Non-selected Lines Research Articles

Transcriptome analysis of the spleen of heterophils to lymphocytes ratio-selected chickens revealed their mechanism of differential resistance to Salmonella

Salmonella is one of the most common food-borne pathogens and its resistance in chicken can be improved through genetic selection. The heterophils/lymphocytes (H/L) ratio in the blood reflects the immune system status of chicken. We compared the genome data and spleen transcriptomes between the H/L ratio-selected and non-selected chickens, after Salmonella infection, aiming to identify the key genes participating in the antibacterial activity in the spleen. The results revealed that, the selected population had stronger (P<0.05) liver resistance to Salmonella typhimurium (ST) than the non-selected population. In the selected and non-selected lines, the identified differentiation genes encode proteins involved in biological processes or metabolic pathways that included the TGF-beta signaling pathway, FoxO signaling pathway, and Salmonella infection pathway. The results of the analysis of all identified differentially expressed genes (DEGs) of spleen revealed that the G protein-coupled receptor (GPCR) and insulin-like growth factor (IGF-I) signaling pathways were involved in the Salmonella infection pathway. Integrated analysis of DEGs and FST (fixation index), identified candidate genes involved in Salmonella infection pathway, such as GPR39, NTRK2, and ANXA1. The extensive genomic changes highlight the polygenic genetic of the immune response in these chicken populations. Numerous genes related to the immune performance are differentially expressed in the selected and non-selected lines and the selected lines has a higher resistance to Salmonella.

Electrocardiograms of mice selectively bred for high levels of voluntary exercise: Effects of short-term exercise training and the mini-muscle phenotype

Changes in cardiac function that occur with exercise training have been studied in detail, but those accompanying evolved increases in the duration or intensity of physical activity are poorly understood. To address this gap, we studied electrocardiograms (ECGs) of mice from an artificial selection experiment in which four replicate lines are bred for high voluntary wheel running (HR) while four non-selected lines are maintained as controls (C). ECGs were recorded using an ECGenie (Mouse Specifics, Inc.) both before and after six days of wheel access (as used in the standard protocol to select breeders). We hypothesized that HR mice would show innate differences in ECG characteristics and that the response to training would be greater in HR mice relative to C mice because the former run more. After wheel access, in statistical analyses controlling for variation in body mass, all mice had lower heart rates, and mice from HR lines had longer PR intervals than C lines. Also after wheel access, male mice had increased heart rate variability, whereas females had decreased heart rate variability. With body mass as a covariate, six days of wheel access significantly increased ventricle mass in both HR and C males. Within the HR lines, a subset of mice known as mini-muscle individuals have a 50% reduction in hindlimb muscle mass and generally larger internal organs, including the heart ventricles. As compared with normal-muscled individuals, mini-muscle individuals had a longer QRS complex, both before and after wheel access. Some studies in other species of mammals have shown correlations between athletic performance and QRS duration. Correlations between wheel running and either heart rate or QRS duration (before wheel running) among the eight individual lines of the HR selection experiment or among 17 inbred mouse strains taken from the literature were not statistically significant. However, total revolutions and average speed were negatively correlated with PR duration among lines of the HR selection experiment for males, and duration of running was negatively correlated with PR duration among 17 inbred strains for females. We conclude that HR mice have enhanced trainability of cardiac function as compared with C mice (as indicated by their longer PR duration after wheel access), and that the mini-muscle phenotype causes cardiac changes that have been associated with increased athletic performance in previous studies of mammals.

Evolution of dispersal syndrome and its corresponding metabolomic changes

Dispersal is one of the strategies for organisms to deal with climate change and habitat degradation. Therefore, investigating the effects of dispersal evolution on natural populations is of considerable interest to ecologists and conservation biologists. Although it is known that dispersal itself can evolve due to selection, the behavioral, life-history and metabolic consequences of dispersal evolution are not well understood. Here, we explore these issues by subjecting four outbred laboratory populations of Drosophila melanogaster to selection for increased dispersal. The dispersal-selected populations had similar values of body size, fecundity, and longevity as the nonselected lines (controls), but evolved significantly greater locomotor activity, exploratory tendency, and aggression. Untargeted metabolomic fingerprinting through NMR spectroscopy suggested that the selected flies evolved elevated cellular respiration characterized by greater amounts of glucose, AMP, and NAD. Concurrent evolution of higher level of Octopamine and other neurotransmitters indicate a possible mechanism for the behavioral changes in the selected lines. We discuss the generalizability of our findings in the context of observations from natural populations. To the best of our knowledge, this is the first report of the evolution of metabolome due to selection for dispersal and its connection to dispersal syndrome evolution.

