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  • 1
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    On the anticorrelation between HFormula temperature and density in giant planet ionospheres (2014)
    Oxford University Press
    Details
    Publication Date: 2014-01-29
    Description: The intensity of H $_3^+$ emission can be driven by both temperature and density, and when fitting a set of infrared H $_3^+$ line spectra, an anticorrelation between the fitted temperatures and densities is commonly observed. The ambiguity present in the existing published literature on how to treat this effect puts into question the physical significance of the derived parameters. Here, we examine the nature of this anticorrelation and quantify the inherent uncertainty in the fitted temperature and density that this produces. We find that the uncertainty produced by the H $_3^+$ temperature and density anticorrelation is to a very good approximation equal to the uncertainties that are derived from the fitting procedure invoking Cramer's rule. This means that any previously observed correlated variability in the observed H $_3^+$ temperature and density outside these errors, in the absence of other error sources, are statistically separated and can be considered physical. These results are compared to recent ground-based infrared Keck Near InfRared echelle SPECtrograph (NIRSPEC) observations of H $_3^+$ emission from Saturn's aurora, which show no clear evidence for large-scale radiative cooling, but do show stark hemispheric differences in temperature.
    Print ISSN: 0035-8711
    Electronic ISSN: 1365-2966
    Topics: Physics
    Published by Oxford University Press
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  • 2
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    Species-Specific Exon Loss in Human Transcriptomes (2015)
    Oxford University Press
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    Publication Date: 2015-01-20
    Description: Changes in exon–intron structures and splicing patterns represent an important mechanism for the evolution of gene functions and species-specific regulatory networks. Although exon creation is widespread during primate and human evolution and has been studied extensively, much less is known about the scope and potential impact of human-specific exon loss events. Historically, transcriptome data and exon annotations are significantly biased toward humans over nonhuman primates. This ascertainment bias makes it challenging to discover human-specific exon loss events. We carried out a transcriptome-wide search of human-specific exon loss events, by taking advantage of RNA sequencing (RNA-seq) as a powerful and unbiased tool for exon discovery and annotation. Using RNA-seq data of humans, chimpanzees, and other primates, we reconstructed and compared transcript structures across the primate phylogeny. We discovered 33 candidate human-specific exon loss events, among which six exons passed stringent experimental filters for the complete loss of splicing activities in diverse human tissues. These events may result from human-specific deletion of genomic DNA, or small-scale sequence changes that inactivated splicing signals. The impact of human-specific exon loss events is predominantly regulatory. Three of the six events occurred in the 5' untranslated region (5'-UTR) and affected cis-regulatory elements of mRNA translation. In SLC7A6 , a gene encoding an amino acid transporter, luciferase reporter assays suggested that both a human-specific exon loss event and an independent human-specific single nucleotide substitution in the 5'-UTR increased mRNA translational efficiency. Our study provides novel insights into the molecular mechanisms and evolutionary consequences of exon loss during human evolution.
    Print ISSN: 0737-4038
    Electronic ISSN: 1537-1719
    Topics: Biology
    Published by Oxford University Press
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  • 3
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    Nuclease-mediated gene editing by homologous recombination of the human globin locus (2014)
    Oxford University Press
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    Publication Date: 2014-01-28
    Description: Tal-effector nucleases (TALENs) are engineered proteins that can stimulate precise genome editing through specific DNA double-strand breaks. Sickle cell disease and β-thalassemia are common genetic disorders caused by mutations in β-globin, and we engineered a pair of highly active TALENs that induce modification of 54% of human β-globin alleles near the site of the sickle mutation. These TALENS stimulate targeted integration of therapeutic, full-length beta-globin cDNA to the endogenous β-globin locus in 19% of cells prior to selection as quantified by single molecule real-time sequencing. We also developed highly active TALENs to human -globin, a pharmacologic target in sickle cell disease therapy. Using the β-globin and -globin TALENs, we generated cell lines that express GFP under the control of the endogenous β-globin promoter and tdTomato under the control of the endogenous -globin promoter. With these fluorescent reporter cell lines, we screened a library of small molecule compounds for their differential effect on the transcriptional activity of the endogenous β- and -globin genes and identified several that preferentially upregulate -globin expression.
