Application Of Selection Pressure Research Articles

Frequency of RPGRIP1 and MAP9 genetic modifiers of canine progressive retinal atrophy, in 132 breeds of dog.

Variants in RPGRIP1 and MAP9, termed RPGRIP1ins44 and MAP9del respectively, are both associated with a form of canine progressive retinal atrophy referred to as RPGRIP1-CRD and have both been demonstrated to modify the development and progression of this disease. In the current study both variants were genotyped in at least 50 dogs of 132 diverse breeds and the data reveal that both segregate in multiple breeds. Individually, each variant is common within largely non-overlapping subsets of breed, and there is a negative correlation between their frequencies within breeds that segregate both variants. The frequency of both variants exceeds 0.05 in a single breed only, the Miniature Longhaired Dachshund. These data indicate that both variants are likely to be ancient and predate the development and genetic isolation of modern dog breeds. That both variants are present individually at high frequency in multiple breeds is consistent with the hypothesis that homozygosity of either variant alone is not associated with a clinically relevant phenotype, whereas the negative correlation between the two variants is consistent with the application of selective pressure, from dog breeders, against homozygosity at both loci, probably due to the more severe phenotype associated with homozygosity at both loci.

Simultaneous saccharification and fermentation of hemicellulose to butanol by a non-sporulating Clostridium species

Production of lignocellulosic butanol has drawn increasing attention. However, currently few microorganisms can produce biofuels, particularly butanol, from lignocellulosic biomass via simultaneous saccharification and fermentation. Here we report discovery of a wild-type, mesophilic Clostridium sp. strain MF28 that ferments xylan to produce butanol (up to 3.2g/L) without the addition of saccharolytic enzymes and without any chemical pretreatments. Application of selective pressure from 2-deoxy-d-glucose facilitated isolation of strain MF28, which exhibits inactivation of genes (gid and ccp genes) responsible for carbon catabolite repression, thus allowing strain MF28 to simultaneously ferment a combination of glucose (30g/L), xylose (15g/L), and arabinose (15g/L) to produce 11.9g/L of butanol. Strain MF28 possesses several unique features: (i) non-sporulating, (ii) no acetone/ethanol, (iii) complete hemicellulose-binding enzymatic domain, and (iv) absence of carbon catabolite repression. These unique characteristics demonstrate the industrial potential of strain MF28 for cost-effective biofuel generation from lignocellulosic biomass.

Effect of Silver Nanoparticles and Antibiotics on Antibiotic Resistance Genes in Anaerobic Digestion

Water resource recovery facilities have been described as creating breeding ground conditions for the selection, transfer, and dissemination of antibiotic resistance genes (ARGs) among various bacteria. The objective of this study was to determine the effect of direct addition of antibiotic and silver nanoparticles (Ag NPs, or nanosilver) on the occurrence of ARGs in thermophilic anaerobic digesters. Test thermophilic digesters were amended with environmentally-relevant concentrations of Ag NP (0.01, 0.1, and 1.0 mg-Ag/L; corresponding to approximately 0.7, 7.0, and 70 mg-Ag/kg total solids) and sulfamethoxazole (SMX) that span susceptible to resistant classifications (1, 5, and 50 mg/L) as potential selection pressures for ARGs. Tetracycline (tet(O), tet(W)) and sulfonamide (sulI, sulII) ARGs and the integrase enzyme gene (intI1) associated with Class 1 integrons were measured in raw sludge, test thermophilic digesters, a control thermophilic digester, and a control mesophilic digester. There was no apparent effect of Ag NPs on thermophilic anaerobic digester performance. The maximum SMX addition (50 mg/L) resulted in accumulation of volatile fatty acids and low pH, alkalinity, and volatile solids reduction. There was no significant difference between ARG gene copy numbers (absolute or normalized to 16S rRNA genes) in amended thermophilic digesters and the control thermophilic digester. Antibiotic resistance gene copy numbers in digested sludge ranged from 10(3) to 10(6) copies per microL (approximately 8 x10(1) to 8 x 10(4) copies per microg) of sludge as result of a 1-log reduction of ARGs (2-log reduction for intI1). Quantities of the five ARGs in raw sludge ranged from 10(4) to 10(8) copies per microL (approximately 4 x 10(2) to 4 x 10(6) per microg) of sludge. Test and control thermophilic digesters (53 degrees C, 12-day solids retention time [SRT]) consistently reduced but did not eliminate levels of all analyzed genes. The mesophilic digester (37 degrees C, 20-day SRT) also reduced levels of sulI, sulII, and intI1 genes, but levels of tet(O) and tet(W) were the same or higher than in raw sludge. Antibiotic resistance gene reductions remained constant despite the application of selection pressures, which suggests that digester operating conditions are a strong governing factor of the bacterial community composition and thus the prevalence of ARGs.

Alternate phase variation in expression of two major surface membrane proteins (MBA and UU376) ofUreaplasma parvumserovar 3

Ureaplasma urealyticum and Ureaplasma parvum are commensals and pathogens of the human urogenital tract and of newborn infants. There are four distinct U. parvum serovars and 10 distinct U. urealyticum serovars. Both species possess a distinct immunodominant variable surface protein, the multiple banded antigen (MBA), which shows size variability among isolates as a result of changes in the number of C-terminal repeating units. Adjacent to the MBA gene (UU375) lies UU376, which was annotated as 'Ureaplasma-specific conserved hypothetical gene'. In four different strains of U. parvum serovar 3, we demonstrated expression of UU376 by Western blot analysis and phase variation between UU376, here designated Upvmp376 (Ureaplasma phase-variable membrane protein 376), and MBA after application of selective pressure with hyperimmune antisera directed against either protein. By Southern blot analysis, we found that the switch between MBA and Upvmp376 expression is associated with a DNA inversion event in which the nonrepetitive region of the MBA gene and its putative promoter region are opposed to either the repetitive region of MBA or UU376. We propose that in U. parvum serovar 3, and presumably in all U. parvum and U. urealyticum, an inversion event at specific sites effects an alternate ON/OFF switching of the genes UU375 and UU376.