Inter- and intra-species variation in genome-wide gene expression of Drosophila in response to parasitoid wasp attack

BackgroundParasitoid resistance in Drosophila varies considerably, among and within species. An immune response, lamellocyte-mediated encapsulation, evolved in a subclade of Drosophila and was subsequently lost in at least one species within this subclade. While the mechanisms of resistance are fairly well documented in D. melanogaster, much less is known for closely related species. Here, we studied the inter- and intra-species variation in gene expression after parasitoid attack in Drosophila. We used RNA-seq after parasitization of four closely related Drosophila species of the melanogaster subgroup and replicated lines of D. melanogaster experimentally selected for increased resistance to gain insights into short- and long-term evolutionary changes.ResultsWe found a core set of genes that are consistently up-regulated after parasitoid attack in the species and lines tested, regardless of their level of resistance. Another set of genes showed no up-regulation or expression in D. sechellia, the species unable to raise an immune response against parasitoids. This set consists largely of genes that are lineage-restricted to the melanogaster subgroup. Artificially selected lines did not show significant differences in gene expression with respect to non-selected lines in their responses to parasitoid attack, but several genes showed differential exon usage.ConclusionsWe showed substantial similarities, but also notable differences, in the transcriptional responses to parasitoid attack among four closely related Drosophila species. In contrast, within D. melanogaster, the responses were remarkably similar. We confirmed that in the short-term, selection does not act on a pre-activation of the immune response. Instead it may target alternative mechanisms such as differential exon usage. In the long-term, we found support for the hypothesis that the ability to immunologically resist parasitoid attack is contingent on new genes that are restricted to the melanogaster subgroup.

An automated method for determining the cytoadhesion of Plasmodium falciparum-infected erythrocytes to immobilized cells.

BackgroundPlasmodium falciparum exports antigens to the surface of infected erythrocytes causing cytoadhesion to the host vasculature. This is central in malaria pathogenesis but in vitro studies of cytoadhesion rely mainly on manual counting methods. The current study aimed at developing an automated high-throughput method for this purpose utilizing the pseudoperoxidase activity of intra-erythrocytic haemoglobin.MethodsChinese hamster ovary (CHO) cells were grown to confluence in chamber slides and microtiter plates. Cytoadhesion of co-cultured P. falciparum, selected for binding to CHO cells, was quantified by microscopy of Giemsa-stained chamber slides. In the automated assay, binding was quantified spectrophotometrically in microtiter plates after cell lysis using tetramethylbenzidine as peroxidase-catalysed substrate. The relevance of the method for binding studies was assessed using: i) binding of P. falciparum-infected erythrocytes to CHO cells over-expressing chondroitin sulfate A and ii) CHO cells transfected with CD36. Binding of infected erythrocytes including field isolates to primary endothelial cells was also performed. Data was analysed using linear regression and Bland-Altman plots.ResultsThe manual and automated quantification showed strong, positive correlation (r2 = 0.959, p <0.001) and with similar detection limit and precision. The automated assay showed the expected dose-dependent reduction in binding to CHO cells when blocking with soluble chondroitin sulfate A or anti-CD36 antibody. Quantification of binding to endothelial cells showed clear distinction between selected vs. non-selected parasite lines. Importantly, the assay was sufficiently sensitive to detect adhesion of field isolates to endothelial cells.ConclusionsThe assay is simple and in a reproducible manner quantifies erythrocyte adhesion to several types of immobilized cells.

Low-Power Drive Method for MIM-Cathode Displays

A low-power drive method for reducing dissipation power by 75% is developed for cathodoluminescence displays using metal-insulator-metal (MIM) cathodes. The dissipation power is the power consumed in driver circuits for charging and discharging capacitive loads of a display panel. The drive method, called high-impedance (high-Z) drive method, reduces the panel's effective capacitance by connecting non-selected scan lines to high-impedance driver outputs. No visual crosstalk occurred in displayed images on a 3.8-cm-diagonal MIM-cathode display, regardless of significant induced voltages observed on the high-impedance scan lines. This insensitivity to the induced voltages is mainly because the polarity of induced voltages is reverse for electron emission. To reduce the induced voltage, an “enhanced high-Z drive method” is also developed. Analytical formulae to calculate the dissipation power and the amplitude of the induced voltages are devised on the basis of a capacitor model of the MIM-cathode array. Excellent agreement between measured and calculated results validates this model. On the basis of the capacitor model, the total power consumption of a 32-inch diagonal MIM-cathode display is estimated. This estimation indicates the total power consumption would be only 24 W including dissipation power at an average brightness of 200 cd/m .

