Plasmid Insertion Research Articles

DNA Polymerase δ and ζ Switch by Sharing Accessory Subunits of DNA Polymerase δ

Translesion DNA synthesis is an important branch of the DNA damage tolerance pathway that assures genomic integrity of living organisms. The mechanisms of DNA polymerase (Pol) switches during lesion bypass are not known. Here, we show that the C-terminal domain of the Pol ζ catalytic subunit interacts with accessory subunits of replicative DNA Pol δ. We also show that, unlike other members of the human B-family of DNA polymerases, the highly conserved and similar C-terminal domains of Pol δ and Pol ζ contain a [4Fe-4S] cluster coordinated by four cysteines. Amino acid changes in Pol ζ that prevent the assembly of the [4Fe-4S] cluster abrogate Pol ζ function in UV mutagenesis. On the basis of these data, we propose that Pol switches at replication-blocking lesions occur by the exchange of the Pol δ and Pol ζ catalytic subunits on a preassembled complex of accessory proteins retained on DNA during translesion DNA synthesis.

Abstract 3325: The role of MyD88 in chemoresistance, differentiation and hypoxia in cancer stem cells

Abstract Ovarian cancer is the leading cause of gynaecological cancer death worldwide. This high mortality is caused in large part by development of chemoresistant recurrent disease. In primary tumours differentiation status of the tumour is considered a key prognostic factor however in recurrent disease this is no longer the case, suggesting that in recurrent tumours the differentiation status of the tumour has no bearing on the response to chemotherapy. The origins of recurrent disease may be explained by the cancer stem cell (CSC) theory. CSCs are a minority population of cancer cells with stem like properties including enhanced proliferation. The adaptor molecule myeloid differentiation-primary response gene (88) (MyD88), is a key constituent of the toll like receptor 4 (TLR4) pathway. In normal circumstances TLR pathways mediate the body's response to pathogens; however MyD88 has been recently suggested as a possible marker of cancer stemness in ovarian cancer. This work investigates the involvement of MyD88 in the CSC response to chemotherapy, differentiation stimulus and hypoxia treatment. Cell line experiments were carried out in two CSC lines: NTera2 and 2102Ep. NTera2 cells readily differentiate in response to retinoic acid (RA) treatment while 2102Ep cells are nullipotent and resist differentiation via RA. NTera2 cells are more chemosensitive than 2102Ep cells. Cells were incubated in the presence of both retinoic acid and a platinum based chemotherapy drug, both in isolation and in various combinations. RNA was isolated from treated cells, cDNA was synthesised and interrogated for TLR4/MyD88 expression via Q-PCR. To assess whether MyD88 was necessary and/or sufficient for the normal cell response, MyD88 was knocked down in cells via siRNA transfection, and overexpressed via insertion of an overexpression plasmid. The effect of these alterations on the cell lines response to chemotherapy, differentiation stimulus and hypoxia was then measured. The downstream effect of ablation of the MyD88 signal was also assessed using an Affymetrix array, and the chemokine and cytokine expression profile of both cell lines in all treatments was assessed using Quantibody arrays from RayBiotech. MyD88 expression was shown to be affected by chemotherapy, differentiation stimulus and hypoxic treatments. Furthermore when cells were pre-treated with differentiation stimulus or chemotherapy, their normal responses were altered. So far, our work has shown that ablation of the MyD88 signal in nullipotent 2102Ep cells removes their ability to resist differentiation via RA. To close this mechanism we have also shown that overexpression of MyD88 in pluripotent NTera2 cells allows them to resist differentiation via RA. Functional analysis of the role of MyD88 in chemotherapy and hypoxia resistance is currently being assessed. This and the mechanisms involved will be detailed, along with the chemokine/cytokine profile of treated cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3325. doi:1538-7445.AM2012-3325

A genome-wide inducible phenotypic screen identifies antisense RNA constructs silencing Escherichia coli essential genes

