28 research outputs found
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Population-Specific Regulation of Chmp2b by Lbx1 during Onset of Synaptogenesis in Lateral Association Interneurons
Chmp2b is closely related to Vps2, a key component of the yeast protein complex that creates the intralumenal vesicles of multivesicular bodies. Dominant negative mutations in Chmp2b cause autophagosome accumulation and neurodegenerative disease. Loss of Chmp2b causes failure of dendritic spine maturation in cultured neurons. The homeobox gene Lbx1 plays an essential role in specifying postmitotic dorsal interneuron populations during late pattern formation in the neural tube. We have discovered that Chmp2b is one of the most highly regulated cell-autonomous targets of Lbx1 in the embryonic mouse neural tube. Chmp2b was expressed and depended on Lbx1 in only two of the five nascent, Lbx1-expressing, postmitotic, dorsal interneuron populations. It was also expressed in neural tube cell populations that lacked Lbx1 protein. The observed population-specific expression of Chmp2b indicated that only certain population-specific combinations of sequence specific transcription factors allow Chmp2b expression. The cell populations that expressed Chmp2b corresponded, in time and location, to neurons that make the first synapses of the spinal cord. Chmp2b protein was transported into neurites within the motor-and association-neuropils, where the first synapses are known to form between E11.5 and E12.5 in mouse neural tubes. Selective, developmentally-specified gene expression of Chmp2b may therefore be used to endow particular neuronal populations with the ability to mature dendritic spines. Such a mechanism could explain how mammalian embryos reproducibly establish the disynaptic cutaneous reflex only between particular cell populations
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The Type II Secretion Pathway in Vibrio cholerae Is Characterized by Growth Phase-Dependent Expression of Exoprotein Genes and Is Positively Regulated by σ[superscript E]
Vibrio cholerae, an etiological agent of cholera, circulates between aquatic reservoirs and the human gastrointestinal tract. The
type II secretion (T2S) system plays a pivotal role in both stages of the lifestyle by exporting multiple proteins, including cholera
toxin. Here, we studied the kinetics of expression of genes encoding the T2S system and its cargo proteins. We have found that
under laboratory growth conditions, the T2S complex was continuously expressed throughout V. cholerae growth, whereas
there was growth phase-dependent transcriptional activity of genes encoding different cargo proteins. Moreover, exposure of V.
cholerae to different environmental cues encountered by the bacterium in its life cycle induced transcriptional expression of T2S.
Subsequent screening of a V. cholerae genomic library suggested that σ[superscript E] stress response, phosphate metabolism, and the second
messenger 3',5'-cyclic diguanylic acid (c-di-GMP) are involved in regulating transcriptional expression of T2S. Focusing on σ[superscript E],
we discovered that the upstream region of the T2S operon possesses both the consensus σ[superscript E] and σ⁷⁰ signatures, and deletion of
the σ[superscript E] binding sequence prevented transcriptional activation of T2S by RpoE. Ectopic overexpression of σ[superscript E] stimulated transcription of T2S in wild-type and isogenic ΔrpoE strains of V. cholerae, providing additional support for the idea that the T2S
complex belongs to the σ[superscript E] regulon. Together, our results suggest that the T2S pathway is characterized by the growth phase-dependent
expression of genes encoding cargo proteins and requires a multifactorial regulatory network to ensure appropriate kinetics of the secretory traffic and the fitness of V. cholerae in different ecological niches
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The Genome of Tolypocladium inflatum: Evolution, Organization, and Expression of the Cyclosporin Biosynthetic Gene Cluster
The ascomycete fungus Tolypocladium inflatum, a pathogen of beetle larvae, is best known as the producer of the immunosuppressant drug cyclosporin. The draft genome of T. inflatum strain NRRL 8044 (ATCC 34921), the isolate from which cyclosporin was first isolated, is presented along with comparative analyses of the biosynthesis of cyclosporin and other secondary metabolites in T. inflatum and related taxa. Phylogenomic analyses reveal previously undetected and complex patterns of homology between the nonribosomal peptide synthetase (NRPS) that encodes for cyclosporin synthetase (simA) and those of other secondary metabolites with activities against insects (e.g., beauvericin, destruxins, etc.), and demonstrate the roles of module duplication and gene fusion in diversification of NRPSs. The secondary metabolite gene cluster responsible for cyclosporin biosynthesis is described. In addition to genes necessary for cyclosporin biosynthesis, it harbors a gene for a cyclophilin, which is a member of a family of immunophilins known to bind cyclosporin. Comparative analyses support a lineage specific origin of the cyclosporin gene cluster rather