Removal Of Selection Pressure Research Articles

Design of a novel switchable antibody display system in Pichiapastoris.

Yeast surface display (YSD) has been shown to represent a powerful tool in the field of antibody discovery and engineering as well as for selection of high producer clones. However, YSD is predominantly applied in Saccharomycescerevisiae, whereas expression of heterologous proteins is generally favored in the non-canonical yeast Pichiapastoris (Komagataellaphaffii). Establishment of surface display in P.pastoris would therefore enable antibody selection and expression in a single host. Here we describe the generation of a Pichia surface display (PSD) system based on antibody expression from episomal plasmids. By screening a diverse set of expression vectors using Design of Experiments(DoE), the effect of different genetic elements on the surface expression of antibody fragments was analyzed. Among the tested genetic elements, we found that the combination of P.pastoris formaldehyde dehydrogenase (FLD1) promoter, S.cerevisiae invertase 2 signal peptide (SUC2), and α-agglutinin cell wall protein (SAG1) including an autonomously replicating sequence of Kluyveromyceslactis (panARS) were contributing most strongly to higher display levels of three tested antibody fragments. Employing this combination resulted in the display of antibody fragments for up to 25% of cells. Despite significantly reduced expression levels in PSD compared to well-established YSD in S.cerevisiae, similar fractions of antigen binding single-chain variable fragments (scFvs) were observed (80% vs. 84%). In addition, plasmid stability assays and flow cytometric analysis demonstrated the efficient plasmid clearance of cells and associated loss of antibody fragment display after removal of selective pressure. KEY POINTS: • First report of antibody display in P.pastoris using episomal plasmids. • Identification of genetic elements conferring highest levels of antibody display. • Comparable antigen binding capacity of displayed scFvs for PSD compared to YSD.

Can parasites adapt to pollutants? A multigenerational experiment with a Daphnia × Metschnikowia model system exposed to the fungicide tebuconazole

There is increasing evidence about negative effects of fungicides on non-target organisms, including parasitic species, which are key elements in food webs. Previous experiments showed that environmentally relevant concentrations of fungicide tebuconazole are toxic to the microparasite Metschnikowia bicuspidata, a yeast species that infects the planktonic crustacean Daphnia spp. However, due to their short-term nature, this and other experimental studies were not able to test if parasites could potentially adapt to these contaminants. Here, we tested if M. bicuspidata parasite can adapt to tebuconazole selective pressure. Infected D. magna lineages were reared under control conditions (no tebuconazole) and environmentally realistic tebuconazole concentrations, for four generations, and their performance was compared in a follow-up reciprocal assay. Additionally, we assessed whether the observed effects were transient (phenotypic) or permanent (genetic), by reassessing parasite fitness after the removal of selective pressure. Parasite fitness was negatively affected throughout the multigenerational exposure to the fungicide: prevalence of infection and spore load decreased, whereas host longevity increased, in comparison to control (naive) parasite lineages. In a follow-up reciprocal assay, tebuconazole-conditioned (TEB) lineages performed worse than naive parasite lineages, both in treatments without and with tebuconazole, confirming the cumulative negative effect of tebuconazole. The underperformance of TEB lineages was rapidly reversed after removing the influence of the selective pressure (tebuconazole), demonstrating that the costs of prolonged exposure to tebuconazole were phenotypic and transient. The microparasitic yeast M. bicuspidata did not reveal potential for rapid evolution to an anthropogenic selective pressure; instead, the long-term exposure to tebuconazole was hazardous to this non-target species. These findings highlight the potential environmental risks of azole fungicides on non-target parasitic fungi. The underperformance of these microbes and their inability to adapt to such stressors can interfere with the key processes where they intervene. Further research is needed to rank fungicides based on the hazard to non-target fungi (parasites, but also symbionts and decomposers), towards more effective management and protective legislation.