Regulated antisense RNA (asRNA) expression has been employed successfully in Gram-positive bacteria for genome-wide essential gene identification and drug target determination. However, there have been no published reports describing the application of asRNA gene silencing for comprehensive analyses of essential genes in Gram-negative bacteria. In this study, we report the first genome-wide identification of asRNA constructs for essential genes in Escherichia coli. We screened 250000 library transformants for conditional growth inhibitory recombinant clones from two shotgun genomic libraries of E.coli using a paired-termini expression vector (pHN678). After sequencing plasmid inserts of 675 confirmed inducer sensitive cell clones, we identified 152 separate asRNA constructs of which 134 inserts came from essential genes, while 18 originated from nonessential genes (but share operons with essential genes). Among the 79 individual essential genes silenced by these asRNA constructs, 61 genes (77%) engage in processes related to protein synthesis. The cell-based assays of an asRNA clone targeting fusA (encoding elongation factor G) showed that the induced cells were sensitized 12-fold to fusidic acid, a known specific inhibitor. Our results demonstrate the utility of the paired-termini expression vector and feasibility of large-scale gene silencing in E.coli using regulated asRNA expression.

Assembly of the Light-Harvesting Chlorophyll Antenna in the Green Alga Chlamydomonas reinhardtii Requires Expression of the TLA2-CpFTSY Gene

The truncated light-harvesting antenna2 (tla2) mutant of Chlamydomonas reinhardtii showed a lighter-green phenotype, had a lower chlorophyll (Chl) per-cell content, and higher Chl a/b ratio than corresponding wild-type strains. Physiological analyses revealed a higher intensity for the saturation of photosynthesis and greater P(max) values in the tla2 mutant than in the wild type. Biochemical analyses showed that the tla2 strain was deficient in the Chl a-b light-harvesting complex, and had a Chl antenna size of the photosystems that was only about 65% of that in the wild type. Molecular and genetic analyses showed a single plasmid insertion in the tla2 strain, causing a chromosomal DNA rearrangement and deletion/disruption of five nuclear genes. The TLA2 gene, causing the tla2 phenotype, was cloned by mapping the insertion site and upon complementation with each of the genes that were deleted. Successful complementation was achieved with the C. reinhardtii TLA2-CpFTSY gene, whose occurrence and function in green microalgae has not hitherto been investigated. Functional analysis showed that the nuclear-encoded and chloroplast-localized CrCpFTSY protein specifically operates in the assembly of the peripheral components of the Chl a-b light-harvesting antenna. In higher plants, a cpftsy null mutation inhibits assembly of both the light-harvesting complex and photosystem complexes, thus resulting in a seedling-lethal phenotype. The work shows that cpftsy deletion in green algae, but not in higher plants, can be employed to generate tla mutants. The latter exhibit improved solar energy conversion efficiency and photosynthetic productivity under mass culture and bright sunlight conditions.

Simple, sensitive, accurate multiplex quantitative competitive PCR with capillary electrophoresis detection for the determination of genetically modified maize

Legislation in the EU requires that foods containing more than 0.9% of genetically modified organisms (GMOs) should be labelled. To this end, we have developed a simple and accurate capillary electrophoresis multiplex quantitative competitive PCR (ce-mqcPCR) method for event-specific quantification of the five novel GM maize events DAS59122, LY038, MON88017, MIR604 and Event 3272. The method combines the simplicity of constructing multiple competitors in silico with the high resolution and sensitivity of fluorescence capillary electrophoresis and the use of an internal template reference amplicon. The competitors are synthesised commercially and added in equal amounts as a restriction enzyme-digested plasmid insert to the multiplex PCR. Quantification is performed by analysing the relative amounts of GMO and GMO competitor fragment pairs after capillary electrophoresis and correcting for differences in maize DNA by comparing with the internal reference gene amplicon. Since the competitors employ the same primers as their corresponding targets, all existing qualitative multiplex PCRs can in principle easily be converted to quantitative assays without changing primer sets or amplification conditions. The ce-mqcPCR method correctly determined 120 GMO templates in known mixed samples. No false-positive or false-negative signals were obtained.

