A mutation in the nuclear-encoded plastid ribosomal protein S9 leads to early embryo lethality in maize.
Plant J.. 37:92–103. Abstract
Seeds of the lethal embryo 1 (lem1) mutant in maize (Zea mays) display a non-concordant lethal phenotype: whereas the embryo aborts very early, before the transition stage, the endosperm develops almost normally. The mutant was identified in a collection of maize lines that carried the transposon Activation (Ac) at different locations in the genome. Co-segregation and reversion analysis showed that lem1 was tagged by Ac. The lem1 gene encodes a protein that is highly similar to the rice plastid 30S ribosomal protein S9 (PRPS9). lem1 maps to chromosome 1L and appears to be the only copy of prps9 in the maize genome. Green fluorescent protein (GFP) fusion constructs containing only the putative transit peptide (TP) of LEM1 localize exclusively to the plastids, confirming that the LEM1 protein is a PRP. In contrast, GFP fusion constructs containing the entire LEM1 protein co-localize to the plastids and to the nucleus, suggesting a possible dual function for this protein. Two alternative, although not mutually exclusive, explanations are considered for the lem phenotype of the lem1 mutant: (i) functional plastids are required for normal embryo development; and (ii) the PRPS9 has an extra-ribosomal function required for embryogenesis.
Drosophila MAD, a member of the Smad family, translocates to the nucleus upon stimulation of the dpp pathway.
Biochem Biophys Res Commun. 238:595-8. Abstract
Smads are a novel group of proteins which act to mediate signaling by members of the TGF-beta superfamily. Seven vertebrate Smad genes, which fall into three classes, have been reported. Members of the Class I Smads have been shown to bind to the cytoplasmic portion of the TGF-beta like receptors, where they become phosphorylated and translocate to the nucleus. Once in the nucleus they may function as transcriptional activators. We wondered if translocation to the nucleus is a general property of the Smads and whether it was evolutionarily conserved. We examined the subcellular localization of Drosophila MAD and found that it is capable of nuclear translocation, in Drosophila S2 cells, when the dpp pathway is stimulated. To prove the functional conservation of receptor/Smad interactions, we used the mouse BMP type I receptor ALK6 to stimulate the pathway and found that it is capable of sending MAD to the nucleus. These results show that cytoplasmic localization with translocation to the nucleus upon stimulation is a feature of the Smads that is conserved through evolution.
C. elegans serine-threonine kinase KIN-29 modulates TGFβ signaling and regulates body size formation.
BMC developmental biology. 5:8. Abstract
BACKGROUND: In C. elegans there are two well-defined TGFbeta-like signaling pathways. The Sma/Mab pathway affects body size morphogenesis, male tail development and spicule formation while the Daf pathway regulates entry into and exit out of the dauer state. To identify additional factors that modulate TGFbeta signaling in the Sma/Mab pathway, we have undertaken a genetic screen for small animals and have identified kin-29. RESULTS: kin-29 encodes a protein with a cytoplasmic serine-threonine kinase and a novel C-terminal domain. The kinase domain is a distantly related member of the EMK (ELKL motif kinase) family, which interacts with microtubules. We show that the serine-threonine kinase domain has in vitro activity. kin-29 mutations result in small animals, but do not affect male tail morphology as do several of the Sma/Mab signal transducers. Adult worms are smaller than the wild-type, but also develop more slowly. Rescue by kin-29 is achieved by expression in neurons or in the hypodermis. Interaction with the dauer pathway is observed in double mutant combinations, which have been seen with Sma/Mab pathway mutants. We show that kin-29 is epistatic to the ligand dbl-1, and lies upstream of the Sma/Mab pathway target gene, lon-1. CONCLUSION: kin-29 is a new modulator of the Sma/Mab pathway. It functions in neurons and in the hypodermis to regulate body size, but does not affect all TGFbeta outputs, such as tail morphogenesis.
Lon-1 regulates Caenorhabditis elegans body size downstream of the Dbl-1 TGFβ signaling pathway.