Transgene-free genome editing in marine algae by bacterial conjugation \u2013 comparison with biolistic CRISPR/Cas9 transformation

The CRISPR/Cas9 technology has opened the possibility for targeted genome editing in various organisms including diatom model organisms. One standard method for delivery of vectors to diatom cells is by biolistic particle bombardment. Recently delivery by conjugation was added to the tool-box. An important difference between these methods is that biolistic transformation results in transgene integration of vector DNA into the algae genome, whereas conjugative transformation allows the vector to be maintained as an episome in the recipient cells. In this study, we have used both transformation methods to deliver the CRISPR/Cas9 system to the marine diatom Phaeodactylum tricornutum aiming to induce mutations in a common target gene. This allowed us to compare the two CRISPR/Cas9 delivery systems with regard to mutation efficiency, and to assess potential problems connected to constitutive expression of Cas9. We found that the percentage of CRISPR-induced targeted biallelic mutations are similar for both methods, but an extended growth period might be needed to induce biallelic mutations when the CRISPR/Cas9 system is episomal. Independent of the CRISPR/Cas9 vector system, constitutive expression of Cas9 can cause re-editing of mutant lines with small indels. Complications associated with the biolistic transformation system like the permanent and random integration of foreign DNA into the host genome and unstable mutant lines caused by constitutive expression of Cas9 can be avoided using the episomal CRISPR/Cas9 system. The episomal vector can be eliminated from the diatom cells by removal of selection pressure, resulting in transient Cas9 expression and non-transgenic mutant lines. Depending on legislation, such lines might be considered as non-GMOs.

Clinically Relevant Plasmid-Host Interactions Indicate that Transcriptional and Not Genomic Modifications Ameliorate Fitness Costs of Klebsiella pneumoniae Carbapenemase-Carrying Plasmids.

ABSTRACTThe rapid dissemination of antimicrobial resistance (AMR) around the globe is largely due to mobile genetic elements, such as plasmids. They confer resistance to critically important drugs, including extended-spectrum beta-lactams, carbapenems, and colistin. Large, complex resistance plasmids have evolved alongside their host bacteria. However, much of the research on plasmid-host evolution has focused on small, simple laboratory plasmids in laboratory-adapted bacterial hosts. These and other studies have documented mutations in both host and plasmid genes which occur after plasmid introduction to ameliorate fitness costs of plasmid carriage. We describe here the impact of two naturally occurring variants of a large AMR plasmid (pKpQIL) on a globally successful pathogen. In our study, after pKpQIL plasmid introduction, no changes in coding domain sequences were observed in their natural host, Klebsiella pneumoniae. However, significant changes in chromosomal and plasmid gene expression may have allowed the bacterium to adapt to the acquisition of the AMR plasmid. We hypothesize that this was sufficient to ameliorate the associated fitness costs of plasmid carriage, as pKpQIL plasmids were maintained without selection pressure. The dogma that removal of selection pressure (e.g., antimicrobial exposure) results in plasmid loss due to bacterial fitness costs is not true for all plasmid/host combinations. We also show that pKpQIL impacted the ability of K. pneumoniae to form a biofilm, an important aspect of virulence. This study used highly relevant models to study the interaction between AMR plasmids and pathogens and revealed striking differences from results of studies done on laboratory-adapted plasmids and strains.

Nontransgenic Marker-Free Gene Disruption by an Episomal CRISPR System in the Oleaginous Microalga, Nannochloropsis oceanica CCMP1779.

Utilization of microalgae has been hampered by limited tools for creating loss-of-function mutants. Furthermore, modified strains for deployment into the field must be free of antibiotic resistance genes and face fewer regulatory hurdles if they are transgene free. The oleaginous microalga, Nannochloropsis oceanica CCMP1779, is an emerging model for microalgal lipid metabolism. We present a one-vector episomal CRISPR/Cas9 system for N.oceanica that enables the generation of marker-free mutant lines. The CEN/ARS6 region from Saccharomyces cerevisiae was included in the vector to facilitate its maintenance as circular extrachromosal DNA. The vector utilizes a bidirectional promoter to produce both Cas9 and a ribozyme flanked sgRNA. This system efficiently generates targeted mutations, and allows the loss of episomal DNA after the removal of selection pressure, resulting in marker-free nontransgenic engineered lines. To test this system, we disrupted the nitrate reductase gene ( NR) and subsequently removed the CRISPR episome to generate nontransgenic marker-free nitrate reductase knockout lines (NR-KO).

Utilising the native plasmid, pCA2.4, from the cyanobacterium Synechocystis sp. strain PCC6803 as a cloning site for enhanced product production.

