Absence Of Selective Pressure Research Articles

Response to lufenuron of OP- and tebufenozide-resistant greenheaded leafroller, Planotortrix octo (Dugdale) (Lepidoptera: Tortricidae), from Central Otago, New Zealand

Dosage-mortality tests were conducted with first-instar larvae of Planotortrix octo to measure their responses to tebufenozide (diet-spray and diet-incorporation tests) and lufenuron (diet-incorporation test). A laboratory colony not exposed to insecticide for many generations (S x S) was compared with three colonies derived from a wild P. octo population from Dumbarton, Central Otago, known to be resistant to azinphos-methyl and cross-resistant to tebufenozide; these colonies were subjected to different selection pressures with tebufenozide. Comparison between these colonies and with earlier diet-spray bioassays showed that resistance to tebufenozide in each colony was very stable, varying from 7-fold to 110-fold depending on selection, even in the later absence of selection pressure. A selected colony with 107-fold resistance to tebufenozide in the diet-spray test was only 7.4-fold resistant in the diet-incorporation bioassay. Lufenuron was equally toxic to the S x S and tebufenozide-resistant colonies, with no evidence of lufenuron resistance. The implications of these results for tebufenozide resistance and insecticide resistance management of leafrollers are discussed.

Pollination ecology, genetic diversity and selection on nectar spur length inPlatanthera lacera(Orchidaceae)

AbstractPlatanthera lacera(Orchidaceae) is a moth‐pollinated, loess prairie orchid producing a raceme of one to many whitish‐green flowers. Field studies on a western Illinois population found the crepuscular visiting noctuid moth,Anagrapha falcifera(Noctuidae), to be the most frequent pollinator with occasional visits fromAllagrapha aerea(Noctuidae). Visitation rates, assessed by removal of at least one pollinium, were relatively high (84.9%) and fruit production on experimentally outcrossed flowers (94.4%) was higher than open‐pollinated plants (71.4%). Experimental pollination showedP. lacerato be highly self‐compatible (94.1%) with a low level of autogamy (8.2%). Measurements taken from 598 spurs on 44 plants indicated that nectar spur length varied significantly among plants (10.9–17.1 mm, mean 14.3 mm), but was not under selective pressure from visitation byAn. falcifera(mean proboscis length 11.1 mm). The absence of selective pressure on nectar spur length is likely to be explained by occasional pollinating visits fromAl. aerea(proboscis length 18 mm) and a limited amount of autogamy. Electrophoretic analysis of 12 enzymes revealed seven polymorphic loci. Mean levels of heterozygosity were He = 0.3384, Ho = 0.3229 and F = 0.0458, indicating thatP. lacerais primarily an outcrossing species dependent on noctuid moth pollination.

Evidence of vancomycin resistance gene transfer between enterococci of human origin in the gut of mice harbouring human microbiota

Potential intra- and inter-species transfers of vancomycin resistance genes (vanA gene cluster) between Enterococcus strains were evaluated in the gut of heteroxenic mice harbouring a human microbiota. Mice colonized with a stable population of E. faecium 64/3 or E. faecalis JH2-2 recipient strain and harbouring an enterococci-free human microbiota were obtained. Donor strain E. faecium HC-VI2 of clinical origin was administered orogastrically to these mice and transfers were evaluated over time in faecal samples. Only intraspecies transfers were detected in the digestive tract (DT) of mice harbouring a human microbiota. E. faecium 64/3 transconjugants were detected at several sampling times over the 60 day experiment to levels up to 10(3) cfu/g of faeces, but they did not steadily colonize the DT. Here, we show for the first time that transfer of the vanA gene cluster can occur between Enterococcus strains in the DT colonized with a human microbiota and in the absence of selective pressure. The colonization properties of other enterococci transconjugants and the influence of vancomycin intake should be further investigated since transfers in the DT of animals and humans might contribute to emergence and dissemination of new vancomycin-resistant bacteria.