Dev Biol. 246:418-428. Abstract
In Caenorhabditis elegans, two well-characterized TGF beta signaling cascades have been identified: the Small/Male tail abnormal (Sma/Mab) and Dauer formation (Daf) pathways. The Sma/Mab pathway regulates body size morphogenesis and male tail development. The ligand of the pathway, dbl-1, transmits its signal through two receptor serine threonine kinases, daf-4 and sma-6, which in turn regulate the activity of the Smads, sma-2, sma-3, and sma-4. In general, Smads have been shown to both positively and negatively regulate the transcriptional activity of downstream target genes in various organisms. In C. elegans, however, target genes have remained elusive. We have cloned and characterized lon-1, a gene with homology to the cysteine-rich secretory protein (CRISP) family of proteins. lon-1 regulates body size morphogenesis, but does not affect male tail development. lon-1 is expressed in hypodermal tissues, which is the focus of body size determination, similar to sma-2, sma-4, and sma-6. Using genetic methods, we show that lon-1 lies downstream of the Sma/Mab signaling cascade and demonstrate that lon-1 mRNA levels are up-regulated in sma-6-null mutant animals. This provides evidence that lon-1 is negatively regulated by Sma/Mab pathway signaling. Taken together, these data identify lon-1 as a novel downstream target gene of the dbl-1 TGF beta-like signaling pathway.
Influence of Notch on dorsoventral compartmentalization and actin organization in the Drosophila wing.
Development (Cambridge, England). 132:3823-33. Abstract
Compartment boundaries play key roles in tissue organization by separating cell populations. Activation of the Notch receptor is required for dorsoventral (DV) compartmentalization of the Drosophila wing, but the nature of its requirement has been controversial. Here, we provide additional evidence that a stripe of Notch activation is sufficient to establish a sharp separation between cell populations, irrespective of their dorsal or ventral identities. We further find that cells at the DV compartment boundary are characterized by a distinct shape, a smooth interface, and an accumulation of F-actin at the adherens junction. Genetic manipulation establishes that a stripe of Notch activation is both necessary and sufficient for this DV boundary cell phenotype, and supports the existence of a non-transcriptional branch of the Notch pathway that influences F-actin. Finally, we identify a distinct requirement for a regulator of actin polymerization, capulet, in DV compartmentalization. These observations imply that Notch effects compartmentalization through a novel mechanism, which we refer to as a fence, that does not depend on the establishment of compartment-specific cell affinities, but does depend on the organization of the actin cytoskeleton.
Localization and requirement for Myosin II at the dorsal-ventral compartment boundary of the Drosophila wing.
Developmental dynamics : an official publication of the American Association of Anatomists. 235:3051-8. Abstract
As organisms develop, their tissues can become separated into distinct cell populations through the establishment of compartment boundaries. Compartment boundaries have been discovered in a wide variety of tissues, but in many cases the molecular mechanisms that separate cells remain poorly understood. In the Drosophila wing, a stripe of Notch activation maintains the dorsal-ventral compartment boundary, through a process that depends on the actin cytoskeleton. Here, we show that the dorsal-ventral boundary exhibits a distinct accumulation of Myosin II, and that this accumulation is regulated downstream of Notch signaling. Conversely, the dorsal-ventral boundary is depleted for the Par-3 homologue Bazooka. We further show that mutations in the Myosin heavy chain subunit encoded by zipper can impair dorsal-ventral compartmentalization without affecting anterior-posterior compartmentalization. These observations identify a distinct accumulation and requirement for Myosin activity in dorsal-ventral compartmentalization, and suggest a novel mechanism in which contractile tension along an F-actin cable at the compartment boundary contributes to compartmentalization.
Engineering the plastid genome of Nicotiana sylvestris, a diploid model species for plastid genetics.