Can Insects Develop Resistance to Insect Pathogenic Fungi?

Microevolutionary adaptations and mechanisms of fungal pathogen resistance were explored in a melanic population of the Greater wax moth, Galleria mellonella. Under constant selective pressure from the insect pathogenic fungus Beauveria bassiana, 25th generation larvae exhibited significantly enhanced resistance, which was specific to this pathogen and not to another insect pathogenic fungus, Metarhizium anisopliae. Defense and stress management strategies of selected (resistant) and non-selected (susceptible) insect lines were compared to uncover mechanisms underpinning resistance, and the possible cost of those survival strategies. We hypothesize that the insects developed a transgenerationally primed resistance to the fungus B. bassiana, a costly trait that was achieved not by compromising life-history traits but rather by prioritizing and re-allocating pathogen-species-specific augmentations to integumental front-line defenses that are most likely to be encountered by invading fungi. Specifically during B. bassiana infection, systemic immune defenses are suppressed in favour of a more limited but targeted repertoire of enhanced responses in the cuticle and epidermis of the integument (e.g. expression of the fungal enzyme inhibitor IMPI, and cuticular phenoloxidase activity). A range of putative stress-management factors (e.g. antioxidants) is also activated during the specific response of selected insects to B. bassiana but not M. anisopliae. This too occurs primarily in the integument, and probably contributes to antifungal defense and/or helps ameliorate the damage inflicted by the fungus or the host’s own immune responses.

Egg production curve fitting using nonlinear models for selected and nonselected lines of White Leghorn hens

Egg production curves describe the laying patterns of hen populations over time. The objectives of this study were to fit the weekly egg production rate of selected and nonselected lines of a White Leghorn hen population, using nonlinear and segmented polynomial models, and to study how the selection process changed the egg-laying patterns between these 2 lines. Weekly egg production rates over 54 wk of egg production (from 17 to 70 wk of age) were measured from 1,693 and 282 laying hens from one selected and one nonselected (control) genetic line, respectively. Six nonlinear and one segmented polynomial models were gathered from the literature to investigate whether they could be used to fit curves for the weekly egg production rate. The goodness of fit of the models was measured using Akaike's information criterion, mean square error, coefficient of determination, graphical analysis of the fitted curves, and the deviations of the fitted curves. The Logistic, Yang, Segmented Polynomial, and Grossman models presented the best goodness of fit. In this population, there were significant differences between the parameter estimates of the curves fitted for the selected and nonselected lines, thus indicating that the effect of selection changed the shape of the egg production curves. The selection for egg production was efficient in modifying the birds' egg production curve in this population, thus resulting in genetic gain from the 5th to the 54th week of egg laying and improved the peak egg production and the persistence of egg laying.

Variability for resistance to Fusarium solani culture filtrate and fusaric acid among somaclones in pea

Pea (Pisum sativum L.) somaclones of cultivars Adept, Komet and Bohatýr were obtained after selection in vitro with Fusarium solani filtrate and fusaric acid (FA). R2 regenerants were analysed by random amplification of polymorphic DNA (RAPD; OPAB4, P-14, UBC-556) and inter-retrotransposon amplification polymorphism (IRAP; Ogre) markers. Marker UBC-556 showed different banding patterns for each cultivar, but without specific bands for selected and control plants. Markers OPAB4, P14 and Ogre were useful for clear discrimination between selected and non-selected variants of all three cultivars. Flow cytometry analysis proved the same genome size of selected and non-selected pea lines. Therefore in vitro selection by pathogen derived agents could be the efficient method for obtaining of pea somaclones with increased resistance to F. solani.

