Normal Chromosomal Location Research Articles

Importance of Real-Time Assays To Distinguish Multidrug Efflux Pump-Inhibiting and Outer Membrane-Destabilizing Activities in Escherichia coli.

The constitutively expressed AcrAB multidrug efflux system of Escherichia coli shows a high degree of homology with the normally silent AcrEF system. Exposure of a strain with acrAB deleted to antibiotic selection pressure frequently leads to the insertion sequence-mediated activation of the homologous AcrEF system. In this study, we used strains constitutively expressing either AcrAB or AcrEF from their normal chromosomal locations to resolve a controversy about whether phenylalanylarginine β-naphthylamide (PAβN) inhibits the activities of AcrAB and AcrEF and/or acts synergistically with antibiotics by destabilizing the outer membrane permeability barrier. Real-time efflux assays allowed a clear distinction between the efflux pump-inhibiting activity of PAβN and the outer membrane-destabilizing action of polymyxin B nonapeptide (PMXBN). When added in equal amounts, PAβN, but not PMXBN, strongly inhibited the efflux activities of both AcrAB and AcrEF pumps. In contrast, when outer membrane destabilization was assessed by the nitrocefin hydrolysis assay, PMXBN exerted a much greater damaging effect than PAβN. Strong action of PAβN in inhibiting efflux activity compared to its weak action in destabilizing the outer membrane permeability barrier suggests that PAβN acts mainly by inhibiting efflux pumps. We concluded that at low concentrations, PAβN acts specifically as an inhibitor of both AcrAB and AcrEF efflux pumps; however, at high concentrations, PAβN in the efflux-proficient background not only inhibits efflux pump activity but also destabilizes the membrane. The effects of PAβN on membrane integrity are compounded in cells unable to extrude PAβN. The increase in multidrug-resistant bacterial pathogens at an alarming rate has accelerated the need for implementation of better antimicrobial stewardship, discovery of new antibiotics, and deeper understanding of the mechanism of drug resistance. The work carried out in this study highlights the importance of employing real-time fluorescence-based assays in differentiating multidrug efflux-inhibitory and outer membrane-destabilizing activities of antibacterial compounds.

The CDK regulators Cdh1 and Sic1 promote efficient usage of DNA replication origins to prevent chromosomal instability at a chromosome arm.

Robustness and completion of DNA replication rely on redundant DNA replication origins. Reduced efficiency of origin licensing is proposed to contribute to chromosome instability in CDK-deregulated cell cycles, a frequent alteration in oncogenesis. However, the mechanism by which this instability occurs is largely unknown. Current models suggest that limited origin numbers would reduce fork density favouring chromosome rearrangements, but experimental support in CDK-deregulated cells is lacking. We have investigated the pattern of origin firing efficiency in budding yeast cells lacking the CDK regulators Cdh1 and Sic1. We show that each regulator is required for efficient origin activity, and that both cooperate non-redundantly. Notably, origins are differentially sensitive to CDK deregulation. Origin sensitivity is independent on normal origin efficiency, firing timing or chromosomal location. Interestingly, at a chromosome arm, there is a shortage of origin firing involving active and dormant origins, and the extent of shortage correlates with the severity of CDK deregulation and chromosome instability. We therefore propose that CDK deregulation in G1 phase compromises origin redundancy by decreasing the number of active and dormant origins, leading to origin shortage and increased chromosome instability.

