scholarly journals Genetic Tools for Allelic Replacement in Burkholderia Species

2008 ◽  
Vol 74 (14) ◽  
pp. 4498-4508 ◽  
Author(s):  
Ashley R. Barrett ◽  
Yun Kang ◽  
Ken S. Inamasu ◽  
Mike S. Son ◽  
Joseph M. Vukovich ◽  
...  
Keyword(s):  
Selectable Marker ◽  
Clinical Importance ◽  
The United States ◽  
Gene Replacement ◽  
Betaine Aldehyde Dehydrogenase ◽  
Selectable Markers ◽  
Α Subunit ◽  
Genetic Traits ◽  
Allelic Replacement ◽  
Resistance Markers

ABSTRACT Allelic replacement in the Burkholderia genus has been problematic due to the lack of appropriate counter-selectable and selectable markers. The counter-selectable marker sacB, commonly used in gram-negative bacteria, is nonselective on sucrose in many Burkholderia species. In addition, the use of antibiotic resistance markers of clinical importance for the selection of desirable genetic traits is prohibited in the United States for two potential bioterrorism agents, Burkholderia mallei and Burkholderia pseudomallei. Here, we engineered a mutated counter-selectable marker based on the B. pseudomallei PheS (the α-subunit of phenylalanyl tRNA synthase) protein and tested its effectiveness in three different Burkholderia species. The mutant PheS protein effectively killed 100% of the bacteria in the presence of 0.1% p-chlorophenylalanine. We assembled the mutant pheS on several allelic replacement vectors, in addition to constructing selectable markers based on tellurite (Telr) and trimethoprim (Tpr) resistance that are excisable by flanking unique FLP recombination target (FRT) sequences. As a proof of concept, we utilized one of these gene replacement vectors (pBAKA) and the Telr-FRT cassette to produce a chromosomal mutation in the Burkholderia thailandensis betBA operon, which codes for betaine aldehyde dehydrogenase and choline dehydrogenase. Chromosomal resistance markers could be excised by the introduction of pFLP-AB5 (Tpr), which is one of two constructed flp-containing plasmids, pFLP-AB4 (Telr) and pFLP-AB5 (Tpr). These flp-containing plasmids harbor the mutant pheS gene and allow self curing on media that contain p-chlorophenylalanine after Flp-FRT excision. The characterization of the ΔbetBA::Telr-FRT and ΔbetBA::FRT mutants indicated a defect in growth with choline as a sole carbon source, while these mutants grew as well as the wild type with succinate and glucose as alternative carbon sources.

scholarly journals Glyphosate Resistance as a Novel Select-Agent-Compliant, Non-Antibiotic-Selectable Marker in Chromosomal Mutagenesis of the Essential Genes asd and dapB of Burkholderia pseudomallei

2009 ◽  
Vol 75 (19) ◽  
pp. 6062-6075 ◽  
Author(s):  
Michael H. Norris ◽  
Yun Kang ◽  
Diana Lu ◽  
Bruce A. Wilcox ◽  
Tung T. Hoang
Keyword(s):  
Burkholderia Pseudomallei ◽  
Selectable Marker ◽  
Genetic Manipulation ◽  
Chromosome 1 ◽  
Chromosome 2 ◽  
Gene Encoding ◽  
Selectable Markers ◽  
Genetic Manipulations ◽  
Semialdehyde Dehydrogenase ◽  
Allelic Replacement

