Song, R, Llaca V, Linton E, Messing J.  2001.  Sequence, regulation, and evolution of the maize 22-kD alpha zein gene family. Genome research. 11:1817-25. AbstractWebsite
We have isolated and sequenced all 23 members of the 22-kD alpha zein (z1C) gene family of maize. This is one of the largest plant gene families that has been sequenced from a single genetic background and includes the largest contiguous genomic DNA from maize with 346,292 bp to date. Twenty-two of the z1C members are found in a roughly tandem array on chromosome 4S forming a dense gene cluster 168,489-bp long. The twenty-third copy of the gene family is also located on chromosome 4S at a site approximately 20 cM closer to the centromere and appears to be the wild-type allele of the floury-2 (fl2) mutation. On the basis of an analysis of maize cDNA databases, only seven of these genes appear to be expressed including the fl2 allele. The expressed genes in the cluster are interspersed with nonexpressed genes. Interestingly, some of the expressed genes differ in their transcriptional regulation. Gene amplification appears to be in blocks of genes explaining the rapid and compact expansion of the cluster during the evolution of maize.
Messing, J.  2001.  The universal primers and the shotgun DNA sequencing method. Methods in molecular biology (Clifton, NJ). 167:13-31.Website
Mathias, JR, Zhong H, Jin Y, Vershon AK.  2001.  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.
Ananyev, GM, Zaltsman L, Vasko C, Dismukes GC.  2001.  The inorganic biochemistry of photosynthetic oxygen evolution/water oxidation. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1503:52-68.Website
Dismukes, GC, Klimov VV, Baranov SV, Kozlov YN, Dasgupta J, Tyryshkin A.  2001.  The origin of atmospheric oxygen on Earth: The innovation of oxygenic photosynthesis. Proceedings of the National Academy of Sciences of the United States of America. 98:2170-2175. AbstractWebsite
The evolution of O-2-producing cyanobacteria that use water as terminal reductant transformed Earth's atmosphere to one suitable for the evolution of aerobic metabolism and complex life. The innovation of water oxidation freed photosynthesis to invade new environments and visibly changed the face of the Earth. We offer a new hypothesis for how this process evolved, which identifies two critical roles for carbon dioxide in the Archean period. First, we present a thermodynamic analysis showing that bicarbonate (formed by dissolution of CO2) is a more efficient alternative substrate than water for O-2 production by oxygenic phototrophs. This analysis clarifies the origin of the long debated "bicarbonate effect" on photosynthetic O-2 production. We propose that bicarbonate was the thermodynamically preferred reductant before water in the evolution of oxygenic photosynthesis. Second, we have examined the speciation of manganese(II) and bicarbonate in water, and find that they form Mn-bicarbonate clusters as the major species under conditions that model the chemistry of the Archean sea. These clusters have been found to be highly efficient precursors for the assembly of the tetramanganese-oxide core of the water-oxidizing enzyme during biogenesis. We show that these clusters can be oxidized at electrochemical potentials that are accessible to anoxygenic phototrophs and thus the most likely building blocks for assembly of the first O-2 evolving photoreaction center, most likely originating from green nonsulfur bacteria before the evolution of cyanobacteria.
Yagi, M, Wolf KV, Baesjou PJ, Bernasek SL, Dismukes CG.  2001.  Selective Photoproduction of O2 from the Mn4O4 Cubane Core: A Structural and Functional Model for the Photosynthetic Water-Oxidizing Complex. Angewandte Chemie. 113:3009-3012.Website
Zimmerman, CM, Padgett RW.  2000.  Transforming growth factor-β signaling mediators and modulators. Gene. 249:17-30. AbstractWebsite
Transforming growth factor beta is a multi-functional growth and differentiation factor responsible for regulating many diverse biological processes in both vertebrate and invertebrate species. Among the most dramatic of TGFbeta's effects are those associated with specification of cell fates during development and inhibition of cell cycle progression. The core TGFbeta signaling pathway has now been described using a synergistic combination of genetic and biochemical approaches. Transmembrane receptors with intrinsic protein serine kinase activity bind ligand in the extracellular milieu and then phosphorylate intracellular proteins known as Smads. Phosphorylated Smads form heterooligomers and translocate into the nucleus where they can modulate transcriptional responses. More recent studies indicate that many other proteins serve as modulators of Smad activity, and utimately define specific cellular responses to TGFbeta. Here we describe both the simplistic core TGFbeta signaling pathway and the growing number of proteins that impinge on this pathway at the level of Smad function to either enhance or inhibit TGFbeta responses.
