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- Liljeruhm, Josefine, et al.
(författare)
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Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology
- 2018
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Ingår i: Journal of Biological Engineering. - : BIOMED CENTRAL LTD. - 1754-1611. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Background: Coral reefs are colored by eukaryotic chromoproteins (CPs) that are homologous to green fluorescent protein. CPs differ from fluorescent proteins (FPs) by intensely absorbing visible light to give strong colors in ambient light. This endows CPs with certain advantages over FPs, such as instrument-free detection uncomplicated by ultra-violet light damage or background fluorescence, efficient Forster resonance energy transfer (FRET) quenching, and photoacoustic imaging. Thus, CPs have found utility as genetic markers and in teaching, and are attractive for potential cell biosensor applications in the field. Most near-term applications of CPs require expression in a different domain of life: bacteria. However, it is unclear which of the eukaryotic CP genes might be suitable and how best to assay them.Results: Here, taking advantage of codon optimization programs in 12 cases, we engineered 14 CP sequences (meffRed, eforRed, asPink, spisPink, scOrange, fwYellow, amilGFP, amajLime, cjBlue, mefiBlue, aeBlue, amilCP, tsPurple and gfasPurple) into a palette of Escherichia coil BioBrick plasmids. BioBricks comply with synthetic biology's most widely used, simplified, cloning standard. Differences in color intensities, maturation times and fitness costs of expression were compared under the same conditions, and visible readout of gene expression was quantitated. A surprisingly large variation in cellular fitness costs was found, resulting in loss of color in some overnight liquid cultures of certain high-copy-plasmid-borne CPs, and cautioning the use of multiple CPs as markers in competition assays. We solved these two problems by integrating pairs of these genes into the chromosome and by engineering versions of the same CP with very different colors.Conclusion: Availability of 14 engineered CP genes compared in E coil, together with chromosomal mutants suitable for competition assays, should simplify and expand CP study and applications. There was no single plasmid-borne CP that combined all of the most desirable features of intense color, fast maturation and low fitness cost, so this study should help direct future engineering efforts.
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| 2. |
- Dodson, Lois M., et al.
(författare)
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From incomplete penetrance with normal telomere length to severe disease and telomere shortening in a family with monoallelic and biallelic PARN pathogenic variants
- 2019
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Ingår i: Human Mutation. - : Hindawi Limited. - 1059-7794 .- 1098-1004. ; 40:12, s. 2414-2429
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Tidskriftsartikel (refereegranskat)abstract
- PARN encodes poly(A)‐specific ribonuclease. Biallelic and monoallelic PARN variants are associated with Hoyeraal‐Hreidarsson syndrome/dyskeratosis congenita and idiopathic pulmonary fibrosis (IPF), respectively. The molecular features associated with incomplete penetrance of PARN‐associated IPF have not been described. We report a family with a rare missense, p.Y91C, and a novel insertion, p.(I274*), PARN variant. We found PARN p.Y91C had reduced deadenylase activity and the p.(I274*) transcript was depleted. Detailed analysis of the consequences of these variants revealed that, while PARN protein was lowest in the severely affected biallelic child who had the shortest telomeres, it was also reduced in his mother with the p.(I274*) variant but telomeres at the 50th percentile. Increased adenylation of telomerase RNA, human telomerase RNA, and certain small nucleolar RNAs, and impaired ribosomal RNA maturation were observed in cells derived from the severely affected biallelic carrier, but not in the other, less affected biallelic carrier, who had less severely shortened telomeres, nor in the monoallelic carriers who were unaffected and had telomeres ranging from the 1st to the 50th percentiles. We identified hsa‐miR‐202‐5p as a potential negative regulator of PARN. We propose one or more genetic modifiers influence the impact of PARN variants on its targets and this underlies incomplete penetrance of PARN‐associated disease.
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| 3. |
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| 4. |
- Nosrati, Masoumeh, et al.
(författare)
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Insights from engineering the Affibody-Fc interaction with a computational-experimental method
- 2017
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press. - 1741-0126 .- 1741-0134. ; 30:9, s. 593-601
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Tidskriftsartikel (refereegranskat)abstract
- The interaction between the Staphylococcal Protein A (SpA) domain B (the basis of the Affibody) molecule and the Fc of IgG is key to the use of Affibodies in affinity chromatography and in potential therapies against certain inflammatory diseases. Despite its importance and four-decade history, to our knowledge this interaction has never been affinity matured. We elucidate reasons why single-substitutions in the SpA which improve affinity to Fc may be very rare, and also discover substitutions which potentially serve several engineering purposes. We used a variation of FoldX to predict changes in protein-protein-binding affinity, and produce a list of 41 single-amino acid substitutions on the SpA molecule, of which four are near wild type (wt) and five are at most a factor of four from wt affinity. The nine substitutions include one which removes lysine, and several others which change charge. Subtle modulations in affinity may be useful for modifying column elution conditions. The method is applicable to other protein-protein systems, providing molecular insights with lower workload than existing experimental techniques.
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| 6. |
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| 7. |
- Perricaudet, Michel, et al.
(författare)
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Structure of two spliced mRNAs from the transforming region of human subgroup C adenoviruses
- 1979
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 281:5733, s. 694-696
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- The papova viruses and the human adenoviruses are widely used as a model system to study cell transformation in vitro. In subgroup C human adenoviruses, fragment HpaI-E, which comprises as little as 4.5% of the adenovirus type 5 (ad5) DNA, is sufficient for transformation of rat embryo cells1. Analysis of messenger RNAs (mRNAs) from the transforming region of adenoviruses type 2 (ad2) has identified several spliced mRN A species2−4. Promoter mapping studies indicate that the leftmost early region contains two separate transcription units, E1A and E1B (ref. 5) (Fig. 1a). Region E1A is approximately equivalent HpaI-E. The complete nucleotide sequence of the HpaI-E fragment of ad5 was recently reported6. However, the spliced nature of early adenovirus mRNAs prevents a prediction of the amino acid sequence of the corresponding polypeptides directly from the DNA sequence. To study the structure of early ad2 mRNAs at the nucleotide level, we have used molecular cloning procedures to amplify the appropriate mRNA sequences. In this report, clones corresponding to the 12S and 13S mRNA from region E1A (Fig. 1c) have been isolated and characterised by hybridisation and sequence analysis. Our results enable us to predict the primary sequence of two related polypeptides from region E1A of human subgroup C adenoviruses.
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| 8. |
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| 9. |
- Virtanen, Anders, 1957-, et al.
(författare)
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An adenovirus agnogene
- 1983
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Ingår i: Nucleic Acids Research. - 0305-1048 .- 1362-4962. ; 10, s. 2539-2548
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Tidskriftsartikel (refereegranskat)
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| 10. |
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