ABSTRACT Genetic manipulation of the category B select agents Burkholderia pseudomallei and Burkholderia mallei has been stifled due to the lack of compliant selectable markers. Hence, there is a need for additional select-agent-compliant selectable markers. We engineered a selectable marker based on the gat gene (encoding glyphosate acetyltransferase), which confers resistance to the common herbicide glyphosate (GS). To show the ability of GS to inhibit bacterial growth, we determined the effective concentrations of GS against Escherichia coli and several Burkholderia species. Plasmids based on gat, flanked by unique flip recombination target (FRT) sequences, were constructed for allelic-replacement. Both allelic-replacement approaches, one using the counterselectable marker pheS and the gat-FRT cassette and one using the DNA incubation method with the gat-FRT cassette, were successfully utilized to create deletions in the asd and dapB genes of wild-type B. pseudomallei strains. The asd and dapB genes encode an aspartate-semialdehyde dehydrogenase (BPSS1704, chromosome 2) and dihydrodipicolinate reductase (BPSL2941, chromosome 1), respectively. Mutants unable to grow on media without diaminopimelate (DAP) and other amino acids of this pathway were PCR verified. These mutants displayed cellular morphologies consistent with the inability to cross-link peptidoglycan in the absence of DAP. The B. pseudomallei 1026b Δasd::gat-FRT mutant was complemented with the B. pseudomallei asd gene on a site-specific transposon, mini-Tn7-bar, by selecting for the bar gene (encoding bialaphos/PPT resistance) with PPT. We conclude that the gat gene is one of very few appropriate, effective, and beneficial compliant markers available for Burkholderia select-agent species. Together with the bar gene, the gat cassette will facilitate various genetic manipulations of Burkholderia select-agent species.

Knockdown Resistance-Associated Mutations Dominate Populations of the Common Bed Bug (Hemiptera: Cimicidae) Across the South Central United States

2019 ◽  
Vol 56 (6) ◽  
pp. 1678-1683 ◽  
Author(s):  
Jeffrey G Holleman ◽  
Grant A Robison ◽  
Ian J Bellovich ◽  
Warren Booth
Keyword(s):  
United States ◽  
Insecticide Resistance ◽  
The United States ◽  
Knockdown Resistance ◽  
The South ◽  
Α Subunit ◽  
Old World ◽  
Bed Bug ◽  
South Central ◽  
Central United States

Abstract Despite awareness of the mutations conferring insecticide resistance in the bed bug, Cimex lectularius L. (Hemiptera: Cimicidae), within the United States few studies address the distribution and frequency of these. Within the United States, studies have focused on collections made along the East Coast and Midwest, documenting the occurrence of two mutations (V419L and L925I) within the voltage-gated sodium channel α-subunit gene shown to be associated with knockdown resistance (kdr) to pyrethroids. Here, the distribution and frequency of the V419L and L925I site variants is reported from infestations sampled within Oklahoma and its immediately adjacent states. Additionally, the presence of a mutation previously undocumented in the United States (I935F) is noted. While novel in the United States, this mutation has previously been reported in Australian and Old World populations. No infestations were found to harbor wild-type individuals, and hence susceptible, at each of the three sites. Instead, ~21% were found to possess the resistant mutation at the L925I site (haplotype B), ~77% had mutations at both the V419L and L925I sites (haplotype C), and 2% possessed the mutation at the L936F site (haplotype Ab). The high frequency of haplotype C corresponds to previous studies in the United States, and contrasts dramatically with those of the Old World and Australia. The data presented here provide insight into the contemporary occurrence of kdr-associated insecticide resistance in the South Central United States, a region for which data have previously been absent. These data suggest that New World and Old World/Australian infestations are likely to have originated from different origins.

scholarly journals Comparison of Two Alternative Dominant Selectable Markers for Wine Yeast Transformation

2004 ◽  
Vol 70 (12) ◽  
pp. 7018-7023 ◽  
Author(s):  
Eduardo Cebollero ◽  
Ramon Gonzalez
Keyword(s):  
Laboratory Strain ◽  
Wine Yeast ◽  
Selection Marker ◽  
Selectable Markers ◽  
Yeast Strains ◽  
Strain Transformation ◽  
Wine Yeast Strain ◽  
Industrial Strains ◽  
Resistance Markers ◽  
Selection For

ABSTRACT Genetic improvement of industrial yeast strains is restricted by the availability of selectable transformation markers. Antibiotic resistance markers have to be avoided for public health reasons, while auxotrophy markers are generally not useful for wine yeast strain transformation because most industrial Saccharomyces cerevisiae strains are prototrophic. For this work, we performed a comparative study of the usefulness of two alternative dominant selectable markers in both episomic and centromeric plasmids. Even though the selection for sulfite resistance conferred by FZF1-4 resulted in a larger number of transformants for a laboratory strain, the p-fluoro-dl-phenylalanine resistance conferred by ARO4-OFP resulted in a more suitable selection marker for all industrial strains tested. Both episomic and centromeric constructions carrying this marker resulted in transformation frequencies close to or above 103 transformants per μg of DNA for the three wine yeast strains tested.

