480 research outputs found
Sequence-Dependent Dynamics of Synthetic and Endogenous RSSs in V(D)J Recombination
Developing lymphocytes of jawed vertebrates cleave and combine distinct gene segments to assemble antigenâreceptor genes. This process called V(D)J recombination that involves the RAG recombinase binding and cutting recombination signal sequences (RSSs) composed of conserved heptamer and nonamer sequences flanking less well-conserved 12- or 23-bp spacers. Little quantitative information is known about the contributions of individual RSS positions over the course of the RAGâRSS interaction. We employ a single-molecule method known as tethered particle motion to track the formation, lifetime and cleavage of individual RAGâ12RSSâ23RSS paired complexes (PCs) for numerous synthetic and endogenous 12RSSs. We reveal that single-bp changes, including in the 12RSS spacer, can significantly and selectively alter PC formation or the probability of RAG-mediated cleavage in the PC. We find that some rarely used endogenous gene segments can be mapped directly to poor RAG binding on their adjacent 12RSSs. Finally, we find that while abrogating RSS nicking with CaÂČâș leads to substantially shorter PC lifetimes, analysis of the complete lifetime distributions of any 12RSS even on this reduced system reveals that the process of exiting the PC involves unidentified molecular details whose involvement in RAGâRSS dynamics are crucial to quantitatively capture kinetics in V(D)J recombination
Fundamental limits on the rate of bacterial growth
Recent years have seen an experimental deluge interrogating the relationship between bacterial growth rate, cell size, and protein content, quantifying the abundance of proteins across growth conditions with unprecedented resolution. However, we still lack a rigorous understanding of what sets the scale of these quantities and when protein abundances should (or should not) depend on growth rate. Here, we seek to quantitatively understand this relationship across a collection of Escherichia coli proteomic data covering â 4000 proteins and 36 growth rates. We estimate the basic requirements for steady-state growth by considering key processes in nutrient transport, cell envelope biogenesis, energy generation, and the central dogma. From these estimates, ribosome biogenesis emerges as a primary determinant of growth rate. We expand on this assessment by exploring a model of proteomic regulation as a function of the nutrient supply, revealing a mechanism that ties cell size and growth rate to ribosomal content
Fundamental limits on the rate of bacterial growth
Recent years have seen an experimental deluge interrogating the relationship between bacterial growth rate, cell size, and protein content, quantifying the abundance of proteins across growth conditions with unprecedented resolution. However, we still lack a rigorous understanding of what sets the scale of these quantities and when protein abundances should (or should not) depend on growth rate. Here, we seek to quantitatively understand this relationship across a collection of Escherichia coli proteomic data covering â 4000 proteins and 36 growth rates. We estimate the basic requirements for steady-state growth by considering key processes in nutrient transport, cell envelope biogenesis, energy generation, and the central dogma. From these estimates, ribosome biogenesis emerges as a primary determinant of growth rate. We expand on this assessment by exploring a model of proteomic regulation as a function of the nutrient supply, revealing a mechanism that ties cell size and growth rate to ribosomal content
Microtesla MRI of the human brain combined with MEG
One of the challenges in functional brain imaging is integration of
complementary imaging modalities, such as magnetoencephalography (MEG) and
functional magnetic resonance imaging (fMRI). MEG, which uses highly sensitive
superconducting quantum interference devices (SQUIDs) to directly measure
magnetic fields of neuronal currents, cannot be combined with conventional
high-field MRI in a single instrument. Indirect matching of MEG and MRI data
leads to significant co-registration errors. A recently proposed imaging method
- SQUID-based microtesla MRI - can be naturally combined with MEG in the same
system to directly provide structural maps for MEG-localized sources. It
enables easy and accurate integration of MEG and MRI/fMRI, because microtesla
MR images can be precisely matched to structural images provided by high-field
MRI and other techniques. Here we report the first images of the human brain by
microtesla MRI, together with auditory MEG (functional) data, recorded using
the same seven-channel SQUID system during the same imaging session. The images
were acquired at 46 microtesla measurement field with pre-polarization at 30
mT. We also estimated transverse relaxation times for different tissues at
microtesla fields. Our results demonstrate feasibility and potential of human
brain imaging by microtesla MRI. They also show that two new types of imaging
equipment - low-cost systems for anatomical MRI of the human brain at
microtesla fields, and more advanced instruments for combined functional (MEG)
and structural (microtesla MRI) brain imaging - are practical.Comment: 8 pages, 5 figures - accepted by JM
Sequence-Dependent Dynamics of Synthetic and Endogenous RSSs in V(D)J Recombination