Urg1: A Uracil-Regulatable Promoter System for Fission Yeast with Short Induction and Repression Times

BackgroundThe fission yeast Schizosaccharomyces pombe is a popular genetic model organism with powerful experimental tools. The thiamine-regulatable nmt1 promoter and derivatives, which take >15 hours for full induction, are most commonly used for controlled expression of ectopic genes. Given the short cell cycle of fission yeast, however, a promoter system that can be rapidly regulated, similar to the GAL system for budding yeast, would provide a key advantage for many experiments.Methodology/Principal FindingsWe used S. pombe microarrays to identify three neighbouring genes (urg1, urg2, and urg3) whose transcript levels rapidly and strongly increased in response to uracil, a condition which otherwise had little effect on global gene expression. We cloned the promoter of urg1 (uracil-regulatable gene) to create several PCR-based gene targeting modules for replacing native promoters with the urg1 promoter (Purg1) in the normal chromosomal locations of genes of interest. The kanMX6 and natMX6 markers allow selection under urg1 induced and repressed conditions, respectively. Some modules also allow N-terminal tagging of gene products placed under urg1 control. Using pom1 as a proof-of-principle, we observed a maximal increase of Purg1-pom1 transcripts after uracil addition within less than 30 minutes, and a similarly rapid decrease after uracil removal. The induced and repressed transcriptional states remained stable over 24-hour periods. RT-PCR comparisons showed that both induced and repressed Purg1-pom1 transcript levels were lower than corresponding P3nmt1-pom1 levels (wild-type nmt1 promoter) but higher than P81nmt1-pom1 levels (weak nmt1 derivative).Conclusions/SignificanceWe exploited the urg1 promoter system to rapidly induce pom1 expression at defined cell-cycle stages, showing that ectopic pom1 expression leads to cell branching in G2-phase but much less so in G1-phase. The high temporal resolution provided by the urg1 promoter should facilitate experimental design and improve the genetic toolbox for the fission yeast community.

Simplified primer design for PCR‐based gene targeting and microarray primer database: two web tools for fission yeast

PCR-based gene targeting is a popular method for manipulating yeast genes in their normal chromosomal locations. The manual design of primers, however, can be cumbersome and error-prone. We have developed a straightforward web-based tool that applies user-specified inputs to automate and simplify the task of primer selection for deletion, tagging and/or regulated expression of genes in Schizosaccharomyces pombe. This tool, named PPPP (for Pombe PCR Primer Programs), is available at http://www.sanger.ac.uk/PostGenomics/S_pombe/software/. We also present a searchable Microarray Primer Database to retrieve the sequences and accompanying information for primers and PCR products used to build our in-house Sz. pombe microarrays. This database contains information on both coding and intergenic regions to provide context for the microarray data, and it should be useful also for other applications, such as quantitative PCR. The database can be accessed at http://www.sanger.ac.uk/PostGenomics/S_pombe/microarray/. Copyright © 2006 John Wiley & Sons, Ltd.

Ultraviolet light selection assay to optimize oligonucleotide correction of mutations in endogenous xeroderma pigmentosum genes.

Various oligonucleotide (ODN)-based approaches have been proposed for their ability to correct mutated genes at the normal chromosomal locations. However, the reported gene correction frequencies of these approaches have varied markedly in different experimental settings, including when different tissues or cell types are targeted. In order to find the optimal ODN-based approach for a specific target tissue, an assay system that allows direct comparison of the different methods on that tissue is necessary. Herein, we describe an XP-UVC selection assay that can be used to evaluate and compare gene correction frequencies in different cell types obtained from a xeroderma pigmentosum (XP) patient, following treatment by different ODN-based approaches. As an experimental example, the XP-UVC selection assay was used to assess the ability of chimeric RNA/DNA ODN to correct point mutations in the XPA gene. This assay can be used to assess and evaluate other types of ODN-based approaches, and to further optimize them.

Extreme nuclear disproportion and constancy of enzyme activity in a heterokaryon of Neurospora crassa.