Moloney, DJ, Panin VM, Johnston SH, Chen J, Shao L, Wilson R, Wang Y, Stanley P, Irvine KD, Haltiwanger RS et al..  2000.  Fringe is a glycosyltransferase that modifies Notch. Nature. 406:369-75. AbstractWebsite
Notch receptors function in highly conserved intercellular signalling pathways that direct cell-fate decisions, proliferation and apoptosis in metazoans. Fringe proteins can positively and negatively modulate the ability of Notch ligands to activate the Notch receptor. Here we establish the biochemical mechanism of Fringe action. Drosophila and mammalian Fringe proteins possess a fucose-specific beta1,3 N-acetylglucosaminyltransferase activity that initiates elongation of O-linked fucose residues attached to epidermal growth factor-like sequence repeats of Notch. We obtained biological evidence that Fringe-dependent elongation of O-linked fucose on Notch modulates Notch signalling by using co-culture assays in mammalian cells and by expression of an enzymatically inactive Fringe mutant in Drosophila. The post-translational modification of Notch by Fringe represents a striking example of modulation of a signalling event by differential receptor glycosylation and identifies a mechanism that is likely to be relevant to other signalling pathways.
Savage-Dunn, C, Tokarz R, Wang H, Cohen S, Giannikas C, Padgett RW.  2000.  SMA-3 smad has specific and critical functions in DBL-1/SMA-6 TGFβ-related signaling. Dev Biol. 223:70-6. AbstractWebsite
A TGFbeta signal transduction cascade controls body size and male tail morphogenesis in the nematode Caenorhabditis elegans. We have analyzed the function of the sma-3 Smad gene, one of three Smad genes that function in this pathway. Null mutations in sma-3 are at least as severe as null mutations in the ligand and type I receptor genes, dbl-1 and sma-6, indicating that the other Smads do not function in the absence of SMA-3. Furthermore, null mutations in sma-3 do not cause defects in egg laying or in regulation of the developmentally arrested dauer larva stage, indicating no overlapping function with another C. elegans TGFbeta signaling pathway. The sma-3 gene is widely expressed at all developmental stages in hermaphrodites and males. The molecular lesions associated with eight sma-3 alleles of varying severity have been determined. The missense mutations cluster in two previously identified regions important for Smad function.
Patterson, G, Padgett R.  2000.  TGFβ-related pathways. Roles in Caenorhabditis elegans development. Trends Genet. 16:27-33. AbstractWebsite
Genetic and molecular analysis in Caenorhabditis elegans has produced new insights into how TGF beta-related pathways transduce signals and the developmental processes in which they function. These pathways are essential regulators of dauer formation, body-size determination, male copulatory structures and axonal guidance. Here, we review the insights that have come from standard molecular genetic experiments and discuss how the recently completed genome sequence has contributed to our understanding of these pathways.
Liu, X, Grammont M, Irvine KD.  2000.  Roles for scalloped and vestigial in regulating cell affinity and interactions between the wing blade and the wing hinge. Developmental biology. 228:287-303. AbstractWebsite
The scalloped and vestigial genes are both required for the formation of the Drosophila wing, and recent studies have indicated that they can function as a heterodimeric complex to regulate the expression of downstream target genes. We have analyzed the consequences of complete loss of scalloped function, ectopic expression of scalloped, and ectopic expression of vestigial on the development of the Drosophila wing imaginal disc. Clones of cells mutant for a strong allele of scalloped fail to proliferate within the wing pouch, but grow normally in the wing hinge and notum. Cells overexpressing scalloped fail to proliferate in both notal and wing-blade regions of the disc, and this overexpression induces apoptotic cell death. Clones of cells overexpressing vestigial grow smaller or larger than control clones, depending upon their distance from the dorsal-ventral compartment boundary. These studies highlight the importance of correct scalloped and vestigial expression levels to normal wing development. Our studies of vestigial-overexpressing clones also reveal two further aspects of wing development. First, in the hinge region vestigial exerts both a local inhibition and a long-range induction of wingless expression. These and other observations imply that vestigial-expressing cells in the wing blade organize the development of surrounding wing-hinge cells. Second, clones of cells overexpressing vestigial exhibit altered cell affinities. Our analysis of these clones, together with studies of scalloped mutant clones, implies that scalloped- and vestigial-dependent cell adhesion contributes to separation of the wing blade from the wing hinge and to a gradient of cell affinities along the dorsal-ventral axis of the wing.