Methods in Molecular Biology. 701:37-50. Abstract
The plastids of higher plants have their own approximately 120-160-kb genome that is present in 1,000-10,000 copies per cell. Engineering of the plastid genome (ptDNA) is based on homologous recombination between the plastid genome and cloned ptDNA sequences in the vector. A uniform population of engineered ptDNA is obtained by selection for marker genes encoded in the vectors. Manipulations of ptDNA include (1) insertion of transgenes in intergenic regions; (2) posttransformation excision of marker genes to obtain marker-free plants; (3) gene knockouts and gene knockdowns, and (4) cotransformation with multiple plasmids to introduce nonselected genes without physical linkage to marker genes. Most experiments on plastome engineering have been carried out in the allotetraploid Nicotiana tabacum. We report here for the first time plastid transformation in Nicotiana sylvestris, a diploid ornamental species. We demonstrate that the protocols and vectors developed for plastid transformation in N. tabacum are directly applicable to N. sylvestris with the advantage that the N. sylvestris transplastomic lines are suitable for mutant screens.
Plastid transformation in flowering plants.
Genomics of Chloroplasts and Mitochondria. 35:393-414. Abstract
The plastid genome of higher plants is relatively small, 120–230-kb in size, and present in up to 10,000 copies per cell. Standard protocols for the introduction of transforming DNA employ biolistic DNA delivery or polyethylene glycol treatment. Genetically stable, transgenic plants are obtained by modification of the plastid genome by homologous recombination, followed by selection for the transformed genome copy by the expression of marker genes that protect the cells from selective agents. Commonly used selective agents are antibiotics, including spectinomycin, streptomycin, kanamycin and chloramphenicol. Selection for resistance to amino acid analogues has also been successful. The types of plastid genome manipulations include gene deletion, gene insertion, and gene replacement, facilitated by specially designed transformation vectors. Methods are also available for post-transformation removal of marker genes. The model species for plastid genetic manipulation is Nicotiana tabacum, in which most protocols have been tested. Plastid transformation is also available in several solanaceous crops (tomato, potato, eggplant) and ornamental species (petunia, Nicotianasylvestris). Significant progress has been made with Brasssicaceae including cabbage, oilseed rape and Arabidopsis. Recent additions to the crops in which plastid transformation is reproducibly obtained are lettuce, soybean and sugar beet. The monocots are a taxonomic group recalcitrant to plastid transformation; initial inroads have been made only in rice.
Kinetics of proton-coupled electron-transfer reactions to the manganese-oxo “cubane” complexes containing the Mn4O and Mn4O core types.
Proceedings of the National Academy of Sciences. 100:3707-3712. Abstract
The kinetics of proton-coupled electron-transfer (pcet) reactions are reported for Mn4O4(O2PPh2)6, 1, and [Mn4O4(O2PPh2)6]+, 1+, with phenothiazine (pzH). Both pcet reactions form 1H, by H transfer to 1 and by hydride transfer to 1+. Surprisingly, the rate constants differ by only 25% despite large differences in the formal charges and driving force. The driving force is proportional to the difference in the bond-dissociation energies (BDE >94 kcal/mol for homolytic, 1H → H + 1, vs. ≈127 kcal/mol for heterolytic, 1H → H− + 1+, dissociation of the O—H bond in 1H). The enthalpy and entropy of activation for the homolytic reaction (ΔH‡ = −1.2 kcal/mol and ΔS‡ = −32 cal/mol⋅K; 25–6.7°C) reveal a low activation barrier and an appreciable entropic penalty in the transition state. The rate-limiting step exhibits no H/D kinetic isotope effect (kH/kD = 0.96) for the first H atom-transfer step and a small kinetic isotope effect (1.4) for the second step (1H + pzH → 1H2 + pz•). These lines of evidence indicate that formation of a reactive precursor complex before atom transfer is rate-limiting (conformational gating), and that little or no N—H bond cleavage occurs in the transition state. H-atom transfer from pzH to alkyl, alkoxyl, and peroxyl radicals reveals that BDEs are not a good predictor of the rates of this reaction. Hydride transfer to 1+ provides a concrete example of two-electron pcet that is hypothesized for the O—H bond cleavage step during catalysis of photosynthetic water oxidation.
Dachs: an unconventional myosin that functions downstream of Fat to regulate growth, affinity and gene expression in Drosophila.