BackgroundThe use of photosynthetic autotrophs and in particular the model organism Synechocystis PCC6803 is receiving much attention for the production of sustainable biofuels and other economically useful products through metabolic engineering. Optimisation of metabolic-engineered organisms for high-level sustained production of product is a key element in the manipulation of this organism. A limitation to the utilisation of metabolically-engineered Synechocystis PCC6803 is the availability of strong controllable promoters and stable gene dosage methods for maximising gene expression and subsequent product formation following genetic manipulation.ResultsA native Synechocystis PCC6803 small plasmid, pCA2.4, is consistently maintained at a copy level of up to 7 times that of the polyploid chromosome. As this plasmid is stable during cell division, it is potentially an ideal candidate for maximising gene dosage levels within the organism. Here, we describe the construction of a novel expression vector generated from the native plasmid, pCA2.4. To investigate the feasibility of this new expression system, a yellow fluorescent protein (YFP) encoding gene was cloned downstream of the strong Ptrc promoter and integrated into a predicted neutral site within the pCA2.4 plasmid. The stability of the integrated construct was monitored over time compared to a control strain containing an identical YFP-expressing construct integrated at a known neutral site in a chromosomal location.ConclusionsA significantly higher fluorescence level of the yellow fluorescent protein was observed when its encoded gene was integrated into the pCA2.4 native plasmid when compared to the isogenic chromosomally integrated control strain. On average, a minimum of 20-fold higher fluorescence level could be achieved from integration into the native plasmid. Fluorescence was also monitored as a function of culture time and demonstrated to be stable over multiple sub-cultures even after the removal of selective pressure. Therefore, the native small plasmid, pCA2.4 may be utilised to stably increase gene expression levels in Synechocystis PCC6803. With the complementary utilisation of an inducible promoter system, rapid generation of commodity-producing Synechocystis PCC6803 strains having high level, controlled expression may be more achievable.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0385-x) contains supplementary material, which is available to authorized users.

Abstract 580: Combined treatment of breast cancer cells with epigallocatechin gallate (EGCG) and Tamoxifen delays cell-growth in long-term culture: Implications for recurrent breast cancer

Abstract Recent health surveys have revealed that green tea is one of the most commonly consumed natural products among breast cancer survivors. Although breast cancer survival has increased during the past two decades due to adjuvant endocrine therapy with antiestrogens (AE) such as Tamoxifen (TAM) and Faslodex (FAS) the risk of acquired endocrine-resistance still persists for many women. This can be mimicked by a prolonged culture of breast cancer cells with sequentially increasing concentrations of 4-hydroxy tamoxifen (4OHT; 1 nM -1 µM), applying a selection pressure for cells to acquire resistance to 4OHT. Such cells maintain their resistant phenotype even after removal of the pressure (Brunner et al., 1993). The aim of this study was to determine whether a green tea polyphenol (EGCG) can affect the development of TAM-resistance in breast cancer. We tested the efficacy of EGCG, in this long-term [LT] cell culture model. Estrogen receptor (ER)α-positive MCF-7 cells (n=2/group) were cultured in media containing: i) Vehicle (0.01% EtOH;, LT-V); ii) LT-4OHT (1 nM to 1 µM; increasing every 3rd passage; LT-4OHT); or iii) 4OHT (1 nM to 1 µM) + EGCG (100 nM;single dose;LT-EGCG). Growth rate was recorded for each passage (P), measured as the time required reach confluence (70%), after which the cells were subcultured. We will present results of this LT study including mechanisms by which EGCG may alter resistance-development, focusing on signaling pathways such as ER, NFkB and unfolded protein response (UPR) known to be involved in endocrine resistance. During the initial passages (P1-P6; [4OHT] ≤ 5 nM) the cells grew at the same rate regardless of the treatment. Starting at P8, time to confluence was delayed for LT-4OHT ([10 nM]; 7d) compared to the LT-V. This delay was even more pronounced at P15 ([100 nM]; ≤ 21d), suggesting that cells were under a growth inhibition/selection pressure. The growth rate of LT-EGCG did not differ from LT-4OHT prior to P15, but in subsequent passages a delay was evident (14 to 18 d). To test whether the cells maintained their (delayed growth) phenotype, they were first cultured in regular media for 24h and tested for growth inhibition by 4OHT (1 nM-1µM) and FAS (100 nM). LT-V cells (P27) were more susceptible to the inhibitory effects of 1 µM 4OHT (73 ± 11% versus 0 µM 4OHT) than LT-4OHT cells (P19; 105 ± 40%), confirming the development of resistance in LT-4OHT cells. In contrast, LT-EGCG cells had retained their sensitivity to 4OHT, similar to LT-V (P19; 75 ± 8%). All treatment groups were equally responsive to FAS (≥30%). These results suggest that EGCG may delay the development of a 4OHT-resistance in breast cancer cells. It will be confirmed whether the effect prevails after removal of selection pressure (4OHT). This data will provide a better understanding of green tea's potential to prevent TAM-resistance in breast cancer patients. 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 580. doi:1538-7445.AM2012-580