Routing Misfolded Proteins through the Multivesicular Body (MVB) Pathway Protects against Proteotoxicity

The secretory pathway maintains multiple quality control checkpoints. Initially, endoplasmic reticulum-associated degradation pathways monitor protein folding to retain and eliminate aberrant products. Despite its broad client range, some molecules escape detection and traffic to Golgi membranes. There, a poorly understood mechanism termed Golgi quality control routes aberrant proteins for lysosomal/vacuolar degradation. To better understand Golgi quality control, we examined the processing of the obligate substrate Wsc1p. Misfolded Wsc1p does not use routes of typical vacuolar membrane proteins. Instead, it partitions into intralumenal vesicles of the multivesicular body (MVB) pathway, mediated by the E3 ubiquitin ligase Rsp5p. Its subsequent transport to the vacuolar lumen is essential for complete molecule breakdown. Surprisingly, the transport mode plays a second crucial function in neutralizing potential substrate toxicity. Eliminating the MVB sorting signal diverted molecules to the vacuolar limiting membrane, resulting in the generation of toxic by-products. These data demonstrate a new role of the MVB pathway in protein quality control.

Examination of a Virulence Mutant Uncovers the Ribosome Biogenesis Regulatory Protein of Toxoplasma gondii

Several insertional mutants identified in a screen for Toxoplasma gondii that were defective in establishing a chronic infection had a common site of plasmid insertion. This insertion site was determined to be 43 bp upstream of the transcription initiation site of a gene whose predicted product has homology to ribosome biogenesis regulatory protein Rrs1p, an essential protein required for ribosome biogenesis in Saccharomyces cerevisiae. Northern blot analysis of this locus, termed TgRRS1 , showed that in the C3 mutant, the full-length transcript is down-regulated and at least 1 new smaller transcript is present. Restoration of the intact predicted promoter and locus to TgRRS1 insertional mutant strain C3 did not restore brain cyst formation to the levels of the parent strain. Epitope-tagged TgRRS1 was found to localize to the parasite nucleolus, in an area corresponding to the granular component region. TgRRS1 can serve as a marker for the sub-nucleolar granular component region of T. gondii.

Analysis of Membrane Topology and Identification of Essential Residues for the Yeast Endoplasmic Reticulum Inositol Acyltransferase Gwt1p

Glycosylphosphatidylinositol (GPI) is a post-translational modification that anchors cell surface proteins to the plasma membrane, and GPI modifications occur in all eukaryotes. Biosynthesis of GPI starts on the cytoplasmic face of the endoplasmic reticulum (ER) membrane, and GPI precursors flip from the cytoplasmic side to the luminal side of the ER, where biosynthesis of GPI precursors is completed. Gwt1p and PIG-W are inositol acyltransferases that transfer fatty acyl chains to the inositol moiety of GPI precursors in yeast and mammalian cells, respectively. To ascertain whether flipping across the ER membrane occurs before or after inositol acylation of GPI precursors, we identified essential residues of PIG-W and Gwt1p and determined the membrane topology of Gwt1p. Guided by algorithm-based predictions of membrane topology, we experimentally identified 13 transmembrane domains in Gwt1p. We found that Gwt1p, PIG-W, and their orthologs shared four conserved regions and that these four regions in Gwt1p faced the luminal side of the ER membrane. Moreover, essential residues of Gwt1p and PIG-W faced the ER lumen or were near the luminal edge of transmembrane domains. The membrane topology of Gwt1p suggested that inositol acylation occurred on the luminal side of the ER membrane. Rather than stimulate flipping of the GPI precursor across the ER membrane, inositol acylation of GPI precursors may anchor the precursors to the luminal side of the ER membrane, preventing flip-flops.

