for repeated administration and minimal immunological response. However, to date, the gene transfer efficiency remains low with this type of vector. This low efficiency is due to several barriers non-viral vectors must overcome prior to transgene expression. Perhaps the most important barrier in non-viral gene transfer is the nuclear envelope. In non-dividing cells, theplasmid DNA has no access to the nucleus where the transcriptional machinery resides, and thus transgenes are not expressed. However, inclusion of specific sequences termed DNA targeting sequences (DTS) on plasmids will mediate nuclear import via the nuclear pore complex and enhance non-viral DNA gene transfer. These sequences, when included on a plasmid, will bind newly-synthesized transcription factors in the cytoplasm and the plasmid DNA will translocate into the nucleus via the nuclear localization signals on the bound transcription factors. Additionally, we can use cell-specific DTSs to mediate nuclear import only in specific cell types, thus limiting transgene expression to the cell type of choice. Using this novel strategy, we can improve both safety and efficiency of non-viral gene therapy by targeting the nucleus of only specific cell types. We have demonstrated previously that the 365 bp fragment of the human SP-C promoter (|[minus]|318 to +47) will mediate nuclear import of plasmid DNA in alveolar epithelial cells but in no other cell types tested to date. Within this 365 nucleotide fragment, there exist several known binding sites for transcription factors. Several truncations of the SP-C promoter have been constructed, as well as mutations that will abrogate transcription factor binding to the SP-C promoter DNA. Using microinjection and in situ hybridization strategy in both A549 and MLE-12 cell lines, we have determined which sequences within the SP-C promoter are required for nuclear import these alveolar epithelial cell lines. These data gives us a further understanding as to the mechanism of DTS function, knowledge that will allow us in the future to predict or create new DTSs for targeted plasmid DNA transfer to many other cell types.

Assessing genetic diversity in plasmids from Escherichia coli and Salmonella enterica using a mixed-plasmid microarray

To compare genetic composition of plasmids using microarrays composed of randomly selected fragments of plasmid DNA. Separate shotgun libraries were constructed from plasmid DNA pooled from Escherichia coli and Salmonella enterica. Cloned fragments were used as probes for microarrays. Plasmid targets were labelled, hybridized overnight, and bound targets were imaged after enzymatic signal amplification. Control hybridizations demonstrated significantly higher signal when probes and targets shared >95% sequence identity. Diagnostic sensitivity and specificity of the assay was 95 and 99%, respectively. Cluster analysis showed close matches for replicate experiments with a high correlation between replicates (r = 0.91) compared with the correlation for nonreplicates (r = 0.09). Analysis of hybridization data from 43 plasmids generated five distinct clusters, two for known serovar-specific plasmids, one for enterohemorrhagic E. coli plasmids, and two for plasmids harboring a recently disseminated antibiotic resistance gene (bla(CMY-2)). Mixed-plasmid microarrays are suitable for comparing genetic content of wild-type plasmids and hybridization results from this study suggest several novel hypotheses about plasmid gene exchange between E. coli and S. enterica. Mixed-plasmid microarrays permit rapid, low cost analysis and comparison of many plasmids. This ability is critical to understanding the source, fate, and transport of plasmids amongst commensal and pathogenic bacteria.

The copy-number of plasmids and other genetic elements can be determined by SYBR-Green-based quantitative real-time PCR

In this study, we explored whether SYBR Green-based quantitative real-time PCR (qPCR) could be used to determine the copy number of a plasmid and whether the method was broadly applicable to chromosomally encoded genetic elements often occurring in multiple copies, such as rRNA genes and insertion sequences (IS). Three different template sources (whole cells, total DNA, and restriction-enzyme digested total DNA) derived from the bacterium Comamonas sp. strain JS46 were analyzed by qPCR using primer-pairs targeting plasmid pJS46 and three chromosomally encoded sequences (16S rDNA, IS Csp1, and IS 1071). The difference between threshold cycle values, C T, of amplicons targeting these elements and of an amplicon targeting the single-copy reference element mnbA (chromosomally encoded) was used to establish Δ C T. Δ C T values were then used to derive copy number. For pJS46, qPCR analyses of whole cells and total DNA underestimated the copy number of pJS46 ∼7-fold and ∼2.5-fold, respectively, whereas copy number values derived from qPCR analyses of digested total DNA were comparable to those derived from Southern blot (SB) analyses. In contrast, for the chromosomally encoded elements, qPCR analyses of all three template sources gave copy number values that were virtually identical to or differed by ∼2 from copy number values derived by SB analysis. These data indicate that qPCR can be used to estimate the copy number of various genetic elements but that the accuracy of qPCR-derived values is affected by the template source.