Insertion-duplication mutagenesis of Salmonella enterica and related species using a novel thermosensitive vector

We constructed a novel temperature-sensitive vector as a tool for gene disruption by insertion-duplication mutagenesis (IDM) in Salmonella enterica and related species. A phoN insertion mutant was proven highly stable during growth in LB medium and during infection of macrophage cells in the absence of selection pressure. By progressive shortening of a phoN fragment, the minimal length for effective insertional mutagenesis driven by homologous recombination was determined to be 50 bp, allowing to disrupt even short genes that could not yet be subjected to site-specific IDM. We also showed that plasmid excision from the chromosome restores the wild-type genotype with a reliability of 98%. Intracellular recovery of the excised vector provides the option to switch between two genotypes and thus to rapidly attribute the observed mutant phenotype to the targeted gene. In addition, a fragment library was used to measure the integration rate at various chromosomal sites that varies greatly by at least 2.5 magnitudes, independently from the length of the cloned fragment.

The susceptibility to growth-promoting antibiotics of Enterococcus faecium isolates from pigs and chickens in Europe

To establish the susceptibility of Enterococcus faecium isolates from pigs and chickens to antimicrobial growth promoters that either were or had been in use in the European Union. Samples were taken at abattoirs in two successive years (mid-1998-mid-1999, year 1; mid-1999-mid-2000, year 2) from chickens (France, The Netherlands, Sweden, UK) and pigs (Denmark, The Netherlands, Spain, Sweden). E. faecium was isolated from faecal samples at national laboratories and sent to a central laboratory where MICs of avilamycin, avoparcin, bacitracin, flavophospholipol, spiramycin, tylosin and virginiamycin were determined. Microbiological breakpoints were allocated on the basis of MIC distributions, and comparison was made between host species, country of origin and year of sample. In total, 2567 isolates were obtained from chickens and 1742 from pigs. In all countries, resistance to avoparcin (banned in 1997) was uncommon, but resistance to bacitracin and flavophospholipol was common (and was probably largely intrinsic). The prevalence of resistance was similar in chicken and pig isolates, with the exception of avilamycin, to which resistance was commoner among chicken isolates. The removal of four compounds as growth promoters (bacitracin, spiramycin, tylosin, virginiamycin) between years 1 and 2 appeared to result in a significant decrease in resistance to three of them-spiramycin, tylosin and virginiamycin, with no change in resistance to bacitracin, but an increase in resistance to avilamycin (not discontinued). Associated resistance was shown between some of the compounds. Resistance prevalence declined rapidly following removal of growth promoters in pigs and chickens, suggesting that in the absence of selective pressure, a susceptible population began to replace phenotypically resistant strains. Associated resistance between different compounds, where seen, could have resulted from either shared resistance mechanisms or from carriage of resistance genes on the same plasmid. Multiresistance to streptogramins, macrolides and glycopeptides was rare.

Stability and persistence of aldrin and methyl-parathion resistance in western corn rootworm populations (Coleoptera: Chrysomelidae)

The susceptibilities of laboratory and field-collected western corn rootworm populations ( Diabrotica virgifera virgifera LeConte) to methyl-parathion and aldrin were estimated by topical application of insecticide during 2002 to determine the stability of resistance in the absence of selective pressures. Most of the laboratory-reared and field-collected populations were significantly resistant to both insecticides. Average LD 50 values of laboratory and field-collected populations were 19- and 13-fold greater than the susceptible population in methyl-parathion bioassays, respectively, and 204- and 125-fold greater in the aldrin bioassays, respectively. The presence of aldrin and methyl-parathion resistance in field-collected populations strongly suggests that both resistance traits are stable in the absence of selection pressure and that neither mechanism is associated with a strong fitness disadvantage.

Identification of a DNA fragment that increases mitotic stability of episomal linear DNAs in Leishmania major

The centromere is a specialized region of eukaryotic chromosomes, the site of kinetochore formation, spindle attachment and regulation of chromosome segregation during mitotic and meiotic cell divisions. To identify sequences which increase mitotic stability and/or act as potential centromeres in Leishmania major, we first generated libraries of Leishmania linear artificial chromosomes (LACs) bearing 30 kb inserts of randomly selected genomic DNAs. These were introduced into parasites, and then their stability was assessed following a period of 10 passages of growth in the absence of selective pressure. Approximately 80% of the 108 transfectants tested lost their LACs promptly and only 20% of the recombinants were retained; of these six showed strong but partial stability (maintained in 30–46% of cells). Mapping and sequencing of one clone (cSC10), which confers the highest degree of maintenance, revealed the presence of a sequence that was found within another stable episome, and which is dispersed in the genome of L. major. The implications of these data to the possible mechanisms of chromosomal maintenance are discussed.