An insertional trap for conditional gene expression in Toxoplasma gondii: Identification of TAF250 as an essential gene

Toxoplasmosis is characterized by fast lytic replication cycles leading to severe tissue lesions. Successful host cell invasion is essential for pathogenesis. The division cycle of Toxoplasma gondii is characterized by an unusual cell cycle progression and a distinct internal budding mechanism. To identify essential genes involved in the lytic cycle we devised an insertional gene trapping strategy using the Tet-transactivator system. In essence, a random, active promoter is displaced with a tetracycline regulatable promoter, which if in an essential gene, will result in a conditionally lethal phenotype upon tetracycline addition. We isolated eight mutants with growth defects, two of which displayed modest invasion defects, one of which had an additional cell cycle defect. The trapped loci were identified using expression microarrays, exploiting the tetracycline dependent expression of the trapped genes. In mutant 3.3H6 we identified TCP-1, a component of the chaperonin protein folding machinery under the control of the Tet promoter. However, this gene was not critical for growth of mutant 3.3H6. Subsequently, we identified a suppressor gene encoding a protein with a hypothetical function by guided cosmid complementation. In mutant 4.3B13, we identified TAF250, an RNA polymerase II complex component, as the trapped, essential gene. Furthermore, by mapping the plasmid insertion boundaries we identified multiple genomic rearrangements, which hint at a potential replication dependent DNA repair mechanism. Furthermore, these rearrangements provide an explanation for inconsistent locus rescue results observed by molecular biological approaches. Taken together, we have added an approach to identify and study essential genes in Toxoplasma.

Deciphering the intracellular metabolism of Listeria monocytogenes by mutant screening and modelling

BackgroundThe human pathogen Listeria monocytogenes resides and proliferates within the cytoplasm of epithelial cells. While the virulence factors essentially contributing to this step of the infection cycle are well characterized, the set of listerial genes contributing to intracellular replication remains to be defined on a genome-wide level.ResultsA comprehensive library of L. monocytogenes strain EGD knockout mutants was constructed upon insertion-duplication mutagenesis, and 1491 mutants were tested for their phenotypes in rich medium and in a Caco-2 cell culture assay. Following sequencing of the plasmid insertion site, 141 different genes required for invasion of and replication in Caco-2 cells were identified. Ten in-frame deletion mutants were constructed that confirmed the data. The genes with known functions are mainly involved in cellular processes including transport, in the intermediary metabolism of sugars, nucleotides and lipids, and in information pathways such as regulatory functions. No function could be ascribed to 18 genes, and a counterpart of eight genes is missing in the apathogenic species L. innocua. Mice infection studies revealed the in vivo requirement of IspE (Lmo0190) involved in mevalonate synthesis, and of the novel ABC transporter Lmo0135-0137 associated with cysteine transport. Based on the data of this genome-scale screening, an extreme pathway and elementary mode analysis was applied that demonstrates the critical role of glycerol and purine metabolism, of fucose utilization, and of the synthesis of glutathione, aspartate semialdehyde, serine and branched chain amino acids during intracellular replication of L. monocytogenes.ConclusionThe combination of a genetic screening and a modelling approach revealed that a series of transporters help L. monocytogenes to overcome a putative lack of nutrients within cells, and that a high metabolic flexibility contributes to the intracellular replication of this pathogen.

Development of a new PCR primer system for selective amplification of Actinobacteria

The occurrence of Actinobacteria in water-damaged building materials as well as the clinical relevance of some Actinobacteria (e.g. Saccharopolyspora spp., Mycobacterium spp., Nocardia spp., etc.), led us to develop a detection system to examine the actinobacterial community. A new primer system, Com2xf/Ac1186r (16S rRNA gene based) specific for Actinobacteria was designed. The adequacy for the intended use of the primer system was first investigated in silico using sequences of 164 different species belonging to 75 different genera of the class Actinobacteria. To test the primer specificity in complex environmental samples, four 16S rRNA gene clone libraries were generated (plaster material, compost material, compost plant- and duck house bioaerosols). Overall, 87% of obtained sequences were assigned to actinobacterial genera. To verify the applicability of the new designed primer system in water-damaged building material, 16S rRNA gene clone libraries of 18 different water-damaged materials were screened for their affiliation to Actinobacteria. A total of 88% of all 'Actinobacteria-positive' detected plasmid inserts were affiliated correctly. Results of SSCP-fingerprinting clearly showed differences of the species detected by the Actinobacteria-specific primer system within the different samples. Overall results obtained in this study indicate the applicability of the developed primer system for its intended use.

