Evolving MRSA: High-level ß-lactam resistance in Staphylococcus aureus is associated with RNA Polymerase alterations and fine tuning of gene expression.

Most clinical MRSA (methicillin-resistant S. aureus) isolates exhibit low-level ß-lactam resistance (oxacillin MIC 2-4 µg/ml) due to the acquisition of a novel penicillin binding protein (PBP2A), encoded by mecA. However, strains can evolve high-level resistance (oxacillin MIC ≥256 µg/ml) by an unknown mechanism. Here we have developed a robust system to explore the basis of the evolution of high-level resistance by inserting mecA into the chromosome of the methicillin-sensitive S. aureus SH1000. Low-level mecA-dependent oxacillin resistance was associated with increased expression of anaerobic respiratory and fermentative genes. High-level resistant derivatives had acquired mutations in either rpoB (RNA polymerase subunit ß) or rpoC (RNA polymerase subunit ß') and these mutations were shown to be responsible for the observed resistance phenotype. Analysis of rpoB and rpoC mutants revealed decreased growth rates in the absence of antibiotic, and alterations to, transcription elongation. The rpoB and rpoC mutations resulted in decreased expression to parental levels, of anaerobic respiratory and fermentative genes and specific upregulation of 11 genes including mecA. There was however no direct correlation between resistance and the amount of PBP2A. A mutational analysis of the differentially expressed genes revealed that a member of the S. aureus Type VII secretion system is required for high level resistance. Interestingly, the genomes of two of the high level resistant evolved strains also contained missense mutations in this same locus. Finally, the set of genetically matched strains revealed that high level antibiotic resistance does not incur a significant fitness cost during pathogenesis. Our analysis demonstrates the complex interplay between antibiotic resistance mechanisms and core cell physiology, providing new insight into how such important resistance properties evolve.

Arginine methylation and citrullination of splicing factor proline- and glutamine-rich (SFPQ/PSF) regulates its association with mRNA.

Splicing factor proline- and glutamine-rich (SFPQ) also commonly known as polypyrimidine tract-binding protein-associated-splicing factor (PSF) and its binding partner non-POU domain-containing octamer-binding protein (NONO/p54nrb), are highly abundant, multifunctional nuclear proteins. However, the exact role of this complex is yet to be determined. Following purification of the endogeneous SFPQ/NONO complex, mass spectrometry analysis identified a wide range of interacting proteins, including those involved in RNA processing, RNA splicing, and transcriptional regulation, consistent with a multifunctional role for SFPQ/NONO. In addition, we have identified several sites of arginine methylation in SFPQ/PSF using mass spectrometry and found that several arginines in the N-terminal domain of SFPQ/PSF are asymmetrically dimethylated. Furthermore, we find that the protein arginine N-methyltransferase, PRMT1, catalyzes this methylation in vitro and that this is antagonized by citrullination of SFPQ. Arginine methylation and citrullination of SFPQ/PSF does not affect complex formation with NONO. However, arginine methylation was shown to increase the association with mRNA in mRNP complexes in mammalian cells. Finally we show that the biochemical properties of the endogenous complex from cell lysates are significantly influenced by the ionic strength during purification. At low ionic strength, the SFPQ/NONO complex forms large heterogeneous protein assemblies or aggregates, preventing the purification of the SFPQ/NONO complex. The ability of the SFPQ/NONO complex to form varying protein assemblies, in conjunction with the effect of post-translational modifications of SFPQ modulating mRNA binding, suggests key roles affecting mRNP dynamics within the cell.

c-IAP1 binds and processes PCSK9 protein: linking the c-IAP1 in a TNF-α pathway to PCSK9-mediated LDLR degradation pathway.

