Project description:Salmonella is an important enteric pathogen that causes a spectrum of diseases varying from mild gastroenteritis to life threatening typhoid fever. Salmonella does not have lac operon. However, E. Coli, Salmonella’s close relative, has lac operon. Being an enteric pathogen like E. coli, Salmonella will also benefit from lac operon. Then, why Salmonella has lost lac operon?. To address this question, lacI, an important component of lac operon was expressed in Salmonella via pTrc99A plasmid. As a control, pTrc99A without lacI was also expressed in Salmonella. The effect of LacI on the transcription profile of Salmonella was analyzed using microarray technique.
Project description:In this work we introduce a new high-throughput method, Spec-seq, that directly measures specificity by sequencing. It has several advantages over existing methods to quantify large collections (thousands) of binding site energies in one experiment. Using lac repressor as an example, we show that this method has excellent reproducibility giving energy measurements generally consistent within about 0.1kT. While not measuring in parallel as many different sequences as some of the higher throughput methods, we obtain high accuracies because we measure exactly what is necessary, the relative affinity to a large collection of sequences, without any mathematical fitting procedures or approximations required. It is similar to MITOMI in the number of different sites that can be analyzed in parallel, but it is much simpler to perform, requiring only a means to separate bound and unbound fractions which are then sequenced using standard high throughput, short read sequencing machines that are now readily available. When applied to the lac repressor we obtain, in a single experiment, data covering a large fraction of all the previous studies, plus thousands of additional variants, allowing us to compile a much more comprehensive profile of its specificity. We learn that the lac repressor can bind to sites of different lengths but that the preferred sequence, and the mode of binding, depends on the length. We also apply the method to two other members of the LacI/GalR protein family, PurR and YcjW, to obtain extensive models of their specificity and test the generality of the lac repressor's ability to bind to operators of variable length. We find that the lac repressor is apparently unique in its ability to bind with high affinity to sites of different lengths and with different modes of binding.
Project description:In this work we introduce a new high-throughput method, Spec-seq, that directly measures specificity by sequencing. It has several advantages over existing methods to quantify large collections (thousands) of binding site energies in one experiment. Using lac repressor as an example, we show that this method has excellent reproducibility giving energy measurements generally consistent within about 0.1kT. While not measuring in parallel as many different sequences as some of the higher throughput methods, we obtain high accuracies because we measure exactly what is necessary, the relative affinity to a large collection of sequences, without any mathematical fitting procedures or approximations required. It is similar to MITOMI in the number of different sites that can be analyzed in parallel, but it is much simpler to perform, requiring only a means to separate bound and unbound fractions which are then sequenced using standard high throughput, short read sequencing machines that are now readily available. When applied to the lac repressor we obtain, in a single experiment, data covering a large fraction of all the previous studies, plus thousands of additional variants, allowing us to compile a much more comprehensive profile of its specificity. We learn that the lac repressor can bind to sites of different lengths but that the preferred sequence, and the mode of binding, depends on the length. We also apply the method to two other members of the LacI/GalR protein family, PurR and YcjW, to obtain extensive models of their specificity and test the generality of the lac repressor's ability to bind to operators of variable length. We find that the lac repressor is apparently unique in its ability to bind with high affinity to sites of different lengths and with different modes of binding. 4 independent experiments.
Project description:Three-spined stickleback (Gasterosteus aculeatus) represents a convenient model to study microevolution - adaptation to freshwater environment. While genetic adaptations to freshwater are well-studied, epigenetic adaptations attracted little attention. In this work, we investigated the role of DNA methylation in the adaptation of marine stickleback population to freshwater conditions. DNA methylation profiling was performed in marine and freshwater populations of sticklebacks, as well as in marine sticklebacks placed into freshwater environment and freshwater sticklebacks placed into seawater. For the first time, we demonstrated that genes encoding ion channels kcnd3, cacna1fb, gja3 are differentially methylated between marine and freshwater populations. We also showed that after placing marine stickleback into fresh water, its DNA methylation profile partially converges to the one of a freshwater stickleback. This suggests that immediate epigenetic response to freshwater conditions can be maintained in freshwater population. Interestingly, we observed enhanced epigenetic plasticity in freshwater sticklebacks that may serve as a compensatory regulatory mechanism for the lack of genetic variation in the freshwater population. Some of the regions that were reported previously to be under selection in freshwater populations also show differential methylation. Thus, epigenetic changes might represent a parallel mechanism of adaptation along with genetic selection in freshwater environment.
Project description:N-lactoyl-phenylalanine (Lac-Phe) is a lactate-derived metabolite that suppresses food intake and body weight. Little is known about the mechanisms that mediate Lac-Phe transport across cell membranes. Here we identify SLC17A1 and SLC17A3, two kidney-restricted plasma membrane-localized solute carriers, as physiologic urine Lac-Phe transporters. In cell culture, SLC17A1/3 exhibit high Lac-Phe efflux activity. In humans, levels of Lac-Phe in urine exhibit a strong genetic association with the SLC17A1-4 locus. Urine Lac-Phe levels are also increased following a Wingate sprint test. In mice, genetic ablation of either SLC17A1 or SLC17A3 reduces urine Lac-Phe levels. Despite these differences, both knockout strains have normal blood Lac-Phe and body weights, demonstrating SLC17-dependent de-coupling of urine and plasma Lac-Phe pools. Together, these data establish SLC17A1/3 family members as the physiologic urine transporters for Lac-Phe and uncover a biochemical pathway for the renal excretion of this signaling metabolite. Our data do not exclude the involvement of other transporters in mediating Lac-Phe transport.
Project description:Salmonella is an important enteric pathogen that causes a spectrum of diseases varying from mild gastroenteritis to life threatening typhoid fever. Salmonella does not have lac operon. However, E. Coli, Salmonellaâs close relative, has lac operon. Being an enteric pathogen like E. coli, Salmonella will also benefit from lac operon. Then, why Salmonella has lost lac operon?. To address this question, lacI, an important component of lac operon was expressed in Salmonella via pTrc99A plasmid. As a control, pTrc99A without lacI was also expressed in Salmonella. The effect of LacI on the transcription profile of Salmonella was analyzed using microarray technique. Overall design The total RNA was isolated from Salmonella using âRNeasy Mini Kitâ (QIAGEN). Organism used: Salmonella enterica serovar Typhimurium LT2 * Slides: Agilentâs arrays (8x15k) AMADID: NO: 17809 * Starting material: Salmonella RNA in nuclease-free water * RNA Samples used: Wild type (WT), WT + pTrc99A+LacI (PTRC), WT + pTrc99A-LacI(Delta lac) * Labeling kit: MessageAmp⢠II-Bacteria RNA Amplification Kit from Ambion Cat # AM1790 * Labeling Method: T7 promoter based-linear amplification to generate labeled complementary RNA * Total RNA and cRNA Purification Kit: Qiagenâs RNeasy minikit Cat#74104 * Hybridization Kit: Agilentâs In situ Hybridzation kit 5184-3568 * RNA quality was checked using Bioanalyzer. The array slides were scanned immediately by PerkinElmer Scan array Gx Microarray scanner. The Scan array software (PerkinElmer) was used for grid wise normalization of array images. Two arrays were used with mutant and rescue experiments and dye swap experiments were included in the final analysis. The data was analysed by GeneSpring GX and Biointerpreter software from Genotypic Technology, Bangalore. The differential expression was considered if the Log 2 mean of at least -1 for the down regulated genes and +1 for the upregulated genes. We considered only the genes that were reproducible from both replicates.