Pig transgenesis by Sleeping Beauty DNA transposition

Modelling of human disease in genetically engineered pigs provides unique possibilities in biomedical research and in studies of disease intervention. Establishment of methodologies that allow efficient gene insertion by non-viral gene carriers is an important step towards development of new disease models. In this report, we present transgenic pigs created by Sleeping Beauty DNA transposition in primary porcine fibroblasts in combination with somatic cell nuclear transfer by handmade cloning. Göttingen minipigs expressing green fluorescent protein are produced by transgenesis with DNA transposon vectors carrying the transgene driven by the human ubiquitin C promoter. These animals carry multiple copies (from 8 to 13) of the transgene and show systemic transgene expression. Transgene-expressing pigs carry both transposase-catalyzed insertions and at least one copy of randomly inserted plasmid DNA. Our findings illustrate critical issues related to DNA transposon-directed transgenesis, including coincidental plasmid insertion and relatively low Sleeping Beauty transposition activity in porcine fibroblasts, but also provide a platform for future development of porcine disease models using the Sleeping Beauty gene insertion technology.

Dynamics of genome evolution in facultative symbionts of aphids

Aphids are sap-feeding insects that host a range of bacterial endosymbionts including the obligate, nutritional mutualist Buchnera plus several bacteria that are not required for host survival. Among the latter, ‘Candidatus Regiella insecticola’ and ‘Candidatus Hamiltonella defensa’ are found in pea aphids and other hosts and have been shown to protect aphids from natural enemies. We have sequenced almost the entire genome of R. insecticola (2.07 Mbp) and compared it with the recently published genome of H. defensa (2.11 Mbp). Despite being sister species the two genomes are highly rearranged and the genomes only have ∼55% of genes in common. The functions encoded by the shared genes imply that the bacteria have similar metabolic capabilities, including only two essential amino acid biosynthetic pathways and active uptake mechanisms for the remaining eight, and similar capacities for host cell toxicity and invasion (type 3 secretion systems and RTX toxins). These observations, combined with high sequence divergence of orthologues, strongly suggest an ancient divergence after establishment of a symbiotic lifestyle. The divergence in gene sets and in genome architecture implies a history of rampant recombination and gene inactivation and the ongoing integration of mobile DNA (insertion sequence elements, prophage and plasmids).

A Partial Chromosomal Deletion Caused by Random Plasmid Integration Resulted in a Reduced Virulence Phenotype in Fusarium graminearum

Fusarium graminearum (teleomorph: Gibberella zeae) is an Ascomycete fungal plant pathogen which infects a range of agriculturally important crops, including wheat, barley, and maize. A random plasmid insertion mutagenesis approach was used to analyze the pathogenicity of the PH-1 strain, for which full genomic information is available. Fungal transformants were initially screened for their ability to infect wheat ears. From a total of 1,170 transformants screened, eight were confirmed to be highly reduced in pathogenicity toward wheat ears and roots. These were designated disease-attenuated F. graminearum (daf) mutants. The in vitro growth rate and appearance of each daf mutant was equivalent to the parental strain. Deoxynivalenol (DON) was not detected in threshed grain recovered from ears inoculated with the daf10 mutant. Plasmid rescue and sequencing of the mutant daf10 revealed a deletion of approximately 350 kb from one end of chromosome 1. This chromosome segment is predicted to contain 146 genes. Microarray analysis of daf10 gene expression during growth in DON-inducing conditions confirmed the large deletion. The identities of the genes deleted and their potential role in DON production, pathogenesis, and other life processes are discussed.