than horizontal gene transfer from bacteria or other fungi. RNA-Seq transcriptome analyses in a cyclosporin-inducing medium delineate the boundaries of the cyclosporin cluster and reveal high levels of expression of the gene cluster cyclophilin. In medium containing insect hemolymph, weaker but significant upregulation of several genes within the cyclosporin cluster, including the highly expressed cyclophilin gene, was observed. T. inflatum also represents the first reference draft genome of Ophiocordycipitaceae, a third family of insect pathogenic fungi within the fungal order Hypocreales, and supports parallel and qualitatively distinct radiations of insect pathogens. The T. inflatum genome provides additional insight into the evolution and biosynthesis of cyclosporin and lays a foundation for further investigations of the role of secondary metabolite gene clusters and their metabolites in fungal biology
DELAY OF GERMINATION 1-LIKE 4 acts as an inducer of seed reserve accumulation
More than 70% of global food supply depends on seeds. The major seed reserves, such as proteins, lipids, and polysaccharides, are produced during seed maturation. Here, we report that DELAY OF GERMINATION 1-LIKE 4 (DOGL4) is a major inducer of reserve accumulation during seed maturation. The DOGL family proteins are plant-specific proteins of largely unknown biochemical function. DOGL4 shares only limited homology in amino acid sequence with DOG1, a major regulator of seed dormancy. DOGL4 was identified as one of the outstanding abscisic acid (ABA)-induced genes in our RNA sequencing analysis, whereas DOG1 was not induced by ABA. Induction of DOGL4 caused the expression of 70 seed maturation-specific genes, even in germinating seeds, including the major seed reserves ALBUMIN, CRUCIFERIN and OLEOSIN. Although DOG1 affects the expression of many seed maturation genes, the major seed reserve genes induced by DOGL4 are not altered by the dog1 mutation. Furthermore, the reduced dormancy and longevity phenotypes observed in the dog1 seeds were not observed in the dogl4 mutants, suggesting that these two genes have limited functional overlap. Taken together, these results suggest that DOGL4 is a central factor mediating reserve accumulation in seeds, and that the two DOG1 family proteins have diverged over the course of evolution into independent regulators of seed maturation, but retain some overlapping function.</p
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ZielkeRyszardPharmacyTypeIISecretion.pdf
Vibrio cholerae, an etiological agent of cholera, circulates between aquatic reservoirs and the human gastrointestinal tract. The
type II secretion (T2S) system plays a pivotal role in both stages of the lifestyle by exporting multiple proteins, including cholera
toxin. Here, we studied the kinetics of expression of genes encoding the T2S system and its cargo proteins. We have found that
under laboratory growth conditions, the T2S complex was continuously expressed throughout V. cholerae growth, whereas
there was growth phase-dependent transcriptional activity of genes encoding different cargo proteins. Moreover, exposure of V.
cholerae to different environmental cues encountered by the bacterium in its life cycle induced transcriptional expression of T2S.
Subsequent screening of a V. cholerae genomic library suggested that σ[superscript E] stress response, phosphate metabolism, and the second
messenger 3',5'-cyclic diguanylic acid (c-di-GMP) are involved in regulating transcriptional expression of T2S. Focusing on σ[superscript E],
we discovered that the upstream region of the T2S operon possesses both the consensus σ[superscript E] and σ⁷⁰ signatures, and deletion of
the σ[superscript E] binding sequence prevented transcriptional activation of T2S by RpoE. Ectopic overexpression of σ[superscript E] stimulated transcription of T2S in wild-type and isogenic ΔrpoE strains of V. cholerae, providing additional support for the idea that the T2S
complex belongs to the σ[superscript E] regulon. Together, our results suggest that the T2S pathway is characterized by the growth phase-dependent
expression of genes encoding cargo proteins and requires a multifactorial regulatory network to ensure appropriate kinetics of the secretory traffic and the fitness of V. cholerae in different ecological niches
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ZielkeRyszardPharmacyTypeIISecretion_SupplementalMaterial.pdf
Vibrio cholerae, an etiological agent of cholera, circulates between aquatic reservoirs and the human gastrointestinal tract. The
type II secretion (T2S) system plays a pivotal role in both stages of the lifestyle by exporting multiple proteins, including cholera
toxin. Here, we studied the kinetics of expression of genes encoding the T2S system and its cargo proteins. We have found that
under laboratory growth conditions, the T2S complex was continuously expressed throughout V. cholerae growth, whereas
there was growth phase-dependent transcriptional activity of genes encoding different cargo proteins. Moreover, exposure of V.
cholerae to different environmental cues encountered by the bacterium in its life cycle induced transcriptional expression of T2S.