Recent genetic studies have shown that PCSK9, one of the key genes in cholesterol metabolism, plays a critical role by controlling the level of low-density lipoprotein receptor. However, how PCSK9 mediates LDLR degradation is still unknown. By combining a shotgun proteomic method and differential analysis of natural occurring mutations of the PCSK9 gene, we found that an E3 ubiquitin ligase c-IAP1 binds and processes PCSK9 protein. One of the 'gain-of-function' mutations, S127R, is defective with respect to binding to c-IAP1, and thus has defective autocatalytic activity. Knockdown of c-IAP1 impairs PCSK9 processing and autocatalytic cleavage. In c-IAP1 null mouse embryonic fibroblasts (MEFs), there is a dramatic decrease in secreted mature PCSK9 protein accompanied by a significant increase in LDLR protein levels compared with matched wild-type MEF cells. c-IAP1 also acts as an E3 ligase for ubiquitination of PCSK9. Ubiquitin containing only lysine-27 mediated PCSK9 ubiquitination by c-IAP1. Given K27-linked polyubiquitination promotes lysosomal localization, the finding indicates the c-IAP1 acts on both secretion of PCSK9 and its lysosomal localization. The novel pathway described here will open new avenues for exploring novel disease treatments.

Quantum dot-conjugated anti-GRP78 scFv inhibits cancer growth in mice.

Semiconductor quantum dots (Qdots) have recently been shown to offer significant advantages over conventional fluorescent probes to image and study biological processes. The stability and low toxicity of QDs are well suited for biological applications. Despite this, the potential of Qdots remains limited owing to the inefficiency of existing delivery methods. By conjugating Qdots with small antibody fragments targeting membrane-bound proteins, such as GRP78, we demonstrate here that the Quantum dot- Anti-GRP78 scFv (Qdot-GRP78) retains its immunospecificity and its distribution can be monitored by visualization of multi-color fluorescence imaging both in vitro and in vivo. Moreover we demonstrate here for the first time that Qdot-GRP78 scFv bioconjugates can be efficiently internalized by cancer cells, thus upregulate phophosphate-AKT-ser473 and possess biological anti-tumour activity as shown by inhibition of breast cancer growth in a xenograft model. This suggests that nanocarrier-conjugated scFvs can be used as a therapeutic antibody for cancer treatment.

Studying the mechanism of RNA separations using RNA chromatography and its application in the analysis of ribosomal RNA and RNA:RNA interactions.

DNA/RNA chromatography presents a versatile platform for the analysis of nucleic acids. Although the mechanism of separation of double stranded (ds) DNA fragments is largely understood, the mechanism by which RNA is separated appears more complicated. To further understand the separation mechanisms of RNA using ion pair reverse phase liquid chromatography, we have analysed a number of dsRNA and single stranded (ss) RNA fragments. The high-resolution separation of dsRNA was observed, in a similar manner to dsDNA under non-denaturing conditions. Moreover, the high-resolution separation of ssRNA was observed at high temperatures (75 degrees C) in contrast to ssDNA. It is proposed that the presence of duplex regions/secondary structures within the RNA remain at such temperatures, resulting in high-resolution RNA separations. The retention time of the nucleic acids reflects the relative hydrophobicity, through contributions of the nucleic sequence and the degree of secondary structure present. In addition, the analysis of RNA using such approaches was extended to enable the discrimination of bacterial 16S rRNA fragments and as an aid to conformational analysis of RNA. RNA:RNA interactions of the human telomerase RNA component (hTR) were analysed in conjunction with the incorporation of Mg2+ during chromatography. This novel chromatographic procedure permits analysis of the temperature dependent formation of dimeric RNA species.

Enrichment and analysis of RNA centered on ion pair reverse phase methodology.

Here we describe a procedure for the rapid enrichment of RNA from cell extracts and the subsequent fractionation and analysis of the "small RNA" population by ion pair reverse phase chromatography. Solid phase extraction procedures have been developed utilizing nonporous alkylated poly(styrene-divinylbenzene) particles in conjunction with ion pair reagents to enrich total RNA. This approach facilitates the selective enrichment and separation of the relatively lower abundance small RNAs, from the more abundant higher molecular weight rRNA species. We also describe the application of monolithic capillaries in conjunction with ion pair reverse phase chromatography to bring increased sensitivity in the analysis of very low abundance RNAs. These approaches will simplify the biochemical analysis of this class of molecules, which are emerging as important regulators of global gene expression in higher organisms.