Development (Cambridge, England). 133:2539-51. Abstract
The dachs gene was first identified almost a century ago based on its requirements for appendage growth, but has been relatively little studied. Here, we describe the phenotypes of strong dachs mutations, report the cloning of the dachs gene, characterize the localization of Dachs protein, and investigate the relationship between Dachs and the Fat pathway. Mutation of dachs reduces, but does not abolish, the growth of legs and wings. dachs encodes an unconventional myosin that preferentially localizes to the membrane of imaginal disc cells. dachs mutations suppress the effects of fat mutations on gene expression, cell affinity and growth in imaginal discs. Dachs protein localization is influenced by Fat, Four-jointed and Dachsous, consistent with its genetic placement downstream of fat. However, dachs mutations have only mild tissue polarity phenotypes, and only partially suppress the tissue polarity defects of fat mutants. Our results implicate Dachs as a crucial downstream component of a Fat signaling pathway that influences growth, affinity and gene expression during development.
Characterization of a Dchs1 mutant mouse reveals requirements for Dchs1-Fat4 signaling during mammalian development.
Development (Cambridge, England). 138:947-57. Abstract
The Drosophila Dachsous and Fat proteins function as ligand and receptor, respectively, for an intercellular signaling pathway that regulates Hippo signaling and planar cell polarity. Although gene-targeted mutations in two mammalian Fat genes have been described, whether mammals have a Fat signaling pathway equivalent to that in Drosophila, and what its biological functions might be, have remained unclear. Here, we describe a gene-targeted mutation in a murine Dachsous homolog, Dchs1. Analysis of the phenotypes of Dchs1 mutant mice and comparisons with Fat4 mutant mice identify requirements for these genes in multiple organs, including the ear, kidney, skeleton, intestine, heart and lung. Dchs1 and Fat4 single mutants and Dchs1 Fat4 double mutants have similar phenotypes throughout the body. In some cases, these phenotypes suggest that Dchs1-Fat4 signaling influences planar cell polarity. In addition to the appearance of cysts in newborn kidneys, we also identify and characterize a requirement for Dchs1 and Fat4 in growth, branching and cell survival during early kidney development. Dchs1 and Fat4 are predominantly expressed in mesenchymal cells in multiple organs, and mutation of either gene increases protein staining for the other. Our analysis implies that Dchs1 and Fat4 function as a ligand-receptor pair during murine development, and identifies novel requirements for Dchs1-Fat4 signaling in multiple organs.
Drosophila lowfat, a novel modulator of Fat signaling.
Development (Cambridge, England). 136:3223-33. Abstract
The Fat-Hippo-Warts signaling network regulates both transcription and planar cell polarity. Despite its crucial importance to the normal control of growth and planar polarity, we have only a limited understanding of the mechanisms that regulate Fat. We report here the identification of a conserved cytoplasmic protein, Lowfat (Lft), as a modulator of Fat signaling. Drosophila Lft, and its human homologs LIX1 and LIX1-like, bind to the cytoplasmic domains of the Fat ligand Dachsous, the receptor protein Fat, and its human homolog FAT4. Lft protein can localize to the sub-apical membrane in disc cells, and this membrane localization is influenced by Fat and Dachsous. Lft expression is normally upregulated along the dorsoventral boundary of the developing wing, and is responsible for elevated levels of Fat protein there. Levels of Fat and Dachsous protein are reduced in lft mutant cells, and can be increased by overexpression of Lft. lft mutant animals exhibit a wing phenotype similar to that of animals with weak alleles of fat, and lft interacts genetically with both fat and dachsous. These studies identify Lft as a novel component of the Fat signaling pathway, and the Lft-mediated elevation of Fat levels as a mechanism for modulating Fat signaling.
Direct observation of abortive initiation and promoter escape within single immobilized transcription complexes..