Evolution of a herbicide-resistant population of Alopecurus myosuroides Huds. in a long-term cropping system experiment

Due to heavy reliance on herbicides and a lack of cultural control measures, herbicide-resistant populations of Alopecurus myosuroides Huds. (blackgrass) appeared in the early 1990s in winter cereal rotations in France and in Europe. The aim of the present study was to analyse the effects of different cropping systems on an aryloxy-phenoxypropionate herbicide-resistant population in a field trial. Two crop rotations, one consisting exclusively of winter crops and another including spring crops, were assessed over a six-year period. The rotations were combined with different cultural practices including mouldboard ploughing, delayed sowing, and efficient herbicides for controlling resistant plants. A. myosuroides densities decreased in all the cropping systems, but the weed management was most effective when herbicides were combined with non-chemical practices. Rotation with an alternation of spring and winter crops was the most efficient solution against A. myosuroides. Moreover, during the six years, the percentage of resistant plants in different crop rotations was estimated independently of the cropping systems. This proportion did not vary during the six years of the experiment, suggesting that the resistance gene persisted, despite the removal of selection pressure by the aryloxy-phenoxypropionate herbicides.

Molecular analysis of chloroquine resistance in Plasmodium falciparum in Yunnan Province, China

Resistance of Plasmodium falciparum to chloroquine (CQ) is determined by the mutation at K76T of the P. falciparum chloroquine resistance transporter (pfcrt) gene and modified by other mutations in this gene and in the P. falciparum multidrug resistance 1 (pfmdr1) gene. To determine the extent of polymorphisms in these genes in field P. falciparum isolates from Yunnan province of China, we genotyped the pfcrt codon 76, pfmdr1 codons 86 and 1246. Our results showed that although CQ has been withdrawn from treating falciparum malaria for over two decades, 90.3% of the parasites still carried the pfcrt K76T mutation. In contrast, mutations at pfmdr1 codons 86 and 1246 were rare. Sequencing analysis of the pfcrt gene in 34 parasite field isolates revealed CVIET at positions 72-76 as the major type, consistent with the theory of Southeast Asian origin of CQ resistance in the parasite. In addition, two novel pfcrt haplotypes (75D/144Y/220A and 75E/144Y/220A) were identified. Real-time polymerase chain reaction was used to determine pfmdr1 gene amplification, which is associated with mefloquine resistance. Our result indicated that in agreement with that mefloquine has not been used in this area, most (>90%) of the parasites had one pfmdr1 copy. Genotyping at two hypervariable loci showed relatively low levels of genetic diversity of the parasite population. Meanwhile, 28.4% of cases were found to contain mixed clones, which favour genetic recombination. Furthermore, despite a unique history of antimalarial drugs in Yunnan, its geographical connections with three malarious countries facilitate gene flow among parasite populations and evolution of novel drug-resistant genotypes. Therefore, continuous surveillance of drug resistance in this area is necessary for timely adjustment of local drug policies and more effective malaria control.

A blasticidin S-resistant Plasmodium falciparum mutant with a defective plasmodial surface anion channel

Erythrocytes infected with malaria parasites exhibit marked increases in permeability to organic and inorganic solutes. The plasmodial surface anion channel (PSAC), an unusual voltage-dependent ion channel induced on the host membrane after infection, may play a central role in these permeability changes. Here, we identified a functional PSAC mutant through in vitro selection with blasticidin S. Resistance to blasticidin S was generated during culture and correlated with significant reductions in permeability to multiple solutes, consistent with uptake via a common pathway. Single channel recordings revealed marked changes in PSAC gating with the addition of a subconductance state not present in wild-type channels. The channel's selectivity profile and pharmacology also were significantly altered. Eventual loss of the mutant phenotype upon removal of selective pressure and slower growth of mutant parasites suggest that PSAC serves an important role in intracellular parasite survival. These findings provide solid evidence for the uptake of diverse solutes via PSAC and implicate one or more parasite genes in expression of this channel.