Do physiological roles foster persistence of drug/multidrug-efflux transporters? A case study

Drug and multidrug resistance have greatly compromised the compounds that were once the mainstays of antibiotic therapy. This resistance often persists despite reductions in the use of antibiotics, indicating that the proteins encoded by antibiotic-resistance genes have alternative physiological roles that can foster such persistence in the absence of selective pressure by antibiotics. The recent observations that Tet(L), a tetracycline-efflux transporter, and MdfA, a multidrug-efflux transporter, both confer alkali tolerance offer a striking case study in support of this hypothesis.

Construction of new stable strain over-expressing the glucose isomerase of the Streptomyces sp. SK strain

In order to over express the xylA gene of Streptomyces sp. SK strain, it was cloned under the control of the constitutive ermE-up promoter. This construct was integrated through site-specific recombination process into the chromosome of a Streptomyces violaceoniger glucose isomerase deficient strain using the non-replicative vector pTS55. The resulting CBS4 strain shows a perfect stability in the absence of selection pressure. Its glucose isomerase activity was about four and nine-fold greater, than that obtained from Streptomyces sp. SK, respectively fully induced or not by xylose.

15. Large-Scale Analysis of Adeno-Associated Virus Vector Integration Sites in Normal Human Cells

Top of pageAbstract Unexpected consequences of MLV retrovirus integration in human clinical trials revealed that vector integration is an important safety concern in the field of gene therapy. However, an extensive evaluation of AAV vector integration site distributions has not been completed despite extensive use in human clinical trials. Here we analyze 977 unique AAV vector:chromosome integration junctions from normal human fibroblasts in the absence of selective pressure, and describe their genomic distribution, relationship to transcription levels, and other important genomic features. We found a significant predilection for the first 1 kb of genes and CpG islands but only a slight preference for transcribed sequences as a whole. Vector integration was significantly enriched in segmental duplications and ribosomal DNA repeats, and frequencies were lower than expected in LTR repeats. We observed statistically significant clustering of integration sites throughout the genome, and found 13 |[ldquo]|hotspots|[rdquo]| that contained three or more proviruses within a 500 kb window. Cloning of both vector:chromosome junctions from 323 proviruses allowed extensive characterization of the size of chromosomal deletions, insertions of nucleotide sequence, and putative translocations associated with vector integration. These studies shed light on the profile of insertional mutagenesis associated with AAV vectors and provide unique insight into the location of DSB's in the human genome.

Nonsense Suppression in Yeast Cells Overproducing Sup35 (eRF3) Is Caused by Its Non-heritable Amyloids

The [PSI+] prion determinant of Saccharomyces cerevisiae causes nonsense suppressor phenotype due to a reduced function of the translation termination factor Sup35 (eRF3) polymerized into amyloid fibrils. Prion state of the Rnq1 protein, [PIN+], is required for the [PSI+] de novo generation but not propagation. Yeast [psi-] [PIN+] cells overproducing Sup35 can exhibit nonsense suppression without generation of a stable [PSI+]. Here, we show that in such cells, most of Sup35 represents amyloid polymers, although the remaining Sup35 monomer is sufficient for normal translation termination. The presence of these polymers strictly depends on [PIN+], suggesting that their maintenance relies on efficient generation de novo rather than inheritance. Sup35 polymers contain Rnq1, confirming a hypothesis that Rnq1 polymers seed Sup35 polymerization. About 10% of cells overproducing Sup35 form colonies on medium selective for suppression, which suggests that the proportion of Sup35 monomers to polymers varies between cells of transformants, allowing selection of cells deficient for soluble Sup35. A hybrid Sup35 with the N-terminal domain replaced for 66 glutamine residues also polymerizes and can cause nonsense suppression when overproduced. The described polymers of these proteins differ from the [PSI+] polymers by poor heritability and very high frequency of the de novo appearance, thus being more similar to amyloids than to prions.