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 4
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    XRCC4's interaction with XLF is required for coding (but not signal) end joining (2012)
    Oxford University Press
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    Publication Date: 2012-02-28
    Description: XRCC4 and XLF are structurally related proteins important for DNA Ligase IV function. XRCC4 forms a tight complex with DNA Ligase IV while XLF interacts directly with XRCC4. Both XRCC4 and XLF form homodimers that can polymerize as heterotypic filaments independently of DNA Ligase IV. Emerging structural and in vitro biochemical data suggest that XRCC4 and XLF together generate a filamentous structure that promotes bridging between DNA molecules. Here, we show that ablating XRCC4's affinity for XLF results in DNA repair deficits including a surprising deficit in VDJ coding, but not signal end joining. These data are consistent with a model whereby XRCC4/XLF complexes hold DNA ends together—stringently required for coding end joining, but dispensable for signal end joining. Finally, DNA-PK phosphorylation of XRCC4/XLF complexes disrupt DNA bridging in vitro , suggesting a regulatory role for DNA-PK's phosphorylation of XRCC4/XLF complexes.
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 5
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    Human ZMPSTE24 disease mutations: residual proteolytic activity correlates with disease severity (2012)
    Oxford University Press
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    Publication Date: 2012-08-28
    Description: The zinc metalloprotease ZMPSTE24 plays a critical role in nuclear lamin biology by cleaving the prenylated and carboxylmethylated 15-amino acid tail from the C-terminus of prelamin A to yield mature lamin A. A defect in this proteolytic event, caused by a mutation in the lamin A gene ( LMNA ) that eliminates the ZMPSTE24 cleavage site, underlies the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS). Likewise, mutations in the ZMPSTE24 gene that result in decreased enzyme function cause a spectrum of diseases that share certain features of premature aging. Twenty human ZMPSTE24 alleles have been identified that are associated with three disease categories of increasing severity: mandibuloacral dysplasia type B (MAD-B), severe progeria (atypical ‘HGPS’) and restrictive dermopathy (RD). To determine whether a correlation exists between decreasing ZMPSTE24 protease activity and increasing disease severity, we expressed mutant alleles of ZMPSTE24 in yeast and optimized in vivo yeast mating assays to directly compare the activity of alleles associated with each disease category. We also measured the activity of yeast crude membranes containing the ZMPSTE24 mutant proteins in vitro . We determined that, in general, the residual activity of ZMPSTE24 patient alleles correlates with disease severity. Complete loss-of-function alleles are associated with RD, whereas retention of partial, measureable activity results in MAD-B or severe progeria. Importantly, our assays can discriminate small differences in activity among the mutants, confirming that the methods presented here will be useful for characterizing any new ZMPSTE24 mutations that are discovered.
    Print ISSN: 0964-6906
    Electronic ISSN: 1460-2083
    Topics: Biology , Medicine
    Published by Oxford University Press
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  • 6
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    N-terminal constraint activates the catalytic subunit of the DNA-dependent protein kinase in the absence of DNA or Ku (2012)
    Oxford University Press
    Details
    Publication Date: 2012-04-15
    Description: The DNA-dependent protein kinase (DNA-PK) was identified as an activity and as its three component polypeptides 25 and 15 years ago, respectively. It has been exhaustively characterized as being absolutely dependent on free double stranded DNA ends (to which it is directed by its regulatory subunit, Ku) for its activation as a robust nuclear serine/threonine protein kinase. Here, we report the unexpected finding of robust DNA-PKcs activation by N-terminal constraint, independent of either DNA or its regulatory subunit Ku. These data suggest that an N-terminal conformational change (likely induced by DNA binding) induces enzymatic activation.
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 7
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    The Evolution of RuBisCO Stability at the Thermal Limit of Photoautotrophy (2013)
    Oxford University Press
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    Publication Date: 2013-03-21
    Description: A long-standing question in evolutionary biology is how organisms adapt to novel environments. In North American hot springs, diversification of a clade of the cyanobacterium Synechococcus into hotter environments has resulted in the unique innovation of a light-driven ecosystem at temperatures up to 74°C, and temperature adaptation of photosynthetic carbon fixation with the Calvin cycle contributed to this process. Here, we investigated the evolution of thermostability of the Calvin cycle enzyme ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) during Synechococcus divergence. Circular dichroism thermal scans revealed that the RuBisCO of the most thermotolerant Synechococcus lineage is more stable than those of other lineages or of resurrected ancestral enzymes. Using site-directed mutagenesis, we next identified four amino acid substitutions that together increased stability and activity of this enzyme at higher temperatures. These are clustered near critical subunit interfaces distant from the active site. Each of the four amino acids is also observed in a less thermostable Synechococcus RuBisCO, and the impact on stability of three of these appears to be epistatic. Recombination analyses that allow for recurrent mutation as well as patterns of synonymous variation surrounding these sites suggest that the evolution of a more thermostable RuBisCO may have involved homologous recombination. Our results provide insights on the molecular evolutionary processes that shape niche differentiation and ecosystem function.