Chicken heterophils from commercially selected and non-selected genetic lines express cytokines differently after in vitro exposure to Salmonella enteritidis

Resistance to pathogens such as Salmonella enteritidis (SE) is a heritable trait important in maintaining the health of chickens and reducing bacterial contamination of poultry products. In chickens, heterophils act as the first responders to bacterial infections and are, therefore, responsible for initiating the immune response against SE challenge. This study measured mRNA expression of several immune response genes [interleukin-6 (IL-6), IL-10, transforming growth factor-β4 (TGF-β4), granulocyte macrophage-colony stimulating factor (GM-CSF), and Toll-like receptor-4 (TLR-4)] by heterophils from broiler, Leghorn, and Fayoumi chickens, either non-stimulated or stimulated in vitro with SE using quantitative reverse-transcriptase PCR. We found that heterophils of commercially selected broiler and Leghorn birds had differing early heterophil responses to SE in comparison with the native Fayoumi line. Heterophil stimulation with SE in vitro increased expression of pro- (IL-6 and GM-CSF) and anti-inflammatory cytokine mRNA (IL-10 and TGF-β4) in the Fayoumi line, while the broiler and Leghorn line heterophils had decreased or no changes in the cytokine gene expression levels. The unique response of the Fayoumi line is in contrast to the lines with a history of genetic selection to increase growth or reproduction, a process which may favor reduced or suppressed inflammatory responses. The findings illustrate the potential value of native lines to provide biodiversity to enhance innate health in commercially selected poultry.

Enhanced Tolerance to Oxidative Stress with Elevated Antioxidant Capacity in Doubled Haploid Maize Derived from Microspores Exposed to Paraquat

To improve the oxidative stress tolerance of maize (Zea mays L.), doubled‐haploid (DH) plants were regenerated from in vitro selected microspores exposed to paraquat. The efficiency of in vitro selection was tested by physiological and biochemical investigations, in which the tolerance to paraquat‐mediated oxidative damage and antioxidant capacity of the second generation of fertile microspore‐selected lines (DH2) were determined. In the leaves of paraquat‐selected DH2 lines, reduced accumulation of reactive oxygen species, lower rates of chlorophyll bleaching, protein breakdown and membrane damage, but higher photosynthetic activity were detected on exposure to paraquat stress than either in the leaves of nonselected DH2 lines or in the original hybrid used as controls. The tolerant DH2 plants showed elevated activities of antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, glutathione reductase, glutathione S‐transferase, and catalase as compared to both controls. These results suggest that elevated antioxidant capacity may contribute to the paraquat tolerance of the paraquat‐selected DH2 lines and that in vitro microspore selection represents a potential way to improve oxidative stress tolerance in maize.

Fine Mapping of "Mini-Muscle," a Recessive Mutation Causing Reduced Hindlimb Muscle Mass in Mice

Prolonged selective breeding of Hsd:ICR mice for high levels of voluntary wheel running has favored an unusual phenotype (mini-muscle [MM]), apparently caused by a single Mendelian recessive allele, in which hindlimb muscle mass is reduced by almost 50%. We recently described the creation and phenotypic characterization of a population suitable for mapping the genomic location of the MM gene. Specifically, we crossed females from a high-runner line fixed for the MM allele with male C57BL/6J. F1 males were then backcrossed to the MM parent females. Backcross (BC) mice exhibited a 50:50 ratio of normal to MM phenotypes. Here, we report on linkage mapping of MM in this BC population to a 2.6335-Mb interval on MMU11. This region harbors approximately 100 expressed or predicted genes, many of which have known roles in muscle development and/or function. Identification of the genetic variation that underlies MM could potentially be very important in understanding both normal muscle function and disregulation of muscle physiology leading to disease.

Phenotypic Effects of the "Mini-Muscle" Allele in a Large HR x C57BL/6J Mouse Backcross

From outbred Hsd:ICR mice, we selectively bred 4 replicate lines for high running (High-Runner [HR] lines) on wheels while maintaining 4 nonselected lines as controls (C lines). An apparent Mendelian recessive, the "mini-muscle" (MM) allele, whose main phenotypic effect is to reduce hindlimb muscle mass by 50%, was discovered in 2 HR lines and 1 C line. This gene of major effect has gone to fixation in one selected line, remains polymorphic in another, and is now undetectable in the one C line. Homozygotes exhibit various pleiotropic effects, including a doubling of mass-specific muscle aerobic capacity, and larger hearts, livers, and spleens. To create a population suitable for mapping the genomic location of the MM allele and to better characterize its pleiotropic effects, we crossed females fixed for the MM allele with male C57BL/6J. F(1) males were then backcrossed to the MM parent females. Backcross (BC) mice (N = 404) were dissected, and a 50:50 ratio of normal to MM phenotype was observed with no overlap in relative muscle mass. In the BC, analysis of covariance revealed that MM individuals ran significantly more on days 5 and 6 of a 6-day exposure to running wheels (as in the routine selective-breeding protocol), were smaller in body mass, and had larger ventricles and spleens.