Kim, TK, Ebright RH, Reinberg D.  2000.  Mechanism of ATP-dependent promoter melting by transcription factor IIH.. Science (New York, N.Y.). 288(5470):1418-22. Abstract
We show that transcription factor IIH ERCC3 subunit, the DNA helicase responsible for adenosine triphosphate (ATP)-dependent promoter melting during transcription initiation, does not interact with the promoter region that undergoes melting but instead interacts with DNA downstream of this region. We show further that promoter melting does not change protein-DNA interactions upstream of the region that undergoes melting but does change interactions within and downstream of this region. Our results rule out the proposal that IIH functions in promoter melting through a conventional DNA-helicase mechanism. We propose that IIH functions as a molecular wrench: rotating downstream DNA relative to fixed upstream protein-DNA interactions, thereby generating torque on, and melting, the intervening DNA.
Naryshkin, N, Revyakin A, Kim Y, Mekler V, Ebright RH.  2000.  Structural organization of the RNA polymerase-promoter open complex.. Cell. 101(6):601-11. Abstract
We have used systematic site-specific protein-DNA photocrosslinking to define interactions between bacterial RNA polymerase (RNAP) and promoter DNA in the catalytically competent RNAP-promoter open complex (RPo). We have mapped more than 100 distinct crosslinks between individual segments of RNAP subunits and individual phosphates of promoter DNA. The results provide a comprehensive description of protein-DNA interactions in RPo, permit construction of a detailed model for the structure of RPo, and permit analysis of effects of a transcriptional activator on the structure of RPo.
Tan, Q, Linask KL, Ebright RH, Woychik NA.  2000.  Activation mutants in yeast RNA polymerase II subunit RPB3 provide evidence for a structurally conserved surface required for activation in eukaryotes and bacteria.. Genes & development. 14(3):339-48. Abstract
We have identified a mutant in RPB3, the third-largest subunit of yeast RNA polymerase II, that is defective in activator-dependent transcription, but not defective in activator-independent, basal transcription. The mutant contains two amino-acid substitutions, C92R and A159G, that are both required for pronounced defects in activator-dependent transcription. Synthetic enhancement of phenotypes of C92R and A159G, and of several other pairs of substitutions, is consistent with a functional relationship between residues 92-95 and 159-161. Homology modeling of RPB3 on the basis of the crystallographic structure of alphaNTD indicates that residues 92-95 and 159-162 are likely to be adjacent within the structure of RPB3. In addition, homology modeling indicates that the location of residues 159-162 within RPB3 corresponds to the location of an activation target within alphaNTD (the target of activating region 2 of catabolite activator protein, an activation target involved in a protein-protein interaction that facilitates isomerization of the RNA polymerase promoter closed complex to the RNA polymerase promoter open complex). The apparent finding of a conserved surface required for activation in eukaryotes and bacteria raises the possibility of conserved mechanisms of activation in eukaryotes and bacteria.
Ebright, RH.  2000.  RNA polymerase: structural similarities between bacterial RNA polymerase and eukaryotic RNA polymerase II.. Journal of molecular biology. 304(5):687-98. Abstract
Bacterial RNA polymerase and eukaryotic RNA polymerase II exhibit striking structural similarities, including similarities in overall structure, relative positions of subunits, relative positions of functional determinants, and structures and folding topologies of subunits. These structural similarities are paralleled by similarities in mechanisms of interaction with DNA.
Meibom, KL, Kallipolitis BH, Ebright RH, Valentin-Hansen P.  2000.  Identification of the subunit of cAMP receptor protein (CRP) that functionally interacts with CytR in CRP-CytR-mediated transcriptional repression.. The Journal of biological chemistry. 275(16):11951-6. Abstract
At promoters of the Escherichia coli CytR regulon, the cAMP receptor protein (CRP) interacts with the repressor CytR to form transcriptionally inactive CRP-CytR-promoter or (CRP)(2)-CytR-promoter complexes. Here, using "oriented heterodimer" analysis, we show that only one subunit of the CRP dimer, the subunit proximal to CytR, functionally interacts with CytR in CRP-CytR-promoter and (CRP)(2)-CytR-promoter complexes. Our results provide information about the architecture of CRP-CytR-promoter and (CRP)(2)-CytR-promoter complexes and rule out the proposal that masking of activating region 2 of CRP is responsible for the transcriptional inactivity of the complexes.