Micro-scale open-tube capillary separations of functional proteins.

This article describes a novel technique whereby fully functional proteins or multiprotein complexes are efficiently extracted from biological samples to chemically derivatized walls of fused-silica open-tube capillary columns. Proteins are eluted with very high yields into elution volumes that are smaller in volume than the internal volume of the open-tube capillary column itself, thereby achieving 100-fold increases in target protein concentrations from starting samples of less than 1 ml. The open-tube capillary columns are designed for single use; combined with the physical and chemical characteristics of the open-tube capillary column, this provides exceptional purity to the eluted proteins. Affinity-based open-tube capillary columns are demonstrated here to purify, enrich, and maintain functionality for a monomeric and dimeric enzyme, a low-abundance HeLa nuclear complex, and a light-harvesting octadecameric membrane protein complex. The design of the open-tube capillary column allows for facile direction of the processed protein sample to any number of final detection techniques and is capable of generating final protein concentrations required for many structural biology experiments. The open-tube capillary columns are also characterized by exceptional ease of use. Current designs allow for up to 10 open-tube capillary columns to be applied simultaneously with no fundamental impediments to even greater parallel operation.

Rapid analysis of protein-nucleic acid complexes using MALDI TOF mass spectrometry and ion pair reverse phase liquid chromatography.

The RuvABC resolvasome of Escherichia coli typifies nucleoprotein complexes involved in genetic transactions. This molecular assembly catalyses the resolution of Holliday junctions that arise during genetic recombination and DNA repair. This process involves two key steps: branch migration, catalysed by the RuvB protein that is targeted to the Holliday junction by the structure specific RuvA protein, and resolution, which is catalysed by the RuvC endonuclease. We have used matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS) to rapidly identify the binding of RuvA to an immobilised synthetic Holliday junction; unambiguous identification was verified using tryptic digest of the bound protein. In conjunction with a novel fluorescent-based technique incorporating ion pair reverse phase liquid chromatography, a "footprint" of the RuvA:Holliday complex was obtained. These two complementary techniques offer a generic approach to the analysis of nucleoprotein complexes.

Recognition of base-pairing by DNA polymerases during nucleotide incorporation: the properties of the mutagenic nucleotide dPTP alphaS.

The highly mutagenic nucleoside dP (6-(2-deoxy-beta-D-erythro-pentofuranosyl)-3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-2-one) is a bicyclic analogue of N4-methoxy-2'-deoxycytidine. It exists as a mixture of its imino and amino tautomers in solution with a ratio of about 10:1 based on its tautomeric constant. The bicyclic nature of the heterocycle P restrains the amino substituent in an anti conformation and permits effective Watson-Crick base-pairing using either tautomer. The specificity of incorporation of dP by the 3'-5'-exonuclease-free Klenow fragment of DNA polymerase I (exo-free Klenow) has been studied using the 5'-(1-thio)triphosphate dPTP alphaS in combination with phosphorothioate-specific sequencing of the DNA products. The method provides a convenient qualitative assay for studying nucleotide incorporation and reveals for the first time a potential role for the minor tautomeric forms of the natural DNA bases in base misinsertion (substitution mutagenesis) during replication.

A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes.

A nomenclature is described for restriction endonucleases, DNA methyltransferases, homing endonucleases and related genes and gene products. It provides explicit categories for the many different Type II enzymes now identified and provides a system for naming the putative genes found by sequence analysis of microbial genomes.

The effect of tautomeric constant on the specificity of nucleotide incorporation during DNA replication: support for the rare tautomer hypothesis of substitution mutagenesis.