Real-Time PCR Assays Targeting a Unique Chromosomal Sequence of Yersinia pestis

Yersinia pestis, the causative agent of the zoonotic infection plague, is a major concern as a potential bioweapon. Current real-time PCR assays used for Y. pestis detection are based on plasmid targets, some of which may generate false-positive results. Using the yp48 gene of Y. pestis, we designed and tested 2 real-time TaqMan minor groove binder (MGB) assays that allowed us to use chromosomal genes as both confirmatory and differential targets for Y. pestis. We also designed several additional assays using both Simple-Probe and MGB Eclipse probe technologies for the selective differentiation of Yersinia pseudotuberculosis from Y. pestis. These assays were designed around a 25-bp insertion site recently identified within the yp48 gene of Y. pseudotuberculosis. The Y. pestis-specific assay distinguished this bacterium from other Yersinia species but had unacceptable low-level detection of Y. pseudotuberculosis, a closely related species. Simple-Probe and MGB Eclipse probes specific for the 25-bp insertion detected only Y. pseudotuberculosis DNA. Probes that spanned the deletion site detected both Y. pestis and Y. pseudotuberculosis DNA, and the 2 species were clearly differentiated by a post-PCR melting temperature (Tm) analysis. The Simple-Probe assay produced an almost 7 degrees C Tm difference and the MGB Eclipse probe a slightly more than 4 degrees C difference. Our method clearly discriminates Y. pestis DNA from all other Yersinia species tested and from the closely related Y. pseudotuberculosis. These chromosomal assays are important both to verify the presence of Y. pestis based on a chromosomal target and to easily distinguish it from Y. pseudotuberculosis.

Identification of PamA as a PII-binding Membrane Protein Important in Nitrogen-related and Sugar-catabolic Gene Expression in Synechocystis sp. PCC 6803

The PII signaling protein plays a pivotal role in the coordination of carbon and nitrogen metabolism in a wide variety of bacteria, Archaea, and plant chloroplasts. By using a yeast two-hybrid screening system, we identified a transmembrane protein, designated PamA (encoded by sll0985), as a PII-binding protein in Synechocystis sp. PCC 6803. The interaction between PII and PamA was confirmed in vitro, and the interaction was inhibited in the presence of ATP and 2-oxoglutarate, whereas the interaction was not influenced by the phosphorylation status of PII. Northern blot analyses revealed that the transcripts of a set of nitrogen-related genes, including nblA, nrtABCD, and ureG, were decreased in a pamA deletion mutant. The mRNA and protein levels of a group 2 sigma factor SigE were also reduced by the pamA mutation, and transcripts for sugar catabolic genes, such as gap1, zwf, and gnd that are under the control of SigE, were consequently decreased in the pamA mutant. In addition, the pamA mutant was found to be unable to grow in glucose-containing media. These results indicate that PamA has a role in the transcript control of genes for nitrogen and sugar metabolism in Synechocystis sp. PCC 6803.

Development of Multiplex PCRs for Detection of Common Viral Pathogens and Agents of Congenital Infections