A novel gene associated with intraspecific predation in Spodoptera litura larvae

We investigated the inheritance of cannibalistic behavior in larvae of the common cutworm, Spodoptera litura. The fact that artificial selection of non-cannibalistic animals over 17 generations resulted in decrease of their cannibalistic behavior is clearly the proof of their inheritance of this behavior. Average frequency of cannibalism in selected larvae (3.2%) was significantly lower than that in non-selected larvae (24.5%). We employed the differential display RT-PCR analysis to evaluate differences in gene expression in the brains between selected and non-selected lines, and identified one gene whose expression was significantly higher in non-selected line larvae than that in selected line larvae. The identified gene with 779 bp was a novel gene without a major sequence homology with any reported genes. This gene was expressed in several tissues, fat body, brain, and hemocytes, among which the highest expression was observed in the brain. Further, the expression of this gene was very low in the brain of normally fed larvae but starvation significantly enhanced its expression only in the non-selected larvae. These results imply the possibility that the gene identified in the present study contributes to initiating cannibalistic behavior of S. litura larvae although the detailed function of this gene is unknown at present.

Effects of self-administered ethanol or water preloads on appetitive and consummatory behavior in the alcohol-preferring (P) rat.

Ethanol intake control in the selected alcohol-preferring lines of rats appeared to have shifted in some lines for both increased ethanol seeking and increased consumption once ethanol was available. It was unknown whether a small preload of ethanol would alter either the seeking or the consumption in a selected line. This study examined this issue. Alcohol-preferring (P) rats from Indiana University School of Medicine were initiated to drink ethanol using a sucrose-substitution procedure and a single daily limited-access trial. Following 30 responses on a lever, a sipper tube containing 10% ethanol extended into the operant chamber for 20 minutes. Self-administered ethanol and water preloads were tested prior to either a regular session or an extinction session. In extinction sessions, no access to the sipper tube occurred, and the number of responses occurring during 20 minutes was taken as a measure of ethanol seeking. The ethanol and water preloads had no effect on the following ethanol consumption at any time during the experiment. However, the first two ethanol preloads significantly reduced extinction responding, which did not recover to the levels observed prior to the preload tests. The data support the conclusion that ethanol seeking in the P rat can be influenced by environmental history, whereas consummatory behavior appears to be under more explicit genetic control. This gene-environment interaction suggests that, in the P rat, seeking behavior, initially set at higher levels than observed for nonselected lines, can be modified by certain environmental experiences.

14.3: Novel Low‐Power Driving Method for MIM‐Cathode Displays

AbstractA new driving method is developed for FEDs using metal‐insulator‐ metal (MIM) cathodes. The “Row Hi‐Z drive” method, in which non‐selected scan lines are connected to high‐impedance driver outputs, reduces the dissipation power by 75%. With a 60×60‐dot MIM‐cathode display, we demonstrated no visual crosstalk occurs regardless of a significant voltage crosstalk.

Stability of soybean yield through different sowing periods

Soybean yield is highly affected by sowing period and there are significant productivity losses when sowings are done outward a relatively restricted period in many regions of Brazil. Breeding cultivars less sensitive to photoperiod and to temperature variations is desirable for adaptation to wider sowing period and wider latitude range and also make irrigated soybean cultivation possible during the fall-winter seasons in frost free regions. The possibility of selecting high yielding and stable lines for yield during various sowing periods was studied by analyzing the behavior of 100 non-selected advanced lines (F9 and F10), from each one of all possible biparental crosses involving the genotypes BR85-29009, OCEPAR 8, FT-2, and BR-13. Experiments were set up in a completely randomized design with single-plant hill plots and received supplementary irrigation. Sowing was on Sept 27, Oct 20, Nov 17, and Dec 17 in 1993/94 and Sept 20, Oct 20, Nov 17, and Dec 14 in 1994/95 at Londrina, PR, Brazil. Procedures of regression analysis and minimum variance among planting date means were efficient for selecting stable lines during the four sowing seasons. It was possible to select stable and high yielding genotypes through the four sowing periods in all the crosses. No specific cross was clearly better to produce a greater number of stable genotypes.

Yield and activity of Autographa californica multicapsid nucleopolyhedrovirus and Phthorimaea operculella granulosis virus in cloned and uncloned cell lines of P. operculella.