    Print ISSN: 0737-4038
    Electronic ISSN: 1537-1719
    Topics: Biology
    Published by Oxford University Press
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  • 8
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    High-efficiency genome editing via 2A-coupled co-expression of fluorescent proteins and zinc finger nucleases or CRISPR/Cas9 nickase pairs (2014)
    Oxford University Press
    Details
    Publication Date: 2014-06-03
    Description: Targeted endonucleases including zinc finger nucleases (ZFNs) and clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas9 are increasingly being used for genome editing in higher species. We therefore devised a broadly applicable and versatile method for increasing editing efficiencies by these tools. Briefly, 2A peptide-coupled co-expression of fluorescent protein and nuclease was combined with fluorescence-activated cell sorting (FACS) to allow for efficient isolation of cell populations with increasingly higher nuclease expression levels, which translated into increasingly higher genome editing rates. For ZFNs, this approach, combined with delivery of donors as single-stranded oligodeoxynucleotides and nucleases as messenger ribonucleic acid, enabled high knockin efficiencies in demanding applications, including biallelic codon conversion frequencies reaching 30–70% at high transfection efficiencies and ~2% at low transfection efficiencies, simultaneous homozygous knockin mutation of two genes with ~1.5% efficiency as well as generation of cell pools with almost complete codon conversion via three consecutive targeting and FACS events. Observed off-target effects were minimal, and when occurring, our data suggest that they may be counteracted by selecting intermediate nuclease levels where off-target mutagenesis is low, but on-target mutagenesis remains relatively high. The method was also applicable to the CRISPR/Cas9 system, including CRISPR/Cas9 mutant nickase pairs, which exhibit low off-target mutagenesis compared to wild-type Cas9.
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
    Published by Oxford University Press
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  • 9
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    Mutation Rate, Spectrum, Topology, and Context-Dependency in the DNA Mismatch Repair-Deficient Pseudomonas fluorescens ATCC948 (2015)
    Oxford University Press
    Details
    Publication Date: 2015-01-24
    Description: High levels of genetic diversity exist among natural isolates of the bacterium Pseudomonas fluorescens , and are especially elevated around the replication terminus of the genome, where strain-specific genes are found. In an effort to understand the role of genetic variation in the evolution of Pseudomonas , we analyzed 31,106 base substitutions from 45 mutation accumulation lines of P. fluorescens ATCC948, naturally deficient for mismatch repair, yielding a base-substitution mutation rate of 2.34 x 10 –8 per site per generation (SE: 0.01 x 10 –8 ) and a small-insertion-deletion mutation rate of 1.65 x 10 –9 per site per generation (SE: 0.03 x 10 –9 ). We find that the spectrum of mutations in prophage regions, which often contain virulence factors and antibiotic resistance, is highly similar to that in the intergenic regions of the host genome. Our results show that the mutation rate varies around the chromosome, with the lowest mutation rate found near the origin of replication. Consistent with observations from other studies, we find that site-specific mutation rates are heavily influenced by the immediately flanking nucleotides, indicating that mutations are context dependent.
    Electronic ISSN: 1759-6653
    Topics: Biology
    Published by Oxford University Press
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  • 10
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    Asymmetric Context-Dependent Mutation Patterns Revealed through Mutation-Accumulation Experiments (2015)
    Oxford University Press
    Details
    Publication Date: 2015-06-22
    Description: Despite the general assumption that site-specific mutation rates are independent of the local sequence context, a growing body of evidence suggests otherwise. To further examine context-dependent patterns of mutation, we amassed 5,645 spontaneous mutations in wild- type (WT) and mismatch-repair deficient (MMR – ) mutation–accumulation (MA) lines of the gram-positive model organism Bacillus subtilis . We then analyzed〉7,500 spontaneous base-substitution mutations across B. subtilis , Escherichia coli , and Mesoplasma florum WT and MMR – MA lines, finding a context-dependent mutation pattern that is asymmetric around the origin of replication. Different neighboring nucleotides can alter site-specific mutation rates by as much as 75-fold, with sites neighboring G:C base pairs or dimers involving alternating pyrimidine–purine and purine–pyrimidine nucleotides having significantly elevated mutation rates. The influence of context-dependent mutation on genome architecture is strongest in M. florum , consistent with the reduced efficiency of selection in organisms with low effective population size. If not properly accounted for, the disparities arising from patterns of context-dependent mutation can significantly influence interpretations of positive and purifying selection.
    Print ISSN: 0737-4038
    Electronic ISSN: 1537-1719
    Topics: Biology
    Published by Oxford University Press
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