Boyer, LA, Shao X, Ebright RH, Peterson CL.  2000.  Roles of the histone H2A-H2B dimers and the (H3-H4)(2) tetramer in nucleosome remodeling by the SWI-SNF complex.. The Journal of biological chemistry. 275(16):11545-52. Abstract
SWI-SNF is an ATP-dependent chromatin remodeling complex required for expression of a number of yeast genes. Previous studies have suggested that SWI-SNF action may remove or rearrange the histone H2A-H2B dimers or induce a novel alteration in the histone octamer. Here, we have directly tested these and other models by quantifying the remodeling activity of SWI-SNF on arrays of (H3-H4)(2) tetramers, on nucleosomal arrays reconstituted with disulfide-linked histone H3, and on arrays reconstituted with histone H3 derivatives site-specifically modified at residue 110 with the fluorescent probe acetylethylenediamine-(1,5)-naphthol sulfonate. We find that SWI-SNF can remodel (H3-H4)(2) tetramers, although tetramers are poor substrates for SWI-SNF remodeling compared with nucleosomal arrays. SWI-SNF can also remodel nucleosomal arrays that harbor disulfide-linked (H3-H4)(2) tetramers, indicating that SWI-SNF action does not involve an obligatory disruption of the tetramer. Finally, we find that although the fluorescence emission intensity of acetylethylenediamine-(1,5)-naphthol sulfonate-modified histone H3 is sensitive to octamer structure, SWI-SNF action does not alter fluorescence emission intensity. These data suggest that perturbation of the histone octamer is not a requirement or a consequence of ATP-dependent nucleosome remodeling by SWI-SNF.
McKim, KS, Jang JK, Sekelsky JJ, Laurencon A, Hawley RS.  2000.  mei-41 is required for precocious anaphase in Drosophila females. Chromosoma. 109:44-49.
Page, SL, McKim KS, Deneen B, Van Hook TL, Hawley RS.  2000.  Genetic studies of mei-P26 reveal a link between the processes that control germ cell proliferation in both sexes and those that control meiotic exchange in Drosophila. Genetics. 155:1757-72.. AbstractWebsite
We present the cloning and characterization of mei-P26, a novel P- element-induced exchange-defective female meiotic mutant in Drosophila melanogaster. Meiotic exchange in females homozygous for mei-P26(1) is reduced in a polar fashion, such that distal chromosomal regions are the most severely affected. Additional alleles generated by duplication of the P element reveal that mei-P26 is also necessary for germline differentiation in both females and males. To further assess the role of mei-P26 in germline differentiation, we tested double mutant combinations of mei-P26 and bag-of-marbles (bam), a gene necessary for the control of germline differentiation and proliferation in both sexes. A null mutation at the bam locus was found to act as a dominant enhancer of mei-P26 in both males and females. Interestingly, meiotic exchange in mei-P26(1); bam(Delta)(86)/+ females is also severely decreased in comparison to mei-P26(1) homozygotes, indicating that bam affects the meiotic phenotype as well. These data suggest that the pathways controlling germline differentiation and meiotic exchange are related and that factors involved in the mitotic divisions of the germline may regulate meiotic recombination.
Kim, J, Bortz E, Zhong H, Leeuw T, Leberer E, Vershon AK, Hirsch JP.  2000.  Localization and Signaling of G(beta) Subunit Ste4p are Controlled by A-factor Receptor and the A-specific Protein Asg7p. Mol Cell Biol. 20:8826-8835. Abstract
Haploid yeast cells initiate pheromone signaling upon the binding of pheromone to its receptor and activation of the coupled G protein. A regulatory process termed receptor inhibition blocks pheromone signaling when the a-factor receptor is inappropriately expressed in MATa cells. Receptor inhibition blocks signaling by inhibiting the activity of the G protein beta subunit, Ste4p. To investigate how Ste4p activity is inhibited, its subcellular location was examined. In wild-type cells, alpha-factor treatment resulted in localization of Ste4p to the plasma membrane of mating projections. In cells expressing the a-factor receptor, alpha-factor treatment resulted in localization of Ste4p away from the plasma membrane to an internal compartment. An altered version of Ste4p that is largely insensitive to receptor inhibition retained its association with the membrane in cells expressing the a-factor receptor. The inhibitory function of the a-factor receptor required ASG7, an a-specific gene of previously unknown function. ASG7 RNA was induced by pheromone, consistent with increased inhibition as the pheromone response progresses. The a-factor receptor inhibited signaling in its liganded state, demonstrating that the receptor can block the signal that it initiates. ASG7 was required for the altered localization of Ste4p that occurs during receptor inhibition, and the subcellular location of Asg7p was consistent with its having a direct effect on Ste4p localization. These results demonstrate that Asg7p mediates a regulatory process that blocks signaling from a G protein beta subunit and causes its relocalization within the cell.
Ruettinger, W, Yagi M, Wolf K, Bernasek S, Dismukes GC.  2000.  O2 Evolution from the Manganese−Oxo Cubane Core Mn4O46+:  A Molecular Mimic of the Photosynthetic Water Oxidation Enzyme? Journal of the American Chemical Society. 122:10353-10357. AbstractWebsite