Effect of gcl, glcB and aceA Disruption on Glyoxylate Conversion by Pseudomonas putida JM37

Pseudomonas putida JM 37 metabolized glyoxylate at a specific rate of 55 g/g dry biomass/day. In order to investigate their role, three genes encoding enzymes that potentially convert glyoxylate were disrupted, namely tartronate semialdehyde synthase (gcl), malate synthase (glcB) and isocitrate lyase (aceA). Strains with transposon insertion in either of these genes were isolated from a 50,000 clone library employing a PCR-guided enrichment strategy. Further, all three respective double mutants were constructed via site-directed insertion of a knock-out plasmid. Neither mutation of gcl, glcB, aceA nor any of the respective double mutation influenced glyoxylic acid conversion, indicating that P. putida JM37 may possess other enzymes and pathways for glyoxylate metabolism.

GGDEF and EAL proteins play different roles in the control of <italic>Sinorhizobium meliloti</italic> growth, motility, exopolysaccharide production, and competitive nodulation on host alfalfa

A new bacterial secondary messenger, bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP), is usually synthesized or decomposed by proteins containing GGDEF or glutamate-alanine-leucine (EAL) domain. They often act as cyclase or phosphodiesterase of c-di-GMP and their genes are distributed among almost all bacteria according to known genomic DNA sequences. However, the systematic identification of GGDEF and EAL genes remains unclear in rhizobia, soil bacteria that interact with compatible legumes to form nitrogen-fixing nodules. In this study, 19 putative GGDEF and EAL genes were identified in a model rhizobium, Sinorhizobium meliloti, by bioinformatic analysis (encoding 5 GGDEF proteins, 4 EAL proteins, and 10 GGDEF and EAL double-domain proteins). Null mutants of 14 genes were constructed through systematic plasmid insertion. All 14 gene mutants showed deficient growth in minimal medium and defective motility, and 11 gene mutants produced a lot more exopolysaccharide and displayed less competitive nodulation on the host plant, alfalfa. Our results suggested that GGDEF and EAL proteins may play different roles in the control of S. meliloti physiology, although they contain conserved catalytic (GGDEF or EAL) domains. Our finding also implied that c-di-GMP may play an important role in the interactions between this rhizobium and its host plants to establish efficient symbiosis.

A Role for Topoisomerase I in Fusarium graminearum and F. culmorum Pathogenesis and Sporulation

Fusarium graminearum and F. culmorum are the causal agents of Fusarium ear blight (FEB) in wheat. A forward genetics approach was taken to discover novel pathogenicity genes in the genome of F. graminearum. A library of transformants created by random plasmid insertional mutagenesis was screened on wheat ears for virulence defects. Plasmid rescue on one of the reduced-virulence mutants revealed a single-copy plasmid insertion in the gene coding for the DNA interacting enzyme, topoisomerase I. Targeted topoisomerase I gene-deletion mutants were created in strains of both F. graminearum and F. culmorum. The top1 mutants of both species exhibited greatly reduced virulence in wheat ear infection assays (GO:0009405 and GO:0044145). Detailed microscopy analyses revealed that top1 hyphal growth was restricted to palea tissue whereas host responses were discernable 1,000 mum further away in the rachis node. Asexual sporulation was reduced in the F. graminearum mutants and was absent from the F. culmorum mutants. The F. graminearum mutant did not develop sexual spores when subjected to an in vitro perithecia production assay. During in vitro growth, the top1 mutants of both species were still able to produce the trichothecene mycotoxin, deoxynivalenol.