Heterokaryons of Neurospora crassa were generated by transformation of multinucleate conidia of a histidine-3 auxotroph with his-3(+) plasmid. In one of the transformants, propagated on a medium with histidine supplementation, a gradual but drastic reduction occurred in the proportion of prototrophic nuclei that contained an ectopically integrated his-3(+) allele. This response was specific to histidine. The reduction in prototrophic nuclei was confirmed by several criteria: inoculum size test, hyphal tip analysis, genomic Southern analysis, and by visual change in colour of the transformant incorporating genetic colour markers. Construction and analyses of three-component heterokaryons revealed that the change in nuclear ratio resulted from interaction of auxotrophic nucleus with prototrophic nucleus that contained an ectopically integrated his-3(+) gene, but not with prototrophic nucleus that contained his-3(+) gene at the normal chromosomal location. The growth rate of heterokaryons and the activity of histidinol dehydrogenase - the protein encoded by the his-3(+) gene - remained unchanged despite prototrophic nuclei becoming very scarce. The results suggest that not all nuclei in the coenocytic fungal mycelium may be active simultaneously, the rare active nuclei being sufficient to confer the wild-type phenotype.

Roles for internal and flanking sequences in regulating the activity of mating-type-silencer-associated replication origins in Saccharomyces cerevisiae.

ARS301 and ARS302 are inactive replication origins located at the left end of budding yeast (Saccharomyces cerevisiae) chromosome III, where they are associated with the HML-E and -I silencers of the HML mating type cassette. Although they function as replication origins in plasmids, they do not serve as origins in their normal chromosomal locations, because they are programmed to fire so late in S phase that they are passively replicated by the replication fork from neighboring early-firing ARS305 before they have a chance to fire on their own. We asked whether the nucleotide sequences required for plasmid origin function of these silencer-associated chromosomally inactive origins differ from the sequences needed for plasmid origin function by nonsilencer-associated chromosomally active origins. We could not detect consistent differences in sequence requirements for the two types of origins. Next, we asked whether sequences within or flanking these origins are responsible for their chromosomal inactivity. Our results demonstrate that both flanking and internal sequences contribute to chromosomal inactivity, presumably by programming these origins to fire late in S phase. In ARS301, the function of the internal sequences determining chromosomal inactivity is dependent on the checkpoint proteins Mec1p and Rad53p.

Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe.

We describe a straightforward PCR-based approach to the deletion, tagging, and overexpression of genes in their normal chromosomal locations in the fission yeast Schizosaccharomyces pombe. Using this approach and the S. pombe ura4+ gene as a marker, nine genes were deleted with efficiencies of homologous integration ranging from 6 to 63%. We also constructed a series of plasmids containing the kanMX6 module, which allows selection of G418-resistant cells and thus provides a new heterologous marker for use in S. pombe. The modular nature of these constructs allows a small number of PCR primers to be used for a wide variety of gene manipulations, including deletion, overexpression (using the regulatable nmt1 promoter), C- or N-terminal protein tagging (with HA, Myc, GST, or GFP), and partial C- or N-terminal deletions with or without tagging. Nine genes were manipulated using these kanMX6 constructs as templates for PCR. The PCR primers included 60 to 80 bp of flanking sequences homologous to target sequences in the genome. Transformants were screened for homologous integration by PCR. In most cases, the efficiency of homologous integration was > or = 50%, and the lowest efficiency encountered was 17%. The methodology and constructs described here should greatly facilitate analysis of gene function in S. pombe.

Gene Trapping Identifies Inhibitors of Oncogenic Transformation: THE TISSUE INHIBITOR OF METALLOPROTEINASES-3 (TIMP3) AND COLLAGEN TYPE I α2 (COL1A2) ARE EPIDERMAL GROWTH FACTOR-REGULATED GROWTH REPRESSORS

A gene trap strategy has been used to identify genes that are repressed in cells transformed by an activated epidermal growth factor (EGF)/EGF receptor signal transduction pathway. EGF receptor-expressing NIH3T3 cells (HER1 cells) were infected with a retrovirus containing coding sequences for the human CD2 antigen and for secreted alkaline phosphatase in the U3 region. By selecting for and against CD2 expression, we obtained clones in which the gene trap had integrated into genes selectively repressed by EGF. Two of these clones encoded for the secreted extracellular matrix proteins TIMP3 and COL1A2. We show here that both genes are downstream targets of RAS and are specifically repressed by EGF-induced transformation. Moreover, this strategy tags tumor suppressor genes in their normal chromosomal location, thereby improving target-specific screens for antineoplastic drugs.