scholarly journals Molecular pathway analysis indicates a distinct metabolic and immune phenotype in women with right-sided colon cancer

2019 ◽  
Author(s):  
Yazhi Sun ◽  
Varvara Mironova ◽  
Ying Chen ◽  
Elliott P.F. Lundh ◽  
Qian Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Colon Cancer ◽  
Transverse Colon ◽  
Clinical Importance ◽  
Bioinformatic Analysis ◽  
The United States ◽  
Anatomical Location ◽  
Nutrient Metabolism ◽  
Immune Phenotype ◽  
Tumor Tissues

AbstractColon cancer is the third most commonly diagnosed cancer in the United States. Recent reports have shown that the location of the primary tumor is of clinical importance. Patients with right-sided cancers (RCCs) (tumors arising between the cecum and proximal transverse colon) have poorer clinical outcomes than those with left-sided colon cancers (LCCs) (tumors arising between the distal transverse colon and sigmoid colon, excluding the rectum). Interestingly, women have a lower incidence of colon cancer than men do. However, women have a higher propensity for RCC than men. Identification of gene expression differences between RCC and LCC is considered a potential means of prognostication. Furthermore, studying colon cancer sidedness could reveal important predictive markers for response to various treatments. This study provides a comprehensive bioinformatic analysis of various genes and molecular pathways that correlated with sex and anatomical location of colon cancer using four publicly available annotated datasets housed in the National Center for Biotechnology Information’s Gene Expression Omnibus (GEO). We identified differentially expressed genes in tumor tissues from women with RCC, which showed attenuated energy and nutrient metabolism when compared to women with LCC. Specifically, we showed that the downregulation of 5’ AMP-activated protein kinase alpha subunit (AMPKα) and downregulated anti-tumor immune response in women with RCC. This difference was not seen when comparing tumor tissues from men with RCC to men with LCC. Therefore, women with RCC may have a specific metabolic and immune phenotype which accounts for differences in prognosis and treatment response.

The Role of Adolescent Health in Adult SES Outcomes

2018 ◽  
Vol 18 (2) ◽  
Author(s):  
Dana C. Andersen ◽  
Pinar Mine Gunes
Keyword(s):  
Life Cycle ◽  
Adolescent Health ◽  
Household Income ◽  
Family Background ◽  
The United States ◽  
Future Health ◽  
Genetic Traits ◽  
Socioeconomic Outcomes ◽  
Income Accounting

AbstractThis paper explores the effects of adolescent health and adolescent mental health on long-term socioeconomic outcomes in the United States. Within-twin estimations are employed to overcome the bias generated by unobserved family background and genetic traits. The results indicate that poor adolescent health reduces long-term health, earnings, and household income. Accounting for life-cycle effects suggests that the effect of poor adolescent health on household income and earnings increases over the life cycle. Finally, we demonstrate that the effects on income are a consequence of the persistence of adolescent health on future health.

scholarly journals Breeding Trait Priorities of the Cranberry Industry in the United States and Canada

HortScience ◽  
2018 ◽  
Vol 53 (10) ◽  
pp. 1467-1474 ◽  
Author(s):  
R. Karina Gallardo ◽  
Parichat Klingthong ◽  
Qi Zhang ◽  
James Polashock ◽  
Amaya Atucha ◽  
...  
Keyword(s):  
Fruit Quality ◽  
Fruit Size ◽  
The United States ◽  
Fruit Rot ◽  
Selection Strategy ◽  
Anthocyanin Content ◽  
Biotic And Abiotic Stresses ◽  
Breeding Programs ◽  
Genetic Traits ◽  
Industry Needs

