The Program of Gene Transcription for a Single Differentiating Cell Type During Sporulation in \u3cem\u3eBacillus subtilis\u3c/em\u3e

Asymmetric division during sporulation by Bacillus subtilis generates a mother cell that undergoes a 5-h program of differentiation. The program is governed by a hierarchical cascade consisting of the transcription factors: σE, σK, GerE, GerR, and SpoIIID. The program consists of the activation and repression of 383 genes. The σE factor turns on 262 genes, including those for GerR and SpoIIID. These DNA-binding proteins downregulate almost half of the genes in the σE regulon. In addition, SpoIIID turns on ten genes, including genes involved in the appearance of σK. Next, σK activates 75 additional genes, including that for GerE. This DNA-binding protein, in turn, represses half of the genes that had been activated by σK while switching on a final set of 36 genes. Evidence is presented that repression and activation contribute to proper morphogenesis. The program of gene expression is driven forward by its hierarchical organization and by the repressive effects of the DNA-binding proteins. The logic of the program is that of a linked series of feed-forward loops, which generate successive pulses of gene transcription. Similar regulatory circuits could be a common feature of other systems of cellular differentiation

The Program of Gene Transcription for a Single Differentiating Cell Type during Sporulation in Bacillus subtilis

Asymmetric division during sporulation by Bacillus subtilis generates a mother cell that undergoes a 5-h program of differentiation. The program is governed by a hierarchical cascade consisting of the transcription factors: σ(E), σ(K), GerE, GerR, and SpoIIID. The program consists of the activation and repression of 383 genes. The σ(E) factor turns on 262 genes, including those for GerR and SpoIIID. These DNA-binding proteins downregulate almost half of the genes in the σ(E) regulon. In addition, SpoIIID turns on ten genes, including genes involved in the appearance of σ(K) (.) Next, σ(K) activates 75 additional genes, including that for GerE. This DNA-binding protein, in turn, represses half of the genes that had been activated by σ(K) while switching on a final set of 36 genes. Evidence is presented that repression and activation contribute to proper morphogenesis. The program of gene expression is driven forward by its hierarchical organization and by the repressive effects of the DNA-binding proteins. The logic of the program is that of a linked series of feed-forward loops, which generate successive pulses of gene transcription. Similar regulatory circuits could be a common feature of other systems of cellular differentiation

APC/CCdh1-Mediated Degradation of the F-Box Protein NIPA Is Regulated by Its Association with Skp1

NIPA (Nuclear Interaction Partner of Alk kinase) is an F-box like protein that targets nuclear Cyclin B1 for degradation. Integrity and therefore activity of the SCFNIPA E3 ligase is regulated by cell-cycle-dependent phosphorylation of NIPA, restricting substrate ubiquitination to interphase. Here we show that phosphorylated NIPA is degraded in late mitosis in an APC/CCdh1-dependent manner. Binding of the unphosphorylated form of NIPA to Skp1 interferes with binding to the APC/C-adaptor protein Cdh1 and therefore protects unphosphorylated NIPA from degradation in interphase. Our data thus define a novel mode of regulating APC/C-mediated ubiquitination

Murine Leukemia Virus Spreading in Mice Impaired in the Biogenesis of Secretory Lysosomes and Ca2+-Regulated Exocytosis

Retroviruses have been observed to bud intracellularly into multivesicular bodies (MVB), in addition to the plasma membrane. Release from MVB is thought to occur by Ca(2+)-regulated fusion with the plasma membrane.To address the role of the MVB pathway in replication of the murine leukemia virus (MLV) we took advantage of mouse models for the Hermansky-Pudlak syndrome (HPS) and Griscelli syndrome. In humans, these disorders are characterized by hypopigmentation and immunological alterations that are caused by defects in the biogenesis and trafficking of MVBs and other lysosome related organelles. Neonatal mice for these disease models lacking functional AP-3, Rab27A and BLOC factors were infected with Moloney MLV and the spread of virus into bone marrow, spleen and thymus was monitored. We found a moderate reduction in MLV infection levels in most mutant mice, which differed by less than two-fold compared to wild-type mice. In vitro, MLV release form bone-marrow derived macrophages was slightly enhanced. Finally, we found no evidence for a Ca(2+)-regulated release pathway in vitro. Furthermore, MLV replication was only moderately affected in mice lacking Synaptotagmin VII, a Ca(2+)-sensor regulating lysosome fusion with the plasma membrane.Given that MLV spreading in mice depends on multiple rounds of replication even moderate reduction of virus release at the cellular level would accumulate and lead to a significant effect over time. Thus our in vivo and in vitro data collectively argue against an essential role for a MVB- and secretory lysosome-mediated pathway in the egress of MLV

Models of classroom assessment for course-based research experiences

Course-based research pedagogy involves positioning students as contributors to authentic research projects as part of an engaging educational experience that promotes their learning and persistence in science. To develop a model for assessing and grading students engaged in this type of learning experience, the assessment aims and practices of a community of experienced course-based research instructors were collected and analyzed. This approach defines four aims of course-based research assessment—(1) Assessing Laboratory Work and Scientific Thinking; (2) Evaluating Mastery of Concepts, Quantitative Thinking and Skills; (3) Appraising Forms of Scientific Communication; and (4) Metacognition of Learning—along with a set of practices for each aim. These aims and practices of assessment were then integrated with previously developed models of course-based research instruction to reveal an assessment program in which instructors provide extensive feedback to support productive student engagement in research while grading those aspects of research that are necessary for the student to succeed. Assessment conducted in this way delicately balances the need to facilitate students’ ongoing research with the requirement of a final grade without undercutting the important aims of a CRE education

Philosophy of Social Science

x.116 hal.;21 c

Gel Electrophoretic Mobility-Shift Analysis of SpoIIID Binding

<div><p>DNA fragments of interest were amplified by PCR, gel-purified, and end-labeled using [γ-³²P]-ATP and polynucleotide kinase. Purified SpoIIID was added at increasing concentrations (0 nM for lanes 1 and 5, 50 nM for lane 2, 100 nM for lane 3, and 200 nM for lane 4) and incubated at room temperature for 30 min before loading on to a nondenaturing gel containing 6% polyacrylamide. With the exception of (D), the DNA fragments corresponded to the upstream regions of the indicated genes. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020328#s4" target="_blank">Materials and Methods</a> for the identity (coordinates) of the specific DNA sequences used in the analyses.</p> <p>(A) Gel shifts for known targets of SpoIIID (<i>bofA</i> and <i>spoIVCA</i>), representing positive controls, and genes (<i>abrB, spoIIGA,</i> and <i>racA</i>) under the control of another DNA-binding protein (Spo0A), representing negative controls.</p> <p>(B) Gel shifts for genes identified as possible targets of SpoIIID by transcriptional profiling.</p> <p>(C) Gel shift for <i>cotE</i>. Expression of <i>cotE</i> from its P2 promoter is strongly dependent on SpoIIID. No binding of SpoIIID to the upstream sequence for <i>cotE</i> is observed, suggesting that the effect of SpoIIID on transcription from the P2 promoter is indirect.</p> <p>(D) Gel shifts for chromosomal regions strongly enriched for SpoIIID binding as judged by ChIP-on-chip analysis. For each region, four consecutive DNA fragments of approximately 400 nucleotides in length were analyzed.</p></div