Identification and characterization of SRS1, a Toxoplasma gondii surface antigen upstream of and related to SAG1

Previous investigations of the major surface antigen (SAG1) promoter of Toxoplasma gondii indicated an ability to function bi-directionally in transient transformation assays at least. This suggests there might be another tachyzoite-specific gene being divergently transcribed from the SAG1 promoter in its normal chromosomal location. To investigate this possibility we have characterized the region upstream of SAG1 and report here a co-directional transcription unit coding for a probable GPI-anchored surface protein with homology to SAG1 and SAG3. This antigen, which had not previously been identified in surface iodination experiments is given the acronym SRS1, for SAG1-related sequence 1. Genomic organization and sequence of a full-length cDNA of SRS1 are presented. Antisera against a recombinant SRS1 protein produced in Escherichia coli, recognize a specific band of ≈46 kDa in parasite lysates which corresponds to the largest of the GPI-anchored proteins by Western blot. The possible role of this previously unidentified surface antigen is discussed.

The Cdc4/34/53 pathway targets Cdc6p for proteolysis in budding yeast.

The budding yeast Cdc6 protein (Cdc6p) is essential for formation of pre-replicative complexes (pre-RCs) at origins of DNA replication. Regulation of pre-RC assembly plays a key role in making initiation of DNA synthesis dependent upon passage through mitosis and in limiting DNA replication to once per cell cycle. Cdc6p is normally only present at high levels during the G1 phase of the cell cycle. This is partly because the CDC6 gene is only transcribed during G1. In this article we show that rapid degradation of Cdc6p also contributes to this periodicity. Cdc6p degradation rates are regulated during the cell cycle, reaching a peak during late G1/early S phase. Removal of a 47-amino-acid domain near the N-terminus of Cdc6p prevents degradation of Cdc6p. Likewise, mutations in the Cdc4/34/53 pathway involved in ubiquitin-mediated degradation block proteolysis and genetic evidence is presented indicating that the N-terminus of Cdc6p interacts with the Cdc4/34/53 pathway, probably through Cdc4p. A stable Cdc6p mutant which is no longer degraded by the Cdc4/34/53 pathway is, none the less, fully functional. Constitutive overexpression of either wild-type or stable Cdc6p does not induce re-replication and does not induce assembly of pre-replicative complexes after DNA replication is complete.

Manipulation of Cloned Yeast DNA

A major advantage of working with yeast is the ability to replace the wild-type chromosomal copy of a gene with a mutant derivative that is constructed in vitro using a cloned copy of the gene. This technique unavailable in most other eukaryotes allows the phenotype of the mutation to be studied under accurate in vivo conditions, with the mutation present in single copy at its normal chromosomal location. In the first protocol, a plasmid harboring both a selectable marker and a cloned gene of interest is integrated at the chromosomal location of the cloned gene via homologous recombination (integrative transformation). Four methods are described for constructing a mutation in vitro in a cloned gene and reintroducing this mutation at the correct chromosomal site. This allows assessment of the genetic consequences of a mutation, and is often used to determine whether or not a gene is essential (by determining if a complete gene deletion is viable). Two of these techniques integrative disruption and one-step gene disruption generate either insertion or deletion mutations. The third technique transplacement is more generally applicable: it can be used to introduce insertion or deletion mutations containing a selectable marker, but it can also be used to introduce nonselectable mutations, such as conditional lethal mutations in an essential gene. Protocols are also provided to allow creation of modified genes by one-step integrative replacement, and also conditional alleles by a copper-inducible double-shutoff procedure.

Angiotensin-converting enzyme gene mutations, blood pressures, and cardiovascular homeostasis.