Pfmdr1 mutations associated with chloroquine resistance incur a fitness cost in Plasmodium falciparum

Efforts to control malaria worldwide have been hindered by the development and expansion of parasite populations resistant to many first-line antimalarial compounds. Two of the best-characterized determinants of drug resistance in the human malaria parasite Plasmodium falciparum are pfmdr1 and pfcrt, although the mechanisms by which resistance is mediated by these genes is still not clear. In order to determine whether mutations in pfmdr1 associated with chloroquine resistance affect the capacity of the parasite to persist when drug pressure is removed, we conducted competition experiments between P. falciparum strains in which the endogenous pfmdr1 locus was modified by allelic exchange. In the absence of selective pressure, the component of chloroquine resistance attributable to mutations at codons 1034, 1042 and 1246 in the pfmdr1 gene also gave rise to a substantial fitness cost in the intraerythrocytic asexual stage of the parasite. The loss of fitness incurred by these mutations was calculated to be 25% with respect to an otherwise genetically identical strain in which wild-type polymorphisms had been substituted at these three codons. At least part of the fitness loss may be attributed to a diminished merozoite viability. These in vitro results support recent in vivo observations that in several countries where chloroquine use has been suspended because of widespread resistance, sensitive strains are re-emerging.

T20-insensitive HIV-1 from naïve patients exhibits high viral fitness in a novel dual-color competition assay on primary cells

The relationship between sensitivity to antiviral drugs and viral fitness is of paramount importance in understanding the long-term implications of clinical resistance. Here we report the development of a novel recombinant virus assay to study entry inhibitor-resistant HIV variants using a biologically relevant cell type, primary CD4 T-cells. We have modified the replication-competent molecular clone HIV NL4-3 to express a reporter protein (Renilla luciferase), Green Fluorescent Protein (EGFP), or Red Fluorescent Protein (DsRed2) upon infection, thus allowing quantification of replication. Luciferase-expressing virus was used to evaluate drug sensitivity, while co-infection with viruses carrying the green and red fluorescent proteins was employed in the competitive fitness assay. Using envelope proteins from three T20 insensitive variants, lower levels of resistance were observed in primary CD4 T-cells than had been previously reported for cell lines. Importantly, dual-color competition assays demonstrated comparable or higher fitness for these variants despite their reduced T20 sensitivity. We conclude that reduced sensitivity to T20 is compatible with high viral fitness in the absence of selection pressure. Thus, simultaneously measuring both resistance and viral fitness using this newly described dual-color competition assay will likely provide important information about resistant viral variants that emerge during therapy with entry inhibitors.

Screening for improved cell performance: Selection of subclones with altered production kinetics or improved stability by cell sorting

One of the major problems in the biotechnology industry is the selection of cell lines well suited for production of biopharmaceutical proteins. Usually, the most important selection criterion is the cell specific production rate. Nevertheless, a good producer cell line should have a number of additional advantageous properties, which allow the cell line to perform well in the type of bioreactor chosen for the process. However, the time and work required to select for high production rates as well as the lack of methods to specifically select for other cellular properties, usually prevents researchers from including such criteria into their screening program. With the Single Cell Secretion Assay it is possible to measure the specific production rates of individual cells by catching secreted product in an artificial matrix applied to the cell surface. Flow cytometric cell sorting then allows selection of rare cells with high production rates, which occur at frequencies as low as 10(-6). By combining this method with culture conditions that bring out a desired cellular property, we were able to isolate subclones with similar production rates, but improved performance from a recombinant Chinese hamster ovary cell line producing a human monoclonal antibody. The two desired cellular properties screened for were a non-growth associated production kinetic and improved stability in the absence of selective pressure.