Informed assessment of priority genetic traits in plant breeding programs is important to improve the efficiency of developing cultivars suited to current climate and industry needs. The efficiency of genetic improvement is critical for perennial crops such as cranberries, as they usually involve more resources, time, and funding compared with other crops. This study investigated the relative importance of cranberry producers’ preferences for breeding traits related to fruit quality, productivity, plant physiology, and resistance to biotic and abiotic stresses. Industry responses revealed that fruit characteristics affecting fruit quality, including firmness, fruit size and anthocyanin content, and resistance to fruit rot, were the most desired traits in new cranberry cultivar release. These traits have the potential to increase the quality standards needed to process high-value sweetened dried cranberry products, positively affecting price premiums received by producers, which is critical for the economic viability of the cranberry industry. Our findings will be useful to breeders and allied scientists seeking to develop an advanced DNA-based selection strategy that would impact the global cranberry industry.

scholarly journals Cloning of Phanerochaete chrysosporium leu2 by Complementation of Bacterial Auxotrophs and Transformation of Fungal Auxotrophs

1998 ◽  
Vol 64 (7) ◽  
pp. 2624-2629 ◽  
Author(s):  
Laura Schick Zapanta ◽  
Takefumi Hattori ◽  
Magarita Rzetskaya ◽  
Ming Tien
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Phanerochaete Chrysosporium ◽  
Expression Vector ◽  
Selectable Marker ◽  
Genomic Clone ◽  
Selectable Markers ◽  
E Coli ◽  
Number Of Genes ◽  
Isopropylmalate Dehydrogenase

ABSTRACT A Phanerochaete chrysosporium cDNA library was constructed in an expression vector that allows expression in bothEscherichia coli and Saccharomyces cerevisiae. This expression vector, λYES, contains the lacZ promoter for expression in E. coli and the GAL1 promoter for expression in yeast. A number of genes were cloned by complementation of bacterial amino acid auxotrophs. The cDNA encoding the β-isopropylmalate dehydrogenase from P. chrysosporiumwas characterized further. The genomic clone (gleu2) was subsequently isolated and was used successfully as a selectable marker to transform P. chrysosporium auxotrophs for LEU2. Protoplasts for transformation were prepared with readily obtained conidiospores rather than with basidiospores, which were used in previous P. chrysosporium transformation procedures. The method described here allows other genes to be isolated from P. chrysosporium for use as selectable markers.

scholarly journals Role of Brain Derived Extracellular Vesicles in Decoding Sex Differences Associated with Nicotine Self-Administration

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1883
Author(s):  
Sneh Koul ◽  
Victoria L. Schaal ◽  
Subhash Chand ◽  
Steven T. Pittenger ◽  
Neetha Nanoth Vellichirammal ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Brain Function ◽  
Cell Communication ◽  
Clinical Importance ◽  
The United States ◽  
Female Rats ◽  
Molecular Pathways ◽  
Self Administration ◽  
Critical Knowledge ◽  
Male And Female Rats

Smoking remains a significant health and economic concern in the United States. Furthermore, the emerging pattern of nicotine intake between sexes further adds a layer of complexity. Nicotine is a potent psychostimulant with a high addiction liability that can significantly alter brain function. However, the neurobiological mechanisms underlying nicotine’s impact on brain function and behavior remain unclear. Elucidation of these mechanisms is of high clinical importance and may lead to improved therapeutics for smoking cessation. To fill in this critical knowledge gap, our current study focused on identifying sex-specific brain-derived extracellular vesicles (BDEV) signatures in male and female rats post nicotine self-administration. Extracellular vesicles (EVs) are comprised of phospholipid nanovesicles such as apoptotic bodies, microvesicles (MVs), and exosomes based on their origin or size. EVs are garnering significant attention as molecules involved in cell–cell communication and thus regulating the pathophysiology of several diseases. Interestingly, females post nicotine self-administration, showed larger BDEV sizes, along with impaired EV biogenesis compared to males. Next, using quantitative mass spectrometry-based proteomics, we identified BDEV signatures, including distinct molecular pathways, impacted between males and females. In summary, this study has identified sex-specific changes in BDEV biogenesis, protein cargo signatures, and molecular pathways associated with long-term nicotine self-administration.

scholarly journals Nourseothricin N-acetyl transferase (NAT), a new selectable marker for nuclear gene expression in Chlamydomonas