519. Development of S/MAR Plasmid Vectors for Persistent Expression and Maintenance, In Vivo

Currently used vectors in human gene therapy suffer from a number of limitations with respect to safety and reproducibility. The ideal vector has to be free of these effects and should allowlong-term expression of a transgene in the absence of selection. Recently a novel non-viral episomal expression system which fulfils these criteria, in principle, was reported. This vector, pEPI, replicates episomally in CHO, HeLa, K562, as well as in primary human fibroblast-like cells, and is mitotically stable in the absence of selection for more than 100 generations. Here we show the long term expression of pEPI in a range of cells including cystic fibrosis airway (IB3) cells to be stable for at least 10 weeks. We demonstrate, however, that antibiotic selection pressure, is necessary for the first weeks following transfection. FACS sorting of CHO and IB3 cells, show that expression and maintenance in cells without initial antibiotic selection diminished rapidly, whereas constructs under selection pressure for an initial period of 2 weeks were mitotically stable and expressed transgene for at least 10 weeks, even following the removal of selection pressure. However, initial in vivo results showed no persistance of vector and limited expression of the reporter gene when using viral promoters. We describe the developments we have made to overcome these limitations and the results of the application of an S/MAR plasmid carrying a human promoter and chromosomal destabilising elements to drive the expression and episomal maintenance of these vectors in vivo. Currently used vectors in human gene therapy suffer from a number of limitations with respect to safety and reproducibility. The ideal vector has to be free of these effects and should allowlong-term expression of a transgene in the absence of selection. Recently a novel non-viral episomal expression system which fulfils these criteria, in principle, was reported. This vector, pEPI, replicates episomally in CHO, HeLa, K562, as well as in primary human fibroblast-like cells, and is mitotically stable in the absence of selection for more than 100 generations. Here we show the long term expression of pEPI in a range of cells including cystic fibrosis airway (IB3) cells to be stable for at least 10 weeks. We demonstrate, however, that antibiotic selection pressure, is necessary for the first weeks following transfection. FACS sorting of CHO and IB3 cells, show that expression and maintenance in cells without initial antibiotic selection diminished rapidly, whereas constructs under selection pressure for an initial period of 2 weeks were mitotically stable and expressed transgene for at least 10 weeks, even following the removal of selection pressure. However, initial in vivo results showed no persistance of vector and limited expression of the reporter gene when using viral promoters. We describe the developments we have made to overcome these limitations and the results of the application of an S/MAR plasmid carrying a human promoter and chromosomal destabilising elements to drive the expression and episomal maintenance of these vectors in vivo.

The potential for enhanced fungicide resistance in Beauveria bassiana through strain discovery and artificial selection

Our objectives were to determine the (1) natural variation in fungicide resistance among Beauveria bassiana strains, (2) potential to increase fungicide resistance in B. bassiana through artificial selection, and (3) stability of virulence in selected B. bassiana strains. Fungicides included dodine, fenbuconazole, and triphenyltin hydroxide, which are commonly used in pecan and other horticultural crops. Comparison of seven B. bassiana strains indicated some are substantially more resistant to fungicides than others; a commercial strain (GHA) was less resistant than all wild strains isolated from pecan orchards. Artificial selection resulted in enhanced fungicide resistance in the GHA strain but not in a mixed wild strain. Removal of selection pressure for three passages did not reduce the enhanced fungicide resistance. Sub-culturing with exposure to fungicides did not affect the GHA strain's virulence to pecan weevil, Curculio caryae, larvae, whereas fungicide exposure increased virulence in a mixed wild population of B. bassiana.

Isolation and analysis of fluP, a gene associated with hyphal growth and sporulation in Aspergillus parasiticus.