The nucleoside analogue dP (6-(2-deoxy-beta-D-ribofuranosyl)-3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-2-one) displays ambivalent hydrogen bonding characteristics whereby the imino tautomer of P can base-pair with adenine and its amino tautomer can base-pair with guanine. Fixed imino and amino tautomers of 6-methyl-3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-2-one (N-methyl P) have been synthesised and their structures obtained by X-ray crystallography. The tautomeric constant of N-methyl P has been calculated from pK(a) values of the fixed tautomers and the kinetic parameters for the incorporation of its 5'-triphosphate (dPTP) by exonuclease-free Klenow fragment of DNA polymerase I have been determined. A strong correlation between the tautomeric constant and the incorporation specificity of dPTP is found. These results lend support to the proposal that the minor tautomeric forms of the natural bases may play an important role in substitution mutagenesis during DNA replication. Furthermore, they imply that DNA polymerases impose specific steric requirements on the base-pair during nucleotide incorporation.

The small subunit of M. AquI is responsible for sequence-specific DNA recognition and binding in the absence of the catalytic domain.

AquI DNA methyltransferase (M. AquI) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the C5 position of the outermost deoxycytidine base in the DNA sequence 5'-CCCGGG-3'. M. AquI is a heterodimer in which the polypeptide chain is separated at the junction between the two equivalent structural domains in the related enzyme M. HhaI. Recently, we reported the subcloning, overexpression, and purification of the subunits (alpha and beta) of M. AquI separately. Here we describe the DNA binding properties of M. AquI. The results presented here indicate that the beta subunit alone contains all of the information for sequence-specific DNA recognition and binding. The first step in the sequence-specific recognition of DNA by M. AquI involves the formation of binary complex with the target recognition domain in conjunction with conserved sequence motifs IX and X, found in all known C5 DNA methyltransferases, contained in the beta subunit. The alpha subunit enhances the binding of the beta subunit to DNA specifically and nonspecifically. It is likely that the addition of the alpha subunit to the beta subunit stabilizes the conformation of the beta subunit and thereby enhances its affinity for DNA indirectly. Addition of S-adenosyl-L-methionine and its analogues S-adenosyl-L-homocysteine and sinefungin enhances binding, but only in the presence of the alpha subunit. These compounds did not have any effect on DNA binding by the beta subunit alone. Using a 30-mer oligodeoxynucleotide substrate containing 5-fluorodeoxycytidine (5-FdC), it was found that the beta subunit alone did not form a covalent complex with its specific sequence in the absence or presence of S-adenosyl-L-methionine. However, the addition of the alpha subunit to the beta subunit led to the formation of a covalent complex with specific DNA sequence containing 5-FdC.

Use of fluorescent DNA-intercalating dyes in the analysis of DNA via ion-pair reversed-phase denaturing high-performance liquid chromatography.

SYBR Green 1 is an asymmetrical cyanine DNA-binding dye that provides an opportunity for increasing the sensitivity of nucleic acid detection when used in conjunction with gel electrophoresis. In this paper, we summarize the general properties and specific uses of SYBR green 1 in ion-pair reversed-phase denaturing high-performance liquid chromatography (IP DHPLC). We describe several applications for the WAVE DHPLC platform that illustrate the generic potential of such intercalating dyes in mutation detection and gene expression profiling. We show that SYBR Green 1 obviates the need to use end-labeled oligodeoxynucleotides for the sensitive detection of nucleic acids during chromatography. Moreover the incorporation of SYBR Green 1 into samples and elution buffers does not impair resolution and has no significant effect on the retention times of DNA fragments compared with dye-free DHPLC.

The RuvABC resolvasome.

The RuvABC resolvasome of Escherichia coli catalyses the resolution of Holliday junctions that arise during genetic recombination and DNA repair. This process involves two key steps: branch migration, catalysed by the RuvB protein that is targeted to the Holliday junction by the structure specific RuvA protein, and resolution, which is catalysed by the RuvC endonuclease. We have quantified the interaction of the RuvA protein with synthetic Holliday junctions and have shown that the binding of the protein is highly structure-specific, and leads to the formation of a complex containing two tetramers of RuvA per Holliday junction. Our data are consistent with two tetramers of RuvA binding to the DNA recombination intermediate in a co-operative manner. Once formed this complex prevents the binding of RuvC to the Holliday junction. However, the formation of a RuvAC complex can be observed following sequential addition of the RuvC and RuvA proteins. Moreover, by examining the DNA recognition properties of a mutant RuvA protein (E55R, D56K) we show that the charge on the central pin is critical for directing the structure-specific binding by RuvA.