Potential causes of congenital infection include Toxoplasma gondii and viruses such as cytomegalovirus (CMV), enterovirus, hepatitis C virus, herpes simplex virus types 1 and 2 (HSV-1 and -2), human herpesvirus types 6, 7, and 8, lymphocytic choriomeningitis virus, parvovirus, rubella virus, and varicella-zoster virus. Testing for each of these agents using nucleic acid tests is time consuming and the availability of clinical samples such as amniotic fluid or neonatal blood is often limited. The aim of this study was to develop multiplex PCRs (mPCRs) for detection of DNA and RNA agents in the investigation of congenital infection and an mPCR for the viruses most commonly requested in a diagnostic virology laboratory (CMV, Epstein-Barr virus, enterovirus, HSV-1, HSV-2, and varicella-zoster virus). The assays were assessed using known pathogen-positive tissues (cultures, placentae, plasma, and amniotic fluid) and limits of detection were determined for all the agents studied using serial dilutions of plasmid targets. Nested PCR was performed as the most sensitive assay currently available, and detection of the amplicons using hybridization to labeled probes and enzyme-linked immunosorbent assay detection was incorporated into three of the four assays. This allowed detection of 10 to 10(2) copies of each agent in the samples processed. In several patients, an unexpected infection was diagnosed, including a case of encephalitis where HSV was the initial clinical suspicion but CMV was detected. In the majority of these cases the alternative agent could be confirmed using reference culture, serology, or fluorescence methods and was of relevance to clinical care of the patient. The methods described here provide useful techniques for diagnosing congenital infections and a paradigm for assessment of new multiplex PCRs for use in the diagnostic laboratory.

Reduction of nucleosome assembly during new DNA synthesis impairs both major pathways of double-strand break repair.

Assembly of new chromatin during S phase requires the histone chaperone complexes CAF-1 (Cac2p, Msi1p and Rlf2p) and RCAF (Asf1p plus acetylated histones H3 and H4). Cells lacking CAF-1 and RCAF are hypersensitive to DNA-damaging agents, such as methyl methanesulfonate and camptothecin, suggesting a possible defect in double-strand break (DSB) repair. Assays developed to quantitate repair of defined, cohesive-ended break structures revealed that DSB-induced plasmid:chromosome recombination was reduced ∼10-fold in RCAF/CAF-1 double mutants. Recombination defects were similar with both chromosomal and plasmid targets in vivo, suggesting that inhibitory chromatin structures were not involved. Consistent with these observations, ionizing radiation-induced loss of heterozygosity was abolished in the mutants. Nonhomologous end-joining (NHEJ) repair proficiency and accuracy were intermediate between wild-type levels and those of NHEJ-deficient yku70 and rad50 mutants. The defects in NHEJ, but not homologous recombination, could be rescued by deletion of HMR-a1, a component of the a1/alpha2 transcriptional repressor complex. The findings are consistent with the observation that silent mating loci are partially derepressed. These results demonstrate that defective assembly of nucleosomes during new DNA synthesis compromises each of the known pathways of DSB repair and that the effects can be indirect consequences of changes in silenced chromatin structure.

Both High Mobility Group (HMG)-boxes and the Acidic Tail of HMGB1 Regulate Recombination-activating Gene (RAG)-mediated Recombination Signal Synapsis and Cleavage in Vitro

RAG-1 and RAG-2 initiate V(D)J recombination through synapsis and cleavage of a 12/23 pair of V(D)J recombination signal sequences (RSS). RAG-RSS complex assembly and activity in vitro is promoted by high mobility group proteins of the "HMG-box" family, exemplified by HMGB1. How HMGB1 stimulates the DNA binding and cleavage activity of the RAG complex remains unclear. HMGB1 contains two homologous HMG-box DNA binding domains, termed A and B, linked by a stretch of basic residues to a highly acidic C-terminal tail. To identify determinants of HMGB1 required for stimulation of RAG-mediated RSS binding and cleavage, we prepared an extensive panel of mutant HMGB1 proteins and tested their ability to augment RAG-mediated RSS binding and cleavage activity. Using a combination of mobility shift and in-gel cleavage assays, we find that HMGB1 promotes RAG-mediated cleavage largely through the activity of box B, but optimal stimulation requires a functional A box tethered in the correct orientation. Box A or B mutants fail to promote RAG synaptic complex formation, but this defect is alleviated when the acidic tail is removed from these mutants.

Bacterial Death Comes Full Circle: Targeting Plasmid Replication in Drug‐Resistant Bacteria

AbstractFor Abstract see ChemInform Abstract in Full Text.