A common polymorphism of the angiotensin-converting enzyme (ACE) gene (ACE in humans, Ace in mice) is associated with differences in circulating ACE levels that may confer a differential risk for cardiovascular diseases. To study the effects of genetically determined changes in Ace gene function within a defined genetic and environmental background, we have studied mice having one, two, or three functional copies of the Ace gene at its normal chromosomal location. ACE activities in the serum increased progressively from 62% of normal in the one-copy animals to 144% of normal in the three-copy animals (P < 10(-15), n = 132). The blood pressures of the mice having from one to three copies of the Ace gene did not differ significantly, but the heart rates, heart weights, and renal tubulointerstitial volumes decreased significantly with increasing Ace gene copy number. The level of kidney renin mRNA in the one-copy mice was increased to 129 +/- 9% relative to that of the normal two-copy mice (100 +/- 4%, P = .01, n = 16). We conclude that significant homeostatic adaptations successfully normalize the blood pressures of mice that have quantitative changes in Ace gene function. Our results suggest only that quantitative changes in expression of the Ace gene will observably affect blood pressures when accompanied by additional environmental or genetic factors that together with Ace exceed the capacity of the homeostatic mechanisms.

ORC- and Cdc6-dependent complexes at active and inactive chromosomal replication origins in Saccharomyces cerevisiae.

We have developed a genomic footprinting protocol which allows us to examine protein-DNA interactions at single copy chromosomal origins of DNA replication in the budding yeast Saccharomyces cerevisiae. We show that active replication origins oscillate between two chromatin states during the cell cycle: an origin recognition complex (ORC)-dependent post-replicative state and a Cdc6p-dependent pre-replicative state. Furthermore, we show that both post- and pre-replicative complexes can form efficiently on closely apposed replicators. Surprisingly, ARS301 which is active as an origin on plasmids but not in its normal chromosomal location, forms ORC- and Cdc6p-dependent complexes in both its active and inactive contexts. Thus, although ORC and Cdc6p are essential for initiation, their binding is not sufficient to dictate origin use.

On the Role of Helix 0 of the Tryptophan Synthetase α Chain of Escherichia coli

The role of helix 0 of the alpha chain (TrpA) of the tryptophan synthetase alpha2beta2 multi-functional enzyme complex of Escherichia coli was examined by deleting amino-terminal residues 2-6, 2-11, or 2-19 of TrpA. Selected substitutions were also introduced at TrpA positions 2-6. The altered genes encoding these polypeptides were overexpressed from a foreign promoter on a multicopy plasmid and following insertion at their normal chromosomal location. Each deletion polypeptide was functional in vivo. However all appeared to be somewhat more labile and insoluble and less active enzymatically than wild type TrpA. The deletion polypeptides were overproduced and solubilized from cell debris by denaturation and refolding. Several were partially purified and assayed in various reactions in the presence of tryptophan synthetase beta2 (TrpB). The purified TrpADelta2-6 and TrpADelta2-11 deletion polypeptides had low activity in both the indole + serine --> tryptophan reaction and the indoleglycerol phosphate + serine --> tryptophan reaction. Poor activity in each reaction was partly due to reduced association of TrpA with TrpB. The addition of the TrpA ligands, alpha-glycerophosphate or indoleglycerol phosphate, during catalysis of the indole + serine --> tryptophan reaction increased association and activity. These findings suggest that removal of helix 0 of TrpA decreases TrpA-TrpB association as well as the activity of the TrpA active site. Alignment of the TrpA sequences from different species indicates that several lack part or all of helix 0. In some of these polypeptides, extra residues at the carboxyl end may substitute for helix 0.

Genetic control of blood pressure and the angiotensinogen locus.