Yeast Two-Hybrid System for Studying Protein-Protein Interactions--Stage 4: Isolation of Library Plasmid Insert and Second Confirmation of Positive Interactions

An important element in the characterization of the function of a protein is the identification of other proteins with which it interacts. A powerful genetic strategy for this purpose, termed the "yeast two-hybrid system," uses transcriptional reporters in yeast to indirectly reflect the interaction between two proteins. The term "two-hybrid" derives from the two classes of chimeric, or "hybrid," proteins used in each screen. The first, commonly referred to as the "bait," is a fusion of a protein of interest "x" with a DNA-binding domain (DBD-x). The second, sometimes called the "prey," is a fusion of a cDNA library "y" to a transcriptional activation domain (AD-y). Together, DBD-x and AD-y provide the basis of the detection system. The two-hybrid approach has gained wide popularity because of the relative ease and speed with which it can be used to identify novel protein-protein interactions and to analyze known interactions. Stage 4, described here, outlines a series of first-order and subsequent control experiments designed to establish whether an interacting protein is likely to be biologically significant. The number of positive interactions obtained will vary drastically from bait to bait. Subsequent processing methods will depend on the number initially obtained and on the preference of the individual investigator.

BAC TG-EMBED: one-step method for high-level, copy-number-dependent, position-independent transgene expression

Chromosome position effects combined with transgene silencing of multi-copy plasmid insertions lead to highly variable and usually quite low expression levels of mini-genes integrated into mammalian chromosomes. Together, these effects greatly complicate obtaining high-level expression of therapeutic proteins in mammalian cells or reproducible expression of individual or multiple transgenes. Here, we report a simple, one-step procedure for obtaining high-level, reproducible mini-gene expression in mammalian cells. By inserting mini-genes at different locations within a BAC containing the DHFR housekeeping gene locus, we obtain copy-number-dependent, position-independent expression with chromosomal insertions of one to several hundred BAC copies. These multi-copy DHFR BAC insertions adopt similar large-scale chromatin conformations independent of their chromosome integration site, including insertions within centromeric heterochromatin. Prevention of chromosome position effects, therefore, may be the result of embedding the mini-gene within the BAC-specific large-scale chromatin structure. The expression of reporter mini-genes can be stably maintained during continuous, long-term culture in the presence of drug selection. Finally, we show that this method is extendable to reproducible, high-level expression of multiple mini-genes, providing improved expression of both single and multiple transgenes.

Genetic and Biochemical Characterization of TRU-1, the Endogenous Class C β-Lactamase from Aeromonas enteropelogenes

Aeromonas enteropelogenes (formerly A. tructi) was described to be an ampicillin-susceptible and cephalothin-resistant Aeromonas species, which suggests the production of a cephalosporinase. Strain ATCC 49803 was susceptible to amoxicillin, cefotaxime, and imipenem but resistant to cefazolin (MICs of 2, 0.032, 0.125, and >256 microg/ml, respectively) and produced an inducible beta-lactamase. Cefotaxime-resistant mutants (MIC, 32 microg/ml) that showed constitutive beta-lactamase production could be selected in vitro. The gene coding for the cephalosporinase of A. enteropelogenes ATCC 49803 was cloned, and its biochemical properties were investigated. Escherichia coli transformants showing resistance to various beta-lactams carried a 3.5-kb plasmid insert whose sequence revealed a 1,146-bp open reading frame (ORF) encoding a class C beta-lactamase, named TRU-1, showing the highest identity scores with A. punctata CAV-1 (75%), A. salmonicida AmpC (75%), and A. hydrophila CepH (71%). The bla(TRU-1) locus includes open reading frames (ORFs) showing significant homology with genes found in the genomes of other Aeromonas species, although it exhibits a different organization, as reflected by the presence of additional ORFs located downstream of the beta-lactamase gene in the A. hydrophila and A. salmonicida genomes. Specific PCR assays were negative for cphA-like and bla(OXA-12)-like genes in three A. enteropelogenes ATCC strains. Purified TRU-1 showed a broad substrate profile, efficiently hydrolyzing benzylpenicillin, cephalothin, cefoxitin, and, although with significantly lower turnover rates, oxyiminocephalosporins. Cephaloridine and cefepime were poorly recognized by the enzyme, as reflected by the high K(m) values observed with these substrates. Thus far, A. enteropelogenes represents the only known example of an Aeromonas species that produces only one beta-lactamase belonging to molecular class C.