Biophysical journal. 90(4):1419-31. Abstract
Using total-internal-reflection fluorescence microscopy equipped with alternating-laser excitation, we were able to detect abortive initiation and promoter escape within single immobilized transcription complexes. Our approach uses fluorescence resonance energy transfer to monitor distances between a fluorescent probe incorporated in RNA polymerase (RNAP) and a fluorescent probe incorporated in DNA. We observe small, but reproducible and abortive-product-length-dependent, decreases in distance between the RNAP leading edge and DNA downstream of RNAP upon abortive initiation, and we observe large decreases in distance upon promoter escape. Inspection of population distributions and single-molecule time traces for abortive initiation indicates that, at a consensus promoter, at saturating ribonucleoside triphosphate concentrations, abortive-product release is rate-limiting (i.e., abortive-product synthesis and RNAP-active-center forward translocation are fast, whereas abortive-product dissociation and RNAP-active-center reverse translocation are slow). The results obtained using this new methodology confirm and extend those obtained from diffusing single molecules, and pave the way for real-time, single-molecule observations of the transitions between various states of the transcription complex throughout transcription.
Altering the DNA-binding Specificity of the Yeast Matalpha 2 Homeodomain Protein.
J Biol Chem. 276:32696-32703. Abstract
Homeodomain proteins are a highly conserved class of DNA-binding proteins that are found in virtually every eukaryotic organism. The conserved mechanism that these proteins use to bind DNA suggests that there may be at least a partial DNA recognition code for this class of proteins. To test this idea, we have investigated the sequence-specific requirements for DNA binding and repression by the yeast alpha2 homeodomain protein in association with its cofactors, Mcm1 and Mata1. We have determined the contribution for each residue in the alpha2 homeodomain that contacts the DNA in the co-crystal structures of the protein. We have also engineered mutants in the alpha2 homeodomain to alter the DNA-binding specificity of the protein. Although we were unable to change the specificity of alpha2 by making substitutions at residues 47, 54, and 55, we were able to alter the DNA-binding specificity by making substitutions at residue 50 in the homeodomain. Since other homeodomain proteins show similar changes in specificity with substitutions at residue 50, this suggests that there is at least a partial DNA recognition code at this position.
Repression of the Yeast Ho gene by the MATalpha2 and MATa1 Homeodomain Proteins.
Nucleic Acids Res. 32:6469-6478. Abstract
The HO gene in Saccharomyces cerevisiae is regulated by a large and complex promoter that is similar to promoters in higher order eukaryotes. Within this promoter are 10 potential binding sites for the a1-alpha2 heterodimer, which represses HO and other haploid-specific genes in diploid yeast cells. We have determined that a1-alpha2 binds to these sites with differing affinity, and that while certain strong-affinity sites are crucial for repression of HO, some of the weak-affinity sites are dispensable. However, these weak-affinity a1-alpha2-binding sites are strongly conserved in related yeast species and have a role in maintaining repression upon the loss of strong-affinity sites. We found that these weak sites are sufficient for a1-alpha2 to partially repress HO and recruit the Tup1-Cyc8 (Tup1-Ssn6) co-repressor complex to the HO promoter. We demonstrate that the Swi5 activator protein is not bound to URS1 in diploid cells, suggesting that recruitment of the Tup1-Cyc8 complex by a1-alpha2 prevents DNA binding by activator proteins resulting in repression of HO.
Rfm1, a Novel Tethering Factor Required to Recruit the Hst1 Histone Deacetylase for Repression of Middle Sporulation Genes.
Mol Cell Biol. 23:2009-2016. Abstract
Transcriptional repression is often correlated with the alteration of chromatin structure through modifications of the nucleosomes in the promoter region, such as by deacetylation of the N-terminal histone tails. This is presumed to make the promoter region inaccessible to other regulatory factors and the general transcription machinery. To accomplish this, histone deacetylases are recruited to specific promoters via DNA-binding proteins and tethering factors. We have previously reported the requirement for the NAD(+)-dependent histone deacetylase Hst1 and the DNA-binding protein Sum1 for vegetative repression of many middle sporulation genes in Saccharomyces cerevisiae. Here we report the identification of a novel tethering factor, Rfm1, that is required for Hst1-mediated repression. Rfm1 interacts with both Sum1 and Hst1 and is required for the Sum1-Hst1 interaction. DNA microarray and Northern blot analyses showed that Rfm1 is required for repression of the same subset of Sum1-repressed genes that require Hst1. These results suggest that Rfm1 is a specificity factor that targets the Hst1 deacetylase to a subset of Sum1-regulated genes.