Recombinant alpha and beta subunits of M.AquI constitute an active DNA methyltransferase.

AquI DNA methyltransferase, M.AquI, catalyses the transfer of a methyl group from S-adenosyl-L-methionine to the C5 position of the outermost deoxycytidine base in the DNA sequence 5'CYCGRG3'. M.AquI is encoded by two overlapping ORFs (termed alpha and beta) instead of the single ORF that is customary for Class II methyltransferase genes. The structural organization of the M.AquI protein sequence is quite similar to that of other bacterial C5-DNA methyltransferases. Ten conserved motifs are also present in the correct order, but only on two polypeptides. We separately subcloned the genes that encode the alpha and beta subunits of M.AquI into expression vectors. The overexpressed His-fusion alpha and beta subunits of the enzyme were purified to homogeneity in a single step by Nickel-chelate affinity chromatography. The purified recombinant proteins were assayed for biological activity by an in vitro DNA tritium transfer assay. The alpha and beta subunits of M.AquI alone have no DNA methyltransferase activity, but when both subunits are included in the assay, an active enzyme that catalyses the transfer of the methyl group from S-adenosyl-Lmethionine to DNA is reconstituted. We also showed that the beta subunit alone contains all of the information that is required to generate recognition of specific DNA duplexes in the absence of the alpha subunit

An analytical method for the detection of methylation differences at specific chromosomal loci using primer extension and ion pair reverse phase HPLC.

We have developed a rapid, accurate, and quantitative method for the detection of methylation differences at specific CpG sites based on bisulfite treatment of DNA followed by primer extension and ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC). The application of the method is illustrated by analysis of differentially imprinted alleles arising from Prader-Willi and Angelman syndromes. In order to convert unmethylated cytosines to uracil, plasmid and genomic DNA samples were treated with sodium bisulfite and the targeted sequence was then amplified using oligodeoxynucleotide primers specific for the bisulfite-deaminated DNA. The PCR product(s) from this step was used as a template for a primer extension reaction and the products were subsequently analyzed chromatographically using IP RP HPLC. This method eliminates the need to use restriction enzymes to determine the methylation status of the amplicon and circumvents the need for radio labeling for the quantitative measurements. Finally, this method removes the need for nucleotide sequencing because it is not solely reliant on the presence or absence of one or more PCR products, as is the case with related methods.

Novel mutations in the BRCA1 and BRCA2 genes in Iranian women with early-onset breast cancer.

BACKGROUND: Breast cancer is the most common female malignancy and a major cause of death in middle-aged women. So far, germline mutations in the BRCA1 and BRCA2 genes in patients with early-onset breast and/or ovarian cancer have not been identified within the Iranian population. METHODS: With the collaboration of two main centres for cancer in Iran, we obtained clinical information, family history and peripheral blood from 83 women under the age of 45 with early-onset breast cancer for scanning of germline mutations in the BRCA1 and BRCA2 genes. We analysed BRCA1 exons 11 and BRCA2 exons 10 and 11 by the protein truncation test, and BRCA1 exons 2, 3, 5, 13 and 20 and BRCA2 exons 9, 17, 18 and 23 with the single-strand conformation polymorphism assay on genomic DNA amplified by polymerase chain reaction. RESULTS: Ten sequence variants were identified: five frameshifts (putative mutations - four novel); three missense changes of unknown significance and two polymorphisms, one seen commonly in both Iranian and British populations. CONCLUSIONS: Identification of these novel mutations suggests that any given population should develop a mutation database for its programme of breast cancer screening. The pattern of mutations seen in the BRCA genes seems not to differ from other populations studied. Early-onset breast cancer (less than 45 years) and a limited family history is sufficient to justify mutation screening with a detection rate of over 25% in this group, whereas sporadic early-onset breast cancer (detection rate less than 5%) is unlikely to be cost-effective.

