Antibiotics are not recommended to eliminate Salmonella organisms from reptiles because of the development of antibiotic resistance. Further studies are necessary to determine whether the use of microcin-producing bacteria will be effective in controlling Salmonella infections in companion reptiles.
Introns can increase gene expression levels using a variety of mechanisms collectively referred to as Intron Mediated Enhancement (IME). To date, the magnitude of IME has been quantified in human cell culture and plant models by comparing intronless reporter gene expression levels to those of intron-bearing reporter genes in vitro (mRNA, Western Blots, protein activity), using genome editing technologies that lacked full control of locus and copy number. Here, for the first time, we quantified IME in vivo, in terms of protein expression levels, using fluorescent reporter proteins expressed from a single, defined locus in Caenorhabditis elegans. To quantify the magnitude of IME, we developed a microfluidic chip-based workflow to mount and image individual animals, including software for operation and image processing. We used this workflow to systematically test the effects of position, number and sequence of introns on two different proteins, mCherry and mEGFP, driven by two different promoters, vit-2 and hsp-90. We found the three canonical synthetic introns commonly used in C. elegans transgenes increased mCherry protein concentration by approximately 50%. The naturally-occurring introns found in hsp-90 also increased mCherry expression level by about 50%. Furthermore, and consistent with prior results examining mRNA levels, protein activity or phenotypic rescue, we found that a single, natural or synthetic, 5' intron was sufficient for the full IME effect while a 3' intron was not. IME was also affected by protein coding sequence (50% for mCherry and 80% for mEGFP) but not strongly affected by promoter 46% for hsp-90 and 54% for the stronger vit-2. Our results show that IME of protein expression in C. elegans is affected by intron position and contextual coding sequence surrounding the introns, but not greatly by promoter strength. Our combined controlled transgenesis and microfluidic screening approach should facilitate screens for factors affecting IME and other intron-dependent processes. + equal contribution
105 replicatively senesce. C) Representative single cell traces of mother Htb2 levels showing missegregation (shaded) 106 and active retrograde correction events. Corrections can occur quickly (top), or can take hours to be completed 107 (middle). A GLM becomes terminal (bottom) if it is not corrected. (*) indicates the formation of new buds, and both 108 cells with RETRN events produce additional daughters. AU indicates arbitrary units. D) Missegregation 109 probabilities increase dramatically near the end of replicative lifespan. n=359 mother cells examined. E) Over their 110 entire replicative lifespan, individual mother cells have a greater than 70% chance of having one or more 111 missegregation events. F) Genomic DNA and histones co-localize during GLM events. Two cells expressing 112 Htb2:mCherry and stained with a live DNA dye Hoechst 3342. G) Time-lapse dynamics of a GLM with RETRN 113 correction (top, mother cell replicative age 14) and a terminal missegregation (bottom, mother cell replicative age 114 12) in cells co-expressing Htb2:mCherry and Nup49:GFP. During both GLMs the nuclear envelope is clearly visible 115 in both mother (M) and daughter (D) cells. See Videos 6 and 7. H) Time-lapse dynamics of a GLM with RETRN 116 correction (top, mother cell replicative age 13) and a terminal missegregation (bottom, mother cell replicative age 117 16) in cells expressing Htb2:mCherry and Spc72:GFP. Both spindle poles can be seen to enter the daughter (D)118 during these events, and during the RETRN event a spindle pole returns to the mother (M). In the terminal 119 missegregation, the spindle pole fails to reenter the mother cell. See Videos 8 and 9. Times are indicated in 120 hours:mins from the start of the displayed time-lapse, not the start of the experiment. Arrows indicate mother cells 121 without visible chromatin. 360 Raghuraman for constructive discussions. We also thank L. Veenhoff and Kaeberlein lab 361 members for feedback and advice. Strains YSI129, AMY914 and AMY1081were generous gifts 362 from Jessica Tyler and Adele Marston. This work was supported by NIH grants T32AG000057, 363 R01AG056359, and P30AG013280. 364 365 References 366 Bakker E. 2016. Quantitative fluorescence microscopy methods for studying transcription with367 application to the yeast GAL1 promoter. The University of Edinburgh. 368 Bakker E, Swain PS, Crane MM. 2017. Morphologically constrained and data informed cell segmentation 369 of budding yeast. Bioinformatics. 384 Tyers M. 2004. CDK activity antagonizes Whi5, an inhibitor of G1/S transcription in yeast. Cell 385 117:899-913. 386 Crane MM, Clark IBN, Bakker E, Smith S, Swain PS. 2014. A microfluidic system for studying ageing 387 and dynamic single-cell responses in budding yeast. PLoS One 9:e100042. 388 D'Amours D, Stegmeier F, Amon A. 2004. Cdc14 and condensin control the dissolution of cohesin-389 independent chromosome linkages at repeated DNA. Cell 117:455-469. Cross FR. 2007. The effects of molecular noise and size 399 control on variability in the budding yeast cell...
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