Candidate, Keck School of Medicine of USC This segregation does not depend on sequences within the mRNA, but on the upstream regulatory region. We focus on mRNA processing, RNA modifications and their roles in development and disease. They also allow an investigation of the regulatory signals that modulate the asymmetric distribution of proteins and their subsequent cell type-specific expression in the initial stages of a developmental program. Point mutations in one of the DnaA boxes abolish replication in C. crescentus. Recent evidence suggests that both localized transcription and protein targeting directed by specific amino acid sequence are involved in the localization. Pasadena, CA, USA 91125, Copyright 2023 Mikhail G. Shapiro | Powered by, Scientists, Postdoc Scholars & Graduate Students. Deletion of the region 5' to the apparent sigma 54 promoter caused a complete loss of transcription activation. Biomedical Engineering, Zhejiang University Dynamic chromosome organization and protein localization coordinate the regulatory circuitry that drives the bacterial cell cycle. 1972-1973 Stanford University Medical School We have identified a single amino acid substitution in the Caulobacter structural maintenance of chromosomes (SMC) protein that disrupts chromosome segregation and cell division. A general mechanism in bacteria to rescue stalled ribosomes and to clear the cell of incomplete polypeptides involves an RNA species, tmRNA (SsrA), which functions as both a tRNA and an mRNA. We isolated 35 unique A22-resistant Caulobacter strains with single amino acid substitutions near the nucleotide binding site of MreB. At normal growth temperature (30 degrees C), a different start site was identified 3' to the heat shock start site that conformed to the E. coli sigma 70 promoter consensus sequence. A. Evidence suggests that the protein product of some fla and che genes is targeted to the incipient swarmer cell pole. While asymmetric division in Caulobacter normally yields larger stalked and smaller swarmer daughters, we observe a loss of asymmetric size distribution among daughter cells when clpA is depleted from a strain in which FtsZ is constitutively produced. We also seek to systematically explore the role of AKAP79/150 in orchestrating transcriptional and regulatory control of M/KCNQ channels in sympathetic and sensory neurons. All four proteins were synthesized in the predivisional cell, but the progeny showed cell type-specific bias in the level of enhanced synthesis after heat shock. For questions or comments, contact the SLAC Office of Communications at communications@slac.stanford.edu. Postdoctoral Scholar (co-advised with Richard Andersen) The eukaryotic mitotic machinery uses the cytoskeleton to move specific chromosomal regions. The in vivo intracellular location of components of the Caulobacter replication apparatus was visualized during the cell cycle. cmao@illinois.edu For more information on our department see: https://devbio.stanford.edu. Model checking also showed that mechanisms involving methylation-state changes in regulatory promoter regions during DNA replication increase the robustness of the cell-cycle control. CcrM is a DNA adenine methyltransferase found in the alpha subdivision of the proteobacteria. Structure (2023). Designed by Elegant Themes | Powered by WordPress. Currently: Research Scientist The actin homolog MreB contributes to bacterial cell shape. Future studies should integrate our knowledge of biochemical activities at Cori with our emerging understanding of cytological dynamics in C. crescentus and other bacteria. Bennett Shapiro is a provider established in Stanford, California and his medical specialization is Orthopaedic Surgery. Cell wall morphogenetic protein RodA and penicillin-binding protein PBP1a also change their spatial distribution by accumulating at the division site in response to external osmotic upshifts. Pilus assembly in CAULOBACTER: crescentus occurs during a short period of the cell cycle and pili are only present at the flagellar pole of the swarmer cell. Research Technician Caulobacter crescentus was found to have a DNA methyltransferase, CcrM, that methylates the adenine base of the HinfI recognition sequence, GANTC. Additionally we also are exploring novel and provocative roles of M/KCNQ channels as a neuroprotective mechanism during cerebrovascular ischemic stroke and traumatic brain injury, and chronic pain, to prevent the development of epilepsy and pathological co-morbidities and to develop novel non-opiate approaches to treating chronic pain. Negative control, as a response to the completion of specific steps in the assembly process, may be an important mechanism used by the cell to turn off flagellar gene expression once the gene product is no longer needed. View