Multidimensional differential display via ion-pair reversed-phase denaturing high-performance liquid chromatography.

Experimental approaches are now available for the analysis of whole transcriptome expression in cells and tissues. Since the introduction of such methods for the investigation of differences in mRNA populations, they have been applied successfully to many areas of biology and medicine including development, differentiation, physiology, pharmacology, and carcinogenesis. Here we describe an improved and automated approach based on the differential mRNA display method developed by Liang and Pardee (P. Liang and A. B. Pardee, 1992, Science 257, 967-971). We report the use of ion-pair reversed-phase denaturing high-performance liquid chromatography (IP RP DHPLC), for the first time, to produce a "fingerprint," after amplification of the cDNA corresponding to the mRNA populations, from two or more of the samples that are to be compared. By overlaying the chromatograms produced from the amplification of different samples derived from the same set of oligodeoxynucleotide primers, those genes that are differentially expressed can be selected and subsequently cloned and sequenced rapidly to establish a profile of differentially expressed genes. In addition, validation of the data obtained is readily achieved by this method using IP RP DHPLC and quantitative RT-PCR. In this study total RNA was prepared from NTERA2 cells before and after differentiation induced by retinoic acid and was reverse-transcribed into cDNA prior to amplification to produce fluorescently tagged products. This methodology facilitates multiple rounds of interrogation of RT-PCR products and we tentatively refer to this approach as Multidimensional Differential Display.

RNA footprinting analysis using ion pair reverse phase liquid chromatography.

Hydroxyl radical footprinting is a powerful technique often employed in characterization of the tertiary interactions between proteins and nucleic acids. Following the generation of a nucleic acid "ladder" either by chemical or enzymatic reactions, the radiolabeled products are traditionally separated by denaturing gel electrophoresis and further quantified by phosphorimaging techniques. Here we report the use of ion pair reverse phase liquid chromatography to analyze the products of an RNA footprinting reaction using fluorescently labeled RNA molecules. This technique offers several advantages over existing procedures, including rapid analysis, automation, and direct quantification of the cleavage products without the need to employ radiolabeling. To illustrate the resolving power of this technique, we have analyzed the products of base hydrolysis, generated from a fluorescently labeled RNA molecule and have subsequently used this method to define the solvent accessibility of the substrate strand as it docks with the hairpin ribozyme.

Holliday junction binding and processing by the RuvA protein of Mycoplasma pneumoniae.

The RuvA, RuvB and RuvC proteins of Escherichia coli act together to process Holliday junctions formed during recombination and DNA repair. RuvA has a well-defined DNA binding surface that is sculptured specifically to accommodate a Holliday junction and allow subsequent loading of RuvB and RuvC. A negatively charged pin projecting from the centre limits binding of linear duplex DNA. The amino-acid sequences forming the pin are highly conserved. However, in certain Mycoplasma and Ureaplasma species the structure is extended by four amino acids and two acidic residues forming a crucial charge barrier are missing. We investigated the significance of these differences by analysing RuvA from Mycoplasma pneumoniae. Gel retardation and surface plasmon resonance assays revealed that this protein binds Holliday junctions and other branched DNA structures in a manner similar to E. coli RuvA. Significantly, it binds duplex DNA more readily. However it does not support branch migration mediated by E. coli RuvB and when bound to junction DNA is unable to provide a platform for stable binding of E. coli RuvC. It also fails to restore radiation resistance to an E. coli ruvA mutant. The data presented suggest that the modified pin region retains the ability to promote junction-specific DNA binding, but acts as a physical obstacle to linear duplex DNA rather than as a charge barrier. They also indicate that such an obstacle may interfere with the binding of a resolvase. Mycoplasma species may therefore process Holliday junctions via uncoupled branch migration and resolution reactions.