Plant Methods ◽  
2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Xinjia Yang ◽  
Jialin Peng ◽  
Junmin Pan
Keyword(s):  
Resistance Genes ◽  
Selectable Marker ◽  
Ectopic Expression ◽  
Nuclear Gene ◽  
Expression Vectors ◽  
Cross Resistance ◽  
Hygromycin B ◽  
Selectable Markers ◽  
Hygromycin B Resistance ◽  
Transgenic Strains

Abstract Background Chlamydomonas reinhardtii is a unicellular green alga, which is a most commonly used model organism for basic research and biotechnological applications. Generation of transgenic strains, which usually requires selectable markers, is instrumental in such studies/applications. Compared to other organisms, the number of selectable markers is limited in this organism. Nourseothricin (NTC) N-acetyl transferase (NAT) has been reported as a selectable marker in a variety of organisms but not including C. reinhardtii. Thus, we investigated whether NAT was useful and effective for selection of transgenic strains in C. reinhardtii. The successful use of NAT would provide alterative choice for selectable markers in this organism and likely in other microalgae. Results C. reinhardtii was sensitive to NTC at concentrations as low as 5 µg/ml. There was no cross-resistance to nourseothricin in strains that had been transformed with hygromycin B and/or paromomycin resistance genes. A codon-optimized NAT from Streptomyces noursei was synthesized and assembled into different expression vectors followed by transformation into Chlamydomonas. Around 500 transformants could be obtained by using 50 ng DNA on selection with 10 µg/ml NTC. The transformants exhibited normal growth rate and were stable at least for 10 months on conditions even without selection. We successfully tested that NAT could be used as a selectable marker for ectopic expression of IFT54-HA in strains with paromomycin and hygromycin B resistance markers. We further showed that the selection rate for IFT54-HA positive clones was greatly increased by fusing IFT54-HA to NAT and processing with the FMDV 2A peptide. Conclusions This work represents the first demonstration of stable expression of NAT in the nuclear genome of C. reinhardtii and provides evidence that NAT can be used as an effective selectable marker for transgenic strains. It provides alterative choice for selectable markers in C. reinhardtii. NAT is compatible with paromomycin and hygromycin B resistance genes, which allows for multiple selections.

scholarly journals Genome-wide patterns of differentiation within and among U.S. commercial honey bee stocks

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Perot Saelao ◽  
Michael Simone-Finstrom ◽  
Arian Avalos ◽  
Lelania Bilodeau ◽  
Robert Danka ◽  
...  
Keyword(s):  
Honey Bee ◽  
Phenotypic Diversity ◽  
The United States ◽  
Population Diversity ◽  
Parasite Resistance ◽  
Genetic Characteristics ◽  
Genetic Traits ◽  
Work Related ◽  
Candidate Regions ◽  
Future Work

Abstract Background The population genetics of U.S. honey bee stocks remain poorly characterized despite the agricultural importance of Apis mellifera as the major crop pollinator. Commercial and research-based breeding programs have made significant improvements of favorable genetic traits (e.g. production and disease resistance). The variety of bees produced by artificial selection provides an opportunity to characterize the genetic diversity and regions of the genome undergoing selection in commonly managed stocks. Results Pooled sequencing of eight honey bee stocks found strong genetic similarity among six of the stocks. Two stocks, Pol-line and Hilo, showed significant differentiation likely due to their intense and largely closed breeding for resistance to the parasitic Varroa mite. Few variants were identified as being specific to any one stock, indicating potential admixture among the sequenced stocks. Juxtaposing the underlying genetic variation of stocks selected for disease- and parasite-resistance behavior, we identified genes and candidate regions putatively associated with resistance regulated by hygienic behavior. Conclusion This study provides important insights into the distinct genetic characteristics and population diversity of honey bee stocks used in the United States, and provides further evidence of high levels of admixture in commercially managed honey bee stocks. Furthermore, breeding efforts to enhance parasite resistance in honey bees may have created unique genetic profiles. Genomic regions of interest have been highlighted for potential future work related to developing genetic markers for selection of disease and parasite resistance traits. Due to the vast genomic similarities found among stocks in general, our findings suggest that additional data regarding gene expression, epigenetic and regulatory information are needed to more fully determine how stock phenotypic diversity is regulated.

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