Subsequent screening of a V. cholerae genomic library suggested that σ[superscript E] stress response, phosphate metabolism, and the second
messenger 3',5'-cyclic diguanylic acid (c-di-GMP) are involved in regulating transcriptional expression of T2S. Focusing on σ[superscript E],
we discovered that the upstream region of the T2S operon possesses both the consensus σ[superscript E] and σ⁷⁰ signatures, and deletion of
the σ[superscript E] binding sequence prevented transcriptional activation of T2S by RpoE. Ectopic overexpression of σ[superscript E] stimulated transcription of T2S in wild-type and isogenic ΔrpoE strains of V. cholerae, providing additional support for the idea that the T2S
complex belongs to the σ[superscript E] regulon. Together, our results suggest that the T2S pathway is characterized by the growth phase-dependent
expression of genes encoding cargo proteins and requires a multifactorial regulatory network to ensure appropriate kinetics of the secretory traffic and the fitness of V. cholerae in different ecological niches
Population-Specific Regulation of Chmp2b by <em>Lbx1</em> during Onset of Synaptogenesis in Lateral Association Interneurons
<div><p>Chmp2b is closely related to Vps2, a key component of the yeast protein complex that creates the intralumenal vesicles of multivesicular bodies. Dominant negative mutations in Chmp2b cause autophagosome accumulation and neurodegenerative disease. Loss of Chmp2b causes failure of dendritic spine maturation in cultured neurons. The homeobox gene <em>Lbx1</em> plays an essential role in specifying postmitotic dorsal interneuron populations during late pattern formation in the neural tube. We have discovered that Chmp2b is one of the most highly regulated cell-autonomous targets of <em>Lbx1</em> in the embryonic mouse neural tube. Chmp2b was expressed and depended on <em>Lbx1</em> in only two of the five nascent, Lbx1-expressing, postmitotic, dorsal interneuron populations. It was also expressed in neural tube cell populations that lacked Lbx1 protein. The observed population-specific expression of Chmp2b indicated that only certain population-specific combinations of sequence specific transcription factors allow Chmp2b expression. The cell populations that expressed Chmp2b corresponded, in time and location, to neurons that make the first synapses of the spinal cord. Chmp2b protein was transported into neurites within the motor- and association-neuropils, where the first synapses are known to form between E11.5 and E12.5 in mouse neural tubes. Selective, developmentally-specified gene expression of Chmp2b may therefore be used to endow particular neuronal populations with the ability to mature dendritic spines. Such a mechanism could explain how mammalian embryos reproducibly establish the disynaptic cutaneous reflex only between particular cell populations.</p> </div
Chmp2b Protein in Soma and Dendrites of Motor Neurons.
<p>Chmp2b and Isl1 are co-expressed in motor columns of the ventral horn. (A, C, E) Isl1 primary antibody was detected with Cy3 (red). Chmp2b antibody was detected with Cy5 (infrared) and is shown in green. (B, D, F) Greyscale images of the green channel of images shown in A, C, and E, respectively. Chmp2b staining within soma can be distinguished from staining of projections between soma. The most sensitive secondary antibody (Cy3) and relatively long image acquisition times are required to detect Chmp2b signal in the motor columns. (G, H) Comparison of the VLF of heterozygotes and mutants stained with Chmp2b (red; Cy3) and tubulin (blue; Cy5). Radial breaks in the tubulin stains of heterozygotes (indicated by pairs of arrowheads) may represent the endfeet of radial glial cells (see inset I) that dendrites of motor neurons have been shown to follow into the VLF during early synaptogenesis (diagrammed by flow arrow). Note the loss of tubulin staining in the LF (†) and in the VLF below the sulcus limitans (horizontal line). Chmp2b staining in the VLF is present but distributed differently in mutants. (I) High magnification image of a putative radial glial endfoot that can be seen by background stain in the green channel. Note that Chmp2b and tubulin staining do not colocalize but appear to associate closely on the surface of the endfoot.</p
Chmp2b Colocalization with MAP2a in DLF and VLF.
<p>(A–I) Comparison of Chmp2b/MAP2a colabeling with Chmp2b/ß-tubulin colabeling in the VLF in adjacent sections (J–R) Comparison of Chmp2b/MAP2a colabeling with Chmp2b/ß-tubulin colabeling in the DLF in adjacent sections. Note that Chmp2b and MAP2a colocalize, while Chmp2b and ß-tubulin are only in close apposition.</p