Aflatoxins (AF) are polyketide-derived mycotoxins that frequently contaminate food and feed crops, causing health risks to animals and humans. The fluP gene was cloned by screening an Aspergillus parasiticus genomic DNA library with a cDNA probe encoding part of a polyketide synthase (PKS), the 6-methylsalicylic acid synthase (MSAS) from Penicillium patulum. FluP was hypothesized to function as a PKS in AF biosynthesis. The predicted amino acid sequence of FluP demonstrated a high degree of identity to MSAS (55%), moderate identity to another fungal PKS protein encoded by wA from A. nidulans (22%) and low identity (<5%) to fungal fatty acid synthase (FAS) proteins. Disruption of fluP in A. parasiticus resulted in the loss of fluP transcript, a 3- to 4-fold reduction in hyphal growth rate, the appearance of a fluffy, cotton-like hyphal morphology, reduction or elimination of asexual spores and spore-bearing structures, and a twofold reduction in aflatoxin accumulation. Removal of selective pressure on fluP knockout transformants resulted in frequent reversion (10%) to the wild-type genotype and phenotype, establishing a direct link between gene disruption and the associated phenotype. The data suggest that fluP encodes a novel PKS associated with hyphal growth and cell development (sporulation), whose activity indirectly influences aflatoxin accumulation in A. parasiticus.

Induced tolerance of the psocid, Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae), to controlled atmosphere

Adult populations of the psocid, Liposcelis bostrychophila, were exposed for 30 generations to 35% CO2, 1% O2, and 64% N2 at 28oC, 80% RH, in order to select a strain resistant to controlled atmosphere (CA). Selection pressure was maintained at around 70% mortality. At the 30th generation, comparison of sensitivity between the selected strain and the original susceptible strain (CA-S) indicated a tolerance factor (TF) at the 50% mortality level (LT50) of 5.6-fold. Throughout the selection process, log-time against probit-mortality lines remained roughly parallel and the slopes remained lower than that of CA-S until the last generation. Implications are that at high levels of selection, multiple genetic factors continued to act together even at the 30th generation to select for adaptation to survival in a CA environment. Removal of selection pressure for five generations from one subpopulation of the selected strains from the 25th generation caused significant reduction in resistance. In the absence of CA exposure, the selected strain possessed the fitness defect. The CA-selected strain was calculated by R0 to have a fitness value of 0.39 relative to CA-S. In addition, the potential effective measures in the slowdown of resistance to CA of stored-product insects are also discussed.

Stable transformation of the moss Physcomitrella patens

We report the stable transformation of Physcomitrella patens to either G418 or hygromycin B resistance following polyethylene glycol-mediated direct DNA uptake by protoplasts. The method described in this paper was used successfully in independent experiments carried out in our two laboratories. Transformation was assessed by the following criteria: selection of antibiotic-resistant plants, mitotic and meiotic stability of phenotypes after removal of selective pressure and stable transmission of the character to the offspring; Southern hybridisation analysis of genomic DNA to show integration of the plasmid DNA; segregation of the resistance gene following crosses with antibiotic-sensitive strains; and finally Southern hybridisation analysis of both resistant and sensitive progeny. In addition to stable transformants, a heterogeneous class of unstable transformants was obtained.

Laboratory selection of resistance by the Red Flour Beetle,Tribolium castaneum (Herbst), To a carbon dioxide-enriched atmosphere

Adult populations of the red flour beetle,Tribolium castaneum (Herbst), were exposed for 40 generations to an atmosphere containing 65% CO2, 20% O2 and 15% N2 at 95% RH, in order to select a strain resistant to a high carbon dioxide content (HCC) atmosphere. Selection pressure was maintained at between 50% and 70% mortality. At the 40th generation, comparison of sensitivity between the selected strain and the original non-selected strain indicated a resistance factor at the 50% mortality level (LT50) of x 9.2. Throughout the selection process, log-time against probit-mortality lines remained roughly parallel and the slope remained low and similar to that of the non-selected strain until the last generation. Implications are that at a high level of selection, multiple genetic factors continued to act together even at the 40th generation to select for adaptation to survival at high CO2 concentrations. Removal of selection pressure from a sub-population of the selected strain from the 13th to 21st generation revealed that resistance was partially retained with a decrease of 17% in the resistance factor. The modified atmosphere chosen in this study does not represent real-life situations where O2 concentrations are reduced by flushing with CO2, and intergranular relative humidities are generally not above 70%.