Antibiotic resistance in Staphylococcus aureus colonising the intestines of Swedish infants

Staphylococcus aureus has become a frequent coloniser of the intestinal tract of infants, but the health effects of such colonisation are not clear. In this study, the antibiotic resistance patterns of 116 S. aureus strains from the commensal intestinal microflora were determined. The strains were obtained from 81 Swedish infants who had been followed with regular stool samples and registration of antibiotic usage during their first year of life. The faecal population levels of the individual strains and the duration of their persistence in the microflora had been determined previously. The prevalence of antibiotic resistance among the 116 strains was modest: methicillin, 0%; penicillin G, 78%; erythromycin A, 3%; tetracycline, 2%; clindamycin, 0.9%; and fusidic acid, 0.9%. Colonisation by antibiotic-resistant strains was unrelated to antibiotic consumption by individual infants. Antibiotic-resistant strains were as capable of persisting in the intestinal microflora and reaching high faecal population levels as fully susceptible strains. No strain lost or acquired resistance during the colonisation period. Thus, antibiotic-resistant strains of S. aureus seem to be as fit for competition in the large bowel microflora as susceptible strains, even in the absence of selective pressure from antibiotics. This may aggravate the ecological consequences of antibiotic resistance development.

A two-plasmid system for stable, selective-pressure-independent expression of multiple extracellular proteins in mycobacteria.

Recombinant mycobacteria expressing Mycobacterium tuberculosis extracellular proteins are leading candidates for new vaccines against tuberculosis and other mycobacterial diseases, and important tools both in antimycobacterial drug development and basic research in mycobacterial pathogenesis. Recombinant mycobacteria that stably overexpress and secrete major extracellular proteins of M. tuberculosis in native form on plasmids pSMT3 and pNBV1 were previously constructed by the authors. To enhance the versatility of this plasmid-based approach for mycobacterial protein expression, the Escherichia coli/mycobacteria shuttle plasmid pGB9 was modified to accommodate mycobacterial genes expressed from their endogenous promoters. Previous studies showed that the modified plasmid, designated pGB9.2, derived from the cryptic Mycobacterium fortuitum plasmid pMF1, was present at a low copy number in both E. coli and mycobacteria, and expression of recombinant M. tuberculosis proteins was found to be at levels paralleling its copy number, that is, approximating their endogenous levels. Plasmid pGB9.2 was compatible with the shuttle vectors pSMT3 and pNBV1 and in combination with them it simultaneously expressed the M. tuberculosis 30 kDa extracellular protein FbpB. Plasmid pGB9.2 was stably maintained in the absence of selective pressure in three mycobacterial species: Mycobacterium bovis BCG, M. tuberculosis and M. smegmatis. Plasmid pGB9.2 was found to be self-transmissible between both fast- and slow-growing mycobacteria, but not from mycobacteria to E. coli or between E. coli strains. The combination of two compatible plasmids in one BCG strain allows expression of recombinant mycobacterial proteins at different levels, a potentially important factor in optimizing vaccine potency.

The two major imatinib resistance mutations E255K and T315I enhance the activity of BCR/ABL fusion kinase

The resistance to the tyrosine kinase inhibitor imatinib in BCR/ABL-positive leukemias is mostly associated with mutations in the kinase domain of BCR/ABL, which include the most prevalent mutations E255K and T315I. Intriguingly, these mutations have also been identified in some patients before imatinib treatment. Here we examined the effects of these mutations on the kinase activity of a BCR/ABL kinase domain construct that also contained the SH3 and SH2 domains. When expressed in COS7 cells, the BCR/ABL construct with either E255K or T315I exhibited not only the resistance to imatinib but also the increase in activity to induce autophosphorylation as well as tyrosine phosphorylation of various cellular proteins, which included STAT5. The mutant kinases also showed increased activities in in vitro kinase assays. These results raise a possibility that the major imatinib resistance mutations E255K and T315I may confer the growth advantage on leukemic cells to expand in the absence of selective pressure from imatinib treatment.

Artificially ambiguous genetic code confers growth yield advantage.

A primitive genetic code is thought to have encoded statistical, ambiguous proteins in which more than one amino acid was inserted at a given codon. The relative vitality of organisms bearing ambiguous proteins and the kinds of pressures that forced development of the highly specific modern genetic code are unknown. Previous work demonstrated that, in the absence of selective pressure, enforced ambiguity in cells leads to death or to sequence reversion to eliminate the ambiguous phenotype. Here, we report the creation of a nonreverting strain of bacteria that produced statistical proteins. Ablating the editing activity of isoleucyl-tRNA synthetase resulted in an ambiguous code in which, through supplementation of a limited supply of isoleucine with an alternative amino acid that was noncoding, the mutant generating statistical proteins was favored over the wild-type isogenic strain. Such organisms harboring statistical proteins could have had an enhanced adaptive capacity and could have played an important role in the early development of living systems.