Yeast as a Tractable Genetic System for Functional Studies of the Insulin-degrading Enzyme

We have developed yeast as an expression and genetic system for functional studies of the insulin-degrading enzyme (IDE), which cleaves and inactivates certain small peptide molecules, including insulin and the neurotoxic A beta peptide. We show that heterologously expressed rat IDE is enzymatically active, as judged by the ability of IDE-containing yeast extracts to cleave insulin in vitro. We also show that IDE can promote the in vivo production of the yeast a-factor mating pheromone, a function normally attributed to the yeast enzymes Axl1p and Ste23p. However, IDE cannot substitute for the function of Axl1p in promoting haploid axial budding and repressing haploid invasive growth, activities that require an uncharacterized activity of Axl1p. Particulate fractions enriched for Axl1p or Ste23p are incapable of cleaving insulin, suggesting that the functional conservation of these enzymes may not be bidirectionally conserved. We have made practical use of our genetic system to confirm that residues composing the extended zinc metalloprotease motif of M16A family enzymes are required for the enzymatic activity of IDE, Ste23p, and Axl1p. We have determined that IDE and Axl1p both require an intact C terminus for optimal activity. We expect that the tractable genetic system that we have developed will be useful for investigating the enzymatic and structure/function properties of IDE and possibly for the identification of novel IDE alleles having altered substrate specificity.

Tumor Targeting of Plasmid DNA by Dextran Conjugation Based on Metal Coordination

Tumor targeting of plasmid DNA was achieved through the conjugation of dextran derivative with chelate residue based on metal coordination. Spermine (Sm) was chemically introduced to the hydroxyl groups of dextran to obtain dextran-Sm derivative. A negative zeta potential of plasmid DNA became almost 0 mV by the Zn2+-coordinated conjugation with the dextran-Sm When the dextran-Sm-plasmid DNA conjugate with Zn2+ coordination was intravenously injected to mice subcutaneously bearing Meth-AR-1 fibrosarcoma, the dextran- Sm-plasmid DNA conjugate significantly enhanced the level of gene expression in the tumor, in contrast to free plasmid DNA..

771. Complexes of Nucleic Acids with Multivalent Cationic Lipids for the Study of Delivery Pathways and Mechanisms: From Plasmid DNA to siRNA

Complexes of nucleic acids (NAs) with cationic lipids (CLs) are promising vectors with numerous potential applications, particularly in medicine. Striking differences are observed in their behavior, depending on the nature of the transported nucleic acid. As synthetic vectors, CL-NA complexes can carry NAs of arbitrary size and are safer and easier to produce than viruses. To design optimized lipid vectors, it is indispensable to better understand the mechanism of transfection and how it is affected by NA size, CL-NA complex structure, lipid structure and lipid composition. Tailored lipid molecules have proven extremely useful for this kind of investigations. We have designed several series of multivalent cationic lipids with systematically varied headgroup charge (+1 up to +16) and lipid tail. Experiments with lamellar DNA complexes of these lipids have identified the complex' membrane charge density |[oacute]|M, a lipid-independent measure of the bilayer composition, as a universal parameter for transfection: efficiency plots for diverse, mono- to pentavalent lipids merge onto a single curve when plotted against |[oacute]|M. We compare those results with recent investigations using the same new lipids complexed with short interfering RNA (siRNA) to effect RNA interference (RNAi). RNAi is a novel pathway for inducing post-transcriptional gene silencing: the introduction of synthetically prepared duplexes of 19-21 bp siRNA into mammalian cells leads to the sequence-specific reduction of target mRNAs by enzymatic cleavage and subsequent knockdown of their encoded proteins. The co-transfection of a target and a non-target reporter plasmid into cells, followed by CL-mediated delivery of a target-specific siRNA allows us to simultaneously probe the specific silencing efficiency (SE) of the target plasmid and the non-specific silencing of both plasmids. Varying the ratio of neutral to cationic lipid as well as the CL:siRNA charge ratio, we have created phase diagrams of specific, non-specific and effective silencing as a function of these parameters for several monovalent to pentavalent lipids. These phase diagrams facilitate the identification of regimes of optimum performance. In addition, x-ray diffraction was used to determine the structure of the CL-siRNA complexes. In the phase diagrams, regions with high levels of non-specific silencing are observed. We have used several cell toxicity and cell viability assays to investigate the causes of this non-specificity. In contrast to DNA complexes, evidence for both lipid and NA-based unspecific knockdown of proteins is found. Funding provided by NIH GM-59288, AI-20611 and AI-12520.