Effect of the kanamycin resistance marker on stability of 2μ-based expression plasmids

In this paper we describe the effect of the kanamycin resistance gene (Kmr) on 2?m-based plasmid maintenance in Saccharomyces cerevisiae. The influence of this marker gene on the loss of the stable model-vectors proved to be constant, as well as independent of carbon source and culture growth rates. In strains for GALUAS - driven heterologous protein production introduction of Kmr resulted in curing of the yeast episomal plasmid (YEp) from the population in a small number of generations. Application of selective pressure on the strain producing recombinant penicillin G amidase (rPGA) did not provide the expected increase of protein yield. The influence of genetic elements for heterologous protein production on vector stability was examined, and the most destabilizing factors prove to be the presence and expression of the foreign gene.

Clinical isolates of HIV-1 contain few pre-existing proteinase inhibitor resistance-conferring mutations

Proteinase inhibitors are an important new class of antiviral agents for AIDS, however, in vitro experiments have identified proteinase mutations that confer resistance to several different families of the inhibitors. This study was undertaken to determine if these resistance-conferring amino-acid substitutions occur in HIV strains before the application of selective pressure. We determined the nucleic acid sequence of the proteinase gene from the 23 clinical isolates of HIV-1 and three laboratory-adapted strains using a method that detects the majority species present in viral populations. Analysis of minor subpopulations will require alternative strategies. The clinical isolates studied contained an average of 3 (range 1–8_amino-acid substitutions as compared to the prototypical BH10 sequence. We did not detect substitutions characteristic of reported highly proteinase-resistant strains. These results suggest significant variation occurs in the HIV-1 proteinase gene but pre-existing highly proteinase-resistant strains are uncommon.

Transient induction of single GST-P positive hepatocytes by DEN.

The single cells positive for placental glutathione S-transferase (GST-P), detectable in livers of rats soon after treatment with hepatocarcinogens, are possible 'initiated cells', the hypothesis tested in the present series of experiments. No low dose threshold was observed in male Sprague-Dawley rats at different single doses of diethylnitrosamine (DEN) although a plateau was reached between 160 and 200 mg/kg body weight. At the latter single dose approximately 12,400 positive cells/cm3 were observed immunohistochemically in rat livers after one week, the numbers then decreasing to week 8 and thereafter rising again. In the early stages single cells predominated but with time a gradual increase in mini-foci and larger lesions became evident. Application of selection pressure (feeding of diet containing 0.02% 2-AAF plus partial hepatectomy) to rats 2-24 weeks after single DEN-treatment resulted in the formation of large foci positive for GST-P, especially in the early stages, the growth response being less pronounced with time. The number of foci, on the other hand, was correlated with the number of single cells/mini-foci detected in hepatectomy tissue of the same individuals. These results suggest that the early GST-P positive populations could be the precursor for preneoplastic foci and nodules.

Persistent effect of a low dose of preadministered diethylnitrosamine on the induction of enzyme-altered foci in rat liver.

The effect of a low dose of preadministered diethylnitrosamine (DEN) on the induction of enzyme-altered foci in the livers of male full-grown Fischer 344 rats was studied. As a pretreatment, DEN at a dose of 10 mg/kg body wt was injected i.p. At various times after DEN pretreatment a complete initiation, consisting of administration of the same dose of DEN by the same route in rats subjected to partial hepatectomy (PH), was performed, followed by application of selection pressure. Enzyme-altered foci stained with gamma-glutamyltranspeptidase (gamma-GTP) and glutathione S-transferase placental form (GST-P) were then assayed. Decreases in the numbers and areas of foci in the rats which received saline + PH 14 or 28 days after DEN pretreatment were observed in comparison with rats which received saline + PH immediately after DEN. On the other hand, the numbers and areas of foci were not decreased in rats which received the complete initiation, consisting of DEN + PH, at various times after DEN pretreatment when compared with rats which received these at the same time as the DEN pretreatment. This persistent effect of DEN pretreatment on the complete initiation lasted up to 182 days after the time of DEN pretreatment. In this experiment, GST-P was found to be a more sensitive marker for the detection of putative preneoplastic liver-cell foci than gamma-GTP.

Amplification of expression of hepatitis B surface antigen in 3T3 cells cotransfected with a dominant-acting gene and cloned viral DNA.

3T3 cells containing hepatitis B virus DNA sequences can be efficiently selected by exposure to methotrexate after cotransfection with cloned viral DNA and DNA coding for a methotrexate-resistant dihydrofolate reductase. More than 75% of methotrexate-resistant cells isolated after cotransfection with a head-to-tail tandem of the hepatitis B virus genome synthesized viral surface antigen. The antigen was released into the culture medium in the form of 22-nm particles with buoyant density of 1.20 g/ml. No other virally coded proteins were detected in the cells or the culture medium. Application of selective pressure by increasing the concentration of methotrexate resulted in an amplification of viral DNA sequences and a concomitant increase in the rate of synthesis and release of hepatitis B surface antigen. The ability to produce large amounts of surface antigen appears to be a stable trait and has been maintained in these cultures through more than 30 passages.