The ribosomal RNA gene promoter and adjacent cis-acting DNA sequences govern plasmid DNA partitioning and stable inheritance in the parasitic protozoan Leishmania.

Detailed analysis of the Leishmania donovani ribosomal RNA (rRNA) gene promoter region has allowed the identification of cis-acting sequences involved in plasmid DNA partitioning and stable plasmid inheritance. We report that plasmids bearing the 350 bp rRNA promoter along with the 200 bp region immediately 3' to the promoter exhibited a 6.5-fold increase in transformation frequency and were transmitted to daughter cells as single-copy molecules. This is in contrast to what has been observed for plasmid molecules in this organism so far. Moreover, we show that these low-copy-number plasmids displayed a remarkable mitotic stability in the absence of selective pressure. The region in the vicinity of the RNA pol I transcription initiation site, and also in the adjacent 200 nt, displays a complex structural organization and shares sequence similarity to the yeast autonomously replicating consensus sequence and centromere DNA elements. Deletion analyses indicated that these elements were necessary but not sufficient for plasmid DNA partitioning and stable inheritance, and that the rRNA promoter region was required for optimal function. These results suggest an interplay between RNA pol I transcription, DNA replication, DNA partitioning and mitotic stability in trypanosomatids. This is the first example of defined DNA elements for plasmid partitioning and stable inheritance in the protozoan parasite Leishmania.

826. Non-Viral Gene Therapy Targeting Fanconi Anemia Hematopoietic Stem Cells: A Realistic Hope?

The life span of patients with Fanconi Anemia (FA) is severely shortened by bone marrow failure and malignancies. Currently, the only efficient therapy is bone-marrow transplantation from a matched sibling donor. Therefore, FA is considered a good candidate disease for hematopoietic gene therapy. Our previous preclinical gene therapy experiments with Fancc knock-out mice have shown that wild-type hematopoietic stem cells (introduced by transplantation or generated by gene transfer) have a strong selective growth advantage in vivo. However, despite the successful modeling of FA gene therapy in the mouse, application of the same approach to humans is complicated by the requirement for ex vivo manipulation and scarcity of HSCs in FA patients. Consequently, the development of new gene transfer methods that support stable integration of therapeutic transgenes in vivo are greatly desired. Here, we have utilized the Sleeping Beauty (SB) transposon system to facilitate stable, transposase-mediated gene transfer of the human FANCC cDNA in vivo from a plasmid-based system. The transposase and FANCC transposon plasmid constructs were co-transfected into bone-marrow cells of Fancc mutant mice by two different methods: 1) via electroporation into enriched hematopoietic cells; 2) via direct injection into the femur cavity. In each case, the presence of the transgene was followed in treated animals by semi-quantitative PCR. Although no transgene was detectable in peripheral blood DNA 2 weeks after injection, 2/4 mice injected into the femur cavity were PCR positive 4 weeks after injection, even in the absence of selective pressure. At this time, a single low dose of cyclophosphamide (CPA) was given to provide a selective advantage for FANCC corrected cells. Four weeks after CPA administration, all the experimental electroporated animals (N=2) and femur injected animals (N=8) were strongly PCR positive in their peripheral blood and have remained stably positive for at least 9 months. Furthermore, upon transplantation of bone marrow from these mice into wild-type lethally irradiated recipients, we observed that 81/110 (74%) of day 12 CFU-S colonies were transgene positive. Similarly, 64/110 (64%) of the 12-day CFU-S colonies were transgene positive from the femur-injected donors, thus confirming that transgene integration had occurred in bona fide stem cells. Collectively, these studies demonstrate that a) hematopoietic stem cells from Fancc mutant mice are proficient in taking up plasmid DNA; b) stable integration of plasmid DNA into the genome of hematopoietic stem cells is possible and c) the efficiency of stable gene transfer using these nonviral approaches compares favorably to that achieved by alternative viral-based methods. We are in the process of analyzing transposon host cellular DNA junctions to determine the mechanism of transgene persistence in each experimental group. The above experiments have been repeated independently with more animals: N=5 for electroporated and N=6 for femur injected. Details of all these results will be discussed.