Three selected uncloned Pop 2, Pop 3, Pop 4 and two cloned cell lines Pop cl1A and Pop cl2B were derived from the original cell line established from Phthorimaea operculella (ORS-Pop-93). Three new non-selected cell lines ORS-Pop-94A, ORS-Pop-94B and ORS-Pop-95 were also established from embryos of the same insect. Differences in morphology, growth rate and polypeptide profile were determined between these cell lines. All the cell lines were susceptible to the Autographa californica nucleopolyhedrovirus (AcMNPV). The cloned cell lines produced higher levels of AcMNPV (TCID-50 and PIB) than the parental cells and at the same rate as the Sf9 reference cell line. Substantial amounts of viral DNA were synthesized in the clone Pop cl 2B after infection with the granulosis virus of the potato tuber moth P. operculella (PTMGV) and a complete multiplication was obtained in the ORS-Pop-95 cell line. The comparison between Pop cell lines which support limited or complete replication of certain baculoviruses can offer insights into some of the molecular barriers which restrict the host range of these viruses. These cell lines with variable susceptibility to baculoviruses could also be used for in vitro recombinations, increasing their virus host range to be used for the control of this pest.

Growth, ion content and proline accumulation in NaCl-selected and non-selected cell lines of lucerne cultured on sodium and potassium salts

Batch suspension cultures of Lucerne ( Medicago media cv Rambler), established directly from surface-sterilized leaflet explants, were subcultured into media of progressively higher salinity to produce a cell line tolerant to 400 mol/m 3 NaCl. After these cells were grown for eight passages (28 days each) on NaCl-free medium, the cells were still more salt-tolerant than non-selected cells when recultured to salt supplemented media. The exposure of the NaCl-tolerant cell line to equimolar concentrations of salts other than NaCl resulted in enhanced tolerance to Na 2SO 4 or K 2SO 4 but limited tolerance to KCl. The salt-tolerant cell line showed enhanced Na + exclusion and was able to maintain higher K + and much lower Na +/K + ratios than the non-selected cell line in response to NaCl or Na 2SO 4 stress. The proline concentration was much higher in the salt-selected compared with the non-selected cell line under NaCl or Na 2SO 4 stress. Proline accumulation did not result from K 2SO 4 or KCl stress in either selected or non-selected cell lines, suggesting this is a response to Na + rather than Cl − or K +. The results suggest that growth inhibition in selected and non-selected cells under KCl stress could result from the toxic effects of Cl −.

Reduced folate carrier gene (RFC1) expression and anti-folate resistance in transfected and non-selected cell lines.

Methotrexate transport deficiency due to decreased reduced folate carrier (RFC) activity has been observed in several cell lines selected for resistance to methotrexate (MTX). Since MTX resistance is multifactorial, however, it is difficult to quantify the relative importance of changes in RFC activity in selected cell lines and even more so to determine the relative contribution of naturally occurring RFC activity in the MTX sensitivity of non-selected cell lines. We examined the role of RFC in MTX resistance by studying a transport-deficient cell line transfected with the gene for human RFC, RFC1, and by correlating relative RFC1 expression with MTX and trimetrexate (TMTX) growth inhibition (GI50) in a panel of cell lines used in the NCI Anticancer Drug Screen. Clones of transport-deficient, MTX-resistant ZR-75-1 human breast cancer cells (MTX(R) ZR-75-1) transfected with RFC1 were 250-fold more sensitive to MTX and 300-fold more resistant to TMTX than control cell clones, showing that restoration of RFC activity has a significant impact on MTX and TMTX cytotoxicity. We also surveyed 40 of the 60 cell lines in the NCI drug screen panel for RFCI RNA levels by a quantitative RT-PCR assay. RFCI RNA levels varied over a range of 15-fold, with only 1 cell line found to be null in expression. Using data from the 6-day drug exposure assay, RFC1 correlated positively with MTX and negatively with TMTX cytotoxicity. As predicted by transfection studies, the calculated difference between MTX and TMTX potency was even more strongly correlated with RFC1 RNA levels of the cell lines. In addition, compounds in the NCI Anticancer Drug Screen database with cytotoxicity profiles which correlated with RFC1 RNA levels or with the calculated difference in MTX-TMTX potency were examined for MTX uptake inhibition and cytotoxicity in the RFC1-transfected MTX(R) ZR-75-1 cell line. Overall, our data demonstrate the importance of RFC1 in MTX resistance both as a transgene and as a constitutively expressed gene in non-selected cell lines.