Variants of the human angiotensinogen gene have been linked in some studies to increased circulating angiotensinogen levels and essential hypertension. To test for direct causality between genotypes at the angiotensinogen locus and blood pressures, we have studied mice carrying zero, one, two, three, or four functional copies of the murine wild-type angiotensinogen gene (Agt) at its normal chromosomal location. Plasma angiotensinogen levels increase progressively, although not linearly, from zero in the zero-copy animals to 145% of normal in the four-copy animals. Mice of all genotypes are normal at birth, but most zero-copy animals die before weaning. The kidneys of the zero-copy animals show pathological changes as adults, but the kidneys are normal in the other genotypes. One adult zero-copy male tested was fertile. The blood pressures of the one-copy through four-copy animals show significant and almost linear increases of approximately 8 mmHg per gene copy despite their normal compensatory mechanisms being intact. These results establish a direct causal relationship between Agt genotypes and blood pressures.

Analysis of a replication initiation sequence from the adenosine deaminase region of the mouse genome.

A 4-kb HindIII fragment that supported the efficient autonomous replication of plasmid vector pDY-, a replication-defective construct based on Epstein-Barr virus sequences, in human K562 cells was rescued from amplified double-minute chromosomes containing the murine adenosine deaminase locus. Polymerase chain reaction assays of size-fractionated nascent strands demonstrated that replication initiation occurred within the same 1- to 2-kb region of this fragment in autonomously replicating plasmids containing the sequence in either orientation, in double-minute chromosomes, and in the single-copy locus at its normal chromosomal location. The complete sequence of this fragment was determined; it contains a 248-bp polypurine tract and consensus binding site sequences for several putative transcription and replication factors.

Analysis of a Replication Initiation Sequence from the Adenosine Deaminase Region of the Mouse Genome

A 4-kb HindIII fragment that supported the efficient autonomous replication of plasmid vector pDY-, a replication-defective construct based on Epstein-Barr virus sequences, in human K562 cells was rescued from amplified double-minute chromosomes containing the murine adenosine deaminase locus. Polymerase chain reaction assays of size-fractionated nascent strands demonstrated that replication initiation occurred within the same 1- to 2-kb region of this fragment in autonomously replicating plasmids containing the sequence in either orientation, in double-minute chromosomes, and in the single-copy locus at its normal chromosomal location. The complete sequence of this fragment was determined; it contains a 248-bp polypurine tract and consensus binding site sequences for several putative transcription and replication factors.

Protein-DNA interactions at a yeast replication origin.

An understanding of the protein-DNA interactions in vivo at origins of DNA replication in eukaryotes is essential to delineate the mechanism of initiation of DNA synthesis and its control in the cell cycle. In the yeast Saccharomyces cerevisiae, a family of sequences known as autonomously replicating sequences (ARSs) function as origins of bidirectional DNA replication on plasmids and, in several instances, also in their normal chromosomal location. Here we use nucleotide resolution genomic footprinting to investigate the association of proteins with ARS1. Nuclease protection patterns indicate that at least two different cellular factors interact with functional elements in ARS1. The first seems to be ARS-binding factor 1. The second seems to be a novel protein that generates extensive protection over the essential ARS consensus sequence and phased DNaseI-sensitive sites across a functionally important flanking sequence. Hypersensitivity of this region to cleavage by copper phenanthroline indicates that it is under torsional strain, analogous to that produced at transcriptional start sites by assembly of an initiation complex. The protection in situ is similar to that generated by the origin recognition complex (ORC) protein.

Function of an Ultrabithorax minigene in imaginal cells.

An Ultrabithorax (Ubx) minigene constructed from three key Ubx control regions is capable of supporting development of Ubx null mutants throughout larval life and beyond to pharate flies, thereby rescuing the larval lethality due to the homeotic mutation. The cuticle of these flies shows that the minigene provides at least partial Ubx function in each of the four compartments whose morphogenetic pathways are determined by Ubx. We analyse beta-galactosidase patterns in imaginal discs conferred by each individual Ubx control region. From the comparison of these patterns with Ubx expression in Cbx mutants, we infer that long-range repressor elements in the chromosomal Ubx gene play an important role in the generation of Ubx expression patterns in imaginal discs. Expression and function of our Ubx minigenes indicate that Ubx control regions are capable of functioning properly out of context and detached from their normal chromosomal location within the homeotic gene complex.