Developing lymphocytes of jawed vertebrates cleave and combine distinct gene segments to assemble antigenâreceptor genes. This process called V(D)J recombination that involves the RAG recombinase binding and cutting recombination signal sequences (RSSs) composed of conserved heptamer and nonamer sequences flanking less well-conserved 12- or 23-bp spacers. Little quantitative information is known about the contributions of individual RSS positions over the course of the RAGâRSS interaction. We employ a single-molecule method known as tethered particle motion to track the formation, lifetime and cleavage of individual RAGâ12RSSâ23RSS paired complexes (PCs) for numerous synthetic and endogenous 12RSSs. We reveal that single-bp changes, including in the 12RSS spacer, can significantly and selectively alter PC formation or the probability of RAG-mediated cleavage in the PC. We find that some rarely used endogenous gene segments can be mapped directly to poor RAG binding on their adjacent 12RSSs. Finally, we find that while abrogating RSS nicking with CaÂČâș leads to substantially shorter PC lifetimes, analysis of the complete lifetime distributions of any 12RSS even on this reduced system reveals that the process of exiting the PC involves unidentified molecular details whose involvement in RAGâRSS dynamics are crucial to quantitatively capture kinetics in V(D)J recombination
The Energetics of Molecular Adaptation in Transcriptional Regulation
Mutation is a critical mechanism by which evolution explores the functional landscape of proteins. Despite our ability to experimentally inflict mutations at will, it remains difficult to link sequence-level perturbations to systems-level responses. Here, we present a framework centered on measuring changes in the free energy of the system to link individual mutations in an allosteric transcriptional repressor to the parameters which govern its response. We find the energetic effects of the mutations can be categorized into several classes which have characteristic curves as a function of the inducer concentration. We experimentally test these diagnostic predictions using the well-characterized LacI repressor of Escherichia coli, probing several mutations in the DNA binding and inducer binding domains. We find that the change in gene expression due to a point mutation can be captured by modifying only a subset of the model parameters that describe the respective domain of the wild-type protein. These parameters appear to be insulated, with mutations in the DNA binding domain altering only the DNA affinity and those in the inducer binding domain altering only the allosteric parameters. Changing these subsets of parameters tunes the free energy of the system in a way that is concordant with theoretical expectations. Finally, we show that the induction profiles and resulting free energies associated with pairwise double mutants can be predicted with quantitative accuracy given knowledge of the single mutants, providing an avenue for identifying and quantifying epistatic interactions
Predictive shifts in free energy couple mutations to their phenotypic consequences
Mutation is a critical mechanism by which evolution explores the functional landscape of proteins. Despite our ability to experimentally inflict mutations at will, it remains difficult to link sequence-level perturbations to systems-level responses. Here, we present a framework centered on measuring changes in the free energy of the system to link individual mutations in an allosteric transcriptional repressor to the parameters which govern its response. We find that the energetic effects of the mutations can be categorized into several classes which have characteristic curves as a function of the inducer concentration. We experimentally test these diagnostic predictions using the well-characterized LacI repressor of Escherichia coli, probing several mutations in the DNA binding and inducer binding domains. We find that the change in gene expression due to a point mutation can be captured by modifying only the model parameters that describe the respective domain of the wild-type protein. These parameters appear to be insulated, with mutations in the DNA binding domain altering only the DNA affinity and those in the inducer binding domain altering only the allosteric parameters. Changing these subsets of parameters tunes the free energy of the system in a way that is concordant with theoretical expectations. Finally, we show that the induction profiles and resulting free energies associated with pairwise double mutants can be predicted with quantitative accuracy given knowledge of the single mutants, providing an avenue for identifying and quantifying epistatic interactions
FUNCTIONAL MR OF BRAIN ACTIVITY AND PERFUSION IN PATIENTS WITH CHRONIC CORTICAL STROKE
PURPOSE: (1) To determine whether functional MR can reliably map functional deficits in patients with stroke in the primary visual cortex; (2) to determine whether functional MR can reliably map perfusion deficits; and (3) to determine whether functional MR can give any additional diagnostic information beyond conventional MR. METHODS: Seven patients who had had a stroke in their primary visual system were examined using two functional MR techniques: (1) dynamic susceptibility contrast imaging, and (2) cortical activation mapping during full-field visual stimulation. Maps of relative cerebral blood volume and activation were created and compared with visual field examinations and conventional T2-weighted images on a quadrant-by-quadrant basis in five of these patients. RESULTS: Visual field mapping matched with both T2-weighted conventional images and activation mapping of 16 of 18 quadrants. In two quadrants, the activation maps detected abnormalities that were present on the visual field examination but not present on the T2-weighted images nor on the relative cerebral blood volume maps, which may indicate abnormal function without frank infarction. In addition, the activation maps demonstrated decreased activation in extrastriate cortex and had normal T2 signal and relative cerebral blood volume but was adjacent to infarcted primary cortex, mapping in vivo how stroke in one location can affect the function of distant tissue. CONCLUSION: Functional MR techniques can accurately map functional and perfusion deficits and thereby provide additional clinically useful information. Additional studies will be needed to determine the prognostic utility of functional MR in stroke patients
- âŠ