Laboratory selection of resistance by the red flour beetle,tribolium castaneum (Herbst), to an atmosphere of low oxygen concentration

Adult populations of the red flour beetle,Tribolium castaneum (Herbst), were exposed for 40 generations to an atmosphere containing 99.5% N2 and 0.5% O2 at 95% RH, in order to select a strain resistant to the low oxygen concentration (LOC) atmosphere. Selection pressure was maintained at between 50% and 70% mortality. At the 40th generation comparison of sensitivity between the selected strain and the original non-selected strain indicated a resistance factor at the 50% mortality level (LT50) of x 5.2. However, throughout the selections, log-time against probit-mortality curves remained roughly parallel from generation to generation and the slope remained low. These findings indicate a multiplicity of genetic factors that at a high level of selection contribute together towards an adaptation of the insects to survival in the LOC atmosphere. Removal of selection pressure from a sub-population of the selected strain from the 13th to 21st generation revealed that resistance was partially retained with a decrease in resistance factor of 23%. Although the study revealed that the insects were able to develop a strain resistant to hypoxia, exposures were at 95% RH to minimize the desiccation effect. This does not reflect field situations, where ambient relative humidities are generally below 70%.

Evidence for a switching mechanism in the invasion of erythrocytes by Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum demonstrates variability in its dependence upon erythrocyte sialic acid residues for invasion. Some lines of P. falciparum invade neuraminidase-treated or glycophorin-deficient red blood cells poorly, or not at all, while other lines invade such cells at substantial rates. To explore the molecular basis of non-sialic acid dependent invasion, we selected parasite lines from a clone (Dd2) that initially exhibited low invasion of neuraminidase-treated erythrocytes. After maintaining Dd2 for several cycles in neuraminidase-treated erythrocytes, parasite lines were recovered that invaded both untreated and neuraminidase-treated erythrocytes at equivalently high rates (Dd2/NM). The change in phenotype was maintained after removal of selection pressure. Four subclones of Dd2 were isolated and each readily converted from sialic acid dependence to non-sialic acid dependence during continuous propagation in neuraminidase-treated erythrocytes. The neuraminidase-selected lines and the Dd2 clone demonstrated identical restriction fragment length polymorphism markers indicating that the Dd2 clone was not contaminated during the selection process. Parasite proteins that bound to neuraminidase-treated and untreated erythrocytes were indistinguishable among the parent Dd2 clone and the neuraminidase-selected lines. The ability of the Dd2 parasite to change its invasion requirements for erythrocyte sialic acid suggests a switch mechanism permitting invasion by alternative pathways.

Clonal gene therapy: transplanted mouse fibroblast clones express human alpha 1-antitrypsin gene in vivo.

A retroviral vector was used to insert human alpha 1-antitrypsin (alpha 1AT) complementary DNA into the genome of mouse fibroblasts to create a clonal population of mouse fibroblasts secreting human alpha 1AT. After demonstrating that this clone of fibroblasts produced alpha 1AT after more than 100 population doublings in the absence of selection pressure, the clone was transplanted into the peritoneal cavities of nude mice. When the animals were evaluated 4 weeks later, human alpha 1AT was detected in both sera and the epithelial surface of the lungs. The transplanted clone of fibroblasts could be recovered from the peritoneal cavities of those mice and demonstrated to still be producing human alpha 1AT. Thus, even after removal of selective pressure, a single clone of retroviral vector-infected cells that expressed an exogenous gene in vitro, continued to do so in vivo, and when recovered, continued to produce the product of the exogenous gene.

Spontaneous amplification of yeast CEN ARS plasmids.

Transformation of Saccharomyces cerevisiae with several yeast CEN4 ARS1 plasmids containing the his3-delta 4 allele (as well as the URA3 and TRP1 markers) yielded His+ transformants at 0.1%-50% the frequency of Ura+ Trp+ transformants. Additional His+ derivatives arose on continuous growth of transformants originally scored as His- Ura+ Trp+. In all cases, the His+ phenotype was not due to plasmid or host mutations but invariably correlated with an up to 12-fold increase in plasmid copy number. On removal of selective pressure, the His+ phenotype was lost more readily than the Ura+ Trp+ markers, with a corresponding decrease in plasmid copy number. Also, the amplification did not decrease the mitotic loss rate of the Ura+ Trp+ markers. These results indicate that CEN ARS plasmids can be spontaneously amplified to higher levels than previously observed. However, when amplified, apparently not all copies exhibit the characteristic stability of CEN ARS plasmids.