details for Web of Science ID A1992HJ50200007. Caulobacter goes to great lengths to control the time and place of the activity of this critical regulatory factor during the cell cycle. B.S. Toro, E., Hong, S., McAdams, H. H., Shapiro, L. SpoT regulates DnaA stability and initiation of DNA replication in carbon-starved Caulobacter crescentus, A polymeric protein anchors the chromosomal origin/ParB complex at a bacterial cell pole. In this paper we report the isolation, characterization and genetic analysis of several C. crescentus mutants altered in membrane lipid synthesis. The genes involved in these processes are widely separated on the chromosome. We further show that CckA oligomerizes through a multidomain interaction that is critical for stimulation of kinase activity by DivL, while DivL stimulation of CckA phosphatase activity is independent of CckA homooligomerization. M.Eng. Postdoctoral Scholar Gober, J. W., CHAMPER, R., Reuter, S., Shapiro, L. IDENTIFICATION OF CIS AND TRANS-ELEMENTS INVOLVED IN THE TIMED CONTROL OF A CAULOBACTER FLAGELLAR GENE. Ph.D. Mech. Genes directly controlled by CtrA, a master regulator of the Caulobacter cell cycle. 2017 Indian Institute of Technology, University of Wisconsin-Madison Thus, two transcriptional feedback loops coupled to cell cycle-regulated proteolysis and phosphorylation of the CtrA protein result in the pattern of CtrA activity required for the temporal and spatial control of multiple cell-cycle events. The phage is similar to the Escherichia coli RNA phages in that it (i) sediments at an S(20, w) of 70.6S, (ii) is composed of a single molecule of single-stranded RNA and a protein coat, (iii) contains two structural proteins, and (iv) apparently contains the genetic capacity to code for a coat protein subunit, a maturation-like protein, and an RNA polymerase. Using plasmids carrying transcriptional fusions of either a neo or a lux reporter gene to the promoters of three flagellar genes representing different ranks in the hierarchy (the hook operon, a basal body gene flbN, and the flaO gene), we have measured the level of chimeric gene expression in 13 flagellar mutant backgrounds. Oscillating levels of a few temporally-controlled master regulator proteins in a cyclical circuit drive cell cycle progression. Ann Zhu, SURF Scholar 2021 BS Chemical Engineering, Caltech 2024 (exp) Currently: Assistant Professor of Imaging Physics SciP overexpression disrupts the balance between activation and repression of the CtrA-SciP coregulated genes yielding filamentous cells and loss of viability. Driks, A., Bryan, R., Shapiro, L., DeRosier, D. J. IMAGE-RECONSTRUCTION OF THE FLAGELLAR BASAL BODY OF CAULOBACTER-CRESCENTUS. We conclude that the chromosome structure is supercoiled locally and elongated at large length scales and that substantial cell-to-cell variability in the interloci distances indicates that in vivo crowding prevents the chromosome from reaching an equilibrium arrangement. The response regulator CtrA, which silences the Caulobacter origin of replication and controls multiple cell cycle events, is specifically proteolyzed in cells preparing to initiate DNA replication. We have also partially purified the Caulobacter homolog of IHF and demonstrate that it can facilitate in vitro integrase-mediated lambda recombination. Brenda Wu, SURF Scholar 2016 BS Chemistry, Caltech 2019 View details for DOI 10.1128/JB.185.11.3384-3391.2003, View details for Web of Science ID 000183100900016, View details for PubMedCentralID PMC155372. Because of its critical role, CtrA activity is temporally regulated by multiple mechanisms including phosphorylation and ClpXP-dependent degradation of CtrA. We have identified a periplasmic protease (PerP) that initiates the proteolytic sequence by truncating PodJ(L) to a form with altered activity (PodJ(S)). Ph.D. Student, Biochemistry & Molecular Biophysics (co-ad. Because cell division then yielded a swarmer cell with a different phospholipid profile than its sibling stalked cell, the cell division process may trigger a mechanism which alters the pattern of phospholipid synthesis. However, in the last decade, cytoskeletal proteins have indeed been found to exist in bacteria where they have an important role in organizing the bacterial cell. We generated strains that carry ccrM either on a low-copy-number plasmid (strain GR131) or on a moderate-copy-number plasmid (strain GR132). The significance of this observation remains unclear. In C. crescentus, the Fix network is required for normal cellular growth during hypoxia and controls expression of genes encoding four distinct aerobic respiratory terminal oxidases and multiple carbon and nitrogen metabolic enzymes. Given numerous examples reported thus far, we propose that bacterial polarity displays specific rules and is a more general phenomenon than has been previously recognized. A third gene, flgJ, is also temporally regulated. Zhou, B., Schrader, J. M., Kalogeraki, V. S., Abeliuk, E., Dinh, C. B., Pham, J. Q., Cui, Z. In Caulobacter crescentus the biosynthesis and assembly of this structure is under temporal and spatial control. Mutant strains produce some stalks that have a flagellum, produce some stalks that have an extra lobe protruding from their sides, have filaments lacking the 29-kilodalton flagellin, and produce several unusual cell types, including filamentous cells as well as predivisional cells with two stalks and predivisional cells with no stalk at all. Currently: Orthopedic Surgery Resident Here we show that the spatial distributions of specific cell wall proteins in Caulobacter crescentus are sensitive to small external osmotic upshifts. View details for DOI 10.1128/mBio.03020-20. Nierman, W. C., Feldblyum, T. V., Laub, M. T., Paulsen, I. T., Nelson, K. E., Eisen, J., Heidelberg, J. F., Alley, M. R., Ohta, N., Maddock, J. R., Potocka, I., Nelson, W. C., Newton, A., Stephens, C., Phadke, N. D., Ely, B., DeBoy, R. T., Dodson, R. J., Durkin, A. S., Gwinn, M. L., Haft, D. H., Kolonay, J. F., Smit, J., Craven, M. B., Khouri, H., Shetty, J., Berry, K., Utterback, T., Tran, K., Wolf, A., Vamathevan, J., Ermolaeva, M., White, O., Salzberg, S. L., Venter, J. C., Shapiro, L., Fraser, C. M. Dynamic localization of a cytoplasmic signal transduction response regulator controls morphogenesis during the Caulobacter cell cycle, Global analysis of the genetic network controlling a bacterial cell cycle. By simply increasing the laser power, single molecules of the fluorescent protein fusion HU2-eYFP can be made to blink on and off to achieve super-resolution imaging with a single excitation source. Copyright 2023 Mikhail G. Shapiro | Powered by. We propose that during segregation PopZ sequesters free ParA and induces target-proximal regeneration of ParA DNA binding activity to enforce processive and pole-directed centromere segregation, preventing segregation reversals. To our knowledge, this is the first example of an essential prokaryotic DNA methyltransferase that is not part of a DNA restriction/modification system. Alternatively, beam scientists can take many measurements of the beam itself and try to reconstruct, sometimes using machine learning, what the beam would look like under different experimental circumstances but those methods require a lot of data and a lot of computational power. WebBio. Search results about individual genes are displayed as tables, graphs of time resolved expression profiles, and schematics of protein localization throughout the cell cycle. Enzyme from pure populations of stalked cells, as well as populations enriched in swarmer and predivisional cells, appeared identical in subunit structure and template requirements. WebShapiro Lab University of Illinois, Department of Biochemistry. Paige, M. F., Judd, E., Shapiro, L., Moerner, W. E. Complete genome sequence of Caulobacter crescentus. The specific cell cycle arrest in the smc null mutant indicates the presence of a cell cycle checkpoint that senses perturbations in chromosome organization or segregation. The sequential activation of these three subgroups of structural genes reflects the order of assembly of their gene products into the flagellum. Cumulatively, this study uncovered novel new layers of transcriptional regulation mediating the bacterial cell cycle. Thus, the MCPs are not specifically localized to the immediate vicinity of the flagellar rotor. Currently: Assistant Professor of Bioengineering David Shapiros lab has spent over a decade studying estrogens, acting via estrogen receptor a (ERa), which plays a key role in most breast cancers. To investigate the interaction between membrane synthesis and DNA replication during a single cell cycle, we moved the gpsA mutation into a synchronizable, but otherwise wild-type, strain. To test their ideas, the team used their model to interpret experimental data from the Argonne Wakefield Accelerator at the DOE's Argonne National Laboratory. The ClpXP protease is required for CtrA proteolysis but is present throughout the cell-cycle, so the mechanism for activating and deactivating CtrA proteolysis is unknown. The kinetic properties of an adenine DNA methyltransferase involved in cell cycle regulation of Caulobacter crescentus have been elucidated by using defined unmethylated or hemimethylated DNA (DNAHM) substrates. Dr. Weissmans laboratory is working on identifying and characterizing the progression of discrete changes, genetic and epigenetic, that leads to the generation of cancer stem cells (CSCs) from